DH5α E. coli transformation with 008_pcDNA_Zeo_mTagBFP2_TGA_RT20s_Int_P2A_mScarlet-i and 009_pcDNA_Zeo_1xMmMT3-PfVlp_FLAG_miRFP670nano constructs

Goal;

Transformation was performed to introduce plasmid DNA construct 008_pcDNA_Zeo_mTagBFP2_TGA_RT20s_Int_P2A_mScarlet-i and 009_pcDNA_Zeo_1xMmMT3-PfVlp_FLAG_miRFP670nano into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the 50μL of the cells (1 - 5 μL), with a summary of 200μL of the construct and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

 

DH5α colony picking of 008_pcDNA_Zeo_mTagBFP2_TGA_RT20s_Int_P2A_mScarlet-i and 009_pcDNA_Zeo_1xMmMT3-PfVlp_FLAG_miRFP670nano constructs

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with 009_pcDNA_Zeo_1xMmMT3-PfVlp_FLAG_miRFP670nano, 008_pcDNA_Zeo_mTagBFP2_TGA_RT20s_Int_P2A_mScarlet-i from selection plate containing Agar+Carb was performed for the replication and expression of the desired genetic material.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Carb) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Plasmid Miniprep of 009_pcDNA_Zeo_1xMmMT3-PfVlp_FLAG_miRFP670nano and 008_pcDNA_Zeo_mTagBFP2_TGA_RT20s_Int_P2A_mScarlet-i

Goal;

A miniprep of 008_pcDNA_Zeo_mTagBFP2_TGA_RT20s_Int_P2A_mScarlet-i and 009_pcDNA_Zeo_1xMmMT3-PfVlp_FLAG_miRFP670nano was performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
008.1	169,4
008.2	170,6
009.1	197,5		0,52
009.2	174,2

 

DH5α colony picking of 002_pcDNA3.4-TOPO_IL23a

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with 010_pcDNA_Zeo_FLAG_miRFP670nano from selection plate containing Agar+Carb was performed for the replication and expression of the desired genetic material.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Carb) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Whole Plasmid Sequencing of 002_pcDNA3.4-TOPO_IL23a

Goal;

To confirm the nucleotide sequences of 002_pcDNA3.4-TOPO_IL23a, whole plasmid sequencing was performed by using Genewiz, Azenta services. 

Protocol;

DNA was sequenced using a Whole Plasmid sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 25 μL of purified plasmid at a concentration of 50 ng/μL The tube was delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis found a match between the experimental and expected sequences. 

Sample	Validation
002.1	Insertion of the IL23A confirmed
002.2	Insertion of the IL23A confirmed
002.3	Insertion of the IL23A confirmed

 

Plasmid Miniprep of 002_pcDNA3.4-TOPO_IL23a

Goal;

A miniprep of 002_pcDNA3.4-TOPO_IL23a was performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
010.1	222,72	1,89	1,93
010.2	257,65	1,90	2,06
010.3	232,47	1,90	1,99

 

LB Agar plate streaking of the 003_pC0039-CMV-dPspCas13b-GS-ADAR2DD(E488Q), 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G), 005_pC0078 RESCUE, 006_pAG1159 TOM20-ECFP-FIREmate and 007_pAG1344 EGFP-FIREtag

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with 003_pC0039-CMV-dPspCas13b-GS-ADAR2DD(E488Q), 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G), 005_pC0078 RESCUE, 006_pAG1159 TOM20-ECFP-FIREmate and 007_pAG1344 EGFP-FIREtag from the provided stock was performed for creating an agar plate stock. Colony selection is to be performed after confirming transformed plasmid with sequencing. 

Protocol;

Using a sterile inoculation loop, confirmed by sequencing colonies were carefully picked from the surface of the selection plate. Each colony was then streaked onto a fresh selection plate with Antibiotic. e.g.:  Carbenicillin and incubated overnight at 37 °C for further growth and propagation of the selected colony. To prevent contamination and stagnate colony overgrowth, the original agar plate was sealed using parafilm and transferred to a 4°C refrigerator.

Results;

Successful inoculation of the LB agar plate with desired colony was confirmed with observation of the bacterial growth on the path of the streak. Plates containing 003 and 004 were overgrown, plates containing 006 and 007 had too little colonies.

LB Agar plate re-streaking of 003_pC0039-CMV-dPspCas13b-GS-ADAR2DD(E488Q), 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G), 006_pAG1159 TOM20-ECFP-FIREmate and 007_pAG1344 EGFP-FIREtag

Goal; 

Colony picking of individual DH5α E. coli bacterial colonies transformed with 003_pC0039-CMV-dPspCas13b-GS-ADAR2DD(E488Q), 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G), 006_pAG1159 TOM20-ECFP-FIREmate and 007_pAG1344 EGFP-FIREtag from selection plate containing Agar+Carb was performed for creating an agar plate stock. 

Protocol;

Using a sterile inoculation loop, confirmed by sequencing colonies were carefully picked from the surface of the selection plate. Each colony was then streaked onto a fresh selection plate with [Antibiotic. e.g.:  Carbenicillin] and incubated overnight at 37 °C for further growth and propagation of the selected colony. To prevent contamination and stagnate colony overgrowth, the original agar plate was sealed using parafilm and transferred to a 4°C refrigerator.

Results;

Successful inoculation of the LB agar plate with desired colony was confirmed with observation of the bacterial growth on the path of the streak. Because of the wrong usage of the antibiotic, no growth was seen on the plates containing 006 and 007, as Kanamycin needed to be used. 

LB Agar plate re-streaking of 003_pC0039-CMV-dPspCas13b-GS-ADAR2DD(E488Q), 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G), 006_pAG1159 TOM20-ECFP-FIREmate and 007_pAG1344 EGFP-FIREtag

Goal; 

Colony picking of individual DH5α E. coli bacterial colonies transformed with 003_pC0039-CMV-dPspCas13b-GS-ADAR2DD(E488Q), 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G), 006_pAG1159 TOM20-ECFP-FIREmate and 007_pAG1344 EGFP-FIREtag from selection plate containing Agar+Carb was performed for creating an agar plate stock. 

Protocol;

Using a sterile inoculation loop, confirmed by sequencing colonies were carefully picked from the surface of the selection plate. Each colony was then streaked onto a fresh selection plate with [Antibiotic. e.g.:  Carbenicillin] and incubated overnight at 37 °C for further growth and propagation of the selected colony. To prevent contamination and stagnate colony overgrowth, the original agar plate was sealed using parafilm and transferred to a 4°C refrigerator.

Results;

Successful inoculation of the LB agar plate with desired colony was confirmed with observation of the bacterial growth on the path of the streak. Because of the wrong usage of the antibiotic, no growth was seen on the plates containing 006 and 007, as Kanamycin needed to be used. 

DH5α colony picking of 003_pC0039-CMV-dPspCas13b-GS-ADAR2DD(E488Q), 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G), 005_pC0078 RESCUE, 006_pAG1159 TOM20-ECFP-FIREmate and 007_pAG1344 EGFP-FIREtag

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with  003_pC0039-CMV-dPspCas13b-GS-ADAR2DD(E488Q), 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G), 005_pC0078 RESCUE, 006_pAG1159 TOM20-ECFP-FIREmate and 007_pAG1344 EGFP-FIREtag from selection plate containing Agar+Carb(for the constructs 003-005) and Agar+Kan(for the constructs 006,007) was performed for the replication and expression of the desired genetic material.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Agar+Carb(for the constructs 003-005) and Agar+Kan(for the constructs 006,007) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Splitting of HEK293T cell line into T75 flask

Goal;

Splitting HEK293t cell line from the flask to the next passage number of 75

Procedure:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet.

Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 37°C for 5 minutes. Upon confirming detachment using a phase-contrast microscope, cells were rescued using 3 ml pre-warmed complete Dulbecco’s modified Eagle medium F12 (DMEM) and transferred to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. The supernatant was decanted using a Pasteur pipette, and the cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 75 cm² flask was inoculated with 1 ml cell suspension into 9 ml pre-warmed DMEM. Cells were grown at 37°C with 5% CO₂ and 90% relative humidity.

Results;

HEK293t cell line from the flask was split to passage number 75.

PCR of 002_pcDNA3.4-TOPO_IL23a, 008_pcDNA_Zeo_mTagBFP2_TGA_RT20s_Int_P2A_mScarlet-i and 009_pcDNA_Zeo_1xMmMT3-PfVlp_FLAG_miRFP670nano

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from:

• 002_pcDNA3.4-TOPO_IL23a, using primers 001_F_WPRE_T7tt and 002_R_CMVp_T7p
• 008_pcDNA_Zeo_mTagBFP2_TGA_RT20s_Int_P2A_mScarlet-i, using primers 013_F_mScarletI and 014_R_T7_mTagBFP2
• 008_pcDNA_Zeo_mTagBFP2_TGA_RT20s_Int_P2A_mScarlet-i, using primers 015_F_BGH_mScarlet-I and 016_R_mTagBFP2_RT20
• 009_pcDNA_Zeo_1xMmMT3-PfVlp_FLAG_miRFP670nano, using primers 011_F_mIRFP670nano and 012_pcDNA_Zeo_lin_rev

for constructs 010_pcDNA_Zeo_FLAG_miRFP670nano, 011_pcDNA_Zeo_mScarlet-i, 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s, and 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR with high fidelity and efficiency.

Protocol;

PCR was carried out using the Platinum™ SuperFi™ II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X Platinum™ SuperFi™ II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5 μM concentration)	0.5 μM	2 μL
Template DNA	10 ng plasmid	1 μL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25-35 cycles of denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template.

Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	30 seconds	1
Denaturation	98°C	5-10 seconds	25-35
Annealing	60°C	10 seconds
Extension	72°C	3.5 min
Final extension	72°C	5 minutes	1
Hold	4°C	–

Amplified DNA products were kept at -20°C until further use.

Results;

The efficiency of PCR was evaluated by downstream experiments.

KLD of 010_pcDNA_Zeo_FLAG_miRFP670nano, 011_pcDNA_Zeo_mScarlet-i and 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s

Goal;

KLD reaction was performed to phosphorylate and circularise the PCR product, and degrade template DNA by Dpn I enzyme treatment. 

Protocol;

KLD reaction was performed using the KLD Enzyme Mix (NEW ENGLAND Biolabs), consisting of Kinase, Ligase and Dpn I enzymes, according to the manufacturer’s instructions. For this, 5 µL of the KLD reaction Buffer (2X) (NEW ENGLAND Biolabs), 1 µL of KLD Enzyme Mix, 2 µL of Nuclease-free water and 2 µL of the PCR product were combined. The final composition of the reaction mixture was defined as follows:

Components	Volume
PCR Product	1 µL
KLD Reaction Buffer (2X)	5 µL
KLD Enzyme Mix (10X)	1 µL
Nuclease-free Water	3 µL
Total Volume	10 µL

The sample was incubated at room temperature (25°C) for 5 minutes, and then placed on ice. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

DH5α E. coli transformation with 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR, 010_pcDNA_Zeo_FLAG_miRFP670nano, 011_pcDNA_Zeo_mScarlet-i and 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s

Goal;

Transformation was performed to introduce plasmid DNA construct 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR, 010_pcDNA_Zeo_FLAG_miRFP670nano, 011_pcDNA_Zeo_mScarlet-i and 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

 

Gibson assembly of 002_pcDNA3.4-TOPO_IL23a

Goal;

The goal was to correctly assemble the PCR-product of the 002_pcDNA3.4-TOPO_IL23a for the further experiments

Procedure;

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Reagent	Amount
NEBuilder HiFi DNA Assembly Master Mix	10 μL
Vector	… ng / … μL
Insert	… ng / … μL
ddH2O	… μL
Total	20 μL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

Results are being evaluated by the downstream experimnets

 

Gibson assembly of the 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR(assembled using DMSO or PEG)

Goal;

The goal is to assemble the 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR construct, transforming it to 014_pcDNA3.4-TOPO_T7p_T7tt.

Procedure;

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Reagent	Amount
NEBuilder HiFi DNA Assembly Master Mix	10 μL
Vector	… ng / … μL
Insert	… ng / … μL
ddH2O	… μL
Total	20 μL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

The results are being evaluated in the downstream experiments

 

DH5α E. coli transformation with 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR

Goal;

Transformation was performed to introduce plasmid DNA construct 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

 

Agarose gel electrophoresis of the 013_pcDNA3.4-TOPO_T7p (PCR with DMSO, PEG protocol)

Goal;

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples from 013_pcDNA3.4-TOPO_T7p (PCR with DMSO, PEG protocol)

Protocol;

Agarose gel electrophoresis was performed using a 1.0% w/v agarose (ROTH) gel prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply (BIORAD, PowerPac™ Basic Power Supply). A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 30 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (BIORAD, ChemiDoc Imaging System).

Results;

The following samples of 013_pcDNA3.4-TOPO_T7p (PCR with DMSO, PEG protocol) were loaded into the gel from left to right: 

with 1%DMSO, 3%DMSO,DNA Ladder, other thing, empty,5% DMSO,1% PEG, 2%PEG 

The agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples. The addition of  2% PEG showed to inefficient, as no real band were seen 

PCR of 002_pcDNA3.4-TOPO_IL23a( with DMSO and PEG)

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 002_pcDNA3.4-TOPO_IL23a using primers 001_F_WPRE_T7tt and 002_R_CMVp_T7p for construct(s) 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR with high fidelity and efficiency. DMSO and PEG were used to decrease formaing of the secondary structures and increasing specificity of the primer binding.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run, DMSO 1%	20-μL run, DMSO 3%	20-μL run, DMSO 5%	20-μL run, PEG 1%	20-μL run, PEG 2%
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL	10 μL	10 μL	10 μL	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration) :	0.5 μM	2 μL	2 μL	4 μL	2 μL	2 μL
Template DNA :	10 ng plasmid	1 μL	1 μL	1 μL	1 μL	1 μL
DMSO 50%	-	0,4 μL	1,2 μL	3,2 μL	-	-
PEG 25%	-	-	-	-	0,8μL	1,6 μL
Nuclease-free water	–	6,6 μL	5,8 μL	3,8 μL	6,2 μL	5,4 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

DH5α colony picking of 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s, 011_pcDNA_Zeo_mScarlet-i, 010_pcDNA_Zeo_FLAG_miRFP670nano and 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s, 011_pcDNA_Zeo_mScarlet-i, 010_pcDNA_Zeo_FLAG_miRFP670nano and 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR from selection plate containing Agar+Carb was performed for the replication and expression of the desired genetic material.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Carb) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Sanger sequencing of 010_pcDNA_Zeo_FLAG_miRFP670nano, 011_pcDNA_Zeo_mScarlet-i, 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s

Goal;

To confirm the nucleotide sequences of 010_pcDNA_Zeo_FLAG_miRFP670nano, 011_pcDNA_Zeo_mScarlet-i, 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s, Sanger sequencing was performed by using Genewiz, Azenta services. 

Protocol;

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 10 μL of purified plasmid at a concentration of 30 - 100 ng/μL was added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis … a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Sample	Primer	Validation
10.1	BGHR	successful sequence confirmed
10.1	CMV-Forward
11.1	BGHR	various point mutations
11.1	CMV-Forward
12.1	BGHR	various point mutations
12.1	CMV-Forward

Although 010_pcDNA_Zeo_FLAG_miRFP670nano was confirmed, other two constructs were need to be redone

Plasmid Miniprep of 010_pcDNA_Zeo_FLAG_miRFP670nano, 011_pcDNA_Zeo_mScarlet-i, 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s and 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR(without DMSO or PEG)

Goal;

A miniprep of 010_pcDNA_Zeo_FLAG_miRFP670nano, 011_pcDNA_Zeo_mScarlet-i, 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s and 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR(without DMSO or PEG) was performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
010.1	180,44	1,90	2,17
010.2	136,72	1,89	2,08
010.3	234,37	1,92	2,21
011.1	71,31	1,98	2,10
011.2	79,90	1,84	2,10
011.3	68,57	1,82	2,22
012.1	57,69	1,87	2,14
012.2	49,47	1,93	2,19
012.3	39,55	1,76	2,19
013.1	147,88	1,90	2,23
013.2	86,72	1,85	2,19
013.3	65,14	1,84	2,14

some of the probes were sent to the wps to approve the right assemble

PCR of 008_pcDNA_Zeo_mTagBFP2_TGA_RT20s_Int_P2A_mScarlet-i

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 008_pcDNA_Zeo_mTagBFP2_TGA_RT20s_Int_P2A_mScarlet-i using primers 015_F_BGH_mScarlet-I and 016_R_mTagBFP2_RT20 for construct(s) 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	12 B 20-μL run, no DMSO	12 A 20-μL run, DMSO 3%
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration) :	0.5 μM	2 μL	2 μL
Template DNA :	10 ng plasmid	1 μL	1 μL
DMSO 50%	-	-	1,2 μL
Nuclease-free water	–	7 μL	5,8 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

DH5α E. coli transformation of 013_pcDNA3.4-TOPO_T7p .2(3% DMSO) and .4(1% PEG)

Goal;

Transformation was performed to introduce plasmid DNA construct 013_pcDNA3.4-TOPO_T7p into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

 

Gibson assembly of 013_pcDNA3.4-TOPO_T7p

Goal;

The goal is to assemble the 013_pcDNA3.4-TOPO_T7p (PCR with DMSO, PEG protocol) construct

Procedure;

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Reagent	Amount
NEBuilder HiFi DNA Assembly Master Mix	10 μL
Vector	… ng / … μL
Insert	… ng / … μL
ddH2O	… μL
Total	20 μL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

Results are being evaluated by the downstream experiments

 

Agarose gel electrophoresis of 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR(with DMSO or PEG) - redo

Goal;

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples from 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR(with DMSO or PEG) 

Protocol;

Agarose gel electrophoresis was performed using a 1.0% w/v agarose (ROTH) gel prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply (BIORAD, PowerPac™ Basic Power Supply). A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 30 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (BIORAD, ChemiDoc Imaging System).

Results;

The following samples of 013_pcDNA3.4-TOPO_T7p (PCR with DMSO, PEG protocol) were loaded into the gel from left to right: 

with DNA ladder, empty, 1%DMSO, 3%DMSO,5% DMSO, empty, 1% PEG, 2%PEG 

The agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

DH5α E. coli transformation with 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s

Goal;

Transformation was performed to introduce plasmid DNA construct 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

 

KLD of 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s

Goal;

KLD reaction was performed to phosphorylate and circularise the PCR product, and degrade template DNA by Dpn I enzyme treatment. 

Protocol;

KLD reaction was performed using the KLD Enzyme Mix (NEW ENGLAND Biolabs), consisting of Kinase, Ligase and Dpn I enzymes, according to the manufacturer’s instructions. For this, 5 µL of the KLD reaction Buffer (2X) (NEW ENGLAND Biolabs), 1 µL of KLD Enzyme Mix, 2 µL of Nuclease-free water and 2 µL of the PCR product were combined. The final composition of the reaction mixture was defined as follows:

Components	Volume
PCR Product	1 µL
KLD Reaction Buffer (2X)	5 µL
KLD Enzyme Mix (10X)	1 µL
Nuclease-free Water	3 µL
Total Volume	10 µL

The sample was incubated at room temperature (25°C) for 5 minutes, and then placed on ice. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

Agarose gel electrophoresis of 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s

Goal;

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples from 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s

Protocol;

Agarose gel electrophoresis was performed using a 1.0% w/v agarose (ROTH) gel prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply (BIORAD, PowerPac™ Basic Power Supply). A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 30 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (BIORAD, ChemiDoc Imaging System).

Results;

The following samples were loaded into the gel from left to right: DNA ladder, 012A, 012B

The agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

DH5α colony picking of 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s & 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with constructs 013_pcDNA3.4-TOPO_T7p_T7tt (2 plates labelled: pRAM_T7_2 and pRAM_T7_4) and 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s (2 plates labelled: 012A and 012B) from selection plates was performed for the replication and expression of the desired genetic material.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Two colonies were inoculated from each plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with 1x carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful transformation was confirmed by the observed turbidity of the inoculated liquid bacterial culture.

KLD of 014_pcDNA3.4-TOPO_T7p_T7tt

Goal;

KLD reaction was performed to phosphorylate and circularise the PCR product, and degrade template DNA by Dpn I enzyme treatment. 

Protocol;

KLD reaction was performed using the KLD Enzyme Mix (NEW ENGLAND Biolabs), consisting of Kinase, Ligase and Dpn I enzymes, according to the manufacturer’s instructions. For this, 5 µL of the KLD reaction Buffer (2X) (NEW ENGLAND Biolabs), 1 µL of KLD Enzyme Mix, 2 µL of Nuclease-free water and 2 µL of the PCR product were combined. The final composition of the reaction mixture was defined as follows:

Components	Volume
PCR Product	1 µL
KLD Reaction Buffer (2X)	5 µL
KLD Enzyme Mix (10X)	1 µL
Nuclease-free Water	3 µL
Total Volume	10 µL

The sample was incubated at room temperature (25°C) for 5 minutes, and then placed on ice. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

Plasmid Miniprep of 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s & 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR

Goal;

A miniprep of 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s & 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR was performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g. The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute. The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 ml eppendorf tubes. DNA was eluted by addition of 30 μl of nuclease-free water to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

Sample	Concentration [ng/μL]	260/280	260/230
13A.1	78.37	1,96	2,26
13A.2	146.10	1,91	2,24
13B.1	110.20	1,85	2,17
13B.2	356.22	1,91	2,23
12B.2	130.32	1,90	1,91
12B.1	126.11	1,92	2,17
12A.1	130,19	1,93	2,27
12A.2	25,81	2,04	1,24

Subsequently, samples 13B.2 and 12B.2 were submitted for Whole Plasmid Sequencing (Genewiz, Azenta).

Troubleshooting:

Miniprep of 12A.2 did not result in required DNA concentration, and sample was subsequently discarded.

Whole Plasmid Sequencing of 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s & 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR

Goal;

To validate cloning of 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR and 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s whole plasmid sequencing has been ordered.

Protocol;

DNA was sequenced using a Whole Plasmid2 sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 25 μL of purified plasmid at a concentration of 50 ng/μL The tube was delivered to a drop-off point.

Results;

	Sample	Validation
1	013B.2	Unsuccessful
2	012B.2	Success

 

Transformation of DH5α E.coli with 014_pcDNA3.4-TOPO_T7p_T7tt KLD product & 015_pcDNA_Zeo_DD-N_NLuc_P10SN

Goal;

E. coli transformation with 014_pcDNA3.4-TOPO_T7p_T7tt KLD product and 015_pcDNA_Zeo_DD-N_NLuc_P10SN.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 350 µL of room temperature LB, 80 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

The following day, single colonies on both plates were observed. Three single colonies from the 014_pcDNA3.4-TOPO_T7p_T7tt plate were further inoculated for minipreps. Subsequently, both plates were transferred to fridge.

PCR of 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR & 015_pcDNA_Zeo_DD-N_NLuc_P10SN backbone

Goal;

Amplify the 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR plasmid with F_SV40pA and R_WPRE primers for subsequent KLD reaction.

Amplify the backbone of the pcDNA_Zeo_DD-N_NLuc_P10SN plasmid with F_pcDNA_ NLuc and R_Kozak_NLuc primers for subsequent Gibson assembly.

Protocol;

The components of PCR reaction were added to the tube as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration) :	0.5 μM	2 μL
Template DNA :	10 ng plasmid	1 μL
Nuclease-free water	–	to 20 μL

The PCR was placed in the thermocycler, and following programme was run:

 

Results;

 

Troubleshooting: 

 

DH5α E. coli transformation with 017_pcDNA_Zeo_NLuc

Goal;

Transformation was performed to introduce plasmid DNA construct 017_pcDNA_Zeo_NLuc into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

 

Agarose gel electrophoresis of 017_pcDNA_Zeo_NLuc

Goal;

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples from 017_pcDNA_Zeo_NLuc

Protocol;

Agarose gel electrophoresis was performed using a 1.0% w/v agarose (ROTH) gel prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply (BIORAD, PowerPac™ Basic Power Supply). A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 30 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (BIORAD, ChemiDoc Imaging System).

Results;

The following samples were loaded into the gel from left to right: empty, Ladder, 017_pcDNA_Zeo_NLuc, Insert, Backbone

The agarose gel electrophoresis analysis did not confirmed the size distribution of the expected DNA fragments in the samples.

Agarose gel electrophoresis of 017_pcDNA_Zeo_NLuc

Goal;

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples from 017_pcDNA_Zeo_NLuc

Protocol;

Agarose gel electrophoresis was performed using a 1.0% w/v agarose (ROTH) gel prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply (BIORAD, PowerPac™ Basic Power Supply). A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 30 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (BIORAD, ChemiDoc Imaging System).

Results;

The following samples were loaded into the gel from left to right: ladder, backbone, empty, Gibson product(017_pcDNA_Zeo_NLuc), ladder

The agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

Gibson assembly of 017_pcDNA_Zeo_NLuc redo

Goal;

The goal was to redo failed Gibson assembly of the 017_pcDNA_Zeo_NLuc

Procedure;

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Reagent	Amount
NEBuilder HiFi DNA Assembly Master Mix	10 μL
Vector	… ng / … μL
Insert	… ng / … μL
ddH2O	… μL
Total	20 μL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

The results were evaluated by the downstream experiments

 

LB Agar plate streaking of 008_pcDNA_Zeo_mTagBFP2_TGA_RT20s_Int_P2A_mScarlet-i

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with 008_pcDNA_Zeo_mTagBFP2_TGA_RT20s_Int_P2A_mScarlet-ifrom selection plate containing Carb was performed for creating an agar plate stock. Colony selection is to be performed after confirming transformed plasmid with sequencing. 

Protocol;

Using a sterile inoculation loop, confirmed by sequencing colonies were carefully picked from the surface of the selection plate. Each colony was then streaked onto a fresh selection plate with [Antibiotic. e.g.:  Carbenicillin] and incubated overnight at 37 °C for further growth and propagation of the selected colony. To prevent contamination and stagnate colony overgrowth, the original agar plate was sealed using parafilm and transferred to a 4°C refrigerator.

Results;

Successful inoculation of the LB agar plate with desired colony was confirmed with observation of the bacterial growth on the path of the streak.

Gibson assembly of 017_pcDNA_Zeo_NLuc

Goal;

The goal was to clean the 015_pcDNA_Zeo_DD-N_NLuc_P10SN construct from the unnecessary components using Gibson assembly

Procedure;

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Reagent	Amount
NEBuilder HiFi DNA Assembly Master Mix	10 μL
Vector	2,5μL
Insert	2,5μL
ddH2O	5 μL
Total	20 μL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

The results are being evaluated by downstream experiments

KLD of 014_pcDNA3.4-TOPO_T7p_T7tt

Goal;

End-to-end-ligation of 014_pcDNA3.4-TOPO_T7p_T7tt.

Protocol;

The reaction was prepared as follows:

Components	Volume
PCR Product	2 µl
KLD Reaction Buffer (2X)	5 µl
KLD Enzyme Mix (10X)	1 µl
Nuclease-free Water	2 µl
Total Volume	10 µl

Subsequently, everything was mixed by pipetting. The sample was incubated at room temperature (25°C) for 5 minutes, and then placed on ice. Subsequently E.coli was transformed. 

Results;

/

PCR of 015_pcDNA_Zeo_DD-N_NLuc_P10SN insert

Goal;

Amplify the insert (Nano-luciferase) of the 015_pcDNA_Zeo_DD-N_NLuc_P10SN plasmid with 019_F_NLuc_Kozak and 020_R_NLuc_pcDNA primers for subsequent Gibson assembly.

Protocol;

1. Add the components of PCR reaction to a tube:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 019_F_NLuc_Kozak 020_R_NLuc_pcDNA	0.5 μM	2 μL
Template DNA: 015_pcDNA_Zeo_DD-N_NLuc_P10SN	10 ng plasmid	1 μL
Nuclease-free water	7 µL	to 20 μL

 

2. Put the PCR tube in a thermocycler and select a correct programme: 

 

3. Run an agarose gel to visualize results of PCR. 

Results;

To be established in downstream experiments.

 

DH5α colony picking of 014_pcDNA3.4-TOPO_T7p_T7tt

Goal;

Colony picking of three individual DH5α E. coli bacterial colonies transformed with 014_pcDNA3.4-TOPO_T7p_T7tt construct.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Troubleshooting:

DH5α colony picking of 015_pcDNA_Zeo_DD-N_NLuc_P10SN

Goal;

Colony picking of one individual DH5α E. coli bacterial colony transformed with 015_pcDNA_Zeo_DD-N_NLuc_P10SN construct.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Carbenicillin) and incubated overnight at 37 °C with continuous shaking at 130 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

/

DH5α colony picking of 018_pcDNA_Zeo_HA_DD-N_mScarlet-I_GeoTag

Goal;

Colony picking of two individual DH5α E. coli bacterial colonies transformed with 018_pcDNA_Zeo_HA_DD-N_mScarlet-I_GeoTag construct.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Carbenicillin) and incubated overnight at 37 °C with continuous shaking at 130 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

/

Extended PCR - 002_pcDNA3.4-TOPO_IL23a backbone with T7 promoter and terminator

Goal;

Amplify 002_pcDNA3.4-TOPO_IL23a with backbone with extended primers F_WPRE_T7tt and R_CMVp_T7p containing T7 promoter and terminator.

Protocol;

1. Add the components of PCR reaction to a tube:

Component	Final concentration	20 μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): Primers: F_WPRE_T7tt R_CMVp_T7p	0.5 μM	2 μL
DMSO (prediluted to 50 %)	3 % (v/v)	1.2 µL
Template DNA : 002_pcDNA3.4-TOPO-IL23a (6kb)	10 ng plasmid	1 μL
Nuclease-free water	–	5.8 µL

 

2. Put the PCR tube in a thermocycler (Miulab, PR-96E Gradient Thermal Cycler) and select a correct programme: 

 

3. Run an agarose gel to visualize results of PCR. 

Results;

 

Troubleshooting: 

 

DH5α E. coli transformation of 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR

Goal;

Transformation was performed to introduce plasmid DNA construct 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

 

Agarose gel electrophoresis of 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR

Goal;

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples from …

Protocol;

Agarose gel electrophoresis was performed using a 1.0% w/v agarose (ROTH) gel prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply (BIORAD, PowerPac™ Basic Power Supply). A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 30 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (BIORAD, ChemiDoc Imaging System).

Results;

The following samples were loaded into the gel from left to right: ladder, empty, Gibson product

The agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

Plasmid Miniprep of 015_pcDNA_Zeo_DD-N_NLuc_P10SN and 018_pcDNA_Zeo_HA_DD-N_mScarlet-I_GeoTag

Goal;

A miniprep of 015_pcDNA_Zeo_DD-N_NLuc_P10SN and 018_pcDNA_Zeo_HA_DD-N_mScarlet-I_GeoTag was performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
015.1	186,6	1,93	2,20
018.1	185	1,93	2,23
018.2	213,4	1,92	2,20

 

Gibson assembly to produce 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR

Goal;

Produce 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR with T7 promoter and terminator.

Procedure;

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Reagent	Amount
NEBuilder HiFi DNA Assembly Master Mix	10 μL
Vector	2.5 μL
Insert	-
ddH2O	7.5 µL
Total	20 μL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

Construct was analyzed by gel electrophoresis. Picture attached.

DH5α E. coli transformation with 018_pcDNA_Zeo_HA_DD-N_mScarlet-I_GeoTag

Goal;

Transformation was performed to introduce plasmid DNA constructs 018_pcDNA_Zeo_HA_DD-N_mScarlet-I_GeoTag into DH5α E. coli cells, allowing for plasmid replication.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

Transfection was successful. Plate is in the +4°C in bacterial culture room.

Troubleshooting:

DH5α colony picking of 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR from selection plate containing Carb was performed for the replication and expression of the desired genetic material.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with …) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

DH5α E. coli transformation with 020_pcDNA_Zeo_HA_mScarlet-I_Geotag

Goal;

Transformation was performed to introduce plasmid DNA construct 020_pcDNA_Zeo_HA_mScarlet-I_Geotaginto DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

 

Whole Plasmid Sequencing of 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR, 017_pcDNA_Zeo_NLuc and 018_pcDNA_Zeo_HA_DD-N_mScarlet-I_GeoTag

Goal;

To confirm the nucleotide sequences of 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR, 017_pcDNA_Zeo_NLuc and 018_pcDNA_Zeo_HA_DD-N_mScarlet-I_GeoTag whole plasmid sequencing was performed by using Genewiz, Azenta services. 

Protocol;

DNA was sequenced using a Whole Plasmid sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 25 μL of purified plasmid at a concentration of 50 ng/μL The tube was delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis … a match between the experimental and expected sequences. 

Sample	Validation
013.2	successful cloning confirmed
017.1	deletion of the part of the ori region
018.1	successful cloning confirmed

 

Plasmid Miniprep of 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR

Goal;

A miniprep of 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR was performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
013.1	64,5	2,06	1,8
013.2	114,9	1,95	2,05
013.3	44,8	1,93	1,44

 

PCR of 018_pcDNA_Zeo_HA_DD-N_mScarlet-I_GeoTag to get 020_pcDNA_Zeo_HA_mScarlet-I_Geotag

Goal;

Deletion of unnecessary linker and DD-N in 018_pcDNA_Zeo_HA_DD-N_mScarlet-I_GeoTag in front of mScarlet-I.

Protocol;

1. Add the components of PCR reaction to a tube:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration):   R_T7_mTagBFP2  F_mScarlet-I	0.5 μM	2 μL
Template DNA : 018_pcDNA_Zeo_HA_DD-N_mScarlet-I_GeoTag	10 ng plasmid	x μ
Nuclease-free water	–	to 20 μL

2. Put the PCR tube in a thermocycler (Miulab, PR-96E Gradient Thermal Cycler) and select a correct programme: 

3. Run an agarose gel to visualize results of PCR. 

Results;

Product was KLDed in linked experiment.

No gel has been done.

Troubleshooting: 

 

KLD of 020_pcDNA_Zeo_HA_mScarlet-I_Geotag

Goal;

Ligation of 020_pcDNA_Zeo_HA_mScarlet-I_Geotag after PCR with R_T7_mTagBFP2, F_mScarlet-I (deletion of linker, DD-Nin 018)

Protocol;

The reaction was prepared as follows:

Components	Volume
PCR Product	2 µl
KLD Reaction Buffer (2X)	5 µl
KLD Enzyme Mix (10X)	1 µl
Nuclease-free Water	2 µl
Total Volume	10 µl

 

Subsequently, everything was mixed by pipetting. The sample was incubated at room temperature (25°C) for 5 minutes, and then placed on ice. Subsequently E.coli was transformed. 

Results;

will be clear in sequencing after miniprep

Troubleshooting:

DH5α colony picking of 020_pcDNA_Zeo_HA_mScarlet-I_Geotag

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with … from selection plate containing 020_pcDNA_Zeo_HA_mScarlet-I_Geotag was performed for the replication and expression of the desired genetic material.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with …) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Changing media of HEK293T cell line

Goal;

Replacing old media of HEK293T cells with fresh media.

Protocol;

All solutions and equipment that come in contact with the cells must be sterile. Always use proper sterile technique and work in a laminar flow hood.

1. Remove culturing media from the 75 cm² culturing flask using Pasteur pipette.
2. Add 5 ml pre-warmed DPBS for washing the adherent cells. Proceed with caution by pipetting towards culture vessel walls and not directly on the cell layer. Slowly make “plus” sign tilting motion to wash effectively.
3. Remove DPBS wash using Pasteur pipette.
4. Add 10 ml of pre-warmed complete DMEM media.
5. Incubate cells at 37°C with 5% CO₂.
6. Engage the UV-light upon cleaning laminar hood for at least 30 minutes.

Results;

Media change successfull. Confluency at 70%

 

Miniprep of 020_pcDNA_Zeo_HA_mScarlet-I_Geotag

Goal;

A miniprep of 020_pcDNA_Zeo_HA_mScarlet-I_Geotag from 3 colonies was performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g. The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 3 minutes, until the pink color disappeared. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute. The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 ml eppendorf tubes. DNA was eluted by addition of 30 μl of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

Sample	Concentration [ng/μL]	260/280	260/230
020.1	117.2	1.97	2.32
020.2	136.9	1.92	1.83
020.3	168.4	1.94	2.30

 

Troubleshooting:

DH5α E. coli transformation with 020_pcDNA_Zeo_HA_mScarlet-I_Geotag

Goal;

Transformation of DH5α E. coli with 020_pcDNA_Zeo_HA_mScarlet-I_Geotag

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

Troubleshooting: streaked on 2 plates

PCR of 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR to get 014_pcDNA3.4-TOPO_T7p_T7tt

Goal;

Polymerase chain reaction using the PlatinumTM SuperFi^TM II DNA polymerase was performed to amplify specific DNA fragments from 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR using primers 003_R_WPRE and 004_F_SV40pA with high fidelity and efficiency. The result should be 014_pcDNA3.4-TOPO_T7p_T7tt.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration):  004_F_SV40pA 003_R_WPRE	0.5 μM	2 μL
Template DNA : 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR	10 ng plasmid	1.75 μL
Nuclease-free water	–	to 20 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at 4°C until further use. 

