Adapted from Zymo Research "Mix and Go E. coli Transformation Kit & Buffer Set" for generation of chemically competent E. coli
Mix and Go E. coli Transformation Kit & Buffer Set

Protocol

Preparation:
Cells:
Day 1

• Prepare an overnight culture of the respective E. coli stem in 0.5 mL LB-medium.

Day 2

• Inoculate 50 mL SOB medium in a 500 mL culture flask with the 0.5 mL overnight culture.
• Incubate at 20-33 °C and 150-250 rpm until an OD of 0.4-0.6 is reached.

Buffer:
Zymo Wash and Competent Buffers are provided as 2X solutions. Dilute each to 1X by adding an equal amount of Dilution Buffer.
1X buffers can be stored for up to 2 days at 4 °C.
Keep buffers on ice!

Further Preparation:

• Get Ice and liquid nitrogen.
• Keep empty 50 mL falcons on ice and prepare centrifuge to be at 4 °C.
• Bring equipment under the clean bench.
• Prepare 10 tubes for each 50 mL culture and store at -20 °C.

Procedure:
Work on ice all times!

1. After reaching an OD of 0.4-0.6, transfer the cell suspension into 50 mL Falcons and incubate it on ice for 10 min.
2. Pellet the cells by centrifuging at 300-3700 rpm (1600-2500 x g) for 10 min at 4 °C.
3. Remove supernatant and resuspend cells gently in 5 mL 1X Wash Buffer.
   Adapt the volume if the measured OD was below 0.4 e.g. with an OD of 0.2, use 1 mL or with OD of 0.3 use 2 mL
4. Re-pellet cells as in step 2.
5. Completely remove supernatant and gently resuspend cells in 5 mL 1X Competent Buffer. If volume was adapted in step 3 adapt the volume here as well.
6. Aliquot 50-100 µL of the cell suspension into sterile 1.5 mL tubes.
7. Shock freeze cells in liquid nitrogen and store them at -80 °C until further usage.

• Thaw 100 µL of competent cells on ice for 15 min.
• Add 100 ng/µl of plasmid to aliquot and resuspend carefully (work on ice and under sterile conditions).
• Incubate DNA and cells for 20 min on ice.
• Incubate the solution for 45 seconds at 42 °C.
• Put the cells on ice for 5 min.
• Add 600-700 µl SOC medium to the cells.
• Incubate the cells for 45 min at 37 °C and 300 rpm.
• Centrifuge for 1 min at 14.000 x g.
• Discard all but 50 µL of the supernatant and resuspend the pellet in the remaining 50 µL.
• Plate the sample on LB-Agar plates containing 100 µg/mL of the respective antibiotic(s).
• Incubate plates at 37 °C overnight.

We used the NEBcloner to find the specific protocol for the desired restriction enzymes: https://nebcloner.neb.com/#!/redigest

Protocol

• Set up reaction as follows:

Component	Volume for 50 µL	Volume for 20 µL
DNA	1 µg	0.5 µg
10 XNEBuffer*	5 µL (1X)	2 µl (1X)
restriction enzyme	0.5	0.25
Nuclease-free Water	up to 50 µL	up to 20 µl

* for all enzymes except BsmBI_V2 the rCutSmart-buffer was used. for BsmBI_V2 the r3.1-buffer was used

• All enzymes except BsmBI_V2 were Incubate at 37 °C for 1h
• BsmBI_V2 was Incubate at 55 °C for 1h

4.1 T4 Ligase NEB

The T4 Ligase from NEB was used:

1. Set up the following reaction in a microcentrifuge tube. Work on ice! We used a molar ratio of 1:1 up to 1:7 of vector to insert.
   For the molar ratio calculation we used the NEBioCalculator
   

   Component	Volume for 20 µL
   Vector DNA	0.020 pmol
   Insert DNA	0.06 pmol
   T4 DNA Ligase Buffer (10X)	2 μl
   T4 DNA Ligase	1 μl
   Nuclease-free water	up to 20 μl

2. Gently mix the reaction by pipetting up and down and microfuge shortly.
3. For cohesive (sticky) ends, incubate at 16 °C overnight or room temperature (RT) for 10 minutes.
4. For blunt ends or single base overhangs, incubate at 16 °C overnight or at RT for 2 hours.
5. To inactivate the ligase, incubate at 65 °C for 10 minutes.
6. For further usage in a transformation, put the tube on ice and transform 1-5 μl of the reaction into 50 μl competent cells (see “2. Transformation”). Alternatively, DNA can be stored at -20 °C until further usage.

4.2 T4 DNA Ligase Jena Bioscience

The protocol was adapted from the T4 DNA Ligase-protocol from https://www.jenabioscience.com/images/PDF/EN-149.0004.pdf

Protocol
set up reaction as follows

Component	20 µl Assay
Standard Ligation Buffer, 10x conc.	2 µl
Vector/Insert DNA	1 µg
T4 DNA Ligase	0.4 µl
PCR-grade Water	Add up to 20 µl

Incubate for 30 min at 16°C.

5.1 Q5 Polymerase from NEB

Adapted protocol from the NEB Q5® High-Fidelity 2X Master Mix

Preparations:

• Work on ice for mixing the components.
• Preheat the thermocycler to 98 °C.

Procedure:

• Mix the following components in PCR tubes.

  Component	Volume for a 50 µl Reaction	Final Concentration
  Q5 High-Fidelity 2X Master Mix	25 µl	1X
  10 µM Forward Primer	2.5 µl	0.5 µM
  10 µM Reverse Primer	2.5 µl	0.5 µM
  Template DNA	variable	1-10 ng
  Nuclease-Free Water	to 50 µl

• Gently mix the components by pipetting up and down.
• If necessary, collect all liquid at the bottom of the tubes by shortly centrifuging them.
• Transfer the PCR tubes to the thermocycler and begin thermocycling.

Thermocycling Conditions for a Routine PCR:

Step	Temperature	Time
Initial denaturation	98 °C	3 min
Denaturation	98 °C	30 sec
Annealing*	TM-5	Experiment dependendent
Elongation	Experiment dependendent	30 seconds/kb
Final Extension	72 °C	3 min
Hold	4-10°C	∞

* We either used SnapGene or the "Takara Primer design"-tool to calculate the annealing temperature.

5.2 Phusion from Thermo Fisher or Promega

Phusion PCR protocol adapted from Thermo Fisher and Promega original Protocol:
Thermo Fisher: https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012393_Phusion_HighFidelity_DNAPolymerase_UG.pdf
Promega: https://www.promega.de/-/media/files/resources/protocols/product-information-sheets/g/pfu-dna-polymerase-protocol.pdf?rev=5680f3dffd8a41449c71d0e50ca27ad4&sc_lang=en

In a sterile, nuclease-free microcentrifuge tube, combine the following Components for a 50µL mix:

Component	Volume
Primer 10 µM	2,5 µL
Primer 10 µM	2,5 µL
Template	1-10 ng, e.g. 1 µL from a 5 ng/µL concentrated Template solution
dNTPs(200 µM)	1 µL
10X Phusion Buffer	5 µL
Phusion Polymerase	0.5 µL
H2O	Add to 50 µL

Thermocycler Program:

Steps	Temperature	Time
Initial denaturation	98 °C	3 minutes
denaturation	98 °C	30 seconds
Annealing*	TM-5	Experiment dependendent
Extension	72 °C	Thermo Fisher: 15-30 seconds/kb Promega: 2 minutes/kb
Final extention	72 °C	3 min
Hold	6 °C	∞

* We either used Snapgene or the “Takara Primer design”-tool to calculate the annealing temperature.

5.3 DreamTaq Mastermix from Thermo Fisher

DreamTaq PCR protocol adapted from Thermo Fisher Link
https://assets.thermofisher.com/TFS-Assets/BID/Reference-Materials/dreamtaq-dna-polymerases-labaid.pdf

In a sterile, nuclease-free microcentrifuge tube, combine the following Components for a 50 µL mix:

Components	Volume
Forward Primer (1 µM)	2.5
Reverse Primer (1 µM)	2.5
Template DNA	10 pg–1 μg
DreamTaq DNA Polymerase/DreamTaq PCR Master Mix (2X)	25 μL
H2O	Ad 50 μL

Thermocycler Program:

Steps	Temperature	Time
Initial denaturation	98 °C	3 minutes
denaturation	98 °C	30 seconds
Annealing*	TM-5	30 seconds
Extension	72 °C	1 min for products up to 2 kb. Longer extensions: 1 min/kb
Final extention	72 °C	3- 10 min
Hold	4 °C	∞

1 cycle for the Initial denaturation.
30 cycles for the steps up to the final extension.
For colony PCR: Pick colony with pipette tip and resuspend in PCR sample.