Results;

PCR in 4°C fridge

 

PCR of 015_pcDNA_Zeo_DD-N_NLuc_P10SNto get 017_pcDNA_Zeo_NLuc (backbone)

Goal;

Polymerase chain reaction using PlatinumTM SuperFi^TM II DNA polymerase was performed to amplify specific DNA fragments from 015_pcDNA_Zeo_DD-N_NLuc_P10SN using primers 017_R_Kozak_NLuc and 018_F_pcDNA_NLuc with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 017_R_Kozak_NLuc  018_F_pcDNA_NLuc	0.5 μM	2 μL
Template DNA: 015_pcDNA_Zeo_DD-N_NLuc_P10SN	10 ng plasmid	1 μL
Nuclease-free water	–	to 20 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at 4°C until further use. 

Results;

 

PCR of 015_pcDNA_Zeo_DD-N_NLuc_P10SN to get 017_pcDNA_Zeo_NLuc (insert)

Goal;

Polymerase chain reaction using PlatinumTM SuperFi^TM II DNA polymerase was performed to amplify specific DNA fragments from 015_pcDNA_Zeo_DD-N_NLuc_P10SN using primers 019_F_NLuc_Kozak and 020_R_NLuc_pcDNA with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration) :	0.5 μM	1 μL
Template DNA :	10 ng plasmid	1 μL
Nuclease-free water	–	to 20 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at 4°C until further use. 

Results;

PCR in 4°C fridge

 

DH5α E. coli transformation with 014_pcDNA3.4-TOPO_T7p_T7tt

Goal;

Transformation was performed to introduce plasmid DNA constructs 014_pcDNA3.4-TOPO_T7p_T7tt into DH5α E.coli cells, allowing for plasmid replication.

Protocol;

An aliquot of competent DH5α E.coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

KLD of 014_pcDNA3.4-TOPO_T7p_T7tt

Goal;

Ligation of the 014_pcDNA3.4-TOPO_T7p_T7tt PCR product. 

Protocol;

KLD reaction was carried out using the (New England Biolabs) according to the modified manufacturer’s instructions. The final composition of the reaction mixture is defined as follows:

Components	Volume
PCR Product	2 µL
KLD Reaction Buffer (2X)	5 µL
KLD Enzyme Mix (10X)	1 µL
Nuclease-free Water	2 µL
Total Volume	10 µL

Subsequently, everything was mixed by pipetting. The sample was incubated at room temperature (25°C) for 5 minutes, and then placed on ice.

Results;

 KLD reaction was performed

Splitting of HEK293T cell line

Goal;

Splitting HEK293T cell line from flask to the next passage.

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 3737°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos’s modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 75 cm² flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO₂ and 90% relative humidity.

Results;

HEK293T cell line from flask was split to passage.

 

DH5α E. coli transformation of 017_pcDNA_Zeo_NLuc

Goal;

transformation with gibson assembled 017_pcDNA_Zeo_NLuc_P10SN

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

Gibson assembly of 017_pcDNA_Zeo_NLuc_P10SN

Goal;

Gibson assembly was performed to assemble the respective vector and insert DNA fragments into 017_pcDNA_Zeo_NLuc_P10SN.

Procedure;

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Ratio Backbone:Insert —> 1:3

Reagent	Amount
NEBuilder HiFi DNA Assembly Master Mix	10 μL
Vector	1 μL
Insert	3 μL
ddH2O	6 μL
Total	20 μL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

Experiment was completed. 017_pcDNA_Zeo_NLuc_P10SN to be evaluated in an agarose gel and to be used in transformation.

Agarose gel electrophoresis of PCR products (014_pcDNA3.4-TOPO_T7p_T7tt, Nluc insert and backbone) and KLD (014_pcDNA3.4-TOPO_T7p_T7tt) + Gibson product (017_pcDNA_Zeo_NLuc)

Goal;

Following the PCR of 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR to 014_pcDNA3.4-TOPO_T7p_T7tt and amplification of 017_pcDNA_Zeo_NLuc backbone and inserts, agarose gel electrophoresis was performed to visualize:

- PCR products of 017_pcDNA_Zeo_NLuc: Insert and backbone 

- Gibson assembly 017_pcDNA_Zeo_NLuc

- KLD of 014_pcDNA3.4-TOPO_T7p_T7tt

 

Protocol;

Agarose gel electrophoresis was performed using a 1.0% w/v agarose (ROTH) gel prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply (BIORAD, PowerPac™ Basic Power Supply). A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 30 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (BIORAD, ChemiDoc Imaging System).

Results;

expected: KLD 014 (4 kb) , Gibson 017 (5.5 kb), bb (4 kb), ins (0.5 kb), mScarlet020 (5.7 kb)observed: all in range BUT KLD 014 (too high: ~6 kb)

FAILURE

Troubleshooting:

Maybe re-do gel and/or Gibson, add DMSO to the Gibson as possible option

DH5α colony picking of Gibson Assembly of 017_pcDNA_Zeo_NLuc_P10SN and of 014_pcDNA3.4-TOPO_T7p_T7tt

Goal;

Pick and inoculate for preparation, Miniprep and sequencing of the Gibson Assembly of 017_pcDNA_Zeo_NLuc_P10SN and 014_pcDNA3.4-TOPO_T7p_T7tt

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with 1% Carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

 

Plasmid Miniprep - 014_pcDNA3.4-TOPO_T7p_T7tt and 017_pcDNA_Zeo_NLuc

Goal;

A miniprep of 014_pcDNA3.4-TOPO_T7p_T7tt and 017_pcDNA_Zeo_NLuc was performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of nuclease-free water to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
pRAM_014	268,2	1,93	2,20
NLuc_017	217,2	1,93	2,23

Samples were subsequently stored at -20ºC, and dilutions were prepared for WPS.

 

Sanger sequencing of 020_pcDNA_Zeo_HA_mScarlet-I_Geotag

Goal;

To confirm the nucleotide sequences of 020_pcDNA_Zeo_HA_mScarlet-I_Geotag Sanger sequencing was performed by using Genewiz, Azenta services. 

Protocol;

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 10 μL of purified plasmid at a concentration of 30 - 100 ng/μL was added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis didn’t  a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Sample	Primer	Validation
020.3	BGHR	GeoTag successfully deleted, but many point mutations in mScarlet

 

Whole Plasmid Sequencing of 017_pcDNA_Zeo_NLuc_P10SN and of 014_pcDNA3.4-TOPO_T7p_T7tt

Goal;

To confirm the nucleotide sequences of 017_pcDNA_Zeo_NLuc_P10SN and of 014_pcDNA3.4-TOPO_T7p_T7tt, whole plasmid sequencing was performed by using Genewiz, Azenta services. 

Protocol;

DNA was sequenced using a Whole Plasmid sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 25 μL of purified plasmid at a concentration of 50 ng/μL The tube was delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis … a match between the experimental and expected sequences. 

Sample	Validation
014_pcDNA3.4-TOPO_T7p_T7tt	Not confirmed
017_pcDNA_Zeo_NLuc	Sequencing confirmed

 

Splitting of HEK293T cell line

Goal;

Cell splitting of HEK293T. 1:10 as they were overgrown.

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 3737°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos’s modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 75 cm² flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO₂ and 90% relative humidity.

Results;

to be seen if they survived

 

DH5α E. coli transformation with 014_pcDNA3.4-TOPO_T7p_T7tt KLD product

Goal;

Transformation was performed to introduce plasmid DNA constructs 014_pcDNA3.4-TOPO_T7p_T7tt into DH5α E. coli cells, allowing for plasmid replication.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

 

Agarose gel electrophoresis of new 014_pcDNA3.4-TOPO_T7p_T7tt

Goal;

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples from 014_pcDNA3.4-TOPO_T7p_T7tt KLD. 

Protocol;

Agarose gel electrophoresis was performed using a 1.0% w/v agarose (ROTH) gel prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply (BIORAD, PowerPac™ Basic Power Supply). A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 30 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (BIORAD, ChemiDoc Imaging System).

Results;

The following samples were loaded into the gel from left to right: 1kb plus, PCR and KLD of 014_pcDNA3.4-TOPO_T7p_T7tt

The agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

DH5α E. coli Retransformation with 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR

Goal;

Re-transformation of E.coli with 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR (sequence confirmed by sequencing) was performed for subsequent culture inoculation and maxiprep of the plasmid. 

Protocol;

A 50µl aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

 

PCR of 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR to get 014_pcDNA3.4-TOPO_T7p_T7tt

Goal

Polymerase chain reaction using the PlatinumTM SuperFi^TM II DNA polymerase was performed to amplify specific DNA fragments from 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR using primers 003_R_WPRE and 004_F_SV40pA with high fidelity and efficiency.

Added 15 sec to elongation step.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration) :  004_F_SV40pA 003_R_WPRE	0.5 μM	2 μL
Template DNA :  013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR diluted plasmid with 18 ng/µL	10 ng plasmid	0.7 μL
Nuclease-free water	–	7.3 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

KLD of 014_pcDNA3.4-TOPO_T7p_T7tt

Goal;

KLD reaction was performed to phosphorylate and circularize the 014_pcDNA3.4-TOPO_T7p_T7tt PCR product, and degrade template DNA by Dpn I enzyme treatment. 

Protocol;

KLD reaction was performed using the KLD Enzyme Mix (NEW ENGLAND Biolabs), consisting of Kinase, Ligase and Dpn I enzymes, according to the manufacturer’s instructions. For this, 5 µL of the KLD reaction Buffer (2X) (NEW ENGLAND Biolabs), 1 µL of KLD Enzyme Mix, 2 µL of Nuclease-free water and 2 µL of the PCR product were combined. The final composition of the reaction mixture was defined as follows:

Components	Volume
PCR Product	1 µL
KLD Reaction Buffer (2X)	5 µL
KLD Enzyme Mix (10X)	1 µL
Nuclease-free Water	3 µL
Total Volume	10 µL

The sample was incubated at room temperature (25°C) for 5 minutes, and then placed on ice. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

Redo of PCR of 018_pcDNA_Zeo_HA_DD-N_mScarlet-I_GeoTag to get 020_pcDNA_Zeo_HA_mScarlet-I_Geotag

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 018_pcDNA_Zeo_HA_DD-N_mScarlet-I_GeoTag using primers 013_F_mScarlet-I & 014_R_T7_mTagBFP2 with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 013_F_mScarlet-I 014_R_T7_mTagBFP2	0.5 μM	2 μL
Template DNA: 018_pcDNA_Zeo_HA_DD-N_mScarlet-I_GeoTag	10 ng plasmid	1 μL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

DH5α colony picking of 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR & 014_pcDNA3.4-TOPO_T7p_T7tt

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR and 014_pcDNA3.4-TOPO_T7p_T7tt from selection plate containing Carb was performed for the replication and expression of the desired genetic material.

2 pickings of 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR

3 pickings of 014_pcDNA3.4-TOPO_T7p_T7tt

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Carbenecillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Splitting of HEK293T cell line

Goal;

Split according to confluence —> 1:5

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 ml Acutase solution at 3737°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos’s modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 75 cm² flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO₂ and 90% relative humidity.

Results;

 

Agarose gel electrophoresis of 020_pcDNA_Zeo_HA_mScarlet-I_Geotag PCR product

Goal;

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples from 020_pcDNA_Zeo_HA_mScarlet-I_Geotag PCR product. 

Protocol;

Agarose gel electrophoresis was performed using a 1.0% w/v agarose (ROTH) gel prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply (BIORAD, PowerPac™ Basic Power Supply). A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 30 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (BIORAD, ChemiDoc Imaging System).

Results;

The following samples were loaded into the gel from left to right:

The agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

Restriction Digest of 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR and 014_pcDNA3.4-TOPO_T7p_T7tt

Goal;

Restriction digest of the 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR and 014_pcDNA3.4-TOPO_T7p_T7tt with HaeIII-HF enzyme for downstream cloning.

Protocol;

During the preparation of the digestion, the following protocol was performed: 1 μL of HaeIII-HF, 1 μg of DNA sample, and 5 μL of rCutSmart™ Buffer were added. Then, nuclease-free water was added until a final volume of 50 μL was reached. In this case, 2.5 μL of pRAM 013.4, 3.5 μL of pRAM 014.1*, and 5 μL of pRAM 014.2 were used. After all reagents were added, the mixture was incubated for 14 minutes at 37°C. The amount of DNA samples added was calculated for a total volume of 25 μL, so a 1:2 dilution was already achieved.

Results;

The efficiency of plasmid digestion was evaluated by downstream experiments.

Agarose gel electrophoresis of digested 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR and 014_pcDNA3.4-TOPO_T7p_T7tt

Goal;

Agarose gel electrophoresis was performed to visualize digested DNA fragments of the selected samples from 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR and 014_pcDNA3.4-TOPO_T7p_T7tt

Protocol;

Agarose gel electrophoresis was performed using a 1.0% w/v agarose (ROTH) gel prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. The gel then was cut and only half of it was used for this experiment. DNA samples mixed with loading dye (20μL DNA sample, 5μL dye; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 3 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply (BIORAD, PowerPac™ Basic Power Supply). A constant voltage of 110 V was applied, and the DNA fragments were allowed to migrate through the gel for 30 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (BIORAD, ChemiDoc Imaging System).

Results;

The following samples were loaded into the gel from left to right: ladder, digested 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR (013.4), digested 014_pcDNA3.4-TOPO_T7p_T7tt (014.2), digested 014_pcDNA3.4-TOPO_T7p_T7tt (014.1*).

The agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples. See the photo attached

Seeding of Ibidi 8 Well Microscopic Slides with HEK293T Cells, p16

Goal;

Seeding ibidi µ-Slide 8 Well microscopic slide for further confocal analysis of mTagBFP2, mScarlet-I, and miRFP670nano plasmids transfected into HEK293T cells with 5 × 10³ HEK293T cells per well.

Procedure:

All solutions and equipment that came in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet.

The culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 mL of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 mL of Accutase solution at 37°C for 5 minutes. Upon confirming detachment using a phase-contrast microscope, cells were rescued using 3 mL of pre-warmed complete Dulbecco’s modified Eagle medium (DMEM) F12 and transferred to a 15 mL tube. The cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. The supernatant was decanted using a Pasteur pipette, and the cell pellet was resuspended in 6 mL of pre-warmed DMEM. A single well of an ibidi µ-Slide 8 Well microscopic slide was inoculated with 300 μL of cell suspension at 5 × 10³ cells per well. Cells were grown at 37°C with 5% CO₂ and 90% relative humidity.

Results;

Cell counting was performed using the Countess™ 2 automated cell counter.

Chamber	Cell Count	Live Cells	Dead Cells
A	6.26 × 106	5.88 × 106	3.75 × 105
B	5.71 × 106	5.31 × 106	4 × 105

Counting cells with Cell countess 2

Goal;

Calculating cell suspension concentration and assessing the quality of the said suspension.

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Cells were counted and analyzed for viability using Countess™ II automated cell counter with 2 replicas and taken as average of two measurements.

Results;

Chamber	Cell count	Live cells	Dead cells
A	6.26x106	5.88x106	3.75x105
B	5.71x106	5.31x106	4x105

Cells are in good condition with enough for further seeding

Splitting of HEK293T cell line into T75 flask

Goal;

Splitting HEK293t cell line from flask to the next passage number of 16.

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 3737°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos’s modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 75 cm² flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO₂ and 90% relative humidity.

Results;

HEK293t cell line from flask was split to passage number 16.

DH5α E. coli transformation with 020_pcDNA_Zeo_HA_mScarlet-I_Geotag

Goal;

Transformation was performed to introduce plasmid DNA construct 020_pcDNA_Zeo_HA_mScarlet-I_Geotag into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

After overnight incubation at 37°C no colonies were observed. 

 

KLD of 020_pcDNA_Zeo_HA_mScarlet-I_Geotag PCR product

Goal;

KLD reaction was performed to phosphorylate and circularise the PCR product of 020_pcDNA_Zeo_HA_mScarlet-I_Geotag, and degrade template DNA by Dpn I enzyme treatment. 

Protocol;

KLD reaction was performed using the KLD Enzyme Mix (NEW ENGLAND Biolabs), consisting of Kinase, Ligase and Dpn I enzymes, according to the manufacturer’s instructions. For this, 5 µL of the KLD reaction Buffer (2X) (NEW ENGLAND Biolabs), 1 µL of KLD Enzyme Mix, 2 µL of Nuclease-free water and 2 µL of the PCR product were combined. The final composition of the reaction mixture was defined as follows:

Components	Volume
PCR Product	1 µL
KLD Reaction Buffer (2X)	5 µL
KLD Enzyme Mix (10X)	1 µL
Nuclease-free Water	3 µL
Total Volume	10 µL

The sample was incubated at room temperature (25°C) for 5 minutes, then incubated for 30 mins at 37°C and then placed on ice. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

Plasmid Miniprep of 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR & 014_pcDNA3.4-TOPO_T7p_T7tt

Goal;

A miniprep of 013_pcDNA3.4-TOPO_T7p_T7tt_HSV_f1ori_SV40ori_NeoR and 014_pcDNA3.4-TOPO_T7p_T7tt was performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of nuclease-free water to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range. Sample 014.1* has been obtained by re-elution of the 014.1 column with 10 μL of nuclease free water.

Sample	Concentration [ng/μL]	260/280	260/230
013.4	202,97	1,97	2,18
013.5	173,32	1,96	2,20
014.1	246,91	1,96	2,20
014.1*	159,68	1,96	2,18
014.2	109,85	2,01	2,15

 

Replacing DMEM media

Goal;

Replace old media in cell flask with new media.

Protocol;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

HEK293T were washed with DPBS and supplied with fresh complete Dulbecco’s modified Eagle medium. Cells were grown at  37°C with 5% CO₂ and 90% relative humidity..

Results;

 

 

Agarose gel electrophoresis of the digested 016_pU6_PspCas13b-DR-empty plasmid

Goal;

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples from 016_pU6_PspCas13b-DR-empty.

Protocol;

Agarose gel electrophoresis was performed using a 1.0% w/v agarose (ROTH) gel prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply (BIORAD, PowerPac™ Basic Power Supply). A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 30 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (BIORAD, ChemiDoc Imaging System).

Results;

The following samples were loaded into the gel from left to right: /

The agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

DNA gel extraction of 016_pU6_PspCas13b-DR-empty

Goal;

 

A gel extraction of 016_pU6_PspCas13b-DR-empty digested with BbsI was performed to isolate and purify a specific DNA fragment from agarose gel for downstream applications.

Protocol;

Following gel electrophoresis, DNA bands of interest were excised from the gel under UV transillumination (BIORAD) using a clean scalpel. The excised gel slices were transferred to a 1.5 mL microcentrifuge tube and weighed to determine the amount of gel present. DNA fragments were then isolated and purified from gel using the Monarch® DNA Gel Extraction Kit Protocol (NEW ENGLAND Biolabs, NEB #T1020) following modified manufacturer’s instructions. For this, the gel slices were first dissolved in 4 volumes of Monarch Gel Dissolving Buffer (NEW ENGLAND Biolabs) at 50 °C. The mixture was then transferred to a provided column placed in a collection tube. The column was centrifuged at 13500 × g for 1 min to allow the DNA to bind to the column. The flow-through was discarded, and the column was washed twice with 200 μL of DNA Wash Buffer (NEW ENGLAND Biolabs) at 13500 × g for 1 min each to remove impurities and contaminants. Purified DNA was eluted into a 1.5 mL microcentrifuge tube by adding 10 μL of elution buffer to the column matrix, incubating at room temperature for 1 min, and centrifuging the column for 1 min at 13500 × g. DNA concentration was determined using a NanoPhotometer N60, and the DNA samples were stored at −20 °C.

Results;

 

The extraction procedure yielded DNA of 60.9 ng/μL.

Restriction digest of 016_pU6_PspCas13b-DR-empty with BbsI

Goal;

 

Restriction digest of the 016_pU6_PspCas13b-DR-empty construct with BbsI enzyme, for downstream cloning of gRNA for Nano-luciferase restoration assay.

Protocol;

Restriction digest was carried out using the BbsI-HF^® enzyme (R3539S, New England Biolabs) according to the protocol developed by Truong et al. (Truong, DJ., Armbrust, N., Geilenkeuser, J., Westmeyer, G. Generation of cell lines for the usage of Intron-encoded Scarless Programmable Extranuclear Cistronic Transcripts (INSPECT), 2023, PROTOCOL available at Protocol Exchange). For this, 1 μg of DNA template, 5 μL CutSmart^® (B7204, New England Biolabs), 1 μL BbsI-HF^® were combined in a 1.5 mL Eppendorf reaction tube. The sample was mixed by pipetting, and incubated at 37°C for 2 hours (including mixing every 30 minutes).

Component	Volume (50 μL)
Template DNA: 016_pU6_PspCas13b-DR-empty	5.2 μL (1μg)
CutSmart®	5 μL
BbsI-HF®	1 μL
Nuclease-free water	38.8 μL

 

Results;

The efficiency of plasmid digestion was evaluated by downstream experiments.

 

 

DH5α E. coli transformation with 023_pU6_PspCas13b-DR-gRNA_NLuc

Goal;

Transformation was performed to introduce plasmid DNA construct 023_pU6_PspCas13b-DR-gRNA_NLuc (016_pU6_PspCas13b-DR-empty ligated with NLuc-gRNA) into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. 2 μL of the ligation mixture was added to 50 μL of the cells and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 30 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 5 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 60 minutes with shaking (300 rpm). 70 µl of the mixture was spread onto a selection plate. In the end, the rest of the sample was centrifuged 10000 × g  for 1 minute, and the pellet was resuspended in 70 µl of LB and plated onto a second selection plate. Both plates were incubated overnight at 37°C.

Results;

Successful transformation was confirmed by the presence of colonies on the selective agar plates.

Heterodimerization of the deoxynucleotides encoding for NLuc_gRNA with 025_F_gRNA_NLuc and 026_R_gRNA_NLuc

Goal;

The procedure was performed to heterodimerize the two deoxynucleotides for the gRNA spacer used in downstream experiments for Nano-luciferase restoration assay. 

025_F_gRNA_NLuc

026_R_gRNA_NLuc

Protocol;

Heterodimerization of the deoxynucleotides was carried out according to the modified protocol developed by Truong et al. (Truong, DJ., Armbrust, N., Geilenkeuser, J., Westmeyer, G. Generation of cell lines for the usage of Intron-encoded Scarless Programmable Extranuclear Cistronic Transcripts (INSPECT), 2023, PROTOCOL available at Protocol Exchange). Primers were designed in a way that they are complementary to each other and have 5’ overhangs for ligation with a pU6 plasmid digested with BbsI. For this, 5 μL from both solubilized deoxynucleotides and 40 μL of TE buffer were combined in a PCR tube. The final composition of the reaction mixture is defined as follows:

Component	Volume (50 μL)
Forward primer (100 μM) 025_F_gRNA_NLuc	5 μL
Reverse primer (100 μM) 026_R_gRNA_NLuc	5 μL
TE buffer (pH 8.0)	40 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), with the following conditions. 

Product was kept at -20°C until further use. 

Results;

The efficiency of the deoxynucleotides heterodimerization was evaluated by downstream experiments.

Ligation of 016_pU6_PspCas13b-DR-empty and heterodimerized NLuc-gRNA

Goal;

The procedure was performed to ligate the 016_pU6_PspCas13b-DR-empty plasmid digested with BbsI and heterodimerized NLuc-gRNA to produce 023_pU6_PspCas13b-DR-gRNA_NLuc.

Protocol;

Ligation was carried out according to the modified protocol developed by Truong et al. (Truong, DJ., Armbrust, N., Geilenkeuser, J., Westmeyer, G. Generation of cell lines for the usage of Intron-encoded Scarless Programmable Extranuclear Cistronic Transcripts (INSPECT), 2023, PROTOCOL available at Protocol Exchange). For this, 50 ng BbsI-digested 016_pU6_PspCas13b-DR-empty plasmid, 0.5 µl Heterodimerized product of the spacer, 10 µl Quick Ligase Reaction Buffer (2X) (M2200S, NEW ENGLAND Biolabs), 1 µl of Quick Ligase (M2200S, NEW ENGLAND Biolabs) were combined in a 1.5 mL Eppendorf reaction tube. The final composition of the reaction mixture is defined as follows:

Component	Volume (20 μL)
BbsI-digested pU6 backbone	0.82 μL (50 ng)
Heterodimerized product of the spacer	0.5 μL
Quick Ligase Reaction Buffer (2X)	10 μL
Quick Ligase	1 μL
Nuclease-free water	7.68 μL

The ligation reaction mixture was incubated for 10 min at room temperature.

Product was kept at -20°C until further use. 

Results;

The efficiency of the ligation was evaluated by downstream experiments.

DH5α E. coli transformation with 016_pU6_PspCas13b-DR-empty

Goal;

Transformation was performed to introduce plasmid DNA construct 016_pU6_PspCas13b-DR-empty into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 189 ng/μL, was added to the cells (1 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 30 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for 5 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 60 minutes with shaking (300 rpm). In the end, 70 µl of the mixture was spread onto a pre-warmed selection plate and incubated overnight at 37°C. 

Results;

Successful transformation was confirmed by the presence of colonies on the selective agar plate.

DH5α E. coli transformation with Redo of KLD 020_pcDNA_Zeo_HA_mScarlet-I_Geotag

Goal;

Transformation was performed to introduce plasmid DNA construct 020_pcDNA_Zeo_HA_mScarlet-I_Geotag into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 60 minutes with shaking (300 rpm). In the end, 70 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

Successful transformation was confirmed by the presence of colonies on the selective agar plate.

Redo of KLD of 020_pcDNA_Zeo_HA_mScarlet-I_Geotag

Goal;

KLD reaction was performed to phosphorylate and circularise the 020_pcDNA_Zeo_HA_mScarlet-I_Geotag PCR product, and degrade template DNA by Dpn I enzyme treatment. 

Protocol;

KLD reaction was performed using the KLD Enzyme Mix (NEW ENGLAND Biolabs), consisting of Kinase, Ligase and Dpn I enzymes, according to the manufacturer’s instructions. For this, 5 µL of the KLD reaction Buffer (2X) (NEW ENGLAND Biolabs), 1 µL of KLD Enzyme Mix, 2 µL of Nuclease-free water and 2 µL of the PCR product were combined. The final composition of the reaction mixture was defined as follows:

Components	Volume
PCR Product	1 µL
KLD Reaction Buffer (2X)	5 µL
KLD Enzyme Mix (10X)	1 µL
Nuclease-free Water	3 µL
Total Volume	10 µL

The sample was incubated at room temperature (25°C) for 5 minutes, and then placed on ice. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

DH5α colony picking of 023_pU6_PspCas13b-DR-gRNA_NLuc transformed cells

Goal;

Colony picking of three individual DH5α E. coli bacterial colonies transformed with 023_pU6_PspCas13b-DR-gRNA_NLuc from selection plate containing Carbenicillin was performed for the replication and expression of the desired genetic material.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

DH5α colony picking of 023_pU6_PspCas13b-DR-gRNA_NLuc transformed cells

Goal;

Colony picking of three individual DH5α E. coli bacterial colonies transformed with 023_pU6_PspCas13b-DR-gRNA_NLuc from selection plate containing Carbenicillin was performed for the replication and expression of the desired genetic material.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

DH5α colony picking of 020_pcDNA_Zeo_HA_mScarlet-I_Geotag

Goal;

Colony picking of three individual DH5α E. coli bacterial colonies transformed with 020_pcDNA_Zeo_HA_mScarlet-I_Geotag from selection plate containing Carbenicillin was performed for the replication and expression of the desired genetic material.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Sanger sequencing - 020_pcDNA_Zeo_HA_mScarlet-I_Geotag & 014_pcDNA3.4-TOPO_T7p_T7tt

Goal;

To confirm the nucleotide sequences of 020_pcDNA_Zeo_HA_mScarlet-I_Geotag and 014_pcDNA3.4-TOPO_T7p_T7tt, Sanger sequencing was performed by using Genewiz, Azenta services. 

Protocol;

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 10 μL of purified plasmid at a concentration of 30 - 100 ng/μL was added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis … a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Sample	Primer	Validation
08657407	020.4	T7	Successful deletion of DD-N domain
08657408	020.4	BGHR	Successful deletion of DD-N domain
08657372	014.2	T7	Successful deletion of HSV, f1ori, SV40ori, NeoR
08657373	014.2	SV40pA-R	non-specific

 

Plasmid Miniprep of 016_pU6_PspCas13b-DR-empty, 020_pcDNA_Zeo_HA_mScarlet-I_Geotag & 023_pU6_PspCas13b-DR-gRNA_NLuc

Goal;

A miniprep of 016_pU6_PspCas13b-DR-empty, 020_pcDNA_Zeo_HA_mScarlet-I_Geotag & 023_pU6_PspCas13b-DR-gRNA_NLuc was performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 50 μL of TE buffer, pH 8.0 (SERVA, cat. no. 39799.01) to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
023.1	55.01	1.89	2.32
023.2	48.59	1.88	2.25
023.3	34.98	1.77	2.29
016.1	133.99	1.93	2.28
016.2	71.46	1.86	2.29
016.3	64.33	1.92	2.32
020.4	86.33	1.90	2.25
020.5	85.35	1.88	2.51
020.6	51.28	1.86	2.22

 

Whole Plasmid Sequencing - 016_pU6_PspCas13b-DR-empty & 023_pU6_PspCas13b-DR-gRNA_NLuc

Goal;

To confirm the nucleotide sequences of 016_pU6_PspCas13b-DR-empty and 023_pU6_PspCas13b-DR-gRNA_NLuc, whole plasmid sequencing was performed by using Genewiz, Azenta services. 

Protocol;

DNA was sequenced using a Whole Plasmid sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 25 μL of purified plasmid at a concentration of 50 ng/μL The tube was delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis … a match between the experimental and expected sequences. 

Sample	Validation
023.1	gRNA was introduced to the plasmid twice
016.1	sequence confirmed

 

Splitting of HEK293T cell line into T75 flask

Goal;

Splitting HEK293t cell line from flask  to the next passage number of 17

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 3737°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos’s modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 75 cm² flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO₂ and 90% relative humidity.

Results;

HEK293t cell line from flask was split to passage number 17

DH5α E. coli transformation with 021_pcDNA_Zeo_HA_mScarlet-I & 024_pcDNA3.4-TOPO_T7p_T7tt

Goal;

Transformation was performed to introduce plasmid DNA construct 021_pcDNA_Zeo_HA_mScarlet-I & 024_pcDNA3.4-TOPO_T7p_T7tt into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

Successful transformation was confirmed by the presence of colonies on the selective agar plate.

KLD of 021_pcDNA_Zeo_HA_mScarlet-I & 024_pcDNA3.4-TOPO_T7p_T7tt

Goal;

KLD reaction was performed to phosphorylate and circularise the PCR product of 021_pcDNA_Zeo_HA_mScarlet-I and 024_pcDNA3.4-TOPO_T7p_T7tt, and degrade template DNA by Dpn I enzyme treatment. 

Protocol;

KLD reaction was performed using the KLD Enzyme Mix (NEW ENGLAND Biolabs), consisting of Kinase, Ligase and Dpn I enzymes, according to the manufacturer’s instructions. For this, 5 µL of the KLD reaction Buffer (2X) (NEW ENGLAND Biolabs), 1 µL of KLD Enzyme Mix, 2 µL of Nuclease-free water and 2 µL of the PCR product were combined. The final composition of the reaction mixture was defined as follows:

Components	Volume
PCR Product	1 µL
KLD Reaction Buffer (2X)	5 µL
KLD Enzyme Mix (10X)	1 µL
Nuclease-free Water	3 µL
Total Volume	10 µL

The sample was incubated at room temperature (25°C) for 5 minutes, and then placed on ice. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

Agarose gel electrophoresis - 021_pcDNA_Zeo_HA_mScarlet-I & 024_pcDNA3.4-TOPO_T7p_T7tt

Goal;

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples from 024_pcDNA3.4-TOPO_T7p_T7tt and 021_pcDNA_Zeo_HA_mScarlet-I PCR products.

Protocol;

Agarose gel electrophoresis was performed using a 1.0% w/v agarose (ROTH) gel prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply (BIORAD, PowerPac™ Basic Power Supply). A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 30 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (BIORAD, ChemiDoc Imaging System).

Results;

The following samples were loaded into the gel from left to right: 1kB Plus DNA ladder, 5 μL of 021_pcDNA_Zeo_HA_mScarlet-I PCR product, 5 μL of 024_pcDNA3.4-TOPO_T7p_T7tt PCR product.

The agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

PCR of 014_pcDNA3.4-TOPO_T7p_T7tt to get 024_pcDNA3.4-TOPO_T7p_T7tt

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 014_pcDNA3.4-TOPO_T7p_T7tt using primers 005_R_SV40pA & 006_F_ori with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 005_R_SV40pA 006_F_ori	0.5 μM	2 μL
Template DNA: 014_pcDNA3.4-TOPO_T7p_T7tt	10 ng plasmid	1 μL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

PCR of 020_pcDNA_Zeo_HA_mScarlet-I_Geotag to get 021_pcDNA_Zeo_HA_mScarlet-I

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 020_pcDNA_Zeo_HA_mScarlet-I_Geotag using primers 023_R_mScarlet-I and 024_F_pcDNA_bGH with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration):  023_R_mScarlet-I  024_F_pcDNA_bGH	0.5 μM	2 μL
Template DNA: 020_pcDNA_Zeo_HA_mScarlet-I_Geotag	10 ng plasmid	1 μL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

Sanger sequencing - 023_pU6_PspCas13b-DR-gRNA_NLuc

Goal;

To confirm the nucleotide sequences of 023_pU6_PspCas13b-DR-gRNA_NLuc (023.2 & 023.3), Sanger sequencing was performed by using Genewiz, Azenta services. 

Protocol;

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 10 μL of purified plasmid at a concentration of 30 - 100 ng/μL was added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis … a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Sample	Primer	Validation
08657409	023.2	M13-40FOR	confirmed insertion of gRNA_NLuc
08657410	023.3	M13-40FOR	confirmed insertion of gRNA_NLuc

 

Replacing DMEM media

Goal;

Replace old media in cell culture with fresh media.

Protocol;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

HEK293T were washed with DPBS and supplied with fresh complete Dulbecco’s modified Eagle medium. Cells were grown at  37°C with 5% CO₂ and 90% relative humidity..

Results;

Media was successfully changed on flasks A and B.

 

 

DH5α colony picking of 021_pcDNA_Zeo_HA_mScarlet-I & 024_pcDNA3.4-TOPO_T7p_T7tt

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with plasmid DNA construct 021_pcDNA_Zeo_HA_mScarlet-I & 024_pcDNA3.4-TOPO_T7p_T7tt from selection plate containing LB was performed for the replication and expression of the desired genetic material.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (5 mL LB medium with 5 µL Cb) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Plasmid Miniprep of 021_pcDNA_Zeo_HA_mScarlet-I

Goal;

A miniprep of 021_pcDNA_Zeo_HA_mScarlet-I was performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL ofelution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure didn’t yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
21.1	59,80	1,94	2,19
21.2	22,95	2,23	2,31
21.3	34,30	1,93	2,18

Plasmid Miniprep of 024_pcDNA3.4-TOPO_T7p_T7tt

Goal;

A miniprep of 024_pcDNA3.4-TOPO_T7p_T7tt was performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL ofelution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
024.1	42,76	1,98	2,24
024.2	114,44	1,93	2,30
024.3	34,52	1,96	2,25

 

DH5α colony picking of 021_pcDNA_Zeo_HA_mScarlet-I

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with 021_pcDNA_Zeo_HA_mScarlet-I from selection plate containing LB was performed for the replication and expression of the desired genetic material.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Carbacilin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Splitting of HEK293T cell line into T75 flask

Goal;

Splitting HEK293t cell line from flask to the next passage number of 18.

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 3737°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos’s modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 75 cm² flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO₂ and 90% relative humidity.

Results;

HEK293t cell line from flask at passage number 18.

Splitting of HEK293T cell line into T25 flask

Goal;

Splitting HEK293t cell line from “Mike” flask  to flask “VD+Nicole+Mike” the next passage number of 18

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Culturing media was decanted from the 25 cm² culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 3737°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos’s modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 25 cm² flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO₂ and 90% relative humidity.

Results;

HEK293t cell line from “Mike” flask  to flask “VD+Nicole+Mike” flask was split to passage number 18

Agarose gel electrophoresis - 035_pU6_PspCas13b-DR-empty

Goal;

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples from 035_pU6_PspCas13b-DR-empty KLD product. 

Protocol;

Agarose gel electrophoresis was performed using a 1.0% w/v agarose (ROTH) gel prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply (BIORAD, PowerPac™ Basic Power Supply). A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 30 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (BIORAD, ChemiDoc Imaging System).

Results;

The following samples were loaded into the gel from left to right: 1kB Plus Ladder; 035_pU6_PspCas13b-DR-empty PCR product (5 μL).

The agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

DH5α E. coli transformation with 035_pU6_PspCas13b-DR-empty

Goal;

Transformation was performed to introduce plasmid DNA construct 035_pU6_PspCas13b-DR-empty into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

 

KLD of 035_pU6_PspCas13b-DR-empty

Goal;

KLD reaction was performed to phosphorylate and circularise the 035_pU6_PspCas13b-DR-empty PCR product, and degrade template DNA by Dpn I enzyme treatment. 

Protocol;

KLD reaction was performed using the KLD Enzyme Mix (NEW ENGLAND Biolabs), consisting of Kinase, Ligase and Dpn I enzymes, according to the manufacturer’s instructions. For this, 5 µL of the KLD reaction Buffer (2X) (NEW ENGLAND Biolabs), 1 µL of KLD Enzyme Mix, 2 µL of Nuclease-free water and 2 µL of the PCR product were combined. The final composition of the reaction mixture was defined as follows:

Components	Volume
PCR Product	1 µL
KLD Reaction Buffer (2X)	5 µL
KLD Enzyme Mix (10X)	1 µL
Nuclease-free Water	3 µL
Total Volume	10 µL

The sample was incubated at room temperature (25°C) for 5 minutes, and then placed on ice. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

Plasmid Miniprep (redo) of 021_pcDNA_Zeo_HA_mScarlet-I

Goal;

A miniprep of 021_pcDNA_Zeo_HA_mScarlet-I was performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL ofTE-buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
021.1	119,85	1,93	2,38
021.2	120,33	1,96	2,35
021.3	167,01	1,94	2,24

 

Whole Plasmid Sequencing - 021_pcDNA_Zeo_HA_mScarlet-I & 024_pcDNA3.4-TOPO_T7p_T7tt

Goal;

To confirm the nucleotide sequences of 021_pcDNA_Zeo_HA_mScarlet-I and 024_pcDNA3.4-TOPO_T7p_T7tt, whole plasmid sequencing was performed by using Genewiz, Azenta services.  Samples 021.3 for mScarlet-I and 024.2 for the pRAM were used.

Protocol;

DNA was sequenced using a Whole Plasmid sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 25 μL of purified plasmid at a concentration of 50 ng/μL The tube was delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis was a match between the experimental and expected sequences. 

Sample	Validation
mScar021.3	cloning confirmed
pRAM024.2	cloning confirmed, 100%

 

PCR of 016_pU6_PspCas13b-DR-empty to delete BsaI RS to get 035_pU6_PspCas13b-DR-empty

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 016_pU6_PspCas13b-DR-empty using primers 007_F_AmpR_717G>A and 008_R_AmpR_717G>A with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 007_F_AmpR_717G>A  008_R_AmpR_717G>A	0.5 μM	2 μL
Template DNA: 016_pU6_PspCas13b-DR-empty	10 ng plasmid	1 μL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

Replacing DMEM media of T75 flask

Goal;

Replace old media in cell culture with fresh media.

Protocol;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

HEK293T were washed with DPBS and supplied with fresh complete Dulbecco’s modified Eagle medium. Cells were grown at  37°C with 5% CO₂ and 90% relative humidity..

Results;

Media was successfully changed.

 

 

Replacing DMEM media of T25 flask

Goal;

Replace old media in cell culture with fresh media.

Protocol;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

HEK293T were washed with DPBS and supplied with fresh complete Dulbecco’s modified Eagle medium. Cells were grown at  37°C with 5% CO₂ and 90% relative humidity..

Results;

Media was successfully changed.

 

DH5α colony picking of 035_pU6_PspCas13b-DR-empty

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with 035_pU6_PspCas13b-DR-empty from selection plate containing Carbenicillin was performed for the replication and expression of the desired genetic material.

Protocol;

Using a sterile inoculation loop or pipette tip, three individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with 5uL of Carbenicillin) and incubated overnight at 37 °C with continuous shaking at120 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Splitting of HEK293T cell line into T75 flask

Goal;

Splitting HEK293t cell line from flask A to the next passage number of 19.

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 3737°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos’s modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 75 cm² flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO₂ and 90% relative humidity.

Results;

HEK293t cell line from flask A was split to passage number 19.

DH5α E. coli transformation with 017_pcDNA_Zeo_NLuc Gibson product

Goal;

Transformation was performed to introduce plasmid DNA construct 017_pcDNA_Zeo_NLuc into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (2 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µL of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

Successful transformation was confirmed by the presence of colonies on the selective agar plate.

Plasmid Miniprep of DNA construct 035_pU6_PspCas13b-DR-empty

Goal;

A miniprep of 035_pU6_PspCas13b-DR-empty was performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of nuclease-free water to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range in samples 035.1 and 035.2. Sample 035.3 was discarded to due to low concentration and high contamination. 

Sample	Concentration [ng/μL]	260/280	260/230
035.1	80,06	1,76	1,65
035.2	70,25	1,92	2,34
035.3	38,31	1,89	5,79

 

PCR Cleanup of 017_pcDNA_Zeo_NLuc_P10SN backbone & insert

Goal;

 

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions of 017_pcDNA_Zeo_NLuc_P10SN backbone and 017_pcDNA_Zeo_NLuc_P10SN insert amplification, for use in downstream applications. 

Protocol; 

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g. The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g. The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 × g. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
017_BB	224,9	1,93	2,23
017_I	116,0	1,86	2,10

Gibson assembly of 017_pcDNA_Zeo_NLuc_P10SN

Goal;

Gibson assembly was performed to assemble the respective vector and insert DNA fragments into 017_pcDNA_Zeo_NLuc_P10SN.

Procedure;

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 & 3-fold molar excess of insert, were combined with 10 μL of master mix.

Reagent	Amount
Ratio	1 to 2	1 to 3
NEBuilder HiFi DNA Assembly Master Mix	10 μL	10 μL
Vector	540.9 ng / 2.4 μL	405.7 ng / 1.8 μL
Insert	114.4 ng / 1 μL	128.7 ng / 1.1 μL
ddH2O	6.6 μL	7.1 μL
Total	20 μL	20 μL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

The efficiency of the assembly reaction was evaluated by subsequent experiments.

 

PCR of 015_pcDNA_Zeo_DD-N_NLuc_P10SN to get 017_pcDNA_Zeo_NLuc insert

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 015_pcDNA_Zeo_DD-N_NLuc_P10SN using primers 019_F_NLuc_Kozak & 020_R_NLuc_pcDNA with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 019_F_NLuc_Kozak 020_R_NLuc_pcDNA	0.5 μM	2 μL
Template DNA: 015_pcDNA_Zeo_DD-N_NLuc_P10SN	10 ng plasmid	1 μL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

PCR of 015_pcDNA_Zeo_DD-N_NLuc_P10SN to get 017_pcDNA_Zeo_NLuc backbone

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 015_pcDNA_Zeo_DD-N_NLuc_P10SN using primers 017_R_Kozak_NLuc & 029_F_pcDNA_NLuc_2 with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 017_R_Kozak_NLuc 029_F_pcDNA_NLuc_2	0.5 μM	2 μL
Template DNA: 015_pcDNA_Zeo_DD-N_NLuc_P10SN	10 ng plasmid	1 μL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

Agarose gel electrophoresis - 036_pU6_PspCas13b-DR-empty & 042_pcDNA3.4-TOPO_T7p_T7tt PCR products

Goal;

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples from 036_pU6_PspCas13b-DR-empty (036.1, 036.2) and 042_pcDNA3.4-TOPO_T7p_T7tt PCR products. 

Protocol;

Agarose gel electrophoresis was performed using a 1.0% w/v agarose (ROTH) gel prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply (BIORAD, PowerPac™ Basic Power Supply). A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 30 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (BIORAD, ChemiDoc Imaging System).

Results;

The following samples were loaded into the gel from left to right: DNA ladder, 036_pU6_PspCas13b-DR-empty (036.1, 036.2) and 042_pcDNA3.4-TOPO_T7p_T7tt PCR products. 

The agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

PCR of 024_pcDNA3.4-TOPO_T7p_T7tt to get 042_pcDNA3.4-TOPO_T7p_T7tt

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 024_pcDNA3.4-TOPO_T7p_T7tt using primers 007_F_AmpR_717G>A & 008_R_AmpR_717G>A with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 007_F_AmpR_717G>A 008_R_AmpR_717G>A	0.5 μM	2 μL
Template DNA: 024_pcDNA3.4-TOPO_T7p_T7tt	10 ng plasmid	1 μL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

PCR of 035_pU6_PspCas13b-DR-empty to get 036_pU6_PspCas13b-DR-empty

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 035_pU6_PspCas13b-DR-empty (035.1 & 035.2) using primers 027_R_pU6_2960A<C & 028_F_pU6_2960A<C for with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 027_R_pU6_2960A<C 028_F_pU6_2960A<C	0.5 μM	2 μL
Template DNA: 035_pU6_PspCas13b-DR-empty	10 ng plasmid	1 μL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

DH5α E. coli transformation with 036 - 042 plasmids

Goal;

Transformation was performed to introduce the plasmid DNA constructs below into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

036_pU6_PspCas13b-DR-empty

037_pU6_PspCas13b-DR-gRNA1_tape1

038_pU6_PspCas13b-DR-gRNA1_tape2

039_pU6_PspCas13b-DR-gRNA1_tape3

040_pU6_PspCas13b-DR-gRNA1_tape4

041_pU6_PspCas13b-DR-gRNA1_tape5

042_pcDNA3.4-TOPO_T7p_T7tt 

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

 

Heterodimerization of the Deoxynucleotides for gRNA1_tape1-5

Goal;

The procedure was performed to heterodimerize the two deoxynucleotides for the gRNA spacers: gRNA1_tape1, gRNA1_tape2, gRNA1_tape3, gRNA1_tape4, and gRNA1_tape5 for use in downstream experiments.

Protocol;

Heterodimerization of the deoxynucleotides was carried out according to a modified protocol developed by Truong et al. (Truong, DJ., Armbrust, N., Geilenkeuser, J., Westmeyer, G. “Generation of cell lines for the usage of Intron-encoded Scarless Programmable Extranuclear Cistronic Transcripts (INSPECT),” 2023, PROTOCOL available at Protocol Exchange). Primers were designed to be complementary to each other and to have 5’ overhangs for ligation with a pU6 plasmid digested with BbsI. For this, 5 μL from both solubilized deoxynucleotides and 40 μL of TE buffer were combined in a PCR tube. The final composition of the reaction mixture was as follows:

Component	1	2	3	4	5	Volume (50 μL)
Forward primer (100 μM)	031_F_gRNA1_tape1	033_F_gRNA1_tape2	035_F_gRNA1_tape3	037_F_gRNA1_tape4	039_F_gRNA1_tape5	5 μL
Reverse primer (100 μM)	032_R_gRNA1_tape1	034_R_gRNA1_tape2	036_R_gRNA1_tape3	038_R_gRNA1_tape4	040_R_gRNA1_tape5	5 μL
TE buffer (pH 8.0)						40 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler) with the following conditions:

Step	Temperature	Time
Initial denaturation	98°C	3 minutes
Annealing	85°C – 25°C	10 seconds -0.1°C/s ramp
Hold	4°C	Hold

The product was kept at -20°C until further use.

Results;

The efficiency of the deoxynucleotide heterodimerization was evaluated by downstream experiments.

Seeding of Ibidi 8-Well Microscopic Slides from T25 Flask B

Goal;

Seeding Ibidi µ-Slide 8 Well microscopic slide with 5×10⁴ HEK293T cells for further transfection.

Procedure:

All solutions and equipment that came in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet.

The culturing medium was decanted from the 75 cm² culturing flask. Cells were washed using 5 mL of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 mL Accutase solution at 37°C for 5 minutes. Upon confirming detachment using a light phase microscope, cells were resuspended in 3 mL of pre-warmed complete Dulbecco’s modified Eagle medium (DMEM) F12 and transferred to a 15 mL tube. The cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. The supernatant was decanted using a Pasteur pipette, and the cell pellet was resuspended in 6 mL of pre-warmed DMEM. A single well of an Ibidi µ-Slide 8 Well microscopic slide was inoculated with 300 µL of cell suspension at 5×10³ cells per well. Cells were further grown at 37°C with 5% CO₂ and 90% relative humidity.

Results;

Countess™ II Automated Cell Counter Results

Chamber	Cell Count	Live Cells	Dead Cells
A	_x106	_x106	_x103
B	_x106	_x106	_x103

Ligation of BbsI-digested pU6 and gRNA1 from tapes 1-5

Goal;

The procedure was performed to ligate the 016_pU6 plasmid digested with BbsI and heterodimerized gRNA spacers: gRNA1_tape1, gRNA1_tape2, gRNA1_tape3, gRNA1_tape4, gRNA1_tape5.

Protocol;

Ligation was carried out according to the modified protocol developed by Truong et al. (Truong, DJ., Armbrust, N., Geilenkeuser, J., Westmeyer, G. Generation of cell lines for the usage of Intron-encoded Scarless Programmable Extranuclear Cistronic Transcripts (INSPECT), 2023, PROTOCOL available at Protocol Exchange). For this, 50 ng BbsI-digested 016_pU6_PspCas13b-DR-empty plasmid, 0.5 µl Heterodimerized product of the spacer, 10 µl Quick Ligase Reaction Buffer (2X) (M2200S, NEW ENGLAND Biolabs), 1 µl of Quick Ligase (M2200S, NEW ENGLAND Biolabs) were combined in a 1.5 mL Eppendorf reaction tube. The final composition of the reaction mixture is defined as follows:

Component	Volume (20 μL)
BbsI-digested pU6 backbone	… μL (50 ng)
Heterodimerized product of the spacer	0.5 μL
Quick Ligase Reaction Buffer (2X)	10 μL
Quick Ligase	1 μL
Nuclease-free water	… μL

The ligation reaction mixture was incubated for 10 min at room temperature.

Product was kept at -20°C until further use. 

Results

The efficiency of the ligation was evaluated by downstream experiments.

DH5α E. coli transformation with 010_pcDNA_Zeo_FLAG_miRFP670nano & 054_pcDNA_Lyn-mCherry-FLAG

Goal;

Transformation was performed to introduce plasmid DNA constructs 010_pcDNA_Zeo_FLAG_miRFP670nano & 054_pcDNA_Lyn-mCherry-FLAG into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results

DH5α colony picking of various plasmids (017, 036-042, 054, 010)

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with various plasmids seen below from selection plate containing Carbenicillin was performed for the replication and expression of the desired genetic material.

017_pcDNA_Zeo_NLuc

010_pcDNA_Zeo_FLAG_miRFP670nano

054_pcDNA_Lyn-mCherry-FLAG

036_pU6_PspCas13b-DR-empty

037_pU6_PspCas13b-DR-gRNA1_tape1

038_pU6_PspCas13b-DR-gRNA1_tape2

039_pU6_PspCas13b-DR-gRNA1_tape3 

040_pU6_PspCas13b-DR-gRNA1_tape4 

041_pU6_PspCas13b-DR-gRNA1_tape5

042_pcDNA3.4-TOPO_T7p_T7tt 

 

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Sample	growth
017.1.1	yes
017.1.2	yes
017.2.1	yes
017.2.2	yes
010.1.1	yes
010.1.2	yes
010.1.3	yes
036.1.1	yes
036.1.2	yes
036.2.2	yes
038.2	yes
041.1	yes
041.2	yes
042.1	yes
042.2	yes
042.3	yes
054.1	yes
054.2	yes
054.3	yes
040.1	no
040.2	no
040.3	no
037.1	no
037.2	no
037.3	no
039.1	no
039.2	no
039.3	no
038.1	no
038.3	no
041.3	no

Sanger sequencing of 010_pcDNA_Zeo_FLAG_miRFP670nano & 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s

Goal;

To confirm the nucleotide sequences of 010_pcDNA_Zeo_FLAG_miRFP670nano & 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s, Sanger sequencing was performed by using Genewiz, Azenta services. 

Protocol;

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 10 μL of purified plasmid at a concentration of 30 - 100 ng/μL was added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis … a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Sample	Primer	Validation
08657467	010.2	CMV-forward	point deletion in CDS
08657468	010.2	BGHR	no priming
08657469	010.3	CMV-forward	successful cloning confirmed
08657470	010.3	BGHR	successful cloning confirmed
08657463	012A-1	CMV-forward	successful cloning confirmed (point mutations are a result of uncertain sanger seq peaks)
08657464	012A-1	BGHR	successful cloning confirmed
08657465	012B-1	CMV-forward	successful cloning confirmed
08657466	012B-1	BGHR	successful cloning confirmed

Seeding of ibidi 8-well microscopic slides

Goal

Seeding ibidi µ-Slide 8 Well microscopic slide for further confocal analysis of Plasmids 010_pcDNA_Zeo_FLAG_miRFP670nano, 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s, 021_pcDNA_Zeo_HA_mScarlet-I, and 054_pcDNA_Lyn-mCherry-FLAG as control, transfected into HEK293T cells with 3×10⁴ HEK293T.

Procedure

All solutions and equipment that came in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet.

The culturing medium was decanted from the 75 cm² culturing flask. Cells were washed using 5 mL of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 mL Accutase solution at 37°C for 5 minutes. Upon confirming detachment using a light phase microscope, cells were rescued using 3 mL pre-warmed complete Dulbecco’s modified Eagle medium F12 (DMEM) and transferred to a 15 mL tube. The cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. The supernatant was decanted using a Pasteur pipette, and the cell pellet was resuspended in 6 mL pre-warmed DMEM. A single well of an ibidi µ-Slide 8 Well microscopic slide was inoculated with 300 µL of cell suspension at 3×10⁴ cells per well. Cells were further grown at 37°C with 5% CO₂ and 90% relative humidity.

Results

Countess™ II automated cell counter Results;

Chamber	Cell count	Live cells	Dead cells
A	5.4×106	4.99×106	4.16×105
B	4.97×106	4.53×106	4.34×105
Mean	5.2×106	4.75×106	4.17×105

Counting cells with Cell countess II

Goal

Calculating cell suspension from flask concentration and assessing the quality of the said suspension

Procedure

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work

Cells were counted and analyzed for viability using Countess™ II automated cell counter with 2 replicas and taken as average of two measurements

Results

Chamber	Cell count	Live cells	Dead cells
A>	5.4x106	4.99x106	4.16x105
B>	4.97x106	4.53x106	4.34x105
Mean	5.2x106	4.75x106	4.17x105

 Cells are in good condition with enough for further seeding

Splitting of HEK293T cell line into T75 flask

Goal;

Splitting HEK293T cell line from flask to the next passage number of 20.

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 3737°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos’s modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 75 cm² flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO₂ and 90% relative humidity.

Results;

HEK293T cell line from flask to the next passage number of 20.

DH5α E. coli transformation with 017_pcDNA_Zeo_NLuc-STOP

Goal;

Transformation was performed to introduce plasmid DNA constructs 017_pcDNA_Zeo_NLuc-STOP into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

 

KLD of 017_pcDNA_Zeo_NLuc-STOP

Goal;

KLD reaction was performed to phosphorylate and circularise the 017_pcDNA_Zeo_NLuc-STOP PCR product, and degrade template DNA by Dpn I enzyme treatment. 

Protocol;

KLD reaction was performed using the KLD Enzyme Mix (NEW ENGLAND Biolabs), consisting of Kinase, Ligase and Dpn I enzymes, according to the manufacturer’s instructions. For this, 5 µL of the KLD reaction Buffer (2X) (NEW ENGLAND Biolabs), 1 µL of KLD Enzyme Mix, 2 µL of Nuclease-free water and 2 µL of the PCR product were combined. The final composition of the reaction mixture was defined as follows:

Components	Volume
PCR Product	1 µL
KLD Reaction Buffer (2X)	5 µL
KLD Enzyme Mix (10X)	1 µL
Nuclease-free Water	3 µL
Total Volume	10 µL

The sample was incubated at room temperature (25°C) for 5 minutes, and then placed on ice. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

PCR of 015_pcDNA_Zeo_DD-N_NLuc_P10SN to get 055_pcDNA_Zeo_DD-N_NLuc_P10SN_W12X

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 015_pcDNA_Zeo_DD-N_NLuc_P10SN using primers 021_F_NLuc_W12X & 022_R_NLuc_W12X with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 021_F_NLuc_W12X 022_R_NLuc_W12X	0.5 μM	2 μL
Template DNA: 015_pcDNA_Zeo_DD-N_NLuc_P10SN	10 ng plasmid	1 μL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

PCR of 017_pcDNA_Zeo_NLuc to get 017_pcDNA_Zeo_NLuc-STOP

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 017_pcDNA_Zeo_NLuc (017.1.1, 017.1.2, 017.2.2) using primers 021_F_NLuc_W12X & 022_R_NLuc_W12X with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 021_F_NLuc_W12X 022_R_NLuc_W12X	0.5 μM	2 μL
Template DNA: 017_pcDNA_Zeo_NLuc	10 ng plasmid	1 μL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

DH5α E. coli transformation 037-041 ligation products & 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s

Goal;

Re-transformation of E.coli with the ligation products seen below & 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s plasmid. 

037_pU6_PspCas13b-DR-gRNA1_tape1

038_pU6_PspCas13b-DR-gRNA1_tape2

039_pU6_PspCas13b-DR-gRNA1_tape3 

040_pU6_PspCas13b-DR-gRNA1_tape4 

041_pU6_PspCas13b-DR-gRNA1_tape5

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

 

Sanger sequencing of 017_pcDNA_Zeo_NLuc, 010_pcDNA_Zeo_FLAG_miRFP670nano, 038_pU6_PspCas13b-DR-gRNA1_tape2, 041_pU6_PspCas13b-DR-gRNA1_tape5

Goal;

To confirm the nucleotide sequences of 017_pcDNA_Zeo_NLuc (017.1.1, 017.1.2, 017.2.2), 010_pcDNA_Zeo_FLAG_miRFP670nano (010.1.2), 038_pU6_PspCas13b-DR-gRNA1_tape2 (038.2), 041_pU6_PspCas13b-DR-gRNA1_tape5 (041.2), Sanger sequencing was performed by using Genewiz, Azenta services. 

Protocol;

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 10 μL of purified plasmid at a concentration of 30 - 100 ng/μL was added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis … a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Sample	Primer	Validation
08657487	017.1.1	CMV-Forward	successful cloning confirmed
08657488	017.1.2	CMV-Forward	successful cloning confirmed
08657489	017.2.2	CMV-Forward	successful cloning confirmed
08657490	010.1.2	CMV-Forward	successful cloning confirmed
08657491	038.2	T3	successful cloning confirmed
08657492	041.2	T3	successful cloning confirmed

 

Whole Plasmid Sequencing - 036_pU6_PspCas13b-DR-empty, 042_pcDNA3.4-TOPO_T7p_T7tt & 054_pcDNA_Lyn-mCherry-FLAG

Goal;

To confirm the nucleotide sequences of 036_pU6_PspCas13b-DR-empty, 042_pcDNA3.4-TOPO_T7p_T7tt and 054_pcDNA_Lyn-mCherry-FLAG whole plasmid sequencing was performed by using Genewiz, Azenta services. 

Protocol;

DNA was sequenced using a Whole Plasmid sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 25 μL of purified plasmid at a concentration of 50 ng/μL The tube was delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis … a match between the experimental and expected sequences. 

Sample	Validation
036.1.2	deletion of BsaI and Sap I RS confirmed; point mutations in ori and lac promoter, 99.90%
042.3	successful cloning confirmed (100.0%)
054.3	99.98%

 

Plasmid Miniprep of various plasmids (017, 036-042, 054, 010)

Goal;

A miniprep of the plasmids shown below was performed to isolate a small amount of plasmid DNA for downstream applications.

017_pcDNA_Zeo_NLuc

010_pcDNA_Zeo_FLAG_miRFP670nano

054_pcDNA_Lyn-mCherry-FLAG

036_pU6_PspCas13b-DR-empty

037_pU6_PspCas13b-DR-gRNA1_tape1

038_pU6_PspCas13b-DR-gRNA1_tape2

039_pU6_PspCas13b-DR-gRNA1_tape3 

040_pU6_PspCas13b-DR-gRNA1_tape4 

041_pU6_PspCas13b-DR-gRNA1_tape5

042_pcDNA3.4-TOPO_T7p_T7tt 

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE- buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
017.1.1	43.4	1.87	2.36
017.1.2	40.1	1.89	2.56
017.2.1	28.8	1.93	2.56
017.2.2	47.9	1.89	2.46
010.1.1	135.5	1.93	2.31
010.1.2	150.2	1.91	2.38
010.1.3	118.6	1.93	2.34
036.1.1	43.0	1.86	2.41
036.1.2	67.1	1.90	2.20
036.2.2	55.6	1.84	2.37
038.2	60.3	1.81	1.95
041.1	133.5	1.95	2.37
041.2	143.8	19.2	2.27
042.1	78.1	1.91	2.36
042.2	84.3	1.96	2.45
042.3	100.3	1.91	2.40
054.1	89.4	1.96	2.43
054.2	81.9	1.88	2.28
054.3	154.6	1.90	2.29

 

DNA Transfection of HEK293T with jetOPTIMUS® for various plasmids (010, 012, 021, 054)

Goal;

Introduction of Plasmids 10.2_pcDNA_Zeo_FLAG_miRFP670nano, 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s, 021_pcDNA_Zeo_HA_mScarlet-I and 54_pcDNA_Lyn-mCherry-FLAG as control into HEK293T cell line at cell partition 20.

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Cells were seeded in 8 well microscopic slides from ibidi at density of 3*10⁴cells per well and grown overnight. Afterwards cells were treated with jetOPTIMUS reagent mixture, including plasmid of interest and fresh DMEM. Afterwards, cells were grown at  37°C with 5% CO₂ and 90% relative humidity. 

Results;

Cells deattached due to vibrations while walking to the CX7 imager.

DH5α E. coli transformation with KLD 017_pcDNA_Zeo_NLuc-STOP (19.11, 19.21 and 19.22)

Goal;

Transformation was performed to introduce plasmid DNA construct 017_pcDNA_Zeo_NLuc-STOP into DH5α E. coli cells, allowing for plasmid replication.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells  (5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 350 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (300 rpm). Cells were spun down at 1350 rpm for 5 min, supernatant discarded and pellet resuspended in 50 µL LB. In the end, 50 µL of the mixture was spread onto a selection plate and incubated overnight at 37°C. Performed with KLD 017_pcDNA_Zeo_NLuc-STOP (19.11, 19.21 and 19.22).

Results;

 

KLD - 017_pcDNA_Zeo_NLuc-STOP (19.11, 19.21 and 19.22)

Goal;

KLD reaction was performed to phosphorylate and circularise the PCR product 017_pcDNA_Zeo_NLuc-STOP and degrade template DNA by Dpn I enzyme treatment. 

Protocol;

KLD reaction was performed using the KLD Enzyme Mix (NEW ENGLAND Biolabs), consisting of Kinase, Ligase and Dpn I enzymes, according to the manufacturer’s instructions. For this, 5 µL of the KLD reaction Buffer (2X) (NEW ENGLAND Biolabs), 1 µL of KLD Enzyme Mix, 2 µL of Nuclease-free water and 2 µL of the PCR product were combined. The final composition of the reaction mixture was defined as follows:

Components	Volume
PCR Product	1 µL
KLD Reaction Buffer (2X)	5 µL
KLD Enzyme Mix (10X)	1 µL
Nuclease-free Water	3 µL
Total Volume	10 µL

The sample was incubated at room temperature (25°C) for 5 minutes, and then placed on ice. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

PCR of 017_pcDNA_Zeo_NLuc to get 017_pcDNA_Zeo_NLuc-STOP

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 017_pcDNA_Zeo_NLuc using primers 021_F_NLuc_W12X and 022_R_NLuc_W12X with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 021_F_NLuc_W12X 022_R_NLuc_W12X	0.5 μM	2 μL
Template DNA: 017_pcDNA_Zeo_NLuc	10 ng plasmid	1 μL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

DH5α colony picking of various plasmids (037-041)

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with 037-041_pU6_PspCas13b-DR-gRNA1_tape1-5 from selection plate containing Carbenicillin was performed for the replication and expression of the desired genetic material.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Carbacenillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Sanger sequencing - 037_pU6_PspCas13b-DR-gRNA1_tape1, 039_pU6_PspCas13b-DR-gRNA1_tape3, 040_pU6_PspCas13b-DR-gRNA1_tape4

Goal;

To confirm the nucleotide sequences of 037_pU6_PspCas13b-DR-gRNA1_tape1, 039_pU6_PspCas13b-DR-gRNA1_tape3, 040_pU6_PspCas13b-DR-gRNA1_tape4 (037.1, 039.3, 040.3) Sanger sequencing was performed by using Genewiz, Azenta services. 

Protocol;

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 10 μL of purified plasmid at a concentration of 30 - 100 ng/μL was added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis … a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Sample	Primer	Validation
08657493	037.1	T3	successful cloning confirmed
08657494	039.3	T3	mutations in gRNA
08657495	040.3	T3	successful cloning confirmed

 

DH5α colony picking of 017_pcDNA_Zeo_NLuc-STOP (019.1.1, 019.2.1, 019.2.2)

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with 017_pcDNA_Zeo_NLuc-STOP (19.1.1, 19.2.1 and 19.2.2) from selection plate containing Cb was performed for the replication and expression of the desired genetic material.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Cb) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge. 3x each

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Plasmid Miniprep of 037_pU6_PspCas13b-DR-gRNA1_tape1, 039_pU6_PspCas13b-DR-gRNA1_tape3 & 040_pU6_PspCas13b-DR-gRNA1_tape4

Goal;

A miniprep of 037_pU6_PspCas13b-DR-gRNA1_tape1, 039_pU6_PspCas13b-DR-gRNA1_tape3 and 040_pU6_PspCas13b-DR-gRNA1_tape4 was performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL TE-buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range. Karl said to runden.

Sample	Concentration [ng/μL]	260/280	260/230
037.1	47	1.9	1.9
037.2	50	1.8	1.6
037.3	39	1.9	1.7
039.1	39	1.8	1.6
039.2	38	1.9	1.8
039.3	52	1.9	1.7
040.1	22	1.9	1.6
040.2	48	1.8	1.5
040.3	43	1.9	1.9

Troubleshooting:

Nuclease-free water was used instead of TE-buffer for elution step. 

Plasmid Miniprep of 017_pcDNA_Zeo_NLuc-STOP

Goal;

A miniprep of 017_pcDNA_Zeo_NLuc-STOP was performed to isolate a small amount of plasmid DNA for downstream applications.

The goal is to introduce a mutation in the NLuc gene for the restoration assay, so that A can be deaminated to I (G).

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of nuclease-free water to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
019.1.1.1	217.1	1.93	2.30
019.1.1.2 (017.3)	258.2	1.93	2.33
019.1.1.3 (017.4)	225.2	1.92	2.30
019.2.1.1	110.0	1.92	2.30
019.2.1.2	123.9	1.92	2.31
019.2.1.3	149.9	1.93	2.30
019.2.2.1	120.5	1.87	2.31
019.2.2.2	163.7	1.93	2.33
019.2.2.3	142.5	1.91	2.33

 

PCR amplification of parts (025-034) for further cloning

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from parts tape gBlocks (025-034) using primers 030_F_tape-amplification and 027_R_pU6_2960A<C).

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration):  030_F_tape-amplification 027_R_pU6_2960A<C	0.5 μM	2 μL
Template DNA: 025_tape_1.0 026_tape_1.1 027_tape_2.0 028_tape_2.1 029_tape_3.0 030_tape_3.1 031_tape_4.0 032_tape_4.1 033_tape_5.0 034_tape_5.1	10 ng plasmid	1 μL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

PCR Cleanup of tape gBlocks (025-034) for further cloning

Goal;

 

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions tape gBlocks (025-034), for use in downstream applications. 

025_tape_1.0

026_tape_1.1

027_tape_2.0

028_tape_2.1

029_tape_3.0

030_tape_3.1

031_tape_4.0

032_tape_4.1

033_tape_5.0

034_tape_5.1

Protocol; 

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratio 1:5 (for dsDNA <2 kb) and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g. The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g. The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of 6 μL of 1 x TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 × g. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
025	197.70	1.95	2.26
026	134.09	1.93	2.25
027	129.19	1.92	2.20
028	180.55	1.95	2.19
029	184.23	1.94	2.23
030	144.32	1.92	2.10
031	177.19	1.93	2.19
032	163.99	1.96	2.18
033	128.19	1.92	2.19
034	158.14	1.91	2.25

 

LB Agar plate streaking of various plasmids (010, 017, 036-042, 054)

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with composite parts seen below from selection plate containing Carbenicillin was performed for creating an agar plate stock. Colony selection is to be performed after confirming transformed plasmid with sequencing. 

017_pcDNA_Zeo_NLuc

010_pcDNA_Zeo_FLAG_miRFP670nano

054_pcDNA_Lyn-mCherry-FLAG

036_pU6_PspCas13b-DR-empty

037_pU6_PspCas13b-DR-gRNA1_tape1

038_pU6_PspCas13b-DR-gRNA1_tape2

040_pU6_PspCas13b-DR-gRNA1_tape4 

041_pU6_PspCas13b-DR-gRNA1_tape5

042_pcDNA3.4-TOPO_T7p_T7tt 

054_pcDNA_Lyn-mCherry-FLAG

Protocol;

Using a sterile inoculation loop, confirmed by sequencing colonies were carefully picked from the surface of the selection plate. Each colony was then streaked onto a fresh selection plate with Carbenicillin and incubated overnight at 37 °C for further growth and propagation of the selected colony. To prevent contamination and stagnate colony overgrowth, the original agar plate was sealed using parafilm and transferred to a 4°C refrigerator.

Results;

Colony	Results
10.1.2	successful streaking
17.1.1	successful streaking
17.1.2	successful streaking
17.2.2	successful streaking
36.1.2	successful streaking
37.1	no growth
38.2	successful streaking
40.3	successful streaking
41.2	successful streaking
42.3	successful streaking
54.3	successful streaking

Successful inoculation of the LB agar plate with desired colony was confirmed with observation of the bacterial growth on the path of the streak.

Sanger sequencing - 039_pU6_PspCas13b-DR-gRNA1_tape3

Goal;

To confirm the nucleotide sequences of 039_pU6_PspCas13b-DR-gRNA1_tape3, Sanger sequencing was performed by using Genewiz, Azenta services. 

Protocol;

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 10 μL of purified plasmid at a concentration of 30 - 100 ng/μL was added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers (T3) were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis … a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Sample	Primer	Validation
08657496	039.2	T3	successful cloning confirmed

 

Whole Plasmid Sequencing - 017_pcDNA_Zeo_NLuc-STOP (019.1.1.2 and 019.1.1.3)

Goal;

To confirm the nucleotide sequences of 017_pcDNA_Zeo_NLuc-STOP (019.1.1.2 and 019.1.1.3), whole plasmid sequencing was performed by using Genewiz, Azenta services.

Protocol;

DNA was sequenced using a Whole Plasmid sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 25 μL of purified plasmid at a concentration of 50 ng/μL The tube was delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis … a match between the experimental and expected sequences. 

Sample	Validation
019.1.1.2 —> 17.3	matched with 017
019.1.1.3 —> 17.4	matched with 017

 

PCR of 042_pcDNA3.4-TOPO_T7p_T7tt to get 043_pcDNA3.4-TOPO_T7p_T7tt

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 042_pcDNA3.4-TOPO_T7p_T7tt using primers 009_F_pcDNA_5881C>G and 010_R_pcDNA_5881C>G with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 009_F_pcDNA_5881C>G 010_R_pcDNA_5881C>G	0.5 μM	2 μL
Template DNA: 042_pcDNA3.4-TOPO_T7p_T7tt	10 ng plasmid	1 μL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

PCR Cleanup - 017_pcDNA_Zeo_NLuc backbone & tapes (025 - 034) inserts

Goal;

 

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions (seen below), for use in downstream applications. 

017_pcDNA_Zeo_NLuc backbone

Inserts:

025_tape_1.0

026_tape_1.1

027_tape_2.0

028_tape_2.1

029_tape_3.0

030_tape_3.1

031_tape_4.0

032_tape_4.1

033_tape_5.0

034_tape_5.1

Protocol; 

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g. The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g. The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 × g. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	Elution volume [μL]	260/280	260/230
025_Gibson	80.03	10	2,14	2,36
026_Gibson	87.29	10	2,05	2,39
027_Gibson	93.71	10	2,05	2,29
028_Gibson	45.64	20	2,20	2,37
029_Gibson	43.08	20	2,09	2,79
030_Gibson	93.94	10	2,07	2,36
031_Gibson	85.53	10	2,05	2,40
032_Gibson	70.70	10	1,91	3,02
033_Gibson	94.68	10	2,04	2,35
034_Gibson	80.03	10	2,06	2,43
017 BB	181,35	20	1,99	2,31

PCR of 017_pcDNA_Zeo_NLuc backbone for Gibson Assembly

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 017_pcDNA_Zeo_NLuc using primers 041_R_pcDNA_tape-amplification & 042_F_tape-amplification_pcDNA with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 041_R_pcDNA_tape-amplification 042_F_tape-amplification_pcDNA	0.5 μM	2 μL
Template DNA: 017_pcDNA_Zeo_NLuc	10 ng plasmid	1 μL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

Sanger sequencing - 017_pcDNA_Zeo_NLuc-STOP

Goal;

To confirm the nucleotide sequences of 017_pcDNA_Zeo_NLuc-STOP (019.1.1.2 and 019.1.1.3), Sanger sequencing was performed by using Genewiz, Azenta services. 

Protocol;

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 10 μL of purified plasmid at a concentration of 30 - 100 ng/μL was added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis was not a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Sample	Primer	Validation
08657497	019.1.1.2	T7	it alignes with 017
08657498	019.1.1.3	T7	it alignes with 017

 

We want point mutation (differing by 1 base to 017_pcDNA_Zeo_NLuc)

Sanger sequencing - 017_pcDNA_Zeo_NLuc-STOP

Goal;

To confirm the nucleotide sequences of 017_pcDNA_Zeo_NLuc-STOP (019.2.1.3, 019.2.2.2 and 019.2.2.3) Sanger sequencing was performed by using Genewiz, Azenta services. 