6.1 Promega gel and PCR clean up

Original “Wizard® SV Gel and PCR Clean-Up System” Protocol

Website: https://www.promega.de/products/nucleic-acid-extraction/clean-up-and-concentration/wizard-sv-gel-and-pcr-clean-up-system/?catNum=A9281
A. Dissolving the Gel Slice

1. Following electrophoresis, excise DNA band from gel and place gel slice in a 1.5ml microcentrifuge tube.
2. Add 10μl of Membrane Binding Solution per 10 mg of gel slice. Vortex and incubate at 65 °C until gel slice is completely dissolved.

B. Processing PCR Amplifications

1. Add an equal volume of Membrane Binding Solution to the PCR amplification.

Binding of DNA

1. Insert SV Minicolumn into Collection Tube.
2. Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly. Incubate at room temperature for 1 minute.
3. Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.

Washing

1. Add 700 μl of Membrane Wash Solution (ethanol added). Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.
2. Repeat Step 4 with 500 μl of Membrane Wash Solution. Centrifuge at 16,000 × g for 5 minutes.
3. Empty the Collection Tube and recentrifuge the column assembly at top speed for 2 minute with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol.

Elution

1. Carefully transfer Minicolumn to a new 1.5 ml microcentrifuge tube.
2. Add 50 µl of Nuclease-Free Water to the Minicolumn. Incubate at room temperature for 1 minute. Centrifuge at 16,000 × g for 1 minute.
3. Discard Minicolumn and store DNA at 4 °C or -20 °C.

Adapted “Wizard® SV Gel and PCR Clean-Up System” Protocol

A. Dissolving the Gel Slice

1. Following electrophoresis, excise DNA band from gel and place gel slice in a 1.5ml microcentrifuge tube.
2. Add 10μl of Membrane Binding Solution per 10 mg of gel slice. Vortex and incubate at 50-65 °C until gel slice is completely dissolved.

Processing PCR Amplifications

1. Add an equal volume of Membrane Binding Solution to the PCR amplification.

Binding of DNA

1. Insert SV Minicolumn into Collection Tube.
2. Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly. Incubate at room temperature for 1 minute.
3. Centrifuge at 9,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.

Washing

1. Add 700 μl of Membrane Wash Solution (ethanol added). Centrifuge at 9,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.
2. Repeat Step 4 with 500 μl of Membrane Wash Solution. Centrifuge at 9,000 × g for 5 minutes.
3. Empty the Collection Tube and recentrifuge the column assembly at top speed for 2 minute with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol.

Elution

1. Carefully transfer Minicolumn to a new 1.5 ml microcentrifuge tube.
2. Add 20-30μl of Nuclease-Free 65 °C Water to the Minicolumn. Incubate at room temperature for 5-10 minutes. Centrifuge at 16,000 × g for 1 minute.
3. Discard Minicolumn and store DNA at 4 °C or -20 °C.

6.2 Quiagen gel clean up

Original "MinElute® Gel Extraction Kit" protocol

https://www.qiagen.com/de/resources/resourcedetail?id=0d0d73a7-e906-4a74-8f11-3b70311d7d66&lang=en

Notes before starting

• This protocol is for cleanup of up to 5 pg DNA fragments (70 bp to 4 kb).
• The yellow color of Buffer QG indicates a pH of <7.5. Adsorption of DNA to the membrane is efficient only at pH <7.5.
• Add ethanol (96-100%) to Buffer PE concentrate before use (see bottle label for volume).
• Isopropanol {100%} and a heating block or water bath at 50 °C are required.
• All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional tabletop microcentrifuge at room temperature (15-25 °C).
• Symbols: @ centrifuge processing; & vacuum processing.