Protocol;

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 7 μL of purified plasmid at a concentration of 30 - 100 ng/μL was added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers (T7 - starting ~200 bp upstream the region of interest, where point mutation was introduced) were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results;

 

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis is maybe a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Sample	Primer	Validation
08657499	019.2.1.3	T7	matched with 017
08657500	019.2.2.2	T7	matched with 017
08657501	019.2.2.3	T7	successful cloning confirmed

 

PCR amplification of gBlock tapes (025-034) for Gibson Assembly

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from parts tape gBlocks (025-034) using primers 042_F_tape-amplification_pcDNA and 043_R_T2A_NanoLuc.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration):  042_F_tape-amplification_pcDNA 043_R_T2A_NanoLuc	0.5 μM	2 μL
Template DNA: PCR-amplified tapes  025_tape_1.0 026_tape_1.1 027_tape_2.0 028_tape_2.1 029_tape_3.0 030_tape_3.1 031_tape_4.0 032_tape_4.1 033_tape_5.0 034_tape_5.1	10 ng plasmid	1 μL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

Gibson assembly of 017_pcDNA_Zeo_NLuc backbone with tapes inserts (025-034)

Goal;

Gibson assembly was performed to assemble the respective vector and inserts DNA fragments into 017_pcDNA_Zeo_NLuc backbone to get:

044_pcDNA_Zeo_NLuc_tape1.0

045_pcDNA_Zeo_NLuc_tape1.1

046_pcDNA_Zeo_NLuc_tape2.0

047_pcDNA_Zeo_NLuc_tape2.1

048_pcDNA_Zeo_NLuc_tape3.0

049_pcDNA_Zeo_NLuc_tape3.1

050_pcDNA_Zeo_NLuc_tape4.0

051_pcDNA_Zeo_NLuc_tape4.1

052_pcDNA_Zeo_NLuc_tape5.0

053_pcDNA_Zeo_NLuc_tape5.1

Inserts:

025_tape_1.0

026_tape_1.1

027_tape_2.0

028_tape_2.1

029_tape_3.0

030_tape_3.1

031_tape_4.0

032_tape_4.1

033_tape_5.0

034_tape_5.1

Procedure;

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 3-fold molar excess of insert(s), were combined with 10 μL of master mix.

Reagent	044	045	046	047	048	049	050	051	052	053
NEBuilder HiFi DNA Assembly Master Mix	10 μL	10 μL	10 μL	10 μL	10 μL	10 μL	10 μL	10 μL	10 μL	10 μL
Vector: 017_pcDNA_Zeo_NLuc	268 ng / 1,5 μL	268 ng / 1,5 μL	268 ng / 1,5 μL	268 ng / 1,5 μL	268 ng / 1,5 μL	268 ng / 1,5 μL	268 ng / 1,5 μL	268 ng / 1,5 μL	268 ng / 1,5 μL	268 ng / 1,5 μL
Insert: tapes (025-034)	28 ng / 0,3 μL	28 ng / 0,3 μL	28 ng / 0,3 μL	28 ng/ 0,6 μL	28 ng/ 0,6 μL	28 ng / 0,3 μL	28 ng / 0,3 μL	28 ng / 0,4 μL	28 ng / 0,3 μL	28 ng / 0,3 μL
ddH2O	8,2 μL	8,2 μL	8,2 μL	7,9 μL	7,9 μL	8,2 μL	8,2 μL	8,1 μL	8,2 μL	8,2 μL
Total	20 μL	20 μL	20 μL	20 μL	20 μL	20 μL	20 μL	20 μL	20 μL	20 μL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

 

DH5α E. coli transformation with 043_pcDNA3.4-TOPO_T7p_T7tt and Gibson Assembly products (044-053)

Goal;

Transformation of E.coli with 043_pcDNA3.4-TOPO_T7p_T7tt and Gibson Assembly products (044-053) was performed for subsequent culture inoculation and Maxiprep of the plasmid. 

044_pcDNA_Zeo_NLuc_tape1.0

045_pcDNA_Zeo_NLuc_tape1.1

046_pcDNA_Zeo_NLuc_tape2.0

047_pcDNA_Zeo_NLuc_tape2.1

048_pcDNA_Zeo_NLuc_tape3.0

049_pcDNA_Zeo_NLuc_tape3.1

050_pcDNA_Zeo_NLuc_tape4.0

051_pcDNA_Zeo_NLuc_tape4.1

052_pcDNA_Zeo_NLuc_tape5.0

053_pcDNA_Zeo_NLuc_tape5.1

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells  (1-5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 30 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 5 minutes with shaking (300 rpm). In the end, all of the mixture was spread onto a selection plate and incubated overnight at 37°C. Performed with KLD 019 (19.11, 19.21 and 19.22).

Results;

Construct	Results
043	colonies
044	no growth
045	no growth
046	no growth
047	colonies
048	colonies
049	colonies
050	colonies
051	colonies
052	colonies
052	colonies

 

Agarose gel electrophoresis of Gibson Assembly products (044 - 053)

Goal;

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples. 

044_pcDNA_Zeo_NLuc_tape1.0

045_pcDNA_Zeo_NLuc_tape1.1

046_pcDNA_Zeo_NLuc_tape2.0

047_pcDNA_Zeo_NLuc_tape2.1

048_pcDNA_Zeo_NLuc_tape3.0

049_pcDNA_Zeo_NLuc_tape3.1

050_pcDNA_Zeo_NLuc_tape4.0

051_pcDNA_Zeo_NLuc_tape4.1

052_pcDNA_Zeo_NLuc_tape5.0

053_pcDNA_Zeo_NLuc_tape5.1

Protocol;

Agarose gel electrophoresis was performed using a 1.0% w/v agarose (ROTH) gel prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply (BIORAD, PowerPac™ Basic Power Supply). A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 30 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (BIORAD, ChemiDoc Imaging System).

Results;

The following samples were loaded into the gel from left to right: DNA Ladder, 044, 045, 046, 047, 048, 049, 050, 051, 052, 053.

The agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples. Due to the small initial amount of 051, it’s brightness is significantly reduced.

LB Agar plate re-streak of 017_pcDNA_Zeo_NLuc-STOP

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with 017_pcDNA_Zeo_NLuc-STOP (019.2.2.3) from selection plate was performed for creating an agar plate stock. Colony selection is to be performed after confirming transformed plasmid with sequencing. 

Protocol;

Using a sterile inoculation loop, confirmed by sequencing colonies were carefully picked from the surface of the selection plate. Each colony was then streaked onto a fresh selection plate with Carbenicillin and incubated overnight at 37 °C for further growth and propagation of the selected colony. To prevent contamination and stagnate colony overgrowth, the original agar plate was sealed using parafilm and transferred to a 4°C refrigerator.

Results;

Successful inoculation of the LB agar plate with desired colony was confirmed with observation of the bacterial growth on the path of the streak.

LB Agar plate streaking of 017_pcDNA_Zeo_NLuc-STOP and 037_pU6_PspCas13b-DR-gRNA1_tape1

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with 017_pcDNA_Zeo_NLuc-STOP (19.1.1.2) & 037_pU6_PspCas13b-DR-gRNA1_tape1 (37.1) from selection plate containing Cb was performed for creating an agar plate stock. Colony selection is to be performed after confirming transformed plasmid with sequencing. 

Protocol;

Using a sterile inoculation loop, confirmed by sequencing colonies were carefully picked from the surface of the selection plate. Each colony was then streaked onto a fresh selection plate with [Antibiotic. e.g.:  Carbenicillin] and incubated overnight at 37 °C for further growth and propagation of the selected colony. To prevent contamination and stagnate colony overgrowth, the original agar plate was sealed using parafilm and transferred to a 4°C refrigerator.

Results;

Successful inoculation of the LB agar plate with desired colony was confirmed with observation of the bacterial growth on the path of the streak.

KLD of 043_pcDNA3.4-TOPO_T7p_T7tt

Goal;

KLD reaction was performed to phosphorylate and circularise the 043_pcDNA3.4-TOPO_T7p_T7tt PCR product, and degrade template DNA by Dpn I enzyme treatment. 

Protocol;

KLD reaction was performed using the KLD Enzyme Mix (NEW ENGLAND Biolabs), consisting of Kinase, Ligase and Dpn I enzymes, according to the manufacturer’s instructions. For this, 5 µL of the KLD reaction Buffer (2X) (NEW ENGLAND Biolabs), 1 µL of KLD Enzyme Mix, 2 µL of Nuclease-free water and 2 µL of the PCR product were combined. The final composition of the reaction mixture was defined as follows:

Components	Volume
PCR Product	1 µL
KLD Reaction Buffer (2X)	5 µL
KLD Enzyme Mix (10X)	1 µL
Nuclease-free Water	3 µL
Total Volume	10 µL

The sample was incubated at room temperature (25°C) for 5 minutes, and then placed on ice. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

PCR Cleanup of 017_pcDNA_Zeo_NLuc

Goal;

 

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications. 

Protocol; 

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g. The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g. The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 × g. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
017	151.4

Colony picking of DH5α transformed with 017_pcDNA_Zeo_NLuc and plasmids with tapes (044-046)

Goal;

Colony picking of one individual DH5α E. coli bacterial colony transformed with 017_pcDNA_Zeo_NLuc construct and plasmids containing NLuc and tapes.

044_pcDNA_Zeo_NLuc_tape1.0

045_pcDNA_Zeo_NLuc_tape1.1

046_pcDNA_Zeo_NLuc_tape2.0

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Carbenicillin) and incubated overnight at 37 °C with continuous shaking at 130 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Turbulent medium showed growth of the E.coli

Troubleshooting:

DH5α colony picking of 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G), 017_pcDNA_Zeo_NLuc & 036_pU6_PspCas13b-DR-empty

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G), 017_pcDNA_Zeo_NLuc & 036_pU6_PspCas13b-DR-empty from selection plate containing Carbenicillin was performed for the replication and expression of the desired genetic material.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

DH5α E. coli transformation of 017_pcDNA_Zeo_NLuc and plasmids with tapes (044-046)

Goal;

Transformation of E.coli with 017_pcDNA_Zeo_NLuc and Gibson Assembly products (044-046) was performed for subsequent culture inoculation and Maxiprep of the plasmid.

044_pcDNA_Zeo_NLuc_tape1.0

045_pcDNA_Zeo_NLuc_tape1.1

046_pcDNA_Zeo_NLuc_tape2.0

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (2 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 15 minutes with shaking (200 rpm). In the end, 50 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

 

Redo of Gibson assembly of 017_pcDNA_Zeo_NLuc backbone and tape inserts (025-027)

Goal;

Gibson assembly was performed to assemble the respective vector and insert DNA fragments into 017_pcDNA_Zeo_NLuc backbone to get:

044_pcDNA_Zeo_NLuc_tape1.0

045_pcDNA_Zeo_NLuc_tape1.1

046_pcDNA_Zeo_NLuc_tape2.0

Inserts:

025_tape_1.0

026_tape_1.1

027_tape_2.0

Procedure;

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 3-fold molar excess of insert(s), were combined with 10 μL of master mix.

Reagent	044	045	046
NEBuilder HiFi DNA Assembly Master Mix	10 μL	10 μL	10 μL
Vector: 017_pcDNA_Zeo_NLuc	268 ng / 1,8 μL	268 ng / 1,8 μL	268 ng / 1,8 μL
Insert: tapes (025-027)	28 ng / 0,3 μL	28 ng / 0,3 μL	28 ng / 0,3 μL
ddH2O	7,9 μL	7,9 μL	7,9 μL
Total	20 μL	20 μL	20 μL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

 

PCR of 017_pcDNA_Zeo_NLuc backbone for redo of Gibson Assembly of plasmids 044-046

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 017_pcDNA_Zeo_NLuc using primers 041_R_pcDNA_tape-amplification & 042_F_tape-amplification_pcDNA with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration):  041_R_pcDNA_tape-amplification 042_F_tape-amplification_pcDNA	0.5 μM	2 μL
Template DNA: 017_pcDNA_Zeo_NLuc	10 ng plasmid	1 μL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

DH5α colony picking of 043_pcDNA3.4-TOPO_T7p_T7tt and Gibson Assembly products (047-053)

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with 043_pcDNA3.4-TOPO_T7p_T7tt and Gibson Assembly products (047-053) from selection plate containing Carbenicillin was performed for the replication and expression of the desired genetic material.

044_pcDNA_Zeo_NLuc_tape1.0

045_pcDNA_Zeo_NLuc_tape1.1

046_pcDNA_Zeo_NLuc_tape2.0

047_pcDNA_Zeo_NLuc_tape2.1

048_pcDNA_Zeo_NLuc_tape3.0

049_pcDNA_Zeo_NLuc_tape3.1

050_pcDNA_Zeo_NLuc_tape4.0

051_pcDNA_Zeo_NLuc_tape4.1

052_pcDNA_Zeo_NLuc_tape5.0

053_pcDNA_Zeo_NLuc_tape5.1

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Cb) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Splitting of HEK293T cell line into T75 flask

Goal;

Splitting HEK293t cell line from flask to the next passage number of 21.

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 3737°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos’s modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 75 cm² flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO₂ and 90% relative humidity.

Results;

HEK293t cell line from flask was split to passage number 21.

Overnight culture inoculation of 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G), 017_pcDNA_Zeo_NLuc and 036_pU6_PspCas13b-DR-empty

Goal;

An overnight culture was inoculated using a starter culture for the replication and expression of 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G), 017_pcDNA_Zeo_NLuc and 036_pU6_PspCas13b-DR-empty.

Protocol;

5 mL of a starter culture was transferred to 200 mL of fresh culture medium (LB medium with Carbenicillin) and incubated overnight at 37°C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful culture inoculation was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Whole Plasmid Sequencing - 043_pcDNA3.4-TOPO_T7p_T7tt and plasmids containing tapes (047-053)

Goal;

To confirm the nucleotide sequences of 043_pcDNA3.4-TOPO_T7p_T7tt and plasmids containing tapes (047-053), whole plasmid sequencing was performed by using Genewiz, Azenta services. 

047_pcDNA_Zeo_NLuc_tape2.1

048_pcDNA_Zeo_NLuc_tape3.0

049_pcDNA_Zeo_NLuc_tape3.1

050_pcDNA_Zeo_NLuc_tape4.0

051_pcDNA_Zeo_NLuc_tape4.1

052_pcDNA_Zeo_NLuc_tape5.0

053_pcDNA_Zeo_NLuc_tape5.1

Protocol;

DNA was sequenced using a Whole Plasmid sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 25 μL of purified plasmid at a concentration of 50 ng/μL The tube was delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis … a match between the experimental and expected sequences. 

Sample	Validation
1 - 043.3	100%, successful cloning confirmed
2 - 047.1	S->W mutation in T2A
3 - 048.1	successful cloning of the tape confirmed
4 - 049.2	cloning of the tape failed
5 - 050.2	successful cloning of the tape confirmed
6 - 051.2	successful cloning of the tape confirmed
7 - 052.1	successful cloning of the tape confirmed
8 - 053.1	successful cloning of the tape confirmed

 

Plasmid Miniprep of 043_pcDNA3.4-TOPO_T7p_T7tt & plasmids with tapes (047-053)

Goal;

A miniprep of 3 colonies from each of contructs 043_pcDNA3.4-TOPO_T7p_T7tt and plasmids with tapes (047-053) was performed to isolate a small amount of plasmid DNA for downstream applications.

047_pcDNA_Zeo_NLuc_tape2.1

048_pcDNA_Zeo_NLuc_tape3.0

049_pcDNA_Zeo_NLuc_tape3.1

050_pcDNA_Zeo_NLuc_tape4.0

051_pcDNA_Zeo_NLuc_tape4.1

052_pcDNA_Zeo_NLuc_tape5.0

053_pcDNA_Zeo_NLuc_tape5.1

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230	seq labelling
043.1	87,3	1,94	2,22
043.2	150,1	1,89	2,27
043.3	156,4	1,94	2,28	1
047.1	192,6	1,96	2,28	2
047.2	181,4	1,93	2,26
047.3	185,3	1,93	2,24
048.1	270,8	1,94	2,28	3
048.2	235,1	1,93	2,30
048.3	232,4	1,94	2,29
049.1	188,9	1,94	2,28
049.2	232,7	1,91	2,26	4
049.3	145,5	1,95	2,26
050.1	207,8	1,93	2,26
050.2	207,6	1,91	2,24	5
050.3	207,9	1,92	2,30
051.1	183,9	1,93	2,20
051.2	228,2	1,94	2,28	6
051.3	195,0	1,94	2,29
052.1	258,9	1,93	2,28	7
052.2	217,1	1,91	2,27
052.3	171,0	1,94	2,26
053.1	206,6	1,94	2,27	8
053.2	190,7	1,91	2,27
053.3	160,7	1,92	2,25

 

Maxiprep (Promega) of 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G), 017_pcDNA_Zeo_NLuc & 036_pU6_PspCas13b-DR-empty

Goal;

 

A maxiprep of 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G), 017_pcDNA_Zeo_NLuc and 036_pU6_PspCas13b-DR-empty was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Protocol; 

DNA plasmids were prepared using the PureYield™ Plasmid Maxiprep System kit according to the modified manufacturer’s instructions (Promega). 200 mL of overnight culture of E. coli was centrifuged at 4500 × g for 15 min at room temperature. The resulting pellet was resuspended in 12 mL of Cell Resuspension Solution (Promega). To lyse the cells, 12 mL of Cell Lysis Solution (Promega) was added and mixed by inverting 4 - 6 times. The lysed cells were neutralized by adding 12 mL of Neutralization Solution (Promega) and mixing by inverting 10 - 15 times. The mixture was centrifuged at 4500 × g (Heraeus Multifuge X1R Centrifuge; Thermo Fisher Scientific) for 1 hour at room temperature. In the meantime, the column stack, consisting of assembled blue PureYield™ Clearing Column and white PureYield™ Maxi Binding Column, were placed on the vacuum manifold. Half of the supernatant was applied to the blue PureYield™ Clearing Column and the maximum vacuum was applied until the lysate has passed through both the clearing and binding columns. The same procedure was conducted with the remaining cell lysate. Once the liquid passed through both columns, vacuum was slowly released and the blue PureYield™ Clearing Columns were discarded. The column was then washed by adding 5mL of Endotoxin Removal Wash to the PureYield™ Maxi Binding Column and applying a vacuum, followed by addition of 20mL of Column Wash to the binding column, and applying the vacuum to pull the solution through the column. The membrane was dried by applying a vacuum for 5 minutes. After careful removing remaining ethanol by tapping the tip of the PureYield™ Maxi Binding Column on a paper towel, the columns were placed in new 50 mL centrifuge tubes. The DNA was eluted by addition of 1 mL of TE buffer and centrifugation at 4500 × g for 5 min. The concentration was determined using a NanoPhotometer N60 (Implen), and the DNA was stored at −20 °C.

Results;

The maxiprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
004	117,1	1,92	2,13
017	66,5	1,93	2,33
036	185,3	1,92	2,29

 

Splitting of HEK293T cell line into T75 flask

Goal;

Splitting HEK293t cell line from flask  to the next passage number of 23.

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 3737°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos’s modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 75 cm² flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO₂ and 90% relative humidity.

Results;

HEK293t cell line from flask was split to passage number 23.

Plasmid Miniprep of plasmids containing tapes (044-046)

Goal;

A miniprep of 3 colonies from each from plasmids containing tapes (044-046) was performed to isolate a small amount of plasmid DNA for downstream applications.

044_pcDNA_Zeo_NLuc_tape1.0

045_pcDNA_Zeo_NLuc_tape1.1

046_pcDNA_Zeo_NLuc_tape2.0

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
044.1	201,2	1,93	2,32
044.2	210,5	1,93	2,3
044.3	118,3	1,95	2,35
045.1	114,9	1,92	2,26
045.2	125,7	1,95	2,33
045.3	135	1,95	2,3
046.1	179	1,94	2,31
046.2	183,8	1,95	2,3
046.3	146,5	1,93	2,31

Seeding of ibidi 8-well microscopic slides for XFPs and NLuc_restoration transfection

Goal

Seeding ibidi µ-Slide 8 Well microscopic slide with 1×10⁴ HEK293T cells for further transfection.

Procedure

All solutions and equipment that came in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet.

The culturing medium was decanted from the 75 cm² culturing flask. Cells were washed using 5 mL of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 mL Accutase solution at 37°C for 5 minutes. Upon confirming detachment using a light phase microscope, cells were rescued using 3 mL pre-warmed complete Dulbecco’s modified Eagle medium F12 (DMEM) and transferred to a 15 mL tube. The cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. The supernatant was decanted using a Pasteur pipette, and the cell pellet was resuspended in 6 mL pre-warmed DMEM. A single well of an ibidi µ-Slide 8 Well microscopic slide was inoculated with 300 µL of cell suspension at 1×10⁴ cells per well. Cells were further grown at 37°C with 5% CO₂ and 90% relative humidity.

Results;

Countess™ II automated cell counter Results;

Chamber	Cell count	Live cells	Dead cells
A	1.85×106	1.72×106	1.29×105
B	2.24×106	2.09×106	1.52×105

DNA Transfection of HEK293T with jetOPTIMUS® for plasmids (010, 012, 021 and 054), Attempt 2

Goal;

Introduction of Plasmids 010_pcDNA_Zeo_FLAG_miRFP670nano (colony 2), 012_pcDNA_Zeo_mTagBFP2_TGA_RT20s, 021_pcDNA_Zeo_HA_mScarlet-I and 054_pcDNA_Lyn-mCherry-FLAG as control into HEK293T cell line at cell partition 23.

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Cells were seeded in 8 well microscopic slides from ibidi at density of 5*10³cells per well and grown overnight. Afterwards cells were treated with jetOPTIMUS reagent mixture, including plasmid of interest and fresh DMEM. Afterwards, cells were grown at  37°C with 5% CO₂ and 90% relative humidity. 

Results;

Results shown on the results page of the wiki

DH5α colony picking of 043_pcDNA3.4-TOPO_T7p_T7tt for setting overnight culture for Maxiprep

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with 043_pcDNA3.4-TOPO_T7p_T7tt from selection plate containing Carbenicillin was performed for the replication and expression of the desired genetic material.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Whole Plasmid Sequencing of various plasmids (044-046, 004, 017, 036)

Goal;

To confirm the nucleotide sequences of plasmids seen below, whole plasmid sequencing was performed by using Genewiz, Azenta services. 

004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G)

017_pcDNA_Zeo_NLuc

036_pU6_PspCas13b-DR-empty

044_pcDNA_Zeo_NLuc_tape1.0

045_pcDNA_Zeo_NLuc_tape1.1

046_pcDNA_Zeo_NLuc_tape2.0

Protocol;

DNA was sequenced using a Whole Plasmid sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 25 μL of purified plasmid at a concentration of 50 ng/μL The tube was delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis was a match between the experimental and expected sequences. 

Sample	seq label	Validation
044.2	1	failed cloning
045.3	2	successful
046.2	3	successful
004 (maxi)	4	successful maxiprep confirmed
017 (maxi)	5	successful
036 (maxi)	6	successful

 

Sanger sequencing of 047_pcDNA_Zeo_NLuc_tape2.1 & 049_pcDNA_Zeo_NLuc_tape3.1

Goal;

To confirm the nucleotide sequences of 047_pcDNA_Zeo_NLuc_tape2.1 & 049_pcDNA_Zeo_NLuc_tape3.1 Sanger sequencing was performed by using Genewiz, Azenta services. 

Protocol;

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 10 μL of purified plasmid at a concentration of 30 - 100 ng/μL was added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis … a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Sample	Primer	Validation
047.2	CMV_Forward	successful cloning confirmed
047.3	CMV_Forward	successful cloning confirmed
049.1	CMV_Forward	successful cloning confirmed
049.3	CMV_Forward	successful cloning confirmed

 

Overnight culture inoculation of 043_pcDNA3.4-TOPO_T7p_T7tt

Goal;

An overnight culture was inoculated using a starter culture for the replication and expression of 043_pcDNA3.4-TOPO_T7p_T7tt.

Protocol;

1 mL of a starter culture was transferred to 200 mL of fresh culture medium (LB medium with Carbenicillin) and incubated overnight at 37°C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful culture inoculation was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

LB Agar plate re-streak of 043_pcDNA3.4-TOPO_T7p_T7tt

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with 043_pcDNA3.4-TOPO_T7p_T7tt (043.3) from selection plate containing Carbenicillin was performed for creating an agar plate stock. 

Protocol;

Using a sterile inoculation loop, overnight culture of E.coli transformed with 043.3 confirmed by sequencing was streaked onto a fresh selection plate with Carbenicillin and incubated overnight at 37 °C for further growth and propagation of the selected colony. To prevent contamination and stagnate colony overgrowth, the original agar plate was sealed using parafilm and transferred to a 4°C refrigerator.

Results;

Successful inoculation of the LB agar plate with desired colony was confirmed with observation of the bacterial growth on the path of the streak.

DNA Transfection of HEK293T with plasmids 4, 19,23 and 17 as control with jetOPTIMUS®

Goal;

Introduction of plasmids 4 (pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G)), 19 (pcDNA_Zeo_DD-N_NLuc_P10SN), 23(pU6_PspCas13b-DR-gRNA_NLuc) and 17(pcDNA_Zeo_DD-N_NLuc_P10SN into HEK293T cell line at cell partition 23

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Cells were seeded in 8 well microscopic slides from ibidi at density of 1*10⁴cells per well and grown overnight. Afterwards cells in each well were treated with jetOPTIMUS reagent mixture, including plasmids 4 (pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G)), 19 (pcDNA_Zeo_DD-N_NLuc_P10SN), 23(pU6_PspCas13b-DR-gRNA_NLuc) and 17(pcDNA_Zeo_DD-N_NLuc_P10SN). Fresh DMEM was supplied together with the transfection mixture. Afterwards, cells were grown at  37°C with 5% CO₂ and 90% relative humidity. 

Results;

Order: 

column 1: experiment (019&004&023)

column 2: neg control (019&023)

column 3: neg control (019&004)

column 4: pos control (017)

Detailed results are displayed on the results wiki

Splitting of HEK293T cell line into T75 flask

Goal;

Splitting HEK293T cell line from flask  to the next passage number of 23.

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 3737°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos’s modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 75 cm² flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO₂ and 90% relative humidity.

Results;

HEK293T cell line from flask was split to passage number 23.

Maxiprep of 043_pcDNA3.4-TOPO_T7p_T7tt

Goal;

 

A maxiprep of 043_pcDNA3.4-TOPO_T7p_T7tt was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Protocol; 

DNA plasmids were prepared using the PureYield™ Plasmid Maxiprep System kit according to the modified manufacturer’s instructions (Promega). 200 mL of overnight culture of E. coli was centrifuged at 4500 × g for 15 min at room temperature. The resulting pellet was resuspended in 12 mL of Cell Resuspension Solution (Promega). To lyse the cells, 12 mL of Cell Lysis Solution (Promega) was added and mixed by inverting 4 - 6 times. The lysed cells were neutralized by adding 12 mL of Neutralization Solution (Promega) and mixing by inverting 10 - 15 times. The mixture was centrifuged at 4500 × g (Heraeus Multifuge X1R Centrifuge; Thermo Fisher Scientific) for 1 hour at room temperature. In the meantime, the column stack, consisting of assembled blue PureYield™ Clearing Column and white PureYield™ Maxi Binding Column, were placed on the vacuum manifold. Half of the supernatant was applied to the blue PureYield™ Clearing Column and the maximum vacuum was applied until the lysate has passed through both the clearing and binding columns. The same procedure was conducted with the remaining cell lysate. Once the liquid passed through both columns, vacuum was slowly released and the blue PureYield™ Clearing Columns were discarded. The column was then washed by adding 5mL of Endotoxin Removal Wash to the PureYield™ Maxi Binding Column and applying a vacuum, followed by addition of 20mL of Column Wash to the binding column, and applying the vacuum to pull the solution through the column. The membrane was dried by applying a vacuum for 5 minutes. After careful removing remaining ethanol by tapping the tip of the PureYield™ Maxi Binding Column on a paper towel, the columns were placed in new 50 mL centrifuge tubes. The DNA was eluted by addition of 1 mL of TE buffer and centrifugation at 4500 × g for 5 min. The concentration was determined using a NanoPhotometer N60 (Implen), and the DNA was stored at −20 °C.

Results;

The maxiprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
043.03	70.1	2.02	2.18

 

Sanger sequencing 044_pcDNA_Zeo_NLuc_tape1.0

Goal;

To confirm the nucleotide sequences of 044_pcDNA_Zeo_NLuc_tape1.0 (044.1, 044.3), Sanger sequencing was performed by using Genewiz, Azenta services. 

Protocol;

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 10 μL of purified plasmid at a concentration of 30 - 100 ng/μL was added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis was a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Sample	Primer	Validation
/	044.1	CMV_Forward	successful cloning confirmed
/	044.3	CMV_Forward	successful cloning confirmed

 

DNA Transfection of HEK293T with various plasmids (004, 019, 023 and 017) as control with jetOPTIMUS® Attempt2

Goal;

Introduction of plasmids 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G), 017_pcDNA_Zeo_NLuc-STOP, 023_pU6_PspCas13b-DR-gRNA_NLuc and 017_pcDNA_Zeo_NLuc into HEK293T cell line at cell partition 23.

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Ibidi 8 well microscopic slides were coated with 100 µl of 1x poly-D-Lysin for 30 minutes. Subsequently, Cells were seeded in coated 8 well slides at density of 1*10⁵cells per well and grown overnight. Following 24 hours of culturing, cells in each well were treated with jetOPTIMUS reagent mixture, including plasmids 4 (pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G)), 19 (pcDNA_Zeo_DD-N_NLuc_P10SN), 23(pU6_PspCas13b-DR-gRNA_NLuc) and 17(pcDNA_Zeo_DD-N_NLuc_P10SN). Fresh DMEM was supplied together with the transfection mixture. Afterwards, cells were grown at  37°C with 5% CO₂ and 90% relative humidity. 

Results;

Order: 

column 1: experiment (019&004&023)

column 2: negative control for unintended operation of gRNA plasmid (019&023)

column 3: negative control for unintended operation of REPAIR construct (019&004)

column 4: positive control of unmutated and functional Nano Luciferase (017)

 

DH5α E. coli re-transformation with 023_pU6_PspCas13b-DR-gRNA_NLuc

Goal;

Transformation was performed to introduce plasmid DNA construct 023_pU6_PspCas13b-DR-gRNA_NLuc into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

 

DH5α colony picking of 023_pU6_PspCas13b-DR-gRNA_NLuc

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with 023_pU6_PspCas13b-DR-gRNA_NLuc (023.2) from selection plate containing Carbenicillin was performed for the replication and expression of the desired genetic material.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

No turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C was observed.

DH5α colony picking of 023_pU6_PspCas13b-DR-gRNA_NLuc

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with 023_pU6_PspCas13b-DR-gRNA_NLuc (023.2, 023) from selection plate containing carbenicillin was performed for the replication and expression of the desired genetic material.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking of 023_pU6_PspCas13b-DR-gRNA_NLuc was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C. There were no pellet in the tubes containing 023_pU6_PspCas13b-DR-gRNA_NLuc, therefore they were discarded.

Splitting of HEK293T cell line into T75 flask

Goal;

Splitting HEK293T cell line from flasks to the next passage number of 26.

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 3737°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos’s modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 75 cm² flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO₂ and 90% relative humidity.

Results;

HEK293T cell line from flasks was split to passage number 26.

DH5α colony picking of 023_pU6_PspCas13b-DR-gRNA_NLuc

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with 023_pU6_PspCas13b-DR-gRNA_NLuc from selection plate containing LB was performed for the replication and expression of the desired genetic material.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

6 tubes were placed to incubate.

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Plasmid Miniprep of 023_pU6_PspCas13b-DR-gRNA_NLuc

Goal;

A miniprep of 023_pU6_PspCas13b-DR-gRNA_NLuc (023.2) was performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of insufficient quantity and purity ratios A260/A280 and A260/A230 outside the acceptable range. All three samples were discarded because of low DNA yield or high A260/230 ratio.

Sample	Concentration [ng/μL]	260/280	260/230
023.2.4	15.05	1.73	2.30
023.2.5	17.81	1.75	3.55
023.2.6	22.71	1.79	2.43

 

Plasmid Miniprep - 023_pU6_PspCas13b-DR-gRNA_NLuc & 056_pcDNA_tdPCP-SpyC3

Goal;

A miniprep of 056_pcDNA_tdPCP-SpyC3 and 023_pU6_PspCas13b-DR-gRNA_NLuc was performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
56.1	172.8	1.92	2.28
56.2	33.5	1.89	2.22
23.2.7	146.2	1.94	2.29
23.2.8	84.9	1.93	2.27
23.2.9	99.9	1.91	2.26
23.7	7.1	/	/
23.8	19.1	/	/
23.9	10.2	1.87	2.06

 

Whole Plasmid Sequencing - 056_pcDNA_tdPCP-SpyC3

Goal;

To confirm the nucleotide sequences of 056_pcDNA_tdPCP-SpyC3, whole plasmid sequencing was performed by using Genewiz, Azenta services. 

Protocol;

DNA was sequenced using a Whole Plasmid sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 25 μL of purified plasmid at a concentration of 50 ng/μL The tube was delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis was match between the experimental and expected sequences. 

Sample	Validation
056.1	plasmid sequence confirmed

 

Splitting of HEK293T cell line into T25 flask

Goal;

Splitting HEK293T cell line from flask  to the next passage number of 27.

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Culturing media was decanted from the 25 cm² culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 3737°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos’s modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 25 cm² flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO₂ and 90% relative humidity.

Results;

HEK293T cell line from flask was split to passage number 27.

Sanger sequencing - 023_pU6_PspCas13b-DR-gRNA_NLuc

Goal;

To confirm the nucleotide sequences of 023_pU6_PspCas13b-DR-gRNA_NLuc, Sanger sequencing was performed by using Genewiz, Azenta services. 

Protocol;

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 10 μL of purified plasmid at a concentration of 30 - 100 ng/μL was added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis … a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Sample	Primer	Validation
/	023.2.7	T3	successful cloning confirmed
/	023.2.8	T3	successful cloning confirmed
/	023.2.9	T3	successful cloning confirmed

 

DH5α colony picking of 037_pU6_PspCas13b-DR-gRNA1_tape1 & 039_pU6_PspCas13b-DR-gRNA1_tape3

Goal;

Colony picking of individual DH5α E. coli bacterial colonies from re-streak of 037_pU6_PspCas13b-DR-gRNA1_tape1 & 039_pU6_PspCas13b-DR-gRNA1_tape3 (037.2, 039.2) from selection plate containing carbenicillin was performed for the replication and expression of the desired genetic material and downstream maxiprep of the plasmid. 

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Overnight culture inoculation of 037_pU6_PspCas13b-DR-gRNA1_tape1 & 039_pU6_PspCas13b-DR-gRNA1_tape3

Goal;

An overnight culture was inoculated using a starter culture for the replication and expression of 037_pU6_PspCas13b-DR-gRNA1_tape1 and 039_pU6_PspCas13b-DR-gRNA1_tape3.

Protocol;

1 mL of a starter culture was transferred to 200 mL of fresh culture medium (LB medium with Carbenicillin) and incubated overnight at 37°C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful culture inoculation was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Splitting of HEK293T cell line into T75 flask

Goal;

Splitting HEK293T cell line from flask to the next passage number of 28.

Procedure:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet.

Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 37°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3 ml pre-warmed complete Dulbecco’s modified Eagle medium F12 (DMEM) and transferred to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using a Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 75 cm² flask was inoculated with the 1 ml cell suspension to 9 ml pre-warmed DMEM. Cells were grown at 37°C with 5% CO₂ and 90% relative humidity.

Results;

HEK293T cell line from flask was split to passage number 28.

Seeding of 2 Nunc™ MicroWell™ 96-Well plates

Goal;

Seeding Nunc™ MicroWell™ 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate, Thermo Scientific™ for further confocal analysis of [Plasmids/Treatments/Controls] transfected into HEK293T cells with 1×10⁴ HEK293T.

Procedure:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet.

Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 37°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3 ml pre-warmed complete Dulbecco’s modified Eagle medium F12 (DMEM) and transferred to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using a Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. Each well of the Nunc™ 96-well plate was inoculated with 100 µl of cell suspension at 1×10⁴ cells per well. Cells were further grown at 37°C with 5% CO₂ and 90% relative humidity.

Counting cells with Cell Countess II

Goal;

Calculating cell suspension concentration and assessing the quality of the said suspension.

Procedure:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet.

Cells were counted and analyzed for viability using Countess™ II automated cell counter with 2 replicas and taken as the average of two measurements.

Results;

Chamber	Cell count	Live cells	Dead cells
A	6.87×106	6.32×106	5.51×105
B	6.42×106	6.12×106	2.99×105
Mean	6.65×106	6.22×106	4.25×103

Results;

Cells are in good condition with enough for further seeding.

Maxiprep (Promega) of 037_pU6_PspCas13b-DR-gRNA1_tape1 & 039_pU6_PspCas13b-DR-gRNA1_tape3

Goal;

 

A maxiprep of 037_pU6_PspCas13b-DR-gRNA1_tape1 (037.2.1) and 039_pU6_PspCas13b-DR-gRNA1_tape3 (039.2.1) was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Protocol; 

DNA plasmids were prepared using the PureYield™ Plasmid Maxiprep System kit according to the modified manufacturer’s instructions (Promega). 200 mL of overnight culture of E. coli was centrifuged at 4500 × g for 15 min at room temperature. The resulting pellet was resuspended in 12 mL of Cell Resuspension Solution (Promega). To lyse the cells, 12 mL of Cell Lysis Solution (Promega) was added and mixed by inverting 4 - 6 times. The lysed cells were neutralized by adding 12 mL of Neutralization Solution (Promega) and mixing by inverting 10 - 15 times. The mixture was centrifuged at 4500 × g (Heraeus Multifuge X1R Centrifuge; Thermo Fisher Scientific) for 1 hour at room temperature. In the meantime, the column stack, consisting of assembled blue PureYield™ Clearing Column and white PureYield™ Maxi Binding Column, were placed on the vacuum manifold. Half of the supernatant was applied to the blue PureYield™ Clearing Column and the maximum vacuum was applied until the lysate has passed through both the clearing and binding columns. The same procedure was conducted with the remaining cell lysate. Once the liquid passed through both columns, vacuum was slowly released and the blue PureYield™ Clearing Columns were discarded. The column was then washed by adding 5mL of Endotoxin Removal Wash to the PureYield™ Maxi Binding Column and applying a vacuum, followed by addition of 20mL of Column Wash to the binding column, and applying the vacuum to pull the solution through the column. The membrane was dried by applying a vacuum for 5 minutes. After careful removing remaining ethanol by tapping the tip of the PureYield™ Maxi Binding Column on a paper towel, the columns were placed in new 50 mL centrifuge tubes. The DNA was eluted by addition of 0.8 mL of TE buffer and centrifugation at 4500 × g for 5 min. The concentration was determined using a NanoPhotometer N60 (Implen), and the DNA was stored at −20 °C.

Results;

The maxiprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
37.2.1	243,6	1,93	2,56
39.2.1	337,7	1,91	2,46

 

DNA Transfection of HEK293T with jetOPTIMUS® – NLuc restoration and tape testing

Goal;

Introduction of plasmids into HEK293T cell line at cell partition 27, according to the chart described below: 

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Cells were seeded in 96-well plates at density of 1*10⁴ cells per well and grown overnight. Afterwards cells were treated with jetOPTIMUS reagent mixture, including plasmid of interest and fresh DMEM. Afterwards, cells were grown at  37°C with 5% CO₂ and 90% relative humidity. 

Results;

seen in results page

 

Splitting of HEK293T cell line into T75 flask

Goal;

Splitting HEK293T cell line from flask to the next passage number of 29.

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 3737°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos’s modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 75 cm² flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO₂ and 90% relative humidity.

Results;

HEK293T cell line from flask was split to passage number 29.

Maxiprep (Qiagen) of various plasmids (036, 040, 041 & 046)

Goal;

 

A maxiprep of 036_pU6_PspCas13b-DR-empty (036.2), 040_pU6_PspCas13b-DR-gRNA1_tape4 (040.3), 041_pU6_PspCas13b-DR-gRNA1_tape5 (041.2) and 046_pcDNA_Zeo_NLuc_tape2.0 (046.2) was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Protocol; 

DNA plasmids were prepared using the QIAGEN^® Plasmid Mini, Midi, and Maxi kit according to the modified manufacturer’s instructions (QIAGEN). … mL of overnight culture of E. coli was centrifuged at 4500× g for 20 min at 4°C. The resulting pellet was resuspended in 10 mL of P1 Buffer (QIAGEN) supplemented with RNase A solution (100 μg/mL; QIAGEN), and LyseBlue reagent (1:1000; QIAGEN). The suspension was transferred to a 50 mL centrifuge tube. To lyse the cells, 10 mL of P2 Buffer (QIAGEN) was added and mixed by inverting 4 - 6 times, followed by incubation at room temperature for 5 min. The lysed cells were neutralized by adding 10 mL of pre-cooled P3 Buffer (QIAGEN) and mixing by inverting 4 - 6 times, followed by incubation on ice for 20 min. Lysis and neutralization were confirmed using the LyseBlue reagent, where the solution turned blue upon adding P2 Buffer and reverted to colorless on addition of P3 buffer (pre-cooled at 4 °C) with thorough mixing. The mixture was centrifuged at 7000 × g for 90 min at 4 °C, then the supernatant was centrifuged again in a new tube at 7000 × g for 45 min at 4 °C. The supernatant was applied to the QIAGEN-tip500 column pre-equilibrated with 10 mL of QBT Buffer (QIAGEN). The column was then washed by adding 2 × 30 mL of QC Buffer (QIAGEN), and DNA was eluted with 15 mL of QF Buffer (QIAGEN) into a new and clean 50 mL centrifuge tube. DNA precipitation was performed by adding 10.5 mL of room-temperature isopropanol and centrifuged at 7000 × g for 75 min at 4 °C. After careful decanting of the supernatant, the DNA pellet was washed with 5 mL of room-temperature 70% v/v ethanol and centrifuged at 7000 × g for 30 min at 4 °C. Finally, the supernatant was carefully decanted without disturbing the pellet, and the pellet was air-dried for 5 - 10 min. The DNA was then redissolved in 500 μL of 1x TE buffer, the concentration was determined using a NanoPhotometer N60 (Implen), and the DNA was stored at −20 °C.

Results;

The maxiprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
036.2	279,98	1,93	2,32
040.3	141,01	1,93	2,30
041.2	303,80	1,93	2,36
046.2	598,10	1,92	2,18

 

 

Overnight culture inoculation - 017_pcDNA_Zeo_NLuc-STOP, 023_pU6_PspCas13b-DR-gRNA_NLuc & 042_pcDNA3.4-TOPO_T7p_T7tt

Goal;

An overnight culture was inoculated using a starter culture for the replication and expression of 017_pcDNA_Zeo_NLuc-STOP (19.2.3), 023_pU6_PspCas13b-DR-gRNA_NLuc (023.2.7) and 042_pcDNA3.4-TOPO_T7p_T7tt (042.3).

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge. After 8 hours, 5 mL of a starter culture was transferred to 200 mL of fresh culture medium (LB medium with Carbenicillin) and incubated overnight at 37°C with continuous shaking at 200 rpm for further growth and amplification. 

Results;

Successful culture inoculation was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Maxiprep (QIAGEN) - various plasmids with tapes (044, 045, 047 & 048)

Goal;

 

A maxiprep of 044_pcDNA_Zeo_NLuc_tape1.0 (044.3), 045_pcDNA_Zeo_NLuc_tape1.1 (045.3), 047_pcDNA_Zeo_NLuc_tape2.1 (047.2) and 048_pcDNA_Zeo_NLuc_tape3.0 (048.1) was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Protocol; 

DNA plasmids were prepared using the QIAGEN^® Plasmid Mini, Midi, and Maxi kit according to the modified manufacturer’s instructions (QIAGEN). 200 mL of overnight culture of E. coli was centrifuged at 4500× g for 20 min at 4°C (Heraeus Multifuge X1R Centrifuge; Thermo Fisher Scientific - east lab, left). The resulting pellet was resuspended in 10 mL of P1 Buffer (QIAGEN) supplemented with RNase A solution (100 μg/mL; QIAGEN), and LyseBlue reagent (1:1000; QIAGEN). The suspension was transferred to a 50 mL centrifuge tube. To lyse the cells, 10 mL of P2 Buffer (QIAGEN) was added and mixed by inverting 4 - 6 times, followed by incubation at room temperature for 5 min. The lysed cells were neutralized by adding 10 mL of pre-cooled P3 Buffer (QIAGEN) and mixing by inverting 4 - 6 times, followed by incubation on ice for 20 min. Lysis and neutralization were confirmed using the LyseBlue reagent, where the solution turned blue upon adding P2 Buffer and reverted to colorless on addition of P3 buffer with thorough mixing. The mixture was centrifuged at 7000 g (eppendorf 5430 R - east lab, right) for 90 min at 4 °C. (Supernatant not clear: re-centrifuge for 45 min). The supernatant was applied to the QIAGEN-tip500 column pre-equilibrated with 10 mL of QBT Buffer (QIAGEN). The column was then washed by adding 2 × 30 mL of QC Buffer (QIAGEN), and DNA was eluted with 15 mL of QF Buffer (QIAGEN) into a 50 mL centrifuge tube. DNA precipitation was performed by adding 10.5 mL of room-temperature isopropanol and centrifuging at 7000 × g for 75 min at 4 °C. After careful decanting of the supernatant, the DNA pellet was washed with 5 mL of room-temperature 70% v/v ethanol. Finally, the supernatant was aspirated with a pipette, without disturbing the pellet, and the pellet was air-dried for 5 - 10 min. The DNA was then redissolved in 300 - 500 μL of TE buffer, the concentration was determined using a NanoPhotometer N60 (Implen), and the DNA was stored at −20 °C.

Results;

The maxiprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
044.3	457	1.92	2.22
045.3	438	1.92	2.24
047.2	710	1.91	2.24
048.1	521	1.93	2.21

 

DH5α E. coli transformation with 072_pcDNA3.4-TOPO_T7p_T7tt KLD product

Goal;

Transformation was performed to introduce plasmid DNA construct 072_pcDNA3.4-TOPO_T7p_T7tt into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

 

KLD of 072_pcDNA3.4-TOPO_T7p_T7tt

Goal;

KLD reaction was performed to phosphorylate and circularise the PCR product of 072_pcDNA3.4-TOPO_T7p_T7tt, and degrade template DNA by Dpn I enzyme treatment.

Protocol;

KLD reaction was performed using the KLD Enzyme Mix (NEW ENGLAND Biolabs), consisting of Kinase, Ligase and Dpn I enzymes, according to the manufacturer’s instructions. For this, 5 µL of the KLD reaction Buffer (2X) (NEW ENGLAND Biolabs), 1 µL of KLD Enzyme Mix, 2 µL of Nuclease-free water and 2 µL of the PCR product were combined. The final composition of the reaction mixture was defined as follows:

Components	Volume
PCR Product	1 µL
KLD Reaction Buffer (2X)	5 µL
KLD Enzyme Mix (10X)	1 µL
Nuclease-free Water	3 µL
Total Volume	10 µL

The sample was incubated at room temperature (25°C) for 5 minutes, and then placed on ice. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

PCR of 043_pcDNA3.4-TOPO_T7p_T7tt to get 072_pcDNA3.4-TOPO_T7p_T7tt

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 043_pcDNA3.4-TOPO_T7p_T7tt (043.3) using primers 053_F_CMV_G<C and 054_R_CMV_G>C with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 053_F_CMV_G<C 054_R_CMV_G>C	0.5 μM	2 μL
Template DNA: 043_pcDNA3.4-TOPO_T7p_T7tt	10 ng plasmid	1 μL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

Maxiprep (QIAGEN) - 017_pcDNA_Zeo_NLuc-STOP, 023_pU6_PspCas13b-DR-gRNA_NLuc & 042_pcDNA3.4-TOPO_T7p_T7tt

Goal;

 

A maxiprep of 017_pcDNA_Zeo_NLuc-STOP (019.2.3), 023_pU6_PspCas13b-DR-gRNA_NLuc (023.2.7) and 042_pcDNA3.4-TOPO_T7p_T7tt (042.3) and was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Protocol; 

DNA plasmids were prepared using the QIAGEN^® Plasmid Mini, Midi, and Maxi kit according to the modified manufacturer’s instructions (QIAGEN). 200 mL of overnight culture of E. coli was centrifuged at 4500× g for 20 min at 4°C. The resulting pellet was resuspended in 10 mL of P1 Buffer (QIAGEN) supplemented with RNase A solution (100 μg/mL; QIAGEN), and LyseBlue reagent (1:1000; QIAGEN). The suspension was transferred to a 50 mL centrifuge tube. To lyse the cells, 10 mL of P2 Buffer (QIAGEN) was added and mixed by inverting 4 - 6 times, followed by incubation at room temperature for 5 min. The lysed cells were neutralized by adding 10 mL of pre-cooled P3 Buffer (QIAGEN) and mixing by inverting 4 - 6 times, followed by incubation on ice for 20 min. Lysis and neutralization were confirmed using the LyseBlue reagent, where the solution turned blue upon adding P2 Buffer and reverted to colorless on addition of P3 buffer with thorough mixing. The mixture was centrifuged at 18000 × g or 12442 rpm (Heraeus Multifuge X1R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. The supernatant was applied to the QIAGEN-tip500 column pre-equilibrated with 10 mL of QBT Buffer (QIAGEN). The column was then washed by adding 2 × 30 mL of QC Buffer (QIAGEN), and DNA was eluted with 15 mL of QF Buffer (QIAGEN) into a 50 mL centrifuge tube. DNA precipitation was performed by adding 10.5 mL of room-temperature isopropanol and centrifuging at 7000 × g for 75 min at 4 °C. After careful decanting of the supernatant, the DNA pellet was washed with 5 mL of room-temperature 70% v/v ethanol. Finally, the supernatant was aspirated using a vacuum pump without disturbing the pellet, and the pellet was air-dried for 5 - 10 min. The DNA was then redissolved in 500 μL of TE buffer, the concentration was determined using a NanoPhotometer N60 (Implen), and the DNA was stored at −20 °C.

Results;

The maxiprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
042.3	390	1.92	2.28
019.2.3	18	1.93	2.28
023.2.7	48	1.93	2.68

 

PCR Cleanup of backbone and inserts for 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G)

Goal;

 

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications. 

Protocol; 

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g. The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g. The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 × g. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) backbone	127,55	1,92	2,11
056_pcDNA_tdPCP-SpyC3 insert	175,00	1,91	2,19

Gibson assembly of 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G)

Goal;

Gibson assembly was performed to assemble the respective vector and inserts DNA fragments into 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G).

Procedure;

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 100 ng of vector with 3-fold molar excess of insert(s), were combined with 10 μL of master mix.

Reagent	Amount
NEBuilder HiFi DNA Assembly Master Mix	10 μL
Vector (127 ng/µL, 9800 bp)	100 ng / 0.8 μL
Insert (175 ng/µL, 790 bp)	168 ng / 1.1 μL
ddH2O	8.1 μL
Total	20 μL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored at −20 °C (box “PCR (finished)”) for subsequent transformation.

Results;

/

PCR of 056_pcDNA_tdPCP-SpyC3 to get 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) insert

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 056_pcDNA_tdPCP-SpyC3 using primers 079_new_F_PCP_pC0054 & 083_R_corrected_PCP_dPspCas13b with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 079_new_F_PCP_pC0054 083_R_corrected_PCP_dPspCas13b	0.5 μM	2 μL
Template: 056_pcDNA_tdPCP-SpyC3	10 ng plasmid	1.2 μL
Nuclease-free water	–	6.8 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

PCR of 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) to get 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) backbone

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) using primers 078_R_pC0054_PCP & 084_F_dPspCas13b_PCP with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 078_R_pC0054_PCP 084_F_dPspCas13b_PCP	0.5 μM	2 μL
Template DNA: 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G)	10 ng plasmid	1,7 μL
Nuclease-free water	–	6,3 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

DH5α E. coli transformation with 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) Gibson assembly product

Goal;

Transformation was performed to introduce plasmid DNA construct 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) Gibson assembly product into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

 

Counting cells with Cell Countess II

Goal;

Calculating cell suspension concentration and assessing the quality of the suspension.

Procedure:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet.

Cells were counted and analyzed for viability using Countess™ II automated cell counter with 2 replicas, and the average of the two measurements was taken.

Results;

Chamber	Cell count	Live cells	Dead cells
A	5.52×106	5.04×106	4.81×105
B	4.97×106	4.59×106	3.81×103
Mean	5.25×106	4.82×106	4.31×103

Cells are in good condition with enough for further seeding.

PCR - addition of pp7 aptamer to the recording tape

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments

• from 017_pcDNA_Zeo_NLuc-STOP using primers 093_F_T7p_PacI and 080_R_NanoLuc_MluI
• from 044-053_pcDNA_Zeo_NLuc_tapeX.X using primers 081_F_tape_PacI and 080_R_NanoLuc_MluI

with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 093_F_T7p_PacI 080_R_NanoLuc_MluI 081_F_tape_PacI	0.5 μM	2 μL
Template DNA: 017_pcDNA_Zeo_NLuc-STOP 044-053_pcDNA_Zeo_NLuc_tapeX.X	10 ng plasmid	1 μL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The PCR failed due to technical issues with the PCR thermocycler.

Splitting of HEK293T cell line into T75 flask

Goal;

Splitting HEK293T cell line from flask  to the next passage number of 30.

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 3737°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos’s modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 75 cm² flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO₂ and 90% relative humidity.

Results;

HEK293t cell line from flask was split to passage number 30.

DH5α colony picking of various plasmids (019, 023, 038, 049, 072)

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with 017_pcDNA_Zeo_NLuc-STOP (019.2.3), 023_pU6_PspCas13b-DR-gRNA_NLuc (023.2.7), 038_pU6_PspCas13b-DR-gRNA1_tape2 (038.2), 049_pcDNA_Zeo_NLuc_tape3.1 (049.1) and 072_pcDNA3.4-TOPO_T7p_T7tt (072.1-3) from selection plate containing Carbecillin was performed for the replication and expression of the desired genetic material.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Carbecillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Plasmid Miniprep of 072_pcDNA3.4-TOPO_T7p_T7tt

Goal;

A miniprep of 072_pcDNA3.4-TOPO_T7p_T7tt (072.1-3) was performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

 

PCR Cleanup of various inserts from plasmids (019, 044-053, 076)

Goal;

 

The procedure was carried out for the purification of up to 5 μg of DNA seen below from PCR reactions, for use in downstream applications. 

017_pcDNA_Zeo_NLuc-STOP 

044_pcDNA_Zeo_NLuc_tape1.0

045_pcDNA_Zeo_NLuc_tape1.1

046_pcDNA_Zeo_NLuc_tape2.0

047_pcDNA_Zeo_NLuc_tape2.1

048_pcDNA_Zeo_NLuc_tape3.0

049_pcDNA_Zeo_NLuc_tape3.1

050_pcDNA_Zeo_NLuc_tape4.0

051_pcDNA_Zeo_NLuc_tape4.1

052_pcDNA_Zeo_NLuc_tape5.0

053_pcDNA_Zeo_NLuc_tape5.1

076_pCAG_NLuc_STOP_spacer-PP7_bpA

Protocol; 

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g. The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g. The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 × g. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
019	78,08	1,91	2,43
044	136,83	1,95	2,3
045	83,18	1,89	2,83
046	155,73	1,95	2,41
047	121,25	1,96	2,3
048	66,59	1,79	2,73
049	155,11	1,94	2,31
050	120,65	1,96	2,37
051	112,84	1,97	2,42
052	119,58	1,96	2,34
053	81,46	1,84	2,95
076	133,88 (1/20 dilution of original)	1,96	2,26

 

DH5α colony picking with 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) Gibson assembly product

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) from selection plate containing carbecillin was performed for the replication and expression of the desired genetic material.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with carbecillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

PCR - addition of pp7 aptamer to the recording tape (redo)

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments

• from 017_pcDNA_Zeo_NLuc-STOP using primers 093_F_T7p_PacI and 080_R_NanoLuc_MluI
• from 044-053_pcDNA_Zeo_NLuc_tapeX.X using primers 081_F_tape_PacI and 080_R_NanoLuc_MluI

with high fidelity and efficiency.

 

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 093_F_T7p_PacI 080_R_NanoLuc_MluI 081_F_tape_PacI	0.5 μM	2 μL
Template DNA:   017_pcDNA_Zeo_NLuc-STOP 044-053_pcDNA_Zeo_NLuc_tapeX.X	10 ng plasmid	1 μL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

Overnight culture inoculation of 017_pcDNA_Zeo_NLuc-STOP , 023_pU6_PspCas13b-DR-gRNA_NLuc, 038_pU6_PspCas13b-DR-gRNA1_tape2 & 049_pcDNA_Zeo_NLuc_tape3.1

Goal;

An overnight culture was inoculated using a starter culture for the replication and expression of 017_pcDNA_Zeo_NLuc-STOP (019.2.3), 023_pU6_PspCas13b-DR-gRNA_NLuc (023.2.7), 038_pU6_PspCas13b-DR-gRNA1_tape2 (038.2) & 049_pcDNA_Zeo_NLuc_tape3.1 (049.1) .

Protocol;

1 mL of a starter culture was transferred to 200 mL of fresh culture medium (LB medium with Carbenicillin) and incubated overnight at 37°C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful culture inoculation was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

DNA Transfection of HEK293T with jetOPTIMUS®

Goal;

Introduction of plasmids seen below into HEK293T cell line at cell partition 32.

004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G)

017_pcDNA_Zeo_NLuc

017_pcDNA_Zeo_NLuc-STOP 

023_pU6_PspCas13b-DR-gRNA_NLuc

043_pcDNA3.4-TOPO_T7p_T7tt  

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Cells were seeded in 96 well square, black border plates from ibidi at density of 3*10⁴cells per well and grown overnight. Afterwards cells were treated with jetOPTIMUS reagent mixture, including plasmid of interest and fresh DMEM. Afterwards, cells were grown at  37°C with 5% CO₂ and 90% relative humidity. 

Transfection layout and calculations are attached in the excel file

Results;

seen in results page

Restriction digest of plasmid 076_pCAG_NLuc_STOP_spacer-PP7_bpA and inserts amplified (019 & 044-053)

Goal;

 

Restriction digest of the plasmids seen below with PacI and MluI-HF enzymes, for downstream cloning of composite parts of 044-053 and 019 into plasmid 076.

076_pCAG_NLuc_STOP_spacer-PP7_bpA

044_pcDNA_Zeo_NLuc_tape1.0

045_pcDNA_Zeo_NLuc_tape1.1

046_pcDNA_Zeo_NLuc_tape2.0

047_pcDNA_Zeo_NLuc_tape2.1

048_pcDNA_Zeo_NLuc_tape3.0

049_pcDNA_Zeo_NLuc_tape3.1

050_pcDNA_Zeo_NLuc_tape4.0

051_pcDNA_Zeo_NLuc_tape4.1

052_pcDNA_Zeo_NLuc_tape5.0

053_pcDNA_Zeo_NLuc_tape5.1

017_pcDNA_Zeo_NLuc-STOP 

Protocol;

Restriction digest was carried out using the PacI and MluI-HF enzymes (R0547, R3198;New England Biolabs) according to the manufacturers protocol. Template DNA was supplied in amount of 1 μg and combined with 5 μL of rCutSmart^® (B7204, New England Biolabs) and 0.5 μL of PacI and MluI-HF^® in a 1.5 mL Eppendorf reaction tube. The sample was mixed by pipetting, and incubated at 37°C for 2 hours (including mixing every 30 minutes at the lowest agitation speed). The reaction was then stopped using heat inactivation (65°C, 20 minutes).

Component	Volume (50 μL)
Template DNA:044-053, 017, 076	X μL (1μg)
rCutSmart®	5 μL
PacI	0.4 μL
MluI-HF®	0.4 μL
Nuclease-free water	X μL

Results;

The efficiency of plasmid digestion was evaluated by downstream experiments.

Only 19 worked, which had around 0,5 ug DNA compared to others. Possible ideas to consider for trouble shooting: reduce ligation time, extend inactivation time.

 

 

Splitting of HEK293T cell line into T75 flask

Goal;

Splitting HEK293T cell line from flask  to the next passage number of 31. 

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 3737°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos’s modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 4 ml pre-warmed DMEM. A new 75 cm² flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO₂ and 90% relative humidity.

Results;

HEK293T cell line from flask was split to passage number 31.

DH5α E. coli transformation with 073_pcDNA3.4-TOPO_T7p_T7tt & 074_pcDNA3.4-TOPO_syntUTR_T7p_T7tt

Goal;

Transformation was performed to introduce plasmid DNA construct 073_pcDNA3.4-TOPO_T7p_T7tt KLD product and 074_pcDNA3.4-TOPO_syntUTR_T7p_T7tt Gibson assembly product into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

 

KLD of 073_pcDNA3.4-TOPO_T7p_T7tt

Goal;

KLD reaction was performed to phosphorylate and circularise the PCR product of 073_pcDNA3.4-TOPO_T7p_T7tt and degrade template DNA by Dpn I enzyme treatment. 

Protocol;

KLD reaction was performed using the KLD Enzyme Mix (NEW ENGLAND Biolabs), consisting of Kinase, Ligase and Dpn I enzymes, according to the manufacturer’s instructions. For this, 5 µL of the KLD reaction Buffer (2X) (NEW ENGLAND Biolabs), 1 µL of KLD Enzyme Mix, 2 µL of Nuclease-free water and 1 µL of the PCR product were combined. The final composition of the reaction mixture was defined as follows:

Components	Volume
PCR Product	1 µL
KLD Reaction Buffer (2X)	5 µL
KLD Enzyme Mix (10X)	1 µL
Nuclease-free Water	3 µL
Total Volume	10 µL

The sample was incubated at room temperature (25°C) for 5 minutes, and then placed on ice. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

The efficiency of KLD reaction was evaluated by downstream experiments.

Gibson assembly of 074_pcDNA3.4-TOPO_syntUTR_T7p_T7tt

Goal;

Gibson assembly was performed to assemble the respective vector and inserts DNA fragments to produce 074_pcDNA3.4-TOPO_syntUTR_T7p_T7tt with a 5’ synthetic UTR.

Procedure;

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Reagent	Amount
NEBuilder HiFi DNA Assembly Master Mix	10 μL
Vector: PCR product	2.5 μL
Insert	-
ddH2O	7.5 µL
Total	20 μL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

Whole Plasmid Sequencing - 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G)

Goal;

To confirm the nucleotide sequences of 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G), whole plasmid sequencing was performed by using Genewiz, Azenta services. 

Protocol;

DNA was sequenced using a Whole Plasmid sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 25 μL of purified plasmid at a concentration of 50 ng/μL The tube was delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis was not a match between the experimental and expected sequences. 

Sample	label	Validation
062.1	1	cloning failed
062.2	2	cloning failed
062.3	3	cloning failed

 

DNA gel extraction of plasmids of various plasmids (019, 044-053 & 076)

Goal;

 

A gel extraction of plasmids seen below was performed to isolate and purify a specific DNA fragment from agarose gel for downstream applications.

017_pcDNA_Zeo_NLuc-STOP 

044_pcDNA_Zeo_NLuc_tape1.0

045_pcDNA_Zeo_NLuc_tape1.1

046_pcDNA_Zeo_NLuc_tape2.0

047_pcDNA_Zeo_NLuc_tape2.1

048_pcDNA_Zeo_NLuc_tape3.0

049_pcDNA_Zeo_NLuc_tape3.1

050_pcDNA_Zeo_NLuc_tape4.0

051_pcDNA_Zeo_NLuc_tape4.1

052_pcDNA_Zeo_NLuc_tape5.0

053_pcDNA_Zeo_NLuc_tape5.1

076_pCAG_NLuc_STOP_spacer-PP7_bpA

Protocol;

Following gel electrophoresis, DNA bands of interest were excised from the gel under UV transillumination (BIORAD) using a clean scalpel. The excised gel slices were transferred to a 1.5 mL microcentrifuge tube and weighed to determine the amount of gel present. DNA fragments were then isolated and purified from gel using the Monarch® DNA Gel Extraction Kit Protocol (NEW ENGLAND Biolabs, NEB #T1020) following modified manufacturer’s instructions. For this, the gel slices were first dissolved in 4 volumes of Monarch Gel Dissolving Buffer (NEW ENGLAND Biolabs) at 50 °C. The mixture was then transferred to a provided column placed in a collection tube. The column was centrifuged at 13500 × g for 1 min to allow the DNA to bind to the column. The flow-through was discarded, and the column was washed twice with 200 μL of DNA Wash Buffer (NEW ENGLAND Biolabs) at 13500 × g for 1 min each to remove impurities and contaminants. Purified DNA was eluted into a 1.5 mL microcentrifuge tube by adding 10 μL of elution buffer to the column matrix, incubating at room temperature for 1 min, and centrifuging the column for 1 min at 13500 × g. DNA concentration was determined using a NanoPhotometer N60, and the DNA samples were stored at −20 °C.

Results;

 

The extraction procedure yielded DNA only of 019 plasmid of 21 ng/μL. However, the 260/230 ratio is too low, indicating a possible guanidine salt carry over

PCR of 072_pcDNA3.4-TOPO_T7p_T7tt to get 074_pcDNA3.4-TOPO_syntUTR_T7p_T7tt

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 072_pcDNA3.4-TOPO_T7p_T7tt (072.1) using primers 075_F_T7p_syntUTR & 076_R_CMVp_synthUTR with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 075_F_T7p_syntUTR 076_R_CMVp_synthUTR	0.5 μM	2 μL
DMSO (prediluted to 50 %)	3 % (v/v)	1.2 µL
Template DNA: 072_pcDNA3.4-TOPO_T7p_T7tt	10 ng plasmid	1 μL
Nuclease-free water	–	5.8 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

PCR of 072_pcDNA3.4-TOPO_T7p_T7tt to get 073_pcDNA3.4-TOPO_T7p_T7tt

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 072_pcDNA3.4-TOPO_T7p_T7tt (072.1) to introduce BsmBI using primers 055_F_T7t_BsmBI & 056_R_T7p_BsmBI with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 055_F_T7t_BsmBI 056_R_T7p_BsmBI	0.5 μM	2 μL
Template DNA: 072_pcDNA3.4-TOPO_T7p_T7tt	10 ng plasmid	1 μL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

Sanger sequencing - 072_pcDNA3.4-TOPO_T7p_T7tt

Goal;

To confirm the nucleotide sequences of 072_pcDNA3.4-TOPO_T7p_T7tt, Sanger sequencing was performed by using Genewiz, Azenta services. 

Protocol;

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 10 μL of purified plasmid at a concentration of 30 - 100 ng/μL was added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis was a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Sample	Primer	Validation
06817163	072.1	T7-Term	successful cloning confirmed
06817162	072.2	T7-Term	successful cloning confirmed
06817161	072.3	T7-Term	cloning failed

Plasmid Miniprep of 017_pcDNA_Zeo_NLuc-STOP & 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G)

Goal;

A miniprep of plasmids 017_pcDNA_Zeo_NLuc-STOP (019.2.3) and 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) (62.1-3), was performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
019	175.95	1.94	2.23
062.1	238.43	1.93	2.25
062.2	283.08	1.91	2.25
062.3	296.89	1.92	2.18

DH5α E. coli transformation with various constructs (078-087)

Goal;

Transformation was performed to introduce plasmid DNA constructs (078-087) and (076+044-053) into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

078_pCAG_tape1.0_NLuc_spacer-PP7_bpA

079_pCAG_tape1.1_NLuc_STOP_spacer-PP7_bpA

080_pCAG_tape2.0_NLuc_spacer-PP7_bpA

081_pCAG_tape2.1_NLuc_spacer-PP7_bpA

082_pCAG_tape3.0_NLuc_spacer-PP7_bpA

083_pCAG_tape3.1_NLuc_spacer-PP7_bpA

084_pCAG_tape4.0_NLuc_spacer-PP7_bpA

085_pCAG_tape4.1_NLuc_STOP_spacer-PP7_bpA

086_pCAG_tape5.0_NLuc_STOP_spacer-PP7_bpA

087_pCAG_tape5.1_NLuc_STOP_spacer-PP7_bpA

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10-100 ng/μL, was added to the cells (1-5 μL) and gently mixed by flicking the tube 4-5 times. The cell-DNA mixture was incubated on ice for 2 minutes. Following the incubation period, heat shock at 42 °C for 30 seconds was performed. Subsequently, the cells were placed on ice for a minimum of 2 minutes. Following the addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). Finally, 50-100 µL of the mixture was spread onto a selection plate and incubated overnight at 37 °C.

Results;

Successful transformation was confirmed by the presence of colonies on the selective agar plate.

Agarose gel electrophoresis of various constructs (076 backbone; 019 & 044-053 inserts)

Maxiprep (QIAGEN) - 017_pcDNA_Zeo_NLuc-STOP , 023_pU6_PspCas13b-DR-gRNA_NLuc, 038_pU6_PspCas13b-DR-gRNA1_tape2 & 049_pcDNA_Zeo_NLuc_tape3.1

Goal;

 

A maxiprep of 017_pcDNA_Zeo_NLuc-STOP (19.2.3), 023_pU6_PspCas13b-DR-gRNA_NLuc (023.2.7), 038_pU6_PspCas13b-DR-gRNA1_tape2 (038.2) & 049_pcDNA_Zeo_NLuc_tape3.1 (049.1), was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Protocol; 

DNA plasmids were prepared using the QIAGEN^® Plasmid Mini, Midi, and Maxi kit according to the modified manufacturer’s instructions (QIAGEN). … mL of overnight culture of E. coli was centrifuged at 4500× g for 20 min at 4°C. The resulting pellet was resuspended in 10 mL of P1 Buffer (QIAGEN) supplemented with RNase A solution (100 μg/mL; QIAGEN), and LyseBlue reagent (1:1000; QIAGEN). The suspension was transferred to a 50 mL centrifuge tube. To lyse the cells, 10 mL of P2 Buffer (QIAGEN) was added and mixed by inverting 4 - 6 times, followed by incubation at room temperature for 5 min. The lysed cells were neutralized by adding 10 mL of pre-cooled P3 Buffer (QIAGEN) and mixing by inverting 4 - 6 times, followed by incubation on ice for 20 min. Lysis and neutralization were confirmed using the LyseBlue reagent, where the solution turned blue upon adding P2 Buffer and reverted to colorless on addition of P3 buffer with thorough mixing. The mixture was centrifuged at 7000 × g (eppendorf 5430 R) for 135 min (if supernatant not clear, optional +30) min at 4 °C. The supernatant was applied to the QIAGEN-tip500 column pre-equilibrated with 10 mL of QBT Buffer (QIAGEN). The column was then washed by adding 2 × 30 mL of QC Buffer (QIAGEN), and DNA was eluted with 15 mL of QF Buffer (QIAGEN) into a 50 mL centrifuge tube. DNA precipitation was performed by adding 10.5 mL of room-temperature isopropanol and centrifuging at 7000 × g for 75 min (if pellet not strong enough, additional 30 min) at 4 °C. After careful decanting of the supernatant, the DNA pellet was washed with 5 mL of room-temperature 70% v/v ethanol. Finally, the supernatant was aspirated using a vacuum pump without disturbing the pellet, and the pellet was air-dried for 5 - 10 min. The DNA was then redissolved in 300 - 500 μL of TE buffer, the concentration was determined using a NanoDrop, and the DNA was stored at −20 °C.

Results;

The maxiprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
019.2.3	579.78	1.91	2.21
023.2.7	1532.29	1.93	2.29
038.2	322.87	1.93	2.30
049.1	721.44	1.93	2.25

 

DH5α colony picking of 073_pcDNA3.4-TOPO_T7p_T7tt & 074_pcDNA3.4-TOPO_syntUTR_T7p_T7tt

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with 073_pcDNA3.4-TOPO_T7p_T7tt and 074_pcDNA3.4-TOPO_syntUTR_T7p_T7tt from selection plate containing carbecillin was performed for the replication and expression of the desired genetic material.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with carbecillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

DH5α E. coli transformation of 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) Gibson assembly

Goal;

Transformation was performed to introduce plasmid DNA construct 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

 

DH5α colony picking of repeat of 073_pcDNA3.4-TOPO_T7p_T7tt and 074_pcDNA3.4-TOPO_syntUTR_T7p_T7tt

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with 073_pcDNA3.4-TOPO_T7p_T7tt and 074_pcDNA3.4-TOPO_syntUTR_T7p_T7tt from selection plate containing LB/Carb was performed for the replication and expression of the desired genetic material.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Carb) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Plasmid Miniprep of 073_pcDNA3.4-TOPO_T7p_T7tt, 074_pcDNA3.4-TOPO_syntUTR_T7p_T7tt

Goal;

A miniprep of plasmids 073_pcDNA3.4-TOPO_T7p_T7tt (073.1, 073.2, 073.3), 074_pcDNA3.4-TOPO_syntUTR_T7p_T7tt (074.1, 074.2 and 074.3) was performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of insufficient quantity and purity ratios A260/A280 and A260/A230 outside the acceptable range - re-do the Miniprep.

Sample	Concentration [ng/μL]	260/280	260/230
073.1	20.3	1.92	2.12
073.2	16.8	1.80	2.05
073.3	22.5	1.93	2.07
074.1	15.4	1.92	2.07
074.2	20.3	1.94	2.34
074.3	26.5	1.95	2.52

 

PCR - addition of restriction sites to 017_pcDNA_Zeo_NLuc-STOP insert

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 017_pcDNA_Zeo_NLuc-STOP using primers 093_F_T7p_PacI & 080_R_NanoLuc_MluI with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 093_F_T7p_PacI  080_R_NanoLuc_MluI	0.5 μM	2 μL
Template DNA: 017_pcDNA_Zeo_NLuc-STOP	10 ng plasmid	1 μL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

PCR of insert to get 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) (re-do)

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 056_pcDNA_tdPCP-SpyC3 using primers 079_F_corrected_PCP_pC0054 & 083_R_corrected_PCP_dPspCas13b with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 079_F_corrected_PCP_pC0054 083_R_corrected_PCP_dPspCas13b	0.5 μM	2 μL
Template DNA: 056_pcDNA_tdPCP-SpyC3	10 ng plasmid	1.2 μL
Nuclease-free water	–	6.8 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

Gibson assembly of backbone and insert to get 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) re-do

Goal;

Gibson assembly was performed to assemble the respective vector and inserts DNA fragments to produce 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G).

Procedure;

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including100 ng of vector with 3-fold molar excess of insert(s), were combined with 10 μL of master mix.

Reagent	Amount
NEBuilder HiFi DNA Assembly Master Mix	10 μL
Vector (127 ng/µL, 9800 bp)	100 ng / 0.8 μL
Insert (175 ng/µL, 790 bp)	168 ng / 1.1 μL
ddH2O	8.1 μL
Total	20 μL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored at −20 °C (box “PCR (finished)”) for subsequent transformation.