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg gel ~ 100 pl). The maximum amount of gel slice per spin column is 400 mg. For >2% agarose gels, add 6 volumes Buffer QG.
3. Incubate at 50 °C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2-3 min during incubation to help dissolve the gel.
4. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 pl 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow.
5. Add 1 gel volume of isopropanol to the sample and mix by inverting.
6. Place a MinElute spin column in a provided 2 ml collection tube or & into a vacuum manifold. For information about set up, see the MinElute Handbook.
7. Apply sample to the MinElute column and centrifuge for 1 min or & apply vacuum until the entire sample has passed through the column. Discard flow-through and place the MinElute column back into the same collection tube. For sample volumes of more than 800 pl, simply load and spin again.
8. Add 500 pl Buffer QG to the MinElute column and centrifuge for 1 min or & apply vacuum. @ Discard flow-through and place the MinElute column back into the same collection tube.
9. Add 750 pl Buffer PE to MinElute column and centrifuge for 1 min or & apply vacuum. - Discard flow-through and place the MinElute column back into the same collection tube. Note: If the DNA will be used for saltsensitive applications, such as direct sequencing and bluntended ligation, let the column stand 2-5 min after addition of Buffer PE.
10. Centrifuge the column in a 2 ml collection tube (provided) for 1 min. Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.
11. Place each MinElute column into a clean 1.5 ml microcentrifuge tube. To elute DNA, add 10 pl Buffer EB (10 mM Tris-Cl, pH 8.5) or water to the center of the MinElute membrane. (Ensure that the elution buffer is dispensed directly onto the membrane for complete elution of bound DNA) Let the column stand for 1 min, and then centrifuge the column for 1 min.
12. If purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA, and mix by pipetting up and down before loading the gel.

Adapted “MinElute® Gel Extraction Kit” protocol

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg gel ~ 100 pl). The maximum amount of gel slice per spin column is 400 mg. For >2% agarose gels, add 6 volumes Buffer QG.
3. Incubate at 50 °C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2-3 min during incubation to help dissolve the gel.
4. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 pl 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow.
5. Add 1 gel volume of isopropanol to the sample and mix by inverting.
6. Place a MinElute spin column in a provided 2 ml collection tube or & into a vacuum manifold. For information about set up, see the MinElute Handbook.
7. Apply sample to the MinElute column and centrifuge for 1 min or & apply vacuum until the entire sample has passed through the column. Discard flow-through and place the MinElute column back into the same collection tube. For sample volumes of more than 800 pl, simply load and spin again.
8. Add 500 pl Buffer QG to the MinElute column and centrifuge for 1 min or & apply vacuum. Discard flow-through and place the MinElute column back into the same collection tube.
9. Add 750 pl Buffer PE to MinElute column and centrifuge for 1 min or & apply vacuum. - Discard flow-through and place the MinElute column back into the same collection tube.
10. Add 750 µL 70% ethanol to MinElute column and centrifuge for 1 min. If the DNA will be used for saltsensitive applications, such as direct sequencing and bluntended ligation, let the column stand 5-10 min after addition of Buffer PE.
11. Centrifuge the column in a 2 ml collection tube (provided) for 1 min. Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.
12. Place each MinElute column into a clean 1.5 ml microcentrifuge tube. To elute DNA, add 10 pl Buffer EB (10 mM Tris-Cl, pH 8.5) or water to the center of the MinElute membrane. (Ensure that the elution buffer is dispensed directly onto the membrane for complete elution of bound DNA) Let the column stand for 1 min, and then centrifuge the column for 1 min.
13. If purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA, and mix by pipetting up and down before loading the gel.

7.1 NEB

We used the original "Monarch® Plasmid DNA Miniprep Kit" Protocol from the webside: https://www.neb.com/en/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010

7.2 Roth

Adapted from the original "ROTI®Prep Plasmid MINI-XL" protocol: https://www.carlroth.com/medias/BA-8456-EN.pdf