Results;

/

PCR of 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) backbone to get 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) re-do

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) using primers 078_R_pC0054_PCP & 079_F_corrected_PCP_pC0054 with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 078_R_pC0054_PCP  079_F_corrected_PCP_pC0054	0.5 μM	2 μL
Template DNA: 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G)	10 ng plasmid	1,7 μL
Nuclease-free water	–	6,3 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

Sanger sequencing of 073_pcDNA3.4-TOPO_T7p_T7tt & 074_pcDNA3.4-TOPO_syntUTR_T7p_T7tt

Goal;

To confirm the nucleotide sequences of 073_pcDNA3.4-TOPO_T7p_T7tt  & 074_pcDNA3.4-TOPO_syntUTR_T7p_T7tt, Sanger sequencing was performed by using Genewiz, Azenta services. 

Protocol;

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 10 μL of purified plasmid at a concentration of 30 - 100 ng/μL was added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis found a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Sample	Primer	Validation
06817164	73.1	CMV-Forward	point mutations
06817167	73.2	CMV-Forward	successful cloning confirmed
06817168	74.1	CMV-Forward	successful cloning confirmed
06817169	74.3	CMV-Forward	successful cloning confirmed

 

Plasmid Miniprep of 073_pcDNA3.4-TOPO_T7p_T7tt & 074_pcDNA3.4-TOPO_syntUTR_T7p_T7tt

Goal;

A miniprep of plasmids 073_pcDNA3.4-TOPO_T7p_T7tt  & 074_pcDNA3.4-TOPO_syntUTR_T7p_T7tt was performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 inside the acceptable range

Sample	Concentration [ng/μL]	260/280	260/230
073.1	112.31	1.93	2.30
073.2	113.35	1.93	2.33
073.3	101.10	1.92	2.28
074.1	143.39	1.90	2.06
074.2	120.41	1.97	2.31
074.3	141.01	1.95	2.31

 

Plasmid Miniprep of 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G)

Goal;

A miniprep of 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) was performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

 

Splitting of HEK293T cell line into T75 flask

Goal;

Splitting HEK293T cell line from one flask into two T75 flasks for the next passage number of 32.

Procedure:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet.

Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 mL of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 mL of Accutase solution at 37 °C for 5 minutes. Upon confirming detachment using a light phase microscope, cells were rescued using 3 mL of pre-warmed complete Dulbecco’s modified Eagle medium F12 (DMEM) and transferred to a 15 mL tube. The cell suspension was centrifuged at 25 °C, 320 RCF for 5 minutes in a swinging bucket centrifuge. The supernatant was decanted using a Pasteur pipette, and the cell pellet was resuspended in 6 mL of pre-warmed DMEM. A new 75 cm² flask was inoculated with 1 mL of the cell suspension to 9 mL of pre-warmed DMEM. Cells were grown at 37 °C with 5% CO₂ and 90% relative humidity.

Results;

HEK293T cell line from the flask was split to passage number 32.

Overnight culture inoculation of 017_pcDNA_Zeo_NLuc & 050_pcDNA_Zeo_NLuc_tape4.0 for maxiprep

Goal;

An overnight culture was inoculated using a starter culture for the replication and expression of 017_pcDNA_Zeo_NLuc and 050_pcDNA_Zeo_NLuc_tape4.0.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a refrigerator. After 8 hours, 5 mL of the starter culture was transferred to 200 mL of fresh culture medium (LB medium with carbenicillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells.

Results;

Successful culture inoculation was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation at 37 °C.

Seeding of 2 ibidi 96-Well plates

Goal;

Seeding Nunc™ MicroWell™ 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate, Thermo Scientific™ for further confocal analysis of plasmids transfected into HEK293T cells with 1 × 10⁴ HEK293T cells.

Procedure:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet.

Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 mL of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 mL of Accutase solution at 37 °C for 5 minutes. Upon confirming detachment using a light phase microscope, cells were rescued using 3 mL of pre-warmed complete Dulbecco’s modified Eagle medium F12 (DMEM) and transferred to a 15 mL tube. The cell suspension was centrifuged at 25 °C, 320 RCF for 5 minutes in a swinging bucket centrifuge. The supernatant was decanted using a Pasteur pipette, and the cell pellet was resuspended in 6 mL of pre-warmed DMEM. Each well of the Nunc™ 96-well plate was inoculated with 100 µL of cell suspension at a density of 1 × 10⁴ cells per well. Cells were further grown at 37 °C with 5% CO₂ and 90% relative humidity.

Maxiprep (Promega) of 017_pcDNA_Zeo_NLuc & 050_pcDNA_Zeo_NLuc_tape4.0

Goal;

 

A maxiprep of 017_pcDNA_Zeo_NLuc & 050_pcDNA_Zeo_NLuc_tape4.0 was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Protocol; 

DNA plasmids were prepared using the PureYield™ Plasmid Maxiprep System kit according to the modified manufacturer’s instructions (Promega). … mL of overnight culture of E. coli was centrifuged at 4500 × g for 15 min at room temperature. The resulting pellet was resuspended in 12 mL of Cell Resuspension Solution (Promega). To lyse the cells, 12 mL of Cell Lysis Solution (Promega) was added and mixed by inverting 4 - 6 times. The lysed cells were neutralized by adding 12 mL of Neutralization Solution (Promega) and mixing by inverting 10 - 15 times. The mixture was centrifuged at 4500 × g (Heraeus Multifuge X1R Centrifuge; Thermo Fisher Scientific) for 1 hour at room temperature. In the meantime, the column stack, consisting of assembled blue PureYield™ Clearing Column and white PureYield™ Maxi Binding Column, were placed on the vacuum manifold. Half of the supernatant was applied to the blue PureYield™ Clearing Column and the maximum vacuum was applied until the lysate has passed through both the clearing and binding columns. The same procedure was conducted with the remaining cell lysate. Once the liquid passed through both columns, vacuum was slowly released and the blue PureYield™ Clearing Columns were discarded. The column was then washed by adding 5mL of Endotoxin Removal Wash to the PureYield™ Maxi Binding Column and applying a vacuum, followed by addition of 20mL of Column Wash to the binding column, and applying the vacuum to pull the solution through the column. The membrane was dried by applying a vacuum for 5 minutes. After careful removing remaining ethanol by tapping the tip of the PureYield™ Maxi Binding Column on a paper towel, the columns were placed in new 50 mL centrifuge tubes. The DNA was eluted by addition of 1 mL of TE buffer and centrifugation at 4500 × g for 5 min. The concentration was determined using a NanoDrop, and the DNA was stored at −20 °C.

Results;

The maxiprep procedure yielded DNA of following quantity and purity ratios A260/A280, A260/A230:

Sample	Concentration [ng/μL]	260/280	260/230
017	85.4	1.92	2.64
050	207.3	1.91	2.68

 

Overnight culture inoculation of 017_pcDNA_Zeo_NLuc, 051_pcDNA_Zeo_NLuc_tape4.1 and 053_pcDNA_Zeo_NLuc_tape5.1 for maxiprep

Goal;

An overnight culture was inoculated using a starter culture for the replication and expression of 017_pcDNA_Zeo_NLuc, 051_pcDNA_Zeo_NLuc_tape4.1 and 053_pcDNA_Zeo_NLuc_tape5.1.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Carbecillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge. After 8 hours, 200 mL of a starter culture was transferred to 200 mL of fresh culture medium (LB medium with Carbenicillin) and incubated overnight at 37°C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful culture inoculation was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

PCR Cleanup of 017_pcDNA_Zeo_NLuc-STOP with insert (for subsequent digestion)

Goal;

 

The procedure was carried out for the purification of up to 5 μg of 017_pcDNA_Zeo_NLuc-STOP from PCR reactions, for use in downstream applications. 

Protocol; 

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g. The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g. The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 × g. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
019	102,1	1,93	1,91

R: Ready for digestion

Maxiprep (Promega) of various plasmids (017, 051, 053)

Goal;

 

A maxiprep of plasmids seen below was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

017_pcDNA_Zeo_NLuc

051_pcDNA_Zeo_NLuc_tape4.1 

053_pcDNA_Zeo_NLuc_tape5.1

Protocol; 

DNA plasmids were prepared using the PureYield™ Plasmid Maxiprep System kit according to the modified manufacturer’s instructions (Promega). 150 mL of overnight culture of E. coli was centrifuged at 4500 × g for 15 min at room temperature. The resulting pellet was resuspended in 12 mL of Cell Resuspension Solution (Promega). To lyse the cells, 12 mL of Cell Lysis Solution (Promega) was added and mixed by inverting 4 - 6 times. The lysed cells were neutralized by adding 12 mL of Neutralization Solution (Promega) and mixing by inverting 10 - 15 times. The mixture was centrifuged at 4500 × g (Heraeus Multifuge X1R Centrifuge; Thermo Fisher Scientific) for 1 hour at room temperature. In the meantime, the column stack, consisting of assembled blue PureYield™ Clearing Column and white PureYield™ Maxi Binding Column, were placed on the vacuum manifold. Half of the supernatant was applied to the blue PureYield™ Clearing Column and the maximum vacuum was applied until the lysate has passed through both the clearing and binding columns. The same procedure was conducted with the remaining cell lysate. Once the liquid passed through both columns, vacuum was slowly released and the blue PureYield™ Clearing Columns were discarded. The column was then washed by adding 5mL of Endotoxin Removal Wash to the PureYield™ Maxi Binding Column and applying a vacuum, followed by addition of 20mL of Column Wash to the binding column, and applying the vacuum to pull the solution through the column. The membrane was dried by applying a vacuum for 5 minutes. After careful removing remaining ethanol by tapping the tip of the PureYield™ Maxi Binding Column on a paper towel, the columns were placed in new 50 mL centrifuge tubes. The DNA was eluted by addition of 1 mL of TE buffer and centrifugation at 4500 × g for 5 min. The concentration was determined using a NanoDrop, and the DNA was stored at −20 °C.

Results;

The maxiprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
017.3 (maxi)	173,8	1,93	2,55
051 (maxi)	269,0	1,94	2,49
053 (maxi)	130,1	/	/

 

Overnight culture inoculation of 052_pcDNA_Zeo_NLuc_tape5.0

Goal;

An overnight culture was inoculated using a starter culture for the replication and expression of 052_pcDNA_Zeo_NLuc_tape5.0.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Carbecillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge. After 8 hours, … mL of a starter culture was transferred to 200 mL of fresh culture medium (LB medium with Carbenicillin) and incubated overnight at 37°C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful culture inoculation was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

PCR of 074_pcDNA3.4-TOPO_syntUTR_T7p_T7tt to get 075_pcDNA3.4-TOPO_syntUTR_T7p_T7tt_BsmBI

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 074_pcDNA3.4-TOPO_syntUTR_T7p_T7tt using primers 055_F_T7t_BsmBI & 077_T7psynt_BsmBI with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 055_F_T7t_BsmBI 077_T7psynt_BsmBI	0.5 μM	2 μL
Template DNA:  074_pcDNA3.4-TOPO_syntUTR_T7p_T7tt	10 ng plasmid	1 μL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

DH5α E. coli transformation with 075_pcDNA3.4-TOPO_syntUTR_T7p_T7tt_BsmBI

If no colonies -> retransform with 5ul of KLD product saved in KLD box in -20 (probably problem with antibiotic) 

Goal;

Transformation was performed to introduce plasmid DNA construct 075_pcDNA3.4-TOPO_syntUTR_T7p_T7tt_BsmBI into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

 

KLD of 075_pcDNA3.4-TOPO_syntUTR_T7p_T7tt_BsmBI

Goal;

KLD reaction was performed to phosphorylate and circularise 075_pcDNA3.4-TOPO_syntUTR_T7p_T7tt_BsmBI, and degrade template DNA by Dpn I enzyme treatment. 

Protocol;

KLD reaction was performed using the KLD Enzyme Mix (NEW ENGLAND Biolabs), consisting of Kinase, Ligase and Dpn I enzymes, according to the manufacturer’s instructions. For this, 5 µL of the KLD reaction Buffer (2X) (NEW ENGLAND Biolabs), 1 µL of KLD Enzyme Mix, 2 µL of Nuclease-free water and 2 µL of the PCR product were combined. The final composition of the reaction mixture was defined as follows:

Components	Volume
PCR Product	1 µL
KLD Reaction Buffer (2X)	5 µL
KLD Enzyme Mix (10X)	1 µL
Nuclease-free Water	3 µL
Total Volume	10 µL

The sample was incubated at room temperature (25°C) for 5 minutes, and then placed on ice. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

Ligation of 076_pCAG_NLuc_STOP_spacer-PP7_bpA backbone and inserts of 017_pcDNA_Zeo_NLuc-STOP

Goal;

The procedure was performed to ligate the 076_pCAG_NLuc_STOP_spacer-PP7_bpA digested with PacI and MluI-HF and insert 017_pcDNA_Zeo_NLuc-STOP.

Protocol;

Do previous calculations using https://www.geneinfinity.org/cc/cc_dnaconverter.html. In this case: 

0,020 pmol of digested_76 (4935bp) equals 65 ng

0,060 pmol of digested_19 (620bp, aprox) equals 25 ng

Ligation was carried out according to Quick Ligation Kit (M2200, New England Biolabs) protocol . For this, 0.020 pmol of PacI-MluI-HF-digested pCAG_NLuc_STOP_spacer-PP7_bpA plasmid, 0.060 pmol Heterodimerized product of the spacer, 10 µl Quick Ligase Reaction Buffer (2X) (M2200S, New England Biolabs), 1 µl of Quick Ligase (M2200S, New England Biolabs) were combined in a 1.5 mL Eppendorf reaction tube. The final composition of the reaction mixture is defined as follows:

Component	Volume (20 μL)
PacI-MluI-HF-digested pCAG_NLuc_STOP_spacer-PP7_bpA (76_digested)	0,72 μL (0.020 pmol)
Insert (19_digested)	0,52 μL (0.060 pmol)
Quick Ligase Reaction Buffer (2X)	10 μL
Quick Ligase	1 μL
Nuclease-free water	7,76 μL

The ligation reaction mixture was incubated for 10 min at room temperature.

Product was kept at -20°C until further use. 

Results;

The efficiency of the ligation was evaluated by downstream experiments.

Restriction digest of 076_pCAG_NLuc_STOP_spacer-PP7_bpA and insert amplified from 017_pcDNA_Zeo_NLuc-STOP

Goal;

 

Restriction digest of 076_pCAG_NLuc_STOP_spacer-PP7_bpA and 017_pcDNA_Zeo_NLuc-STOP with PacI and MluI-HF enzymes, for downstream cloning of composite part 077_pCAG_NLuc_STOP_spacer-PP7_bpA.

Protocol;

Restriction digest was carried out using the PacI and MluI-HF enzymes (R0547, R3198;New England Biolabs) according to the manufacturers protocol. Template DNA was supplied in amount of 2 μg (backbone)/ 1 μg (insert) and combined with 5 μL of rCutSmart^® (B7204, New England Biolabs) and 1 μL of PacI and MluI-HF^® in a PCR tube. The sample was mixed by pipetting, and incubated at 37°C for 1 hour. The reaction was then stopped using heat inactivation (65°C, 20 minutes).

Component	Volume (50 μL)
Backbone DNA	11,2 μL (3μg) - original concentration of 134 ng/ul (1/10 dilution)
rCutSmart®	5 μL
PacI	1 μL
MluI-HF®	1 μL
Nuclease-free water	31,8 μL

 

Component	Volume (50 μL)
Insert DNA	9,8 μL (1μg) - original concentration is 102 ng/ul
rCutSmart®	5 μL
PacI	1 μL
MluI-HF®	1 μL
Nuclease-free water	33,2 μL

Results;

The efficiency of plasmid digestion was evaluated by downstream experiments.

 

 

 

DNA gel extraction of 076_pCAG_NLuc_STOP_spacer-PP7_bpA and DNA Cleanup of 017_pcDNA_Zeo_NLuc-STOP

Goal;

 

A gel extraction of (digested) plasmid 076_pCAG_NLuc_STOP_spacer-PP7_bpA was performed to isolate and purify a specific DNA fragment from agarose gel for ligation. Additionally, a PCR cleanup of digested 017_pcDNA_Zeo_NLuc-STOP was performed to isolate and purify a specific DNA fragment after digestion for ligation.

Protocol;

For Gel Extraction

Following gel electrophoresis, DNA bands of interest were excised from the gel under UV transillumination (BIORAD) using a clean scalpel. The excised gel slices were transferred to a 1.5 mL microcentrifuge tube and weighed to determine the amount of gel present. DNA fragments were then isolated and purified from gel using the Monarch® DNA Gel Extraction Kit Protocol (NEW ENGLAND Biolabs, NEB #T1020) following modified manufacturer’s instructions. For this, the gel slices were first dissolved in 4 volumes of Monarch Gel Dissolving Buffer (NEW ENGLAND Biolabs) at 50 °C. The mixture was then transferred to a provided column placed in a collection tube. The column was centrifuged at 13500 × g for 1 min to allow the DNA to bind to the column. The flow-through was discarded, and the column was washed twice with 200 μL of DNA Wash Buffer (NEW ENGLAND Biolabs) at 13500 × g for 1 min each to remove impurities and contaminants. Purified DNA was eluted into a 1.5 mL microcentrifuge tube by adding 10 μL of elution buffer to the column matrix, incubating at room temperature for 1 min, and centrifuging the column for 1 min at 13500 × g. DNA concentration was determined using a NanoPhotometer N60, and the DNA samples were stored at −20 °C.

For DNA Cleanup

Digested products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g. The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g. The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 × g. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

 

The procedures yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
019_digested	48,35	1,89	2,19
076_digested	90,52	2,02	2,05

Agarose gel electrophoresis of digested 076_pCAG_NLuc_STOP_spacer-PP7_bpA

Goal;

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples from 076_pCAG_NLuc_STOP_spacer-PP7_bpA076_pCAG_NLuc_STOP_spacer-PP7_bpA.

Protocol;

Agarose gel electrophoresis was performed using a 1.0% w/v agarose (ROTH) gel prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply (BIORAD, PowerPac™ Basic Power Supply). A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 30 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (BIORAD, ChemiDoc Imaging System).

Results;

The following samples were loaded into the gel from left to right: 1KB ladder - (empty) - 55 uL of 76 after diegestion - (empty lanes) 5 uL of 76 after digestion.

The agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

Sanger sequencing of 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G)

Goal;

To confirm the nucleotide sequences of 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G), Sanger sequencing was performed by using Genewiz, Azenta services. 

Protocol;

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 10 μL of purified plasmid at a concentration of 30 - 100 ng/μL was added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis … a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Sample	Primer	Validation
/	062v2.1	CMV-Forward	many mutations on the
/	062v2.2	CMV-Forward	wrong portion was checked for sequencing?
/	062v2.3	CMV-Forward	various base insertion in Kozak region

 

Maxiprep (QIAGEN) of 052_pcDNA_Zeo_NLuc_tape5.0

Goal;

 

A maxiprep of 052_pcDNA_Zeo_NLuc_tape5.0 was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Protocol; 

DNA plasmids were prepared using the QIAGEN^® Plasmid Mini, Midi, and Maxi kit according to the modified manufacturer’s instructions (QIAGEN). 200 mL of overnight culture of E. coli was centrifuged at 4500× g for 20 min at 4°C. The resulting pellet was resuspended in 10 mL of P1 Buffer (QIAGEN) supplemented with RNase A solution (100 μg/mL; QIAGEN), and LyseBlue reagent (1:1000; QIAGEN). The suspension was transferred to a 50 mL centrifuge tube. To lyse the cells, 10 mL of P2 Buffer (QIAGEN) was added and mixed by inverting 4 - 6 times, followed by incubation at room temperature for 5 min. The lysed cells were neutralized by adding 10 mL of pre-cooled P3 Buffer (QIAGEN) and mixing by inverting 4 - 6 times, followed by incubation on ice for 20 min. Lysis and neutralization were confirmed using the LyseBlue reagent, where the solution turned blue upon adding P2 Buffer and reverted to colorless on addition of P3 buffer with thorough mixing. The mixture was centrifuged at 7000 × g (eppendorf 5430 R) for 135 min (if supernatant not clear, optional +30) min at 4 °C. The supernatant was applied to the QIAGEN-tip500 column pre-equilibrated with 10 mL of QBT Buffer (QIAGEN). The column was then washed by adding 2 × 30 mL of QC Buffer (QIAGEN), and DNA was eluted with 15 mL of QF Buffer (QIAGEN) into a 50 mL centrifuge tube. DNA precipitation was performed by adding 10.5 mL of room-temperature isopropanol and centrifuging at 7000 × g for 75 min (if pellet not strong enough, additional 30 min) at 4 °C. After careful decanting of the supernatant, the DNA pellet was washed with 5 mL of room-temperature 70% v/v ethanol. Finally, the supernatant was aspirated using a vacuum pump without disturbing the pellet, and the pellet was air-dried for 5 - 10 min. The DNA was then redissolved in 300 μL of TE buffer, the concentration was determined using a NanoDrop, and the DNA was stored at −20 °C.

Results;

The maxiprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
052 (maxi)	1332	1.93	2.27

 

DH5α E. coli transformation of 075_pcDNA3.4-TOPO_syntUTR_T7p_T7tt_BsmBI & 077_pCAG_NLuc_STOP_spacer-PP7_bpA

Goal;

Transformation was performed to introduce plasmid DNA construct 075_pcDNA3.4-TOPO_syntUTR_T7p_T7tt_BsmBI & 077_pCAG_NLuc_STOP_spacer-PP7_bpA into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

 

DNA gel extraction of 036_pU6_PspCas13b-DR-empty

Goal;

 

A gel extraction of 036_pU6_PspCas13b-DR-empty digested with BbsI was performed to isolate and purify a specific DNA fragment from agarose gel for downstream applications.

Protocol;

Following gel electrophoresis, DNA bands of interest were excised from the gel under UV transillumination (BIORAD) using a clean scalpel. The excised gel slices were transferred to a 1.5 mL microcentrifuge tube and weighed to determine the amount of gel present. DNA fragments were then isolated and purified from gel using the Monarch® DNA Gel Extraction Kit Protocol (NEW ENGLAND Biolabs, NEB #T1020) following modified manufacturer’s instructions. For this, the gel slices were first dissolved in 4 volumes of Monarch Gel Dissolving Buffer (NEW ENGLAND Biolabs) at 50 °C. The mixture was then transferred to a provided column placed in a collection tube. The column was centrifuged at 13500 × g for 1 min to allow the DNA to bind to the column. The flow-through was discarded, and the column was washed twice with 200 μL of DNA Wash Buffer (NEW ENGLAND Biolabs) at 13500 × g for 1 min each to remove impurities and contaminants. Purified DNA was eluted into a 1.5 mL microcentrifuge tube by adding 10 μL of elution buffer to the column matrix, incubating at room temperature for 1 min, and centrifuging the column for 1 min at 13500 × g. DNA concentration was determined using a NanoPhotometer N60, and the DNA samples were stored at −20 °C.

Results;

 

The extraction procedure yielded DNA of 45,8 ng/μL.

DH5α colony picking of 075_pcDNA3.4-TOPO_syntUTR_T7p_T7tt_BsmBI & 077_pCAG_NLuc_STOP_spacer-PP7_bpA

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with 075_pcDNA3.4-TOPO_syntUTR_T7p_T7tt_BsmBI and 077_pCAG_NLuc_STOP_spacer-PP7_bpA from selection plate containing Carbecillin was performed for the replication and expression of the desired genetic material.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Carbecillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Restriction digest of 036_pU6 with BbsI

Goal;

 

Restriction digest of the 036_pU6 construct with BbsI enzyme, for downstream cloning of gRNA for Nano-luciferase restoration assay.

Protocol;

Restriction digest was carried out using the BbsI-HF^® enzyme (R3539S, New England Biolabs) according to the protocol developed by Truong et al. (Truong, DJ., Armbrust, N., Geilenkeuser, J., Westmeyer, G. Generation of cell lines for the usage of Intron-encoded Scarless Programmable Extranuclear Cistronic Transcripts (INSPECT), 2023, PROTOCOL available at Protocol Exchange). For this, 1 μg of DNA template, 5 μL CutSmart^® (B7204, New England Biolabs), 1 μL BbsI-HF^® were combined in a 1.5 mL Eppendorf reaction tube. The sample was mixed by pipetting, and incubated at 37°C for 2 hours (including mixing every 30 minutes).

Component	Volume (46,7 μL)
Template DNA: 036_pU6_PspCas13b-DR-empty	10,7 μL (3 μg)
CutSmart®	5 μL
BbsI-HF®	1 μL
Nuclease-free water	30 μL

 

Results;

The efficiency of plasmid digestion was evaluated by downstream experiments.

 

 

Agarose gel electrophoresis of the digested 036_pU6 plasmid and 062 insert and backbone

Goal;

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples from …

Protocol;

Agarose gel electrophoresis was performed using a 1.0% w/v agarose (ROTH) gel prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply (BIORAD, PowerPac™ Basic Power Supply). A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 30 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (BIORAD, ChemiDoc Imaging System).

Results;

The following samples were loaded into the gel from left to right: ladder, 062, backbone for 062, insert for 062, 036.2 digested, 036.2 digested,036.2 digested,036.2 digested

The agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

Golden Gate Assembly of XFPs (063-065) and tapes (025-034) and backbone (073) into 119-128

Goal;

 

Golden Gate Assembly of XFPs (063-065) and tapes (025-034) into 073_pcDNA3.4-TOPO_T7p_T7tt backbone.

063_T2A_miRFP670_P2A_eUnaG 

064_T2A_mScarlet3_P2A_eUnaG

065_T2A_mTagBFP2_P2A_eUnaG

025_tape_1.0

026_tape_1.1

027_tape_2.0

028_tape_2.1

029_tape_3.0

030_tape_3.1

031_tape_4.0

032_tape_4.1

033_tape_5.0

034_tape_5.1

Protocol;

Restriction digest was carried out using the BsmBI-v2 and T4 DNA Ligase enzymes (R0739, M0202;New England Biolabs) according to the manufacturers protocol. Template DNA was supplied in amount of 75 ng (backbone), while insert was supplied in a 2:1 molar ratio(use calculator below). The DNAs were combined with 2 μL of T4 Ligase Buffer^® (B0202, New England Biolabs) and 1 μL of T4 DNA Ligase and BsmBI-v2 in a PCR tube, to a final volume of 20 uL. The sample was mixed by pipetting, and incubated according in 30 cycles of 1 min at 42ºC and 1 min at 16ºC, followed by a single 5 min cycle at 60ºC. The reaction was then stored at -20C unless used for transformation.

Component	Volume (20 μL)	119	120	121	122	123	124	125	126	127	128
Backbone DNA (073.2 - 75 ng/μl)	… μl (75 ng)	1 μl	1 μl	1 μl	1 μl	1 μl	1 μl	1 μl	1 μl	1 μl	1 μl
Insert DNA (063 - 25 ng/μl)	… μl (2:1 molar ratio, 42.76 ng)	1,71 μl	1,71 μl	1,71 μl	1,71 μl	1,71 μl	1,71 μl	1,71 μl	1,71 μl	1,71 μl	1,71 μl
Insert DNA (064 - 23,7 ng/μl)	… μl (2:1 molar ratio, 52.97 ng)	2,24 μl	2,24 μl	2,24 μl	2,24 μl	2,24 μl	2,24 μl	2,24 μl	2,24 μl	2,24 μl	2,24 μl
Insert DNA (065 - 25,3 ng/μl)	… μl (2:1 molar ratio, 57,09 ng)	2,26 μl	2,26 μl	2,26 μl	2,26 μl	2,26 μl	2,26 μl	2,26 μl	2,26 μl	2,26 μl	2,26 μl
Insert DNA (tape - 5 ng/μl)	… μl (2:1 molar ratio, 7,47 ng)	1,5 μl (025)	1,5 μl (026)	1,5 μl (027)	1,5 μl (028)	1,5 μl (029)	1,5 μl (030)	1,5 μl (031)	1,5 μl (032)	1,5 μl (033)	1,5 μl (034)
T4 Ligase Reaction Buffer (10X)	2 μl	2 μl	2 μl	2 μl	2 μl	2 μl	2 μl	2 μl	2 μl	2 μl	2 μl
T4 DNA Ligase	1 μl	1 μl	1 μl	1 μl	1 μl	1 μl	1 μl	1 μl	1 μl	1 μl	1 μl
BsmBI-v2	1 μl	1 μl	1 μl	1 μl	1 μl	1 μl	1 μl	1 μl	1 μl	1 μl	1 μl
Nuclease-free H20	… μl	7,29 μl	7,29 μl	7,29 μl	7,29 μl	7,29 μl	7,29 μl	7,29 μl	7,29 μl	7,29 μl	7,29 μl

Program for 2-10 inserts: (42°C, 1 min → 16°C,1 min) x 30 → 60°C, 5 min  

For reference, 073_pcDNA3.4-TOPO_T7p_T7tt is 3676bp.

Results;

The efficiency of Golden Gate Assembly was evaluated by downstream experiments.

 

 

 

Sanger sequencing of 075_pcDNA3.4-TOPO_syntUTR_T7p_T7tt_BsmBI & 077_pCAG_NLuc_STOP_spacer-PP7_bpA

Goal;

To confirm the nucleotide sequences of 075_pcDNA3.4-TOPO_syntUTR_T7p_T7tt_BsmBI (075/075v2) and 077_pCAG_NLuc_STOP_spacer-PP7_bpA (077Lig1/077Lig2), Sanger sequencing was performed by using Genewiz, Azenta services. 

Protocol;

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 10 μL of purified plasmid at a concentration of 30 - 100 ng/μL was added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis found a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Sample	Primer	Validation
/	75.2.1	CMV-Forward	successful cloning confirmed
/	75.2.2	CMV-Forward	successful cloning confirmed
/	77.1.1	BGHR	successful cloning confirmed
/	77.1.3	BGHR	successful cloning confirmed
/	77.2.1	BGHR	successful cloning confirmed
/	77.2.2	BGHR	successful cloning confirmed

 

Plasmid Miniprep of 075_pcDNA3.4-TOPO_syntUTR_T7p_T7tt_BsmBI & 077_pCAG_NLuc_STOP_spacer-PP7_bpA

Goal;

A miniprep of 075_pcDNA3.4-TOPO_syntUTR_T7p_T7tt_BsmBI and 077_pCAG_NLuc_STOP_spacer-PP7_bpA was performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
075.1.1	140,40	1,93	2,23
075.1.2	124,39	1,92	2,22
075.2.1	112,53	1,93	2,18
075.2.2	120,82	1,92	2,20
077.1.1	201,53	1,94	2,19
077.1.2	170,27	1,92	2,21
077.1.3	181,00	1,92	2,16
077.2.1	178,61	1,92	2,21
077.2.2	197,57	1,92	2,15
077.2.3	171,54	1,91	2,21

 

PCR of XFP gBlocks (063-065)

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from gBlocks (063-065) resuspended in TE buffer using primers 030_F_tape-amplification & 027_R_pU6_2960A<C for constructs with high fidelity and efficiency.

063_T2A_miRFP670_P2A_eUnaG 

064_T2A_mScarlet3_P2A_eUnaG

065_T2A_mTagBFP2_P2A_eUnaG

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	50-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	25 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 030_F_tape-amplification 027_R_pU6_2960A<C	0.5 μM	5 μL
Template DNA: 063_T2A_miRFP670_P2A_eUnaG  064_T2A_mScarlet3_P2A_eUnaG 065_T2A_mTagBFP2_P2A_eUnaG	30 ng plasmid	1,5 μL (20 ng/ul stock)
Nuclease-free water	–	18,5 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

PCR Cleanup of various gBlocks (063-065)

Goal;

 

The procedure was carried out for the purification of up to 5 μg of DNA from PCR reactions, for use in downstream applications. 

063_T2A_miRFP670_P2A_eUnaG 

064_T2A_mScarlet3_P2A_eUnaG

065_T2A_mTagBFP2_P2A_eUnaG

Protocol; 

PCR products were purified using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030) according to manufacturer’s instructions (NEW ENGLAND BioLabs). Samples (starting volume: 20–100 μL) were diluted with DNA Cleanup Binding Buffer in ratios 1:2 for dsDNA > 2 kb and 1:5 for dsDNA <2 kb and mixed by pipetting up and down. The suspension was loaded onto the columns, and centrifuged for 1 minute at 13000 × g. The flow-through was discarded. The column was washed twice by adding 200 μL of DNA Wash Buffer and centrifuging for 1 minute at 13000 × g. The columns were transferred to clean 1.5 mL eppendorf tubes and centrifuged at 13000 × g for 1 minute. DNA was eluted by addition of ≥ 6 μL of elution buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute at 13000 × g. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
063	74.7	/	/
064	70.0	/	/
065	75.0	/	/

After this an aliquot of samples was diluted to the concentrations of 25ng/ul, 23ng/ul and 25ng/ul respectfully 

DH5α E. coli transformation with Golden Gate assembly (119 - 128)

Goal;

Transformation was performed to introduce plasmid final constructs (119-128) into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

119_pcDNA3.4-TOPO_T7p-tape_1.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

120_pcDNA3.4-TOPO_T7p-tape_1.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

121_pcDNA3.4-TOPO_T7p-tape_2.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

122_pcDNA3.4-TOPO_T7p-tape_2.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

123_pcDNA3.4-TOPO_T7p-tape_3.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

124_pcDNA3.4-TOPO_T7p-tape_3.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

125_pcDNA3.4-TOPO_T7p-tape_4.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

126_pcDNA3.4-TOPO_T7p-tape_4.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

127_pcDNA3.4-TOPO_T7p-tape_5.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

128_pcDNA3.4-TOPO_T7p-tape_5.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

 

DH5α colony picking of 119-128 (golden gate assembly of the final construct)

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with the results of the golden gate assembly (119-128) from selection plate containing carbecillin was performed for the replication and expression of the desired genetic material. 3 colonies were picked from each plate.

119_pcDNA3.4-TOPO_T7p-tape_1.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

120_pcDNA3.4-TOPO_T7p-tape_1.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

121_pcDNA3.4-TOPO_T7p-tape_2.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

122_pcDNA3.4-TOPO_T7p-tape_2.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

123_pcDNA3.4-TOPO_T7p-tape_3.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

124_pcDNA3.4-TOPO_T7p-tape_3.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

125_pcDNA3.4-TOPO_T7p-tape_4.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

126_pcDNA3.4-TOPO_T7p-tape_4.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

127_pcDNA3.4-TOPO_T7p-tape_5.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

128_pcDNA3.4-TOPO_T7p-tape_5.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with carbecillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Plasmid Miniprep of various plasmids (119-128)

Goal;

A miniprep of the plasmids below was performed to isolate a small amount of plasmid DNA for downstream applications.

119_pcDNA3.4-TOPO_T7p-tape_1.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

120_pcDNA3.4-TOPO_T7p-tape_1.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

121_pcDNA3.4-TOPO_T7p-tape_2.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

122_pcDNA3.4-TOPO_T7p-tape_2.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

123_pcDNA3.4-TOPO_T7p-tape_3.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

124_pcDNA3.4-TOPO_T7p-tape_3.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

125_pcDNA3.4-TOPO_T7p-tape_4.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

126_pcDNA3.4-TOPO_T7p-tape_4.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

127_pcDNA3.4-TOPO_T7p-tape_5.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

128_pcDNA3.4-TOPO_T7p-tape_5.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
119.3	120,5	1,92	2,36
120,2	105,1	1,94	2,18
120,1	123,3	1,93	2,23
124,1	126	1,95	2,27
122,1	93,6	1,94	2,21
119,2	96,6	1,91	2,16
122,2	137	1,94	2,25
122,3	81,8	1,89	2,90
124,3	79,7	1,79	1,83
120,3	84,1	1,83	2,28
126,2	123	1,93	2,15
128,3	102	1,94	2,23
124,2	125	1,95	2,23
126,1	111,6	1,95	2,26
128,2	89,5	1,94	2,16
128,1	107,2	1,95	2,22
127,3	147,2	1,94	2,26
127,2	112,8	1,91	2,23
127,1	92,1	1,95	2,20
119,1	86,8	1,94	2,18
121,1	106,2	1,95	2,29
121,2	91,6	1,95	2,27
121,3	137,6	1,92	2,25
122,1	95	1,96	2,37
123,2	124,5	1,95	2,27
123,3	122,8	1,98	2,27
125,1	146,6	1,93	2,23
125,2	137	1,92	2,29
125,3	108,4	1,93	2,30
126,3	97,6	1,96	2,32

 

Sanger sequencing of various plasmids (119-128)

Goal;

To confirm the nucleotide sequences of the plasmids below, Sanger sequencing was performed by using Genewiz, Azenta services. 

119_pcDNA3.4-TOPO_T7p-tape_1.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

120_pcDNA3.4-TOPO_T7p-tape_1.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

121_pcDNA3.4-TOPO_T7p-tape_2.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

122_pcDNA3.4-TOPO_T7p-tape_2.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

123_pcDNA3.4-TOPO_T7p-tape_3.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

124_pcDNA3.4-TOPO_T7p-tape_3.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

125_pcDNA3.4-TOPO_T7p-tape_4.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

126_pcDNA3.4-TOPO_T7p-tape_4.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

127_pcDNA3.4-TOPO_T7p-tape_5.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

128_pcDNA3.4-TOPO_T7p-tape_5.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

Protocol;

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 10 μL of purified plasmid at a concentration of 30 - 100 ng/μL was added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis was not a match between the experimental and expected sequences. In addition, the sequencing data has not showed a high quality score and a continuous read length of over 1 kb. Cloning has failed.

Gibson assembly of 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G)

Goal;

The goal is to assemble 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G)

Procedure;

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Reagent	Amount
NEBuilder HiFi DNA Assembly Master Mix	10 μL
Vector	X ng / X μL
Insert	X ng / X μL
ddH2O	X μL
Total	20 μL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

/

 

Heterodimerization of deoxynucleotides (112&113, 114&115, 116&117, 118&119, 120&121, 122&123, 126&127, 128&129)

Goal;

The procedure was performed to heterodimerize the two deoxynucleotides for the gRNA spacer used in downstream experiments for 

gRNA2, tape1_30bp: 112_F_gRNA2_tape1& 113_R_gRNA2_tape1

gRNA3, tape1_30bp: 114_F_gRNA3_tape1 & 115_R_gRNA3_tape1

gRNA2, tape2_30bp: 116_F_gRNA2_tape2 & 117_R_gRNA2_tape2

gRNA3, tape2_30bp: 118_F_gRNA3_tape2 & 119_R_gRNA3_tape2

gRNA2, tape3_30bp: 120_F_gRNA2_tape3 & 121_R_gRNA2_tape3

gRNA3, tape3_30bp: 122_F_gRNA3_tape3 & 123_R_gRNA3_tape3

gRNA3, tape4_30bp: 126_F_gRNA3_tape4 & 127_R_gRNA3_tape4

gRNA2, tape5_30bp: 128_F_gRNA2_tape5 & 129_R_gRNA2_tape5

gRNA3, tape5_30bp: 130_F_gRNA3_tape5 & 131_R_gRNA3_tape5

Protocol;

Heterodimerization of the deoxynucleotides was carried out according to the modified protocol developed by Truong et al. (Truong, DJ., Armbrust, N., Geilenkeuser, J., Westmeyer, G. Generation of cell lines for the usage of Intron-encoded Scarless Programmable Extranuclear Cistronic Transcripts (INSPECT), 2023, PROTOCOL available at Protocol Exchange). Primers were designed in a way that they are complementary to each other and have 5’ overhangs for ligation with a pU6 plasmid digested with BbsI. For this, 5 μL from both solubilized deoxynucleotides and 40 μL of TE buffer were combined in a PCR tube. The final composition of the reaction mixture is defined as follows:

Component	Volume (50 μL)
Forward primer (100 μM)	5 μL
Reverse primer (100 μM)	5 μL
TE buffer (pH 8.0)	40 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), with the following conditions. 

Product was kept at -20°C until further use. 

Results;

The efficiency of the deoxynucleotides heterodimerization was evaluated by downstream experiments.

Ligation of 036_pU6_PspCas13b-DR-empty and heterodimerized gRNA

Goal;

The procedure was performed to ligate the 036_pU6_PspCas13b-DR-empty plasmid digested with BbsI and heterodimerized gRNAs.

Protocol;

Ligation was carried out according to the modified protocol developed by Truong et al. (Truong, DJ., Armbrust, N., Geilenkeuser, J., Westmeyer, G. Generation of cell lines for the usage of Intron-encoded Scarless Programmable Extranuclear Cistronic Transcripts (INSPECT), 2023, PROTOCOL available at Protocol Exchange). For this, 50 ng BbsI-digested 016_pU6_PspCas13b-DR-empty plasmid, 0.5 µl Heterodimerized product of the spacer, 10 µl Quick Ligase Reaction Buffer (2X) (M2200S, NEW ENGLAND Biolabs), 1 µl of Quick Ligase (M2200S, NEW ENGLAND Biolabs) were combined in a 1.5 mL Eppendorf reaction tube. The final composition of the reaction mixture is defined as follows:

Component	Volume (20 μL)
BbsI-digested pU6 backbone	1 μL (46 ng)
Heterodimerized product of the spacer	0.5 μL
Quick Ligase Reaction Buffer (2X)	10 μL
Quick Ligase	1 μL
Nuclease-free water	7.5 μL

The ligation reaction mixture was incubated for 10 min at room temperature.

Product was kept at -20°C until further use. 

Results;

The efficiency of the ligation was evaluated by downstream experiments.

PCR of 056_pcDNA_tdPCP-SpyC3 to get 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) insert

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 056_pcDNA_tdPCP-SpyC3 using primers 079_new_F_PCP_pC0054 & 083_R_corrected_PCP_dPspCas13b with high fidelity and efficiency.

Protocol;

PCR was carried out using the Q5 Polymerase (Thermo Fisher Scientific) according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 12.5 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	25-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	12.5 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 079_new_F_PCP_pC0054 083_R_corrected_PCP_dPspCas13b	0.5 μM	2.5 μL
Template DNA: 056_pcDNA_tdPCP-SpyC3	10 ng plasmid	1 μL
Nuclease-free water	–	9 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

DH5α colony picking of 023_pU6_PspCas13b-DR-gRNA_NLuc

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with 023_pU6_PspCas13b-DR-gRNA_NLuc (023.2.9) from selection plate containing Carbecillin was performed for the replication and expression of the desired genetic material.

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Carbecillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

DH5α E. coli transformation of various plasmids (099-104, 106-108, 062)

Goal;

Transformation was performed to introduce plasmid DNA construct into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) 

099_pU6_PspCas13b-DR-gRNA2_tape1

101_pU6_PspCas13b-DR-gRNA2_tape2

102_pU6_PspCas13b-DR-gRNA3_tape2

103_pU6_PspCas13b-DR-gRNA2_tape3

104_pU6_PspCas13b-DR-gRNA3_tape3

106_pU6_PspCas13b-DR-gRNA3_tape4 

107_pU6_PspCas13b-DR-gRNA2_tape5 

108_pU6_PspCas13b-DR-gRNA3_tape5

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

 

Agarose gel electrophoresis of 062 ( backbone insert )

Goal;

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples from 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) backbone and insert

Protocol;

Agarose gel electrophoresis was performed using a 1.0% w/v agarose (ROTH) gel prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply (BIORAD, PowerPac™ Basic Power Supply). A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 30 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (BIORAD, ChemiDoc Imaging System).

Results;

The following samples were loaded into the gel from left to right: 1k DNA Ladder, empty, 062 insert, 062 backbone, empty

The agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

KLD of 061_pcDNA_Zeo_DD-N_NLuc_pp7

Goal;

KLD reaction was performed to phosphorylate and circularise 061_pcDNA_Zeo_DD-N_NLuc_pp7, and degrade template DNA by Dpn I enzyme treatment. 

Protocol;

KLD reaction was performed using the KLD Enzyme Mix (NEW ENGLAND Biolabs), consisting of Kinase, Ligase and Dpn I enzymes, according to the manufacturer’s instructions. For this, 5 µL of the KLD reaction Buffer (2X) (NEW ENGLAND Biolabs), 1 µL of KLD Enzyme Mix, 2 µL of Nuclease-free water and 2 µL of the PCR product were combined. The final composition of the reaction mixture was defined as follows:

Components	Volume
PCR Product	1 µL
KLD Reaction Buffer (2X)	5 µL
KLD Enzyme Mix (10X)	1 µL
Nuclease-free Water	3 µL
Total Volume	10 µL

The sample was incubated at room temperature (25°C) for 5 minutes, and then placed on ice. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

Restriction digest of 036_pU6_PspCas13b-DR-empty with BbsI I

Goal;

 

Restriction digest of the 036_pU6_PspCas13b-DR-empty construct with BbsI enzyme, for downstream cloning of gRNA for Nano-luciferase restoration assay.

Protocol;

Restriction digest was carried out using the BbsI-HF^® enzyme (R3539S, New England Biolabs) according to the protocol developed by Truong et al. (Truong, DJ., Armbrust, N., Geilenkeuser, J., Westmeyer, G. Generation of cell lines for the usage of Intron-encoded Scarless Programmable Extranuclear Cistronic Transcripts (INSPECT), 2023, PROTOCOL available at Protocol Exchange). For this, 1 μg of DNA template, 5 μL CutSmart^® (B7204, New England Biolabs), 1 μL BbsI-HF^® were combined in a 1.5 mL Eppendorf reaction tube. The sample was mixed by pipetting, and incubated at 37°C for 2 hours (including mixing every 30 minutes).

Component	Volume (49.75 μL)
Template DNA: 016_pU6_PspCas13b-DR-empty	18 μL (1 μg, 274 ng/ul)
CutSmart®	5 μL
BbsI-HF®	0,75 μL
Nuclease-free water	26 μL

Results;

The efficiency of plasmid digestion was evaluated by downstream experiments.

 

 

DNA gel extraction of 036_pU6_PspCas13b-DR-empty

Goal;

 

A gel extraction of 036_pU6_PspCas13b-DR-empty digested with BbsI was performed to isolate and purify a specific DNA fragment from agarose gel for downstream applications.

Protocol;

Following gel electrophoresis, DNA bands of interest were excised from the gel under UV transillumination (BIORAD) using a clean scalpel. The excised gel slices were transferred to a 1.5 mL microcentrifuge tube and weighed to determine the amount of gel present. DNA fragments were then isolated and purified from gel using the Monarch® DNA Gel Extraction Kit Protocol (NEW ENGLAND Biolabs, NEB #T1020) following modified manufacturer’s instructions. For this, the gel slices were first dissolved in 4 volumes of Monarch Gel Dissolving Buffer (NEW ENGLAND Biolabs) at 50 °C. The mixture was then transferred to a provided column placed in a collection tube. The column was centrifuged at 13500 × g for 1 min to allow the DNA to bind to the column. The flow-through was discarded, and the column was washed twice with 200 μL of DNA Wash Buffer (NEW ENGLAND Biolabs) at 13500 × g for 1 min each to remove impurities and contaminants. Purified DNA was eluted into a 1.5 mL microcentrifuge tube by adding 10 μL of elution buffer to the column matrix, incubating at room temperature for 1 min, and centrifuging the column for 1 min at 13500 × g. DNA concentration was determined using a NanoPhotometer N60, and the DNA samples were stored at −20 °C.

Results;

 

The extraction procedure yielded DNA of 55 ng/μL (20 μL) .

Agarose gel electrophoresis of digested 036_pU6_PspCas13b-DR-empty

Goal;

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples from 036_pU6_PspCas13b-DR-empty.

Protocol;

Agarose gel electrophoresis was performed using a 1.0% w/v agarose (ROTH) gel prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply (BIORAD, PowerPac™ Basic Power Supply). A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 30 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (BIORAD, ChemiDoc Imaging System).

Results;

The following samples were loaded into the gel from left to right: /

The agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

Plasmid Miniprep of 023_pU6_PspCas13b-DR-gRNA_NLuc

Goal;

A miniprep of 023_pU6_PspCas13b-DR-gRNA_NLuc (023.2.9) was performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
023.2.9.1	210.9	1.96	2.28
023.2.9.2	213.0	1.96	2.30

 

DH5α colony picking of various plasmids (099-104, 106-108, 062)

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with plasmids seen below from selection plate containing Carbecillin was performed for the replication and expression of the desired genetic material.

062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) 

099_pU6_PspCas13b-DR-gRNA2_tape1

100_pU6_PspCas13b-DR-gRNA3_tape1

101_pU6_PspCas13b-DR-gRNA2_tape2

102_pU6_PspCas13b-DR-gRNA3_tape2

103_pU6_PspCas13b-DR-gRNA2_tape3

104_pU6_PspCas13b-DR-gRNA3_tape3

106_pU6_PspCas13b-DR-gRNA3_tape4 

107_pU6_PspCas13b-DR-gRNA2_tape5 

108_pU6_PspCas13b-DR-gRNA3_tape5

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with Carbecillin) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Ligation of 036_pU6_PspCas13b-DR-empty and heterodimerized gRNA

Goal;

The procedure was performed to ligate the 036_pU6_PspCas13b-DR-empty digested with BbsI and heterodimerized gRNAs.

Protocol;

Ligation was carried out according to the modified protocol developed by Truong et al. (Truong, DJ., Armbrust, N., Geilenkeuser, J., Westmeyer, G. Generation of cell lines for the usage of Intron-encoded Scarless Programmable Extranuclear Cistronic Transcripts (INSPECT), 2023, PROTOCOL available at Protocol Exchange). For this, 50 ng BbsI-digested 016_pU6_PspCas13b-DR-empty plasmid, 0.5 µl Heterodimerized product of the spacer, 10 µl Quick Ligase Reaction Buffer (2X) (M2200S, NEW ENGLAND Biolabs), 1 µl of Quick Ligase (M2200S, NEW ENGLAND Biolabs) were combined in a 1.5 mL Eppendorf reaction tube. The final composition of the reaction mixture is defined as follows:

Component	Volume (20 μL)
BbsI-digested pU6 backbone	X μL (50 ng)
Heterodimerized product of the spacer	0.5 μL
Quick Ligase Reaction Buffer (2X)	10 μL
Quick Ligase	1 μL
Nuclease-free water	X μL

The ligation reaction mixture was incubated for 10 min at room temperature.

Product was kept at -20°C until further use. 

Results;

The efficiency of the ligation was evaluated by downstream experiments.

PCR of 017_pcDNA_Zeo_NLuc-STOP to get 061_pcDNA_Zeo_DD-N_NLuc_pp7

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 017_pcDNA_Zeo_NLuc-STOP using primers 146_R_NanoLuc_pp7 & 147_F_bGH_pp7 with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 146_R_NanoLuc_pp7 147_F_bGH_pp7	0.5 μM	2 μL
Template DNA: 017_pcDNA_Zeo_NLuc-STOP	10 ng plasmid	1 μL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

Sanger sequencing of various plasmids (062, 099, 101-104)

Goal;

To confirm the nucleotide sequences of the plasmids seen below, Sanger sequencing was performed by using Genewiz, Azenta services. 

062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) 

099_pU6_PspCas13b-DR-gRNA2_tape1

101_pU6_PspCas13b-DR-gRNA2_tape2

102_pU6_PspCas13b-DR-gRNA3_tape2

103_pU6_PspCas13b-DR-gRNA2_tape3

104_pU6_PspCas13b-DR-gRNA3_tape3

Protocol;

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 10 μL of purified plasmid at a concentration of 30 - 100 ng/μL was added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis … a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Sample	Validation
062.1	no evaluation possible
062.2	no evaluation possible
062.3	no evaluation possible
073.3	successful cloning confirmed
099.1	unsuccessful cloning
101.1	unsuccessful cloning
102.1	unsuccessful cloning
103.1	unsuccessful cloning
104.1	unsuccessful cloning
104.2	unsuccessful cloning

 

Golden Gate Assembly of XFPs (063-065) and tapes (025-034) + backbone (073) to get 119, 121, 123

Goal;

 

Golden Gate Assembly of XFPs (063_T2A_miRFP670_P2A_eUnaG, 064_T2A_mScarlet3_P2A_eUnaG and 065_T2A_mTagBFP2_P2A_eUnaG) and tapes (025-034) into 073_pcDNA3.4-TOPO_T7p_T7tt backbone.

025_tape_1.0

026_tape_1.1

027_tape_2.0

028_tape_2.1

029_tape_3.0

030_tape_3.1

031_tape_4.0

032_tape_4.1

033_tape_5.0

034_tape_5.1

Expected

Results;

119_pcDNA3.4-TOPO_T7p-tape_1.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

121_pcDNA3.4-TOPO_T7p-tape_2.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

123_pcDNA3.4-TOPO_T7p-tape_3.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

Protocol;

Restriction digest was carried out using the BsmBI-v2 and T4 DNA Ligase enzymes (R0739, M0202;New England Biolabs) according to the manufacturers protocol. Template DNA was supplied in amount of 75 ng (backbone), while insert was supplied in a 2:1 molar ratio(use calculator below). The DNAs were combined with 2 μL of T4 Ligase Buffer^® (B0202, New England Biolabs) and 1 μL of T4 DNA Ligase and BsmBI-v2 in a PCR tube, to a final volume of 20 uL. The sample was mixed by pipetting, and incubated according in 30 cycles of 1 min at 42ºC and 1 min at 16ºC, followed by a single 5 min cycle at 60ºC. The reaction was then stored at -20C unless used for transformation.

Component	Volume (20 μL)	119	121	123
Backbone DNA (073.2 - 75 ng/μl)	75 ng	1 μl	1 μl	1 μl
Insert DNA (063_gblock - 20 ng/μl)	2:1 molar ratio, 42.76 ng	2,1	2,1	2,1
Insert DNA (064_gblock - 20 ng/μl)	2:1 molar ratio, 52.97 ng	2,65	2,65	2,65
Insert DNA (065_gblock - 20 ng/μl)	2:1 molar ratio, 57,09 ng	2,9	2,9	2,9
Insert DNA (tape - 5 ng/μl)	2:1 molar ratio, 7,47 ng	1,5 μl (025)	1,5 μl (027)	1,5 μl (029)
T4 Ligase Reaction Buffer (10X)	2 μl	2 μl	2 μl	2 μl
BsmBI-v2	1 μl	1 μl	1 μl	1 μl
Nuclease-free H20	X μl	6,85 μl	6,85 μl	6,85 μl

Program for 2-10 inserts: (42°C, 1 min → 16°C,1 min) x 30 → 60°C, 5 min  

For reference, 073_pcDNA3.4-TOPO_T7p_T7tt is 3676bp.

Results;

The efficiency of Golden Gate Assembly was evaluated by downstream experiments.

 

 

 

Heterodimerization of deoxynucleotides of (124&125, 132&133, 134&135, 136&137)

Goal;

The procedure was performed to heterodimerize the two deoxynucleotides for the gRNA spacer used in downstream experiments for 

gRNA2, tape4_30bp: 124_F_gRNA2_tape4 & 125_R_gRNA2_tape4

gRNA1, tape M1_50bp: 132_F_gRNA1_tapeM1_50bp & 133_R_gRNA1_tapeM1_50bp

gRNA1, tape M2_50bp: 134_F_gRNA1_tapeM2_50bp & 135_R_gRNA1_tapeM2_50bp

gRNA1, tape NM1_50bp: 136_F_gRNA1_tapeNM1_50bp & 137_R_gRNA1_tapeNM1_50bp

Protocol;

Heterodimerization of the deoxynucleotides was carried out according to the modified protocol developed by Truong et al. (Truong, DJ., Armbrust, N., Geilenkeuser, J., Westmeyer, G. Generation of cell lines for the usage of Intron-encoded Scarless Programmable Extranuclear Cistronic Transcripts (INSPECT), 2023, PROTOCOL available at Protocol Exchange). Primers were designed in a way that they are complementary to each other and have 5’ overhangs for ligation with a pU6 plasmid digested with BbsI. For this, 5 μL from both solubilized deoxynucleotides and 40 μL of TE buffer were combined in a PCR tube. The final composition of the reaction mixture is defined as follows:

Component	Volume (50 μL)
Forward primer (100 μM)	5 μL
Reverse primer (100 μM)	5 μL
TE buffer (pH 8.0)	40 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), with the following conditions. 

Product was kept at -20°C until further use. 

Results;

The efficiency of the deoxynucleotides heterodimerization was evaluated by downstream experiments.

DH5α E. coli transformation of various plasmids (109-111)

Goal;

Transformation was performed to introduce plasmid DNA constructs seen below into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

109_pU6_PspCas13b-DR-gRNA1_tapeM1_50bp

110_pU6_PspCas13b-DR-gRNA1_tapeM2_50bp

111_pU6_PspCas13b-DR-gRNA1_tapeNM1_50bp

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

 

PCR of XFP gblocks (063-065)

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from gBlocks seen below resuspended in TE buffer using primers 030_F_tape-amplification & 027_R_pU6_2960A<C with high fidelity and efficiency.

063_T2A_miRFP670_P2A_eUnaG 

064_T2A_mScarlet3_P2A_eUnaG

065_T2A_mTagBFP2_P2A_eUnaG

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	50-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	25 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 030_F_tape-amplification 027_R_pU6_2960A<C	0.5 μM	5 μL
Template DNA: 063_T2A_miRFP670_P2A_eUnaG  064_T2A_mScarlet3_P2A_eUnaG 065_T2A_mTagBFP2_P2A_eUnaG	30 ng plasmid	1,5 μL (20 ng/ul stock)
Nuclease-free water	–	18,5 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

Plasmid Miniprep of various plasmids (062, 099, 101-104)

Goal;

A miniprep of the plasmids below was performed to isolate a small amount of plasmid DNA for downstream applications.

062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) 

099_pU6_PspCas13b-DR-gRNA2_tape1

101_pU6_PspCas13b-DR-gRNA2_tape2

102_pU6_PspCas13b-DR-gRNA3_tape2

103_pU6_PspCas13b-DR-gRNA2_tape3

104_pU6_PspCas13b-DR-gRNA3_tape3

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
062.1	277,1	1,91	2,18
062.2	230,6	1,90	2,20
062.3	253,5	1,91	2,19
103.1	176,8	1,91	2,18
099.1	191,1	1,92	2,20
104.2	140,4	1,92	2,13
102.1	223,6	1,93	2,25
101.1	197,9	1,91	2,20
104.1	195,3	1,92	2,22

 

Splitting of HEK293T cell line into T75 flask

Goal;

Splitting HEK293T cell line from “A” and “B” flasks to the next passage number of 36.

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 3737°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos’s modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 75 cm² flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO₂ and 90% relative humidity.

Results;

HEK293T cell line from “A” and “B” flasks were split to passage number 36.

DH5α colony picking of various plasmids (099-111, 119-121)

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with plasmids seen below from selection plate containing Carbecillin was performed for the replication and expression of the desired genetic material.

One tube for each of the constructs 099-111, 129 and two tubes for 119, 121 and 123 were put to incubate.

099_pU6_PspCas13b-DR-gRNA2_tape1

100_pU6_PspCas13b-DR-gRNA3_tape1

101_pU6_PspCas13b-DR-gRNA2_tape2

102_pU6_PspCas13b-DR-gRNA3_tape2

103_pU6_PspCas13b-DR-gRNA2_tape3

104_pU6_PspCas13b-DR-gRNA3_tape3

105_pU6_PspCas13b-DR-gRNA2_tape4 

106_pU6_PspCas13b-DR-gRNA3_tape4 

107_pU6_PspCas13b-DR-gRNA2_tape5 

108_pU6_PspCas13b-DR-gRNA3_tape5

109_pU6_PspCas13b-DR-gRNA1_tapeM1_50bp

110_pU6_PspCas13b-DR-gRNA1_tapeM2_50bp

111_pU6_PspCas13b-DR-gRNA1_tapeNM1_50bp

129_pcDNA_Zeo_DD-N_NLuc_PP7_P10SN 

119_pcDNA3.4-TOPO_T7p-tape_1.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

121_pcDNA3.4-TOPO_T7p-tape_2.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

123_pcDNA3.4-TOPO_T7p-tape_3.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 5 mL of fresh culture medium (LB medium with …) and incubated overnight at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after overnight incubation in 37°C.

Plasmid Miniprep of various plasmids (099-108 (new gRNA, 30bp tape))

Goal;

A miniprep of various plasmids seen below was performed to isolate a small amount of plasmid DNA for downstream applications.

099_pU6_PspCas13b-DR-gRNA2_tape1

100_pU6_PspCas13b-DR-gRNA3_tape1

101_pU6_PspCas13b-DR-gRNA2_tape2

102_pU6_PspCas13b-DR-gRNA3_tape2

103_pU6_PspCas13b-DR-gRNA2_tape3

104_pU6_PspCas13b-DR-gRNA3_tape3

105_pU6_PspCas13b-DR-gRNA2_tape4 

106_pU6_PspCas13b-DR-gRNA3_tape4 

107_pU6_PspCas13b-DR-gRNA2_tape5 

108_pU6_PspCas13b-DR-gRNA3_tape5

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of mostly sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
099	40,26	1,94	1,97
100	41,84	1,99	2,15
101	44,14	2,03	2,01
102	28,24	2,07	2,26
103	53,17	1,99	2,12
104	18,99	2,10	1,77
105	23,40	1,98	1,70
106	92,82	1,94	2,18
107	69,41	1,96	2,18
108	33,32	1,99	1,99

Samples 102, 104, 105 were discarded due to low concentration.

Plasmid Miniprep of various plasmids (109-111, 119, 121, 123, 129)

Goal;

A miniprep of the plasmids seen below was performed to isolate a small amount of plasmid DNA for downstream applications.

109_pU6_PspCas13b-DR-gRNA1_tapeM1_50bp

110_pU6_PspCas13b-DR-gRNA1_tapeM2_50bp  

111_pU6_PspCas13b-DR-gRNA1_tapeNM1_50bp

119_pcDNA3.4-TOPO_T7p-tape_1.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt (119.1-119.2)

121_pcDNA3.4-TOPO_T7p-tape_2.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt (121.1-121.2)

123_pcDNA3.4-TOPO_T7p-tape_3.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt (123.1-123.2 (GG tape 1 - tape3))

129_pcDNA_Zeo_DD-N_NLuc_PP7_P10SN (129.1)

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of mostly sufficient quantity and sometimes of purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
109.1	19,24	1,88	1,65
110.1	18,57	1,85	1,56
111.1	33,82	1,92	1,90
119.1	32,93	1,87	1,87
119.2	34,72	1,87	1,93
121.1	43,24	2,02	2,10
121.2	30,16	1,94	1,98
123.1	26,33	2,00	1,94
123.2	9,13	2,11	2,19
129.1	56,62	1,89	2,08

Samples 199.1, 110.1, 123.1, 123.2 were discarded due to insufficient concentration.

Sanger sequencing of various plasmids (099-101, 103, 106-108)

Goal;

To confirm the nucleotide sequences of the plasmids seen below, Sanger sequencing was performed by using Genewiz, Azenta services. 

099_pU6_PspCas13b-DR-gRNA2_tape1

100_pU6_PspCas13b-DR-gRNA3_tape1

101_pU6_PspCas13b-DR-gRNA2_tape2

103_pU6_PspCas13b-DR-gRNA2_tape3

106_pU6_PspCas13b-DR-gRNA3_tape4 

107_pU6_PspCas13b-DR-gRNA2_tape5 

108_pU6_PspCas13b-DR-gRNA3_tape5

Protocol;

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 10 μL of purified plasmid at a concentration of 30 - 100 ng/μL was added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis … a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Sample	Primer	Validation
/	99.1	T3	Point mutations in the tape region
/	100.1	T3	Point mutations in the tape region
/	101.1	T3	Point mutations in the tape region
/	103.1	T3	Point mutations in the tape region
/	106.1	T3	Point mutations in the tape region
/	107.1	T3	Point mutations in the tape region
/	108.1	T3	Point mutations in the tape region

All gRNA constructs that we cloned have the same sequence. Check the primers that were used for digestion or the plasmid from which it was used (036_pU6_PspCas13b-DR-empty).

Colony PCR of plasmids 119-128; Attempt 1

Goal;

Colony polymerase chain reaction (PCR) was performed to verify if insert has been successfully clone into the vector. Primers were designed in a way that forward primer 144_F_T7p_cPCR binds to the backbone, and reverse primer 058_R_WPRE_seq binds to an insert, to ensure orientation-specific amplification of DNA. 

119_pcDNA3.4-TOPO_T7p-tape_1.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

120_pcDNA3.4-TOPO_T7p-tape_1.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

121_pcDNA3.4-TOPO_T7p-tape_2.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

122_pcDNA3.4-TOPO_T7p-tape_2.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

123_pcDNA3.4-TOPO_T7p-tape_3.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

124_pcDNA3.4-TOPO_T7p-tape_3.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

125_pcDNA3.4-TOPO_T7p-tape_4.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

126_pcDNA3.4-TOPO_T7p-tape_4.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

127_pcDNA3.4-TOPO_T7p-tape_5.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

128_pcDNA3.4-TOPO_T7p-tape_5.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

Protocol;

First, colony picking of individual DH5α E. coli bacterial colonies transformed with plasmids seen in goal from selection plate containing LB was performed. Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 400 μL of fresh culture medium (LB medium with Carbecillin in a 96-well plate and carefully mixed by pipetting. This material has further been used for colony PCR. PCR was carried out using the Q5^® High-Fidelity polymerase (NEW ENGLAND Biolabs) according to the manufacturer’s instructions. For this, 2 μL of bacteria culture, 0,64 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
Q5 High-Fidelity 2X Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 144_F_T7p_cPCR 058_R_WPRE_seq	0.5 μM	0.8 μL
Bacterial culture	–	2 μL
Nuclease-free water	–	7.2 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

Agarose gel electrophoresis of colony PCR (119-128 + 035 as pos. contr.) - Attempt 1

Goal;

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples from the following:

119_pcDNA3.4-TOPO_T7p-tape_1.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

120_pcDNA3.4-TOPO_T7p-tape_1.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

121_pcDNA3.4-TOPO_T7p-tape_2.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

122_pcDNA3.4-TOPO_T7p-tape_2.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

123_pcDNA3.4-TOPO_T7p-tape_3.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

124_pcDNA3.4-TOPO_T7p-tape_3.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

125_pcDNA3.4-TOPO_T7p-tape_4.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

126_pcDNA3.4-TOPO_T7p-tape_4.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

127_pcDNA3.4-TOPO_T7p-tape_5.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

128_pcDNA3.4-TOPO_T7p-tape_5.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

035_pU6_PspCas13b-DR-empty

Protocol;

Agarose gel electrophoresis was performed using a 1.0% w/v agarose (ROTH) gel prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply (BIORAD, PowerPac™ Basic Power Supply). A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 30 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (BIORAD, ChemiDoc Imaging System).

Results;

The agarose gel electrophoresis analysisdid not confirmed the presence and size distribution of the expected DNA fragments in the samples.

Colony PCR of plasmids 119-128; Attempt 2

Goal;

Colony polymerase chain reaction (PCR) was performed to verify if insert has been successfully clone into the vector. Primers were designed in a way that forward primer 144_F_T7p_cPCR binds to the backbone, and reverse primer 058_R_WPRE_seq binds to an insert, to ensure orientation-specific amplification of DNA. 

119_pcDNA3.4-TOPO_T7p-tape_1.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

120_pcDNA3.4-TOPO_T7p-tape_1.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

121_pcDNA3.4-TOPO_T7p-tape_2.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

122_pcDNA3.4-TOPO_T7p-tape_2.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

123_pcDNA3.4-TOPO_T7p-tape_3.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

124_pcDNA3.4-TOPO_T7p-tape_3.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

125_pcDNA3.4-TOPO_T7p-tape_4.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

126_pcDNA3.4-TOPO_T7p-tape_4.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

127_pcDNA3.4-TOPO_T7p-tape_5.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

128_pcDNA3.4-TOPO_T7p-tape_5.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

Protocol;

First, colony picking of individual DH5α E. coli bacterial colonies transformed with plasmids (goal) from selection plate containing Carbecillin was performed. Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 1 mL of fresh culture medium (LB medium with Carbecillin) in a 96-well plate and carefully mixed by pipetting. This material has further been used for colony PCR. PCR was carried out using the Q5^® High-Fidelity polymerase (NEW ENGLAND Biolabs) according to the manufacturer’s instructions. For this, 2.5 μL of bacteria culture, 1.25 pmol forward and reverse primers, and 12.5 μL of the master mix were combined in a PCR tube. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	25-μL run
Q5 High-Fidelity 2X Master Mix	1X	12.5 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 144_F_T7p_cPCR 058_R_WPRE_seq	0.5 μM	2.5 μL
Bacterial culture	–	2.5 μL
Nuclease-free water	–	7.5 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

Sanger sequencing of various plasmids (109, 110, 111, 129, 101, 102, 104, 105)

Goal;

To confirm the nucleotide sequences of the plasmids seen below, Sanger sequencing was performed by using Genewiz, Azenta services. 

109_pU6_PspCas13b-DR-gRNA1_tapeM1_50bp

110_pU6_PspCas13b-DR-gRNA1_tapeM2_50bp

111_pU6_PspCas13b-DR-gRNA1_tapeNM1_50bp

129_pcDNA_Zeo_DD-N_NLuc_PP7_P10SN 

101_pU6_PspCas13b-DR-gRNA2_tape2

102_pU6_PspCas13b-DR-gRNA3_tape2

104_pU6_PspCas13b-DR-gRNA3_tape3

Protocol;

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 10 μL of purified plasmid at a concentration of 30 - 100 ng/μL was added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis … a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Sample	Validation
101	unsuccessful cloning
102	unsuccessful cloning
104	unsuccessful cloning
109	unsuccessful cloning
110	unsuccessful cloning
111	unsuccessful cloning
129	successful cloning

 

Agarose gel electrophoresis of colony PCR (119-128 + 035 as pos. contr.) - Attempt 2

Goal;

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples from the following:

119_pcDNA3.4-TOPO_T7p-tape_1.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

120_pcDNA3.4-TOPO_T7p-tape_1.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

121_pcDNA3.4-TOPO_T7p-tape_2.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

122_pcDNA3.4-TOPO_T7p-tape_2.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

123_pcDNA3.4-TOPO_T7p-tape_3.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

124_pcDNA3.4-TOPO_T7p-tape_3.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

125_pcDNA3.4-TOPO_T7p-tape_4.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

126_pcDNA3.4-TOPO_T7p-tape_4.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

127_pcDNA3.4-TOPO_T7p-tape_5.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

128_pcDNA3.4-TOPO_T7p-tape_5.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

035_pU6_PspCas13b-DR-empty

Protocol;

Agarose gel electrophoresis was performed using a 1.0% w/v agarose (ROTH) gel prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply (BIORAD, PowerPac™ Basic Power Supply). A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 30 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (BIORAD, ChemiDoc Imaging System).

Results;

The agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

Splitting of HEK293T cell line into T75 flask

Goal;

Splitting HEK293T cell line from flask A & B to the next passage number of 37.

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 3737°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos’s modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 4 ml pre-warmed DMEM. A new 75 cm² flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO₂ and 90% relative humidity.

Results;

HEK293T cell line from flasks A & B was split to passage number 37.

Restriction digest of 036_pU6_PspCas13b-DR-empty with BbsI I

Goal;

 

Restriction digest of the 036_pU6_PspCas13b-DR-empty construct with BbsI enzyme, for downstream cloning of gRNA for Nano-luciferase restoration assay.

Protocol;

Restriction digest was carried out using the BbsI-HF^® enzyme (R3539S, New England Biolabs) according to the protocol developed by Truong et al. (Truong, DJ., Armbrust, N., Geilenkeuser, J., Westmeyer, G. Generation of cell lines for the usage of Intron-encoded Scarless Programmable Extranuclear Cistronic Transcripts (INSPECT), 2023, PROTOCOL available at Protocol Exchange). For this, 1 μg of DNA template, 5 μL CutSmart^® (B7204, New England Biolabs), 1 μL BbsI-HF^® were combined in a 1.5 mL Eppendorf reaction tube. The sample was mixed by pipetting, and incubated at 37°C for 2 hours (including mixing every 30 minutes).

Component	Volume (50 μL)
Template DNA: 016_pU6 (1928 ng/μL)	1,55 μL (3 μg)
CutSmart®	5 μL
BbsI-HF®	2 μL
Nuclease-free water	41.45 μL

Results;

The efficiency of plasmid digestion was evaluated by downstream experiments.

 

 

Plasmid Miniprep of gRNA constructs (101, 102, 104, 105, 109, 110, 111)

Goal;

A miniprep of the constructs seen below was performed to isolate a small amount of plasmid DNA for downstream applications.

101_pU6_PspCas13b-DR-gRNA2_tape2 

102_pU6_PspCas13b-DR-gRNA3_tape2, 104_pU6_PspCas13b-DR-gRNA3_tape3 

105_pU6_PspCas13b-DR-gRNA2_tape4, 109_pU6_PspCas13b-DR-gRNA1_tapeM1_50bp 

110_pU6_PspCas13b-DR-gRNA1_tapeM2_50bp 

111_pU6_PspCas13b-DR-gRNA1_tapeNM1_50bp

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
101.1	336,5	2,13	1,93
101.2	335,3	2,20	1,93
102.1	163,7	2,34	1,99
102.2	406,6	2,30	1,92
102.3	376,8	2,31	1,92
104.1	392,9	2,31	1,92
104.2	375,8	2,30	1,92
105.1	357,4	2,32	1,92
105.2	334,1	2,32	1,92
109.1	375,7	2,20	1,92
109.2	278,7	2,31	1,93
110.1	325,2	2,34	1,94
110.2	136,9	2,37	1,94
111.1	360,8	2,30	1,92
111.2	306,2	2,26	1,91

 

Plasmid Miniprep of various plasmids (119-128)

Goal;

A miniprep of plasmids seen below was performed to isolate a small amount of plasmid DNA for downstream applications.

119_pcDNA3.4-TOPO_T7p-tape_1.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

120_pcDNA3.4-TOPO_T7p-tape_1.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

121_pcDNA3.4-TOPO_T7p-tape_2.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

122_pcDNA3.4-TOPO_T7p-tape_2.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

123_pcDNA3.4-TOPO_T7p-tape_3.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

124_pcDNA3.4-TOPO_T7p-tape_3.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

125_pcDNA3.4-TOPO_T7p-tape_4.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

126_pcDNA3.4-TOPO_T7p-tape_4.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

127_pcDNA3.4-TOPO_T7p-tape_5.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

128_pcDNA3.4-TOPO_T7p-tape_5.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
122.9	474,8	1,90	2,21
123.7	523,9	1,92	2,22
121.8	568,6	1,91	2,21
120.7	521,2	1,90	2,20
120.8	492,1	1,91	2,23
126.8	535,2	1,91	2,25
119.8	568,2	1,91	2,22
124.7	248,3	1,92	2,25
123.8	761,2	1,91	2,23
123.6	578,3	1,91	2,21

 

DH5α colony picking of various plasmids (099-108); right backbone (fast protocol)

Goal;

Colony picking of individual DH5α E. coli bacterial colonies transformed with 099-108 constructs from selection plate containing Carbecillin was performed for the replication and expression of the desired genetic material.