1. Cell Harvest and Resuspension Harvest the bacterial cell culture (5-15 ml) by centrifugation for 10 mins at approx. 4.500 rpm (RT). Discard supernatant and remove remaining culture medium carefully. Resuspend pelleted bacterial cells completely in 550 μl Resuspension Buffer RB (incl. RNAse A). as the a original Resuspension Buffer has expired and was stored at RT and we didn't know how long it had been there, we took a different lysis buffer from the fridge.
2. Cell Lysis and Neutralization. Add 550 μl of Lysis Buffer LS. Mix gently by inverting the tube 10 times (Do not vortex!). Incubate for 5 mins at room temperature. Add 750 μl of Neutralization Buffer NB and immediately mix by inverting the tube 10x. Centrifuge at 12.000-14.000 rpm (or full speed) for 10 mins/RT.
3. Column Loading. Place each Spin Column into a 2 ml collection tube. Apply 750 μl of the clearified supernatant from step 2 into the spin column. Be sure to omit as much of the white precipitant as possible. Centrifuge at 10.000 g (or 12.000 rpm) for 1 min/RT and discard the flow-through*. Apply the residual clearified supernatant from step 2 into the spin column. Be sure to omit as much of the white precipitant as possible. Centrifuge at 10.000 g (or 12.000 rpm) for 1 min/RT and discard the flow-through*
4. Column Washing. Add 650 μl of Washing Buffer PA to the Spin Column. Centrifuge at 10.000 g (or 12.000 rpm) for 1 min/RT and discard the flow-through*. Add 750 μl of Washing Solution PB to the Spin Column. Centrifuge at 10.000 g (or 12.000 rpm) for 1 min/RT and discard the flow-through*. Again centrifuge columns at 12.000-14.000 rpm (or full speed) for 2 mins/RT in order to remove residual ethanol.
5. Elution. Place the Spin Column into a clean 1.5 ml elution tube. Add 100 μl Elution Buffer XEB or sterile, distilled water to the centre of the membrane**. Incubate for 3 mins at room temperature. Centrifuge at 6.000 g (or 8.000 rpm) for 1 min/RT to elute DNA.

*If the solution has not completely passed through the spin column, centrifuge again at higher speed or prolong the centrifugation time.
** Elution with lower volumes of Elution Buffer increases the final concentration of DNA. In case DNA shall be eluted in 50 μl only, preheating of the elution buffer is obligatory. Using heated Elution Buffer (approx. 50 °C) will increase the DNA yield. Alternatively, DNA may be eluted in 2x 50 μl buffer. If so, use 50 μl preheated buffer for elution, centrifuge. Then repeat this elution process with another 50 μl and finally pool both fractions.

• for a 100 ml 1% agarose gel 100 ml TAE buffer was mixed with 1 g of agarose.
• 1 µL of "midori Green Advance" was addet per 50 µL of agarose gel.
• for the ladder "NEB Quick-Load Purple 1 kb Plus DNA Ladder" was used. here we used 8 µL for a 100 mL medium geltray.
• as loading dye "Gel Loading Dye, Purple (6X)" was used.

Make the SDS-Gel

1. Clean a big and a small glass plate with 70% ethanol.
   • leave them on the bench and wipe in one direction
2. Place the small glass plate on the big 1.5 mm glass plate (cleaned surfaces to each other).
3. Place the sandwich in the green clamps (small glass plate facing outwards).
4. Check for leakage with water (remove in the sink with paper tissues).
5. Acrylamide concentration depends on the molecular weight of the target protein.

Running gels. Volumes per 10 mL-gels.

	7.5%	10%	15%
Acrylamide (30% w/v) !!shake	2.5 mL	3.33 mL	5 mL
4x running buffer	2.5 mL	2.5 mL	2.5 mL
H2O	4.89 mL	4.06 mL	2.39 mL
APS (10% w/v)	100 µL	100 µL	100 µL
TEMED	10 µL	10 µL	10 µL

Stacking gel

	10 mL (1 oder 2 Gele)	20 mL	30 mL
Acrylamide (30% w/v) !!shake	1.16 mL	2.32 mL	3.48 mL
4x stacking buffer	2.5 mL	5 mL	7.5 mL
H2O	6.23 mL	12.46 mL	18.69 mL
APS (10% w/v)	100 µL	200 µL	300 µL
TEMED	10 µL	20 µL	30 µL