099_pU6_PspCas13b-DR-gRNA2_tape1

100_pU6_PspCas13b-DR-gRNA3_tape1

101_pU6_PspCas13b-DR-gRNA2_tape2

102_pU6_PspCas13b-DR-gRNA3_tape2

103_pU6_PspCas13b-DR-gRNA2_tape3

104_pU6_PspCas13b-DR-gRNA3_tape3

105_pU6_PspCas13b-DR-gRNA2_tape4 

106_pU6_PspCas13b-DR-gRNA3_tape4 

107_pU6_PspCas13b-DR-gRNA2_tape5 

108_pU6_PspCas13b-DR-gRNA3_tape5

Protocol;

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 2,5 mL of fresh preheated culture medium (LB medium with 0,5x Carb (=1,25ul) ) and incubated for 7 hours at 37 °C with continuous shaking at 200 rpm for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination and transferred to a fridge.

Results;

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture after 7h incubation in 37°C.

Plasmid Miniprep of various plasmids (099-108)

Goal;

A miniprep of the plasmids seen below was performed to isolate a small amount of plasmid DNA for downstream applications. These are the colonies that were grown and attempted to be sequenced in 1-day but failed.

099_pU6_PspCas13b-DR-gRNA2_tape1

100_pU6_PspCas13b-DR-gRNA3_tape1

101_pU6_PspCas13b-DR-gRNA2_tape2

102_pU6_PspCas13b-DR-gRNA3_tape2

103_pU6_PspCas13b-DR-gRNA2_tape3

104_pU6_PspCas13b-DR-gRNA3_tape3

105_pU6_PspCas13b-DR-gRNA2_tape4 

106_pU6_PspCas13b-DR-gRNA3_tape4 

107_pU6_PspCas13b-DR-gRNA2_tape5 

108_pU6_PspCas13b-DR-gRNA3_tape5

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
99.1	213,2	1,89	2,11
99.2	160,8	1,90	2,20
100.1	277	1,8	1,4
100.2	59.2	1,47	1,92
101.1	107,3	1,87	2,17
101.2	114,9	1,89	2,58
102.1	96,3	1,93	2,11
102.2	181	1,88	2,16
103,1	88,7	1,91	3
104,1	49,5	1,84	2,50
104,2	224,2	1,85	1,73
105,2	210,5	1,91	2,30
106.1	115,7	1,86	2,25
106,2	215	1,90	2,22
107,1	92,7	1,86	2,12
107,2	247	1,85	1,76
108,1	43,6	1,88	1,91
108,2	180,8	1,88	1,99

 

PCR of 056_pcDNA_tdPCP-SpyC3 to get insert for 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G)

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 056_pcDNA_tdPCP-SpyC3 (056.1) using primers 079_F_corrected_PCP_pC0054 & 083_R_corrected_PCP_dPspCas13b with high fidelity and efficiency.

Protocol;

PCR was carried out using the Q5 Polymerase (Thermo Fisher Scientific) according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 12.5 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration, BOTH CORRECTED VERSIONS): 079_F_corrected_PCP_pC0054 083_R_corrected_PCP_dPspCas13b	0.5 μM	2 μL
Template DNA: 056_pcDNA_tdPCP-SpyC3	10 ng plasmid	1 μL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

Golden Gate Assembly of 073_pcDNA3.4-TOPO_T7p with tapes 2, 3 state .1, .2

Goal;

 

Golden Gate Assembly of 063_T2A_miRFP670_P2A_eUnaG, 064_T2A_mScarlet3_P2A_eUnaG, 065_T2A_mTagBFP2_P2A_eUnaG and tapes (122, 124, 130, 131) into 073_pcDNA3.4-TOPO_T7p_T7tt (073.2) backbone to get the following:

122_pcDNA3.4-TOPO_T7p-tape_2.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

124_pcDNA3.4-TOPO_T7p-tape_3.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

130_pcDNA3.4-TOPO_T7p-tape_2.2-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

131_pcDNA3.4-TOPO_T7p-tape_3.2-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

Protocol;

Restriction digest was carried out using the BsmBI-v2 and T4 DNA Ligase enzymes (R0739, M0202;New England Biolabs) according to the manufacturers protocol. Template DNA was supplied in amount of 75 ng (backbone), while insert was supplied in a 2:1 molar ratio(use calculator below). The DNAs were combined with 2 μL of T4 Ligase Buffer^® (B0202, New England Biolabs) and 1 μL of T4 DNA Ligase and BsmBI-v2 enzyme mix in a PCR tube, to a final volume of 20 uL. The sample was mixed by pipetting, and incubated according in 30 cycles of 1 min at 42ºC and 1 min at 16ºC, followed by a single 5 min cycle at 60ºC. The reaction was then stored at -20C unless used for transformation.

Component	Volume (20 μL)	122 (tape 2.1)	130 (tape 2.2)	124 (tape 3.1)	131 (tape 3.2)
Backbone DNA (073 - 75 ng/μl)	75 ng	1 μl	1 μl	1 μl	1 μl
Insert DNA (063 - 20 ng/μl)	2:1 molar ratio, 42.76 ng	2,1 μl	2,1 μl	2,1 μl	2,1 μl
Insert DNA (064 - 20 ng/μl)	2:1 molar ratio, 52.97 ng	2,65 μl	2,65 μl	2,65 μl	2,65 μl
Insert DNA (065 - 20 ng/μl)	2:1 molar ratio, 57.09 ng	2,9 μl	2,9 μl	2,9 μl	2,9 μl
Insert DNA (tape - 10 ng/μl)	2:1 molar ratio, 7.47 ng	0,75 μl	0,75 μl	0,75 μl	0,75 μl
T4 Ligase Reaction Buffer (10X)	2 μl	2 μl	2 μl	2 μl	2 μl
BsmBI-v2 enzyme mix	1 μl	1 μl	1 μl	1 μl	1 μl
Nuclease-free H20	X μl	7,6 μl	7,6 μl	7,6 μl	7,6 μl

 

Program for 2-10 inserts: (42°C, 1 min → 16°C,1 min) x 30 → 60°C, 5 min  

 

Results;

The efficiency of Golden Gate Assembly was evaluated by downstream experiments.

Seeding of ibiTreat µ-Plate 24 Well, Ibidi

Goal;

Seeding of two ibidi µ-Plate 24 Well Black ID 14 mm, ibidi-Treated, for further confocal analysis of composite parts encoding 121_pcDNA3.4-TOPO_T7p-tape_2.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt and 123_pcDNA3.4-TOPO_T7p-tape_3.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt transfected into HEK293T cells with 5 × 10⁴ HEK293T cells.

Procedure:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet. Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 mL of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 4 mL of Accutase solution at 37 °C for 5 minutes. Upon confirming detachment using a light phase microscope, cells were rescued using 4 mL of pre-warmed complete Dulbecco’s modified Eagle medium F12 (DMEM) and transferred to a 15 mL tube. The cell suspension was centrifuged at 25 °C, 320 RCF for 5 minutes in a swinging bucket centrifuge. The supernatant was decanted using a Pasteur pipette, and the cell pellet was resuspended in 4 mL of pre-warmed DMEM.

Cells were counted and analyzed for viability using the Countess™ II automated cell counter with 2 replicas, taking the average of the two measurements.

Each well of the 24-well µ-Plate was inoculated with 500 µL of cell suspension at 5 × 10⁴ cells per well. Cells were further grown at 37 °C with 5% CO₂ and 90% relative humidity.

Results;

Cells are in good condition with enough for further seeding.

Agarose gel electrophoresis of colony PCR

Goal;

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples from colony PCR conducted on Golden Gate assembled plasmids: 122_pcDNA3.4-TOPO_T7p-tape_2.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt, 124_pcDNA3.4-TOPO_T7p-tape_3.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt, 130_pcDNA3.4-TOPO_T7p-tape_2.2-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt, 131_pcDNA3.4-TOPO_T7p-tape_3.2-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt.

Protocol;

Agarose gel electrophoresis was performed using a 1.0% w/v agarose (ROTH) gel prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 5 μl of SYBR™ Safe DNA Gel Stain (Thermo Fisher) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply (BIORAD, PowerPac™ Basic Power Supply). A constant voltage of 100 V was applied, and the DNA fragments were allowed to migrate through the gel for 30 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (BIORAD, ChemiDoc Imaging System).

Results;

The following samples were loaded into the gel from left to right: 1kB Plus DNA ladder, 130 (1-8), 131 (1-6) on smaller gel, and 1kB Plus DNA ladder, 122 (1-8), 124 (1-8), 131 (7-8).

The agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in some of the samples.

Plasmid Miniprep of XFPs, 103_pU6_PspCas13b-DR-gRNA2_tape3 &121_pcDNA3.4-TOPO_T7p-tape_2.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

Goal;

A miniprep of 103_pU6_PspCas13b-DR-gRNA2_tape3 and 121_pcDNA3.4-TOPO_T7p-tape_2.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt was performed to isolate a small amount of plasmid DNA for downstream applications.

063_T2A_miRFP670_P2A_eUnaG 

064_T2A_mScarlet3_P2A_eUnaG

065_T2A_mTagBFP2_P2A_eUnaG

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
RFP1	81,58	2,08	2,20
RFP2	148,46	2,07	2,34
RFP3	109,8	2,04	2,18
RFP4	147,33	2,06	2,32
RFP5	99,64	2,07	2,23
BFP1	165	1,06	2,32
BFP2	106,57	2,03	2,24
BFP3	120,36	2,03	2,22
BFP4	92,38	1,99	2,15
BFP5	88,5	2,03	2,22
mScarlet1	155,69	2,04	2,23
mScarlet2	142,2	2,03	2,21
mScarlet3	94	2,02	2,26
mScarlet4	118,68	2,03	2,23
mScarlet5	129,68	2,02	2,21
103.1	121,77	1,97	2,26
121.1	106,33	1,96	2,16

 

Colony PCR for 119_pcDNA3.4-TOPO_T7p-tape_1.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

Goal;

Colony polymerase chain reaction (PCR) was performed to verify if insert has been successfully clone into 119_pcDNA3.4-TOPO_T7p-tape_1.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt. Primers were designed in a way that forward primer 144_F_T7p_cPCR binds to the backbone, and reverse primer 145_R_mTagBFP2_cPCR binds to an insert, to ensure orientation-specific amplification of DNA. 

Protocol;

First, colony picking of individual DH5α E. coli bacterial colonies transformed with … from selection plate containing … was performed. Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 400 μL of fresh culture medium (LB medium with Carbenicillin) in a 96-well plate and carefully mixed by pipetting. This material has further been used for colony PCR. PCR was carried out using the Q5^® High-Fidelity polymerase (NEW ENGLAND Biolabs) according to the manufacturer’s instructions. For this, 2.5 μL of bacteria culture, 1.25 pmol forward and reverse primers, and 12.5 μL of the master mix were combined in a PCR tube. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	25-μL run
Q5 High-Fidelity 2X Master Mix	1X	12.5 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration): 144_F_T7p_cPCR 145_R_mTagBFP2_cPCR	0.5 μM	2.5 μL
Bacteria culture	–	2.5 μL
Nuclease-free water	–	7.5 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

Gibson assembly of 132_pcDNA3.4-TOPO_5’synthUTR-T7p-_tape_2.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt (A and B) and 133_pcDNA3.4-TOPO_5’synthUTR-T7p-tape_3.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2

Goal;

Gibson assembly was performed to assemble the respective vector and inserts DNA fragments to produce 132_pcDNA3.4-TOPO_5’synthUTR-T7p-_tape_2.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt (A and B) and 133_pcDNA3.4-TOPO_5’synthUTR-T7p-tape_3.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt.

Procedure;

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Reagent	Amount
NEBuilder HiFi DNA Assembly Master Mix	10 μL
Vector: PCR product	2,5 μL
Insert	–
ddH2O	7,5 μL
Total	20 μL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

/

 

Gibson assembly of 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) (with corrected primers)

Goal;

Gibson assembly was performed to assemble the respective vector and inserts DNA fragments to produce 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G).

Procedure;

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Reagent	Amount
NEBuilder HiFi DNA Assembly Master Mix	10 μL
Vector: 062_I_PCRclean (20 ng/μL)	24,5 ng / 1,23 μL
Insert: 062_BB_PCRclean (100 ng/μL)	100 ng / 1 μL
ddH2O	7,77 μL
Total	20 μL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

/

DNA Transfection of HEK293T with jetOPTIMUS® - tapes 2.0 and 3.0

Goal;

Introduction of different groups of plasmids depending on the experiment group into HEK293T cell line to conduct ProgRAM tape switching assay. 

Experiment groups:

1. Switching state zero to one: 123_pcDNA3.4-TOPO_T7p-tape_3.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt + 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) + 037_pU6_PspCas13b-DR-gRNA1_tape1

2. Off-target control with gRNA2: 123_pcDNA3.4-TOPO_T7p-tape_3.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt + 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) + 099_pU6_PspCas13b-DR-gRNA2_tape1

3. Off-target control with gRNA3: 123_pcDNA3.4-TOPO_T7p-tape_3.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt + 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) + 100_pU6_PspCas13b-DR-gRNA3_tape1

4. Switching state zero to one and one to two: 123_pcDNA3.4-TOPO_T7p-tape_3.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt + 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) + 037_pU6_PspCas13b-DR-gRNA1_tape1 + 099_pU6_PspCas13b-DR-gRNA2_tape1

5. Switching state zero to one and testing for off-target with gRNA3: 123_pcDNA3.4-TOPO_T7p-tape_3.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt + 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) + 037_pU6_PspCas13b-DR-gRNA1_tape1 + 100_pU6_PspCas13b-DR-gRNA3_tape1

6. Switching state zero to one and one to two, and two to three: 123_pcDNA3.4-TOPO_T7p-tape_3.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt + 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) + 037_pU6_PspCas13b-DR-gRNA1_tape1 + 099_pU6_PspCas13b-DR-gRNA2_tape1 + 100_pU6_PspCas13b-DR-gRNA3_tape1

7. Editor off-target control: 123_pcDNA3.4-TOPO_T7p-tape_3.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt + 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G)

8.  Tape .0 only transfection: 123_pcDNA3.4-TOPO_T7p-tape_3.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

To compensate for missing pU6 plasmids in the groups 016_pU6_PspCas13b-DR-empty has been added. To compensate for missing pC0054 plasmids in the groups 002_pcDNA3.4-TOPO_IL23a has been added. 

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Cells were seeded in 8 well microscopic slides from ibidi at density of 5*10³cells per well and grown overnight. Afterwards cells were treated with jetOPTIMUS reagent mixture, including plasmid of interest and fresh DMEM. Afterwards, cells were grown at  37°C with 5% CO₂ and 90% relative humidity. 

Results;

Images have been obtained with CX7. Cells died before 48 time-point. Experiment was repeated. 

Seeding of ibiTreat µ-Plate 24 Well, Ibidi

Goal;

Seeding of two ibidi µ-Plate 24 Well Black ID 14 mm, ibidi-Treated, for further confocal analysis of composite parts 134_pcDNA3.4-TOPO_T7p-miRFP670nano3-T7tt, 135_pcDNA3.4-TOPO_T7p-mScarlet3-T7tt, and 136_pcDNA3.4-TOPO_T7p-mTagBFP2-T7tt transfected into HEK293T cells with 5 × 10⁴ HEK293T cells.

Procedure:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet. Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 mL of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 4 mL of Accutase solution at 37 °C for 5 minutes. Upon confirming detachment using a light phase microscope, cells were rescued using 4 mL of pre-warmed complete Dulbecco’s modified Eagle medium F12 (DMEM) and transferred to a 15 mL tube. The cell suspension was centrifuged at 25 °C, 320 RCF for 5 minutes in a swinging bucket centrifuge. The supernatant was decanted using a Pasteur pipette, and the cell pellet was resuspended in 4 mL of pre-warmed DMEM.

Cells were counted and analyzed for viability using the Countess™ II automated cell counter with 2 replicas, and the average of the two measurements was taken.

Each well of the 24-well µ-Plate was inoculated with 500 µL of cell suspension at 5 × 10⁴ cells per well. Cells were further grown at 37 °C with 5% CO₂ and 90% relative humidity.

Results;

Cells are in good condition with enough for further seeding.

Plasmid Miniprep of various plasmids (062, 122, 124, 130, 131, 132, 133)

Goal;

A miniprep of the plasmids seen below was performed to isolate a small amount of plasmid DNA for downstream applications.

062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G)

122_pcDNA3.4-TOPO_T7p-tape_2.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

124_pcDNA3.4-TOPO_T7p-tape_3.1-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

130_pcDNA3.4-TOPO_T7p-tape_2.2-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt 

131_pcDNA3.4-TOPO_T7p-tape_3.2-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt

132_pcDNA3.4-TOPO_5’synthUTR-T7p-_tape_2.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt 

133_pcDNA3.4-TOPO_5’synthUTR-T7p-tape_3.0-T2A-miRFP670-P2A-eUnaG-T2A-mScarlet3-P2A-eUnaG-T2A-mTagBFP2-P2A-eUnaG-T7tt 

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230		Sample	Concentration [ng/μL]	260/280	260/230
062.1	277,7	1,92	2,28		131.7	314,8	1,93	2,28
062.2	288,1	1,93	2,25		131.8	312,6	1,92	2,25
062.3	231,8	1,93	2,28		132 A.1	341,2	1,91	2,26
062.4	260,5	1,93	2,28		132 A.2	356,6	1,92	2,27
062.5	370,9	1,92	2,27		132 A.3	280,7	1,93	2,29
122.1	290,2	1,93	2,26		132 A.4	282,8	1,94	2,30
122.3	248,6	1,92	2,28		132 A.5	348,9	1,92	2,21
124.1	302,5	1,93	2,27		132 B.1	201,2	1,94	2,29
124.3	290,8	1,92	2,25		132 B.2	195,0	1,93	2,27
124.4	190,1	1,94	2,30		132 B.3	140,4	1,93	2,42
130.1	214,5	1,93	2,31		132 B.4	125,3	1,92	2,24
130.2	183,5	1,94	2,25		132 B.5	130,8	1,95	2,29
130.3	205,7	1,92	2,28		133.1	129,3	1,95	2,25
130.4	299,6	1,93	2,23		133.2	260,1	1,92	2,27
130.7	190,0	1,92	2,26		133.3	181,1	1,96	2,31
131.1	283,8	1,92	2,24		133.4	72,2	1,93	2,36
131.2	256,6	1,92	2,28		133.5	200,7	1,94	2,31
131.5	277,6	1,92	2,26		/	/	/	/

 

PCR of 136_pcDNA3.4-TOPO_T7p-mTagBFP2-T7tt

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 136_pcDNA3.4-TOPO_T7p-mTagBFP2-T7tt using primers 073_F_mTagBFP2_R16K and 074_R_mTagBFP2_R16K for construct 144_pcDNA3.4-TOPO_T7p-mTagBFP2-T7tt.

with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1 x	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration) :	0.5 μM	2 μL
Template DNA :	10 ng	1 μL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 37 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

PCR for 071_pC0054-CMV-tdPCP_(G4S)3_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G)

Goal;

Polymerase chain reaction (PCR) was performed to amplify specific DNA fragments from 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) using primers 082_R_PCP_(G4S)3 and 085_F_dPspCas13b_(G4S)3 for construct 071_pC0054-CMV-tdPCP_(G4S)3_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G)  with high fidelity and efficiency.

Protocol;

PCR was carried out using the PlatinumTM SuperFi^TM II (Thermo Fisher Scientific) polymerase according to the modified manufacturer’s instructions. For this, 10 ng DNA template, 1 pmol forward and reverse primers, and 10 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoDrop. The final composition of the reaction mixture is defined as follows:

Component	Final concentration	20-μL run
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	10 μL
Forward + Reverse Primers (pre-mixed in 5μM concentration) : 082_R_PCP_(G4S)3 and 085_F_dPspCas13b_(G4S)3	0.5 μM	2 μL
Template DNA : 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G)	10 ng plasmid	1 μL
Nuclease-free water	–	7 μL

The reaction mixture was subjected to thermal cycling (Miulab, PR-96E Gradient Thermal Cycler), which included 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. 

Amplified DNA products were kept at -20°C until further use. 

Results;

The efficiency of PCR was evaluated by downstream experiments.

DNA Transfection of HEK293T with jetOPTIMUS® to image single XFPs

Goal;

Introduction of 134_pcDNA3.4-TOPO_T7p-miRFP670nano3-T7tt, 135_pcDNA3.4-TOPO_T7p-mScarlet3-T7tt, 136_pcDNA3.4-TOPO_T7p-mTagBFP2-T7tt into HEK293T cell line. Each plasmid has been transfected to a different well of a 24-well plate. Each experiment has been performed in a technical triplicate. 

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Cells were seeded in 8 well microscopic slides from ibidi at density of 15*10³cells per well and grown overnight. Afterwards cells were treated with jetOPTIMUS reagent mixture, including plasmid of interest and fresh DMEM. Afterwards, cells were grown at  37°C with 5% CO₂ and 90% relative humidity. 

Results;

The results are presented on the Results page of our wiki. 

DNA Transfection of HEK293T with jetOPTIMUS® with 134_pcDNA3.4-TOPO_T7p-miRFP670nano3-T7tt

Goal;

Introduction of 134_pcDNA3.4-TOPO_T7p-miRFP670nano3-T7tt, 135_pcDNA3.4-TOPO_T7p-mScarlet3-T7tt, 136_pcDNA3.4-TOPO_T7p-mTagBFP2-T7tt, 002_pcDNA3.4-TOPO_IL23a (control) into HEK293T cell line at cell partition 30.

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Cells were seeded in 24 well plate from ibidi at density of 5*10⁴ cells per well and grown overnight. Afterwards cells were treated with jetOPTIMUS reagent mixture, including 500 ng of plasmid of interest. Afterwards, cells were grown at  37°C with 5% CO₂ and 90% relative humidity. 

Each transfection mix was calculated per 3.5 replicas (including pipetting error), the final composition was: 500 ng of desired plasmid, 175 μL of jetOptimus buffer, 1.75 μL of jetOptimus reagent.

The transfection scheme was as follows:

	1	2	3	4	5	6
A	134_pcDNA3.4-TOPO_T7p-miRFP670nano3-T7tt (500 ng)	135_pcDNA3.4-TOPO_T7p-mScarlet3-T7tt (500 ng)	136_pcDNA3.4-TOPO_T7p-mTagBFP2-T7tt (500 ng)	002_pcDNA3.4-TOPO_IL23a (500 ng)
B	134_pcDNA3.4-TOPO_T7p-miRFP670nano3-T7tt (500 ng)	135_pcDNA3.4-TOPO_T7p-mScarlet3-T7tt (500 ng)	136_pcDNA3.4-TOPO_T7p-mTagBFP2-T7tt (500 ng)	002_pcDNA3.4-TOPO_IL23a (500 ng)
C	134_pcDNA3.4-TOPO_T7p-miRFP670nano3-T7tt (500 ng)	135_pcDNA3.4-TOPO_T7p-mScarlet3-T7tt (500 ng)	136_pcDNA3.4-TOPO_T7p-mTagBFP2-T7tt (500 ng)	002_pcDNA3.4-TOPO_IL23a (500 ng)
D

Results;

 

Splitting of HEK293T cell line into T75 flask B

Goal;

Splitting HEK293T cell line from flask B to the next passage number of 31.

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 ml of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 3 ml Accutase solution at 3737°C for 5 minutes. Upon confirming detachment using light phase microscope, cells were rescued using 3ml pre-warmed complete Dulbeccos’s modified Eagle medium F12 (DMEM) and transfered to a 15 ml tube. Cell suspension was centrifuged at 25°C, 320 RCF for 5 minutes in a swinging bucket centrifuge. Supernatant was decanted using Pasteur pipette and cell pellet was resuspended in 6 ml pre-warmed DMEM. A new 75 cm² flask was inoculated with the 1ml cell suspension to 9ml pre-warmed DMEM. Cells were grown at  37°C with 5% CO₂ and 90% relative humidity.

Results;

HEK293T cell line from flask B was split to passage number 31.

Replacing DMEM media in flask A

Goal;

Replace old media in cell culture flask with fresh media.

Protocol;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

HEK293T were washed with DPBS and supplied with fresh complete Dulbecco’s modified Eagle medium. Cells were grown at  37°C with 5% CO₂ and 90% relative humidity..

Results;

DMEM media was successfully replaced in flask A. 

 

Seeding of four 24-well ibidi plates

Goal;

Seeding of four ibidi µ-Plate 24 Well Black ID 14 mm, ibidi-Treated plates for further confocal analysis of composite parts 121_pcDNA3.4-TOPO_T7p_T7tt-tape_2.0-T2A_miRFP670_P2A_eUnaG and 123_pcDNA3.4-TOPO_T7p_T7tt-tape_3.0-T2A_miRFP670_P2A_eUnaG transfected into HEK293T cells with 5 × 10⁴ HEK293T cells.

Procedure:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet. Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 mL of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 4 mL of Accutase solution at 37 °C for 5 minutes. Upon confirming detachment using a light phase microscope, cells were rescued using 4 mL of pre-warmed complete Dulbecco’s modified Eagle medium F12 (DMEM) and transferred to a 15 mL tube. The cell suspension was centrifuged at 25 °C, 320 RCF for 5 minutes in a swinging bucket centrifuge. The supernatant was decanted using a Pasteur pipette, and the cell pellet was resuspended in 4 mL of pre-warmed DMEM.

Cells were counted and analyzed for viability using the Countess™ II automated cell counter with 2 replicas, taking the average of the two measurements.

Each well of the 24-well plate was inoculated with 500 µL of cell suspension at 5 × 10⁴ cells per well. Cells were further grown at 37 °C with 5% CO₂ and 90% relative humidity.

Results;

Chamber	Cell count	Live cells	Dead cells
A	5.31 × 106	5.00 × 106	3.05 × 105
B	5.95 × 106	5.55 × 106	3.99 × 105
Mean	5.63 × 106	5.28 × 106	3.52 × 103

Cells are in good condition with enough for further seeding.

Gibson assembly of single XFPs under 5’ synthetic UTR (141-143, 071)

Goal;

Gibson assembly of single XFPs under 5’ synthetic UTR: 141_pcDNA3.4-TOPO_5’synthUTR-T7p-miRFP670nano3-T7tt, 142_pcDNA3.4-TOPO_5’synthUTR-T7p-mScarlet3-T7tt, 143_pcDNA3.4-TOPO_5’synthUTR-T7p-mTagBFP2-T7tt; 071_pC0054-CMV-tdPCP_(G4S)3_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G)

Procedure;

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with 10 μL of master mix.

Reagent	Amount
NEBuilder HiFi DNA Assembly Master Mix	10 μL
Vector	X ng / X μL
Insert	X ng / X μL
ddH2O	X μL
Total	20 μL

The reaction mixture was then incubated at 50 °C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

/

DH5α E. coli transformation with 145_pcDNA3.4-TOPO_T7p-mScarlet3-T7tt

Goal;

 

Transformation was performed to introduce KLD product of 144_pcDNA3.4-TOPO_T7p-mTagBFP2-T7tt

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

 

DH5α E. coli transformation of of 002_pcDNA3.4-TOPO_IL23a

Goal;

Re-transformation of 002_pcDNA3.4-TOPO_IL23a into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Protocol;

An aliquot of competent DH5α E. coli cells stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 2 min. Following the incubation period, heatshock at 42°C for 30 seconds was performed. Subsequently, the cells were placed on ice for minimum 2 minutes. Following addition of 450 µL of room temperature LB, the tubes were placed at 37 °C and incubated for 30 minutes with shaking (200 rpm). In the end, 50-100 µl of the mixture was spread onto a selection plate and incubated overnight at 37°C. 

Results;

 

KLD of 145_pcDNA3.4-TOPO_T7p-mScarlet3-T7tt

Goal;

KLD reaction was performed to phosphorylate and circularise the 145_pcDNA3.4-TOPO_T7p-mScarlet3-T7tt PCR product, and degrade template DNA by Dpn I enzyme treatment. 

Protocol;

KLD reaction was performed using the KLD Enzyme Mix (NEW ENGLAND Biolabs), consisting of Kinase, Ligase and Dpn I enzymes, according to the manufacturer’s instructions. For this, 5 µL of the KLD reaction Buffer (2X) (NEW ENGLAND Biolabs), 1 µL of KLD Enzyme Mix, 2 µL of Nuclease-free water and 2 µL of the PCR product were combined. The final composition of the reaction mixture was defined as follows:

Components	Volume
PCR Product	1 µL
KLD Reaction Buffer (2X)	5 µL
KLD Enzyme Mix (10X)	1 µL
Nuclease-free Water	3 µL
Total Volume	10 µL

The sample was incubated at room temperature (25°C) for 5 minutes, and then placed on ice. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

KLD to revert point mutation in mTagBFP2

Goal;

KLD reaction was performed to phosphorylate and circularise the PCR product of 144_pcDNA3.4-TOPO_T7p-mTagBFP2-T7tt, and degrade template DNA by Dpn I enzyme treatment. 

Protocol;

KLD reaction was performed using the KLD Enzyme Mix (NEW ENGLAND Biolabs), consisting of Kinase, Ligase and Dpn I enzymes, according to the manufacturer’s instructions. For this, 5 µL of the KLD reaction Buffer (2X) (NEW ENGLAND Biolabs), 1 µL of KLD Enzyme Mix, 2 µL of Nuclease-free water and 2 µL of the PCR product were combined. The final composition of the reaction mixture was defined as follows:

Components	Volume
PCR Product	1 µL
KLD Reaction Buffer (2X)	5 µL
KLD Enzyme Mix (10X)	1 µL
Nuclease-free Water	3 µL
Total Volume	10 µL

The sample was incubated at room temperature (25°C) for 5 minutes, and then placed on ice. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

Gibson assembly of single XFPs under 5’ synthetic UTR and introduction of a (G4S)3 linker into tdPCP_dPspCas13b-longlinker-ADAR2DD

Goal;

Gibson assembly of single XFPs (141_pcDNA3.4-TOPO_5’synthUTR-T7p-miRFP670nano3-T7tt, 142_pcDNA3.4-TOPO_5’synthUTR-T7p-mScarlet3-T7tt, 143_pcDNA3.4-TOPO_5’synthUTR-T7p-mTagBFP2-T7tt) under 5’ synthetic UTR and cloning a (G4S)3 linker into 062_pC0054-CMV-tdPCP_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) (colonies .4 and .5).

Procedure;

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. The final composition of reaction was as follows.

Reagent	Amount
NEBuilder HiFi DNA Assembly Master Mix	10 μL
Vector	2,5 μL
ddH2O	7,5 μL
Total	20 μL

The reaction mixture was then incubated at 50°C for 60 min, allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results;

Results of Gibson assembly were verified by downstream experiments. 

Plasmid Miniprep of 002_pcDNA3.4-TOPO_IL23a, 071_pC0054-CMV-tdPCP_(G4S)3_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G), 144_pcDNA3.4-TOPO_T7p-mTagBFP2-T7tt

Goal;

A miniprep of 002_pcDNA3.4-TOPO_IL23a, 071_pC0054-CMV-tdPCP_(G4S)3_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G), 144_pcDNA3.4-TOPO_T7p-mTagBFP2-T7tt was performed to isolate a small amount of plasmid DNA for downstream applications.

Protocol;

DNA plasmids were prepared using the Plasmid Miniprep Kit Protocol #T1010 (NEW ENGLAND BioLabs) following modified manufacturer’s instructions. The overnight culture was pelleted by centrifugation for 15 minutes at 4000 × g (eppendorf, Centrifuge 5804R). The supernatant was discarded. Pellet was resuspended in 200 μL of Plasmid Resuspension Buffer. Cell lysis was achieved by addition of 200 μL of Plasmid Lysis Buffer, followed by 5 - 6 times gentle tube inversions, and 1 minute incubation at room temperature. The lysed cells were neutralized with 400 μL of Plasmid Neutralization Buffer. The tubes were gently inverted, and incubated at room temperature for 2 minutes. Lysate was then centrifuged for 5 minutes. The supernatant was carefully transfered to the spin column and centrifuged for 1 minute (witeg, Centrifuge CF-10 High-Performance). The flow-through was discarded. The column was washed by adding 200 μL of Plasmid Wash Buffer 1 and centrifuging for 1 minute, followed by 400 μL of Plasmid Wash Buffer 2 and another centrifugation for 1 minute. The columns were transferred to clean 1.5 mL eppendorf tubes. DNA was eluted by addition of 30 μL of TE buffer to the column. Following 1 minute incubation, the samples were centrifuged for 1 minute. DNA concentration was determined using a NanoDrop, and the DNA samples were stored at −20 °C.

Results;

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Sample	Concentration [ng/μL]	260/280	260/230
002A	88,70	1,92	2,17
002B	84,38	1,97	2,51
144.1	141,47	1,98	2,31
144.2	130,28	1,97	2,15
144.3	144,7	2,01	2,19
071.4.1	149,17	1,99	2,28
071.4.2	250,16	1,91	1,88
071.5.1	230,51	1,89	1,88
071.5.2	256,44	1,93	2,07

 

Sanger sequencing of 071_pC0054-CMV-tdPCP_(G4S)3_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) & 144_pcDNA3.4-TOPO_T7p-mTagBFP2-T7tt

Goal;

To confirm the nucleotide sequences of 071_pC0054-CMV-tdPCP_(G4S)3_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) and 144_pcDNA3.4-TOPO_T7p-mTagBFP2-T7tt, Sanger sequencing was performed by using Genewiz, Azenta services. 

Protocol;

DNA was sequenced using a Sanger sequencing service (GENEWIZ, Azenta) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 10 μL of purified plasmid at a concentration of 30 - 100 ng/μL was added to a 1.5 mL flip cap reaction tube. Appropriate sequencing primers were chosen. The tube was labeled with a barcode and delivered to a drop-off point.

Results;

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence in Geneious software. The alignment analysis was a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Sample	Primer	Validation
06817356	144.1	CMV-Forward	successful cloning confirmed
06817357	144.2	CMV-Forward	successful cloning confirmed
06817358	144.3	CMV-Forward	point deletion in mTagBFP2, cloning failed
06817359	071.4.1	T7	successful cloning confirmed
06817360	071.4.2	T7	successful cloning confirmed
06817361	071.5.1	T7	successful cloning confirmed
06817362	071.5.2	T7	successful cloning confirmed

 

Seeding of three 24-well ibidi plates

Goal;

Seeding of four ibidi µ-Plate 24 Well Black ID 14 mm, collagen I-coated, with 5 × 10⁴ HEK293T cells for further transfection.

Procedure:

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet. Culturing media was decanted from the 75 cm² culturing flask. Cells were washed using 5 mL of pre-warmed Dulbecco’s phosphate-buffered saline (DPBS) and detached using 4 mL of Accutase solution at 37 °C for 5 minutes. Upon confirming detachment using a light phase microscope, cells were rescued using 4 mL of pre-warmed complete Dulbecco’s modified Eagle medium F12 (DMEM) and transferred to a 15 mL tube. The cell suspension was centrifuged at 25 °C, 320 RCF for 5 minutes in a swinging bucket centrifuge. The supernatant was decanted using a Pasteur pipette, and the cell pellet was resuspended in 6 mL of pre-warmed DMEM.

Cells were counted and analyzed for viability using the Countess™ II automated cell counter with 2 replicas, and the average of the two measurements was taken.

Each well of the 24-well plate was inoculated with 500 µL of cell suspension at 5 × 10⁴ cells per well. Cells were further grown at 37 °C with 5% CO₂ and 90% relative humidity.

Results;

Chamber	Cell count	Live cells	Dead cells
A	5.95 × 106	5.30 × 106	6.45 × 105
B	6.40 × 106	5.88 × 106	5.28 × 105
Mean	6.18 × 106	5.59 × 106	5.87 × 105

Cells are in good condition with enough for further seeding.

DNA Transfection of HEK293T with jetOPTIMUS® - NLuc restoration

Goal;

Introduction of different groups of plasmids depending on the experiment group into HEK293T cell line to conduct NLuc restoration assay. 

Experiment groups: 019_pcDNA_NLuc-STOP (target), 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G), 023_pU6_PspCas13b-DR-gRNA_NLuc at molar ratios of 1:1:1, 1:2:2, 1:3:3, 1:4:4, 1:5:5, respectively. 

REPAIR off-target controls: 019_pcDNA_NLuc-STOP (target), 004_pC0054-CMV-dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) at molar ratios of 1:1:1, 1:2:2, 1:3:3, 1:4:4, 1:5:5, respectively. 

gRNA off-target controls: 019_pcDNA_NLuc-STOP (target), 023_pU6_PspCas13b-DR-gRNA_NLuc at molar ratios of 1:1:1, 1:2:2, 1:3:3, 1:4:4, 1:5:5, respectively. 

Positive control: 017_pcDNA_Zeo_NLuc.

Experiment tdPCP:PP7:  129_pcDNA_NLuc_PP7 (target), 071_pC0054-CMV-tdPCP_(G4S)3_dPspCas13b-longlinker-ADAR2DD(E488Q/T375G) , 023_pU6_PspCas13b-DR-gRNA_NLuc at molar ratio of 1:5:5. 

Each experiment group has been obtained in a technical triplicate. 

To compensate for missing plasmids in the groups, 043_pcDNA3.4-TOPO_T7p_T7tt was added.

Procedure;

All solutions and equipment that come in contact with the cells were sterile. Proper sterile technique was used in a laminar flow cabinet work.

Cells were seeded in 96-well plate from ibidi at density of 15*10³ cells per well and grown overnight. Afterwards cells were treated with jetOPTIMUS reagent mixture, including plasmid of interest and fresh DMEM. Afterwards, cells were grown at  37°C with 5% CO₂ and 90% relative humidity. 

Results;

The results are presented on the Results page of our wiki.