1. Label two 50 mL tubes (stacking and running gel).
2. Add Acrylamide, 4X running buffer, and H2O.
3. With the help of the comb, mark where the running buffer should be poured (5 mm below the end of the comb).
4. Take up the volume of TEMED with the pipette and set aside.
5. Pipette the volume of APS into the tube and quickly after that also TEMED.
6. Invert the tube 4 times and pour it between the glass plates until the mark is reached.
7. Add quickly a thin layer of isopropanol.
8. Let it polymerize for 15-30 min.
9. Pour off isopropanol and wash twice with ddHO (wipe with tissue paper).
10. Clean the comb from both sides with 70% ethanol.
11. Add Acrylamide, 4X stacking buffer, and H2O.
12. With the help of the comb, mark where the running buffer should be poured (5 mm below the end of the comb).
13. Take up the volume of TEMED with the pipette and set aside.
14. Pipette the volume of APS into the tube and quickly after that also TEMED.
15. Invert the tube 4 times and pour it till it overflows.
16. Insert the comb initially at a 45-degree angle.
17. Let it polymerize for 15-30 min.
18. Check if there is enough reservoir buffer (1800 mL ddHO and 200 mL 10x Resevoir buffer).

Load & run SDS-PAGE

1. Take the gels out of the fridge so they can warm up a bit if you prepared them the day before.
2. Prepare the running chamber.
3. Centrifuge the samples for 15 s (sort them in order).
4. Boil the REDUCING samples at 95 degrees for 5 minutes.
5. Unwrap the gels from plastic wrap & paper towels.
6. Clamp & position the gels in the electrophoresis tank (pay attention to the electrode color!).
   • the small glass plate should face the inner side
7. Label the gels with (NR/R) on the side of the tank with some tape.
8. Fill the running chamber with reservoir buffer by first filling the chamber between the gels and then overflowing and filling up to approx. 1 cm under the upper edge of the large glass plate.
9. Remove the comb by lifting it straight upwards.
10. Load the gels with the sample.
11. Close the lid, switch on the device, and plug in the cable (pay attention to colors!).
12. Set 200 V, press "run" & check if air bubbles are rising.
13. After 5 min, check whether the samples are migrating.
14. Allow running until the blue band has run out.

10.1 SLiCE

Adapted from the paper "Seamless Ligation Cloning Extract (SLiCE) Cloning Method"
doi: 10.1007/978-1-62703-764-8_16

SLiCE Reaction protocol

1. Set up the SLiCE reaction in a 0.2 mL microcentrifuge tube and mix well by vortexing.
   SLiCE reaction conditions

   Components	Standard cloning
   Vector DNA	Purified, 50-200 ng
   Insert DNA	Purified, 1:1-10:1**
   10× SLiCE buffer*	1 μL
   PPY SLiCE extract*	1 μL
   ddH2O	to 10 μL
   Reaction conditions
   Incubation temperature	37 °C
   Incubation time	55 min

   * The SLiCE buffer and Extract was provided by the Kolbe Working Group
   ** Molar ratio of insert to vector
2. Incubate the SLiCE reaction mix at 37 °C for 15 min to 1 h using a PCR machine or water bath.
3. Transfrom 1 μL SLiCE reaction mix into 50-100 μL Competent Cells (see 2. Transformation).
4. Plate the transformed cells onto agar plates containing appropriate antibiotics.

10.2 NEB-builder

The “NEBuilder® Protocol Calculator” was used to adapt the protocol.
Web: https://nebuildercalculator.neb.com/

Protocol for homolog recombination
Shown here using the example of a homologous recombination of EGFP with the pUC_hasA vector (length and concentration may differ from the original).

Name	Length (basepairs)	Concentration (ng/µl)
pUC_HasA	5200	100
EGFP	750	35

• Set up the reaction on ice (see table).

  Component	Volume	Amount
  pUC_HasA	1.6 μl*	0.050 pmoles*
  EGFP	1.3 μl*	0.100 pmoles*
  Deionized H2O	7.1 μl	-
  NEBuilder HiFi DNA Assembly Master Mix	10.0 μl	-
  Total	20.0 μl	0.150 pmoles

  * to calcualte the vector and insert volumes the "NEBuilder® Protocol Calculator" was used. The molar ratio varied between 1:1 and 1:10
• Incubate samples in a thermocycler at 50 °C for 15 minutes. Following incubation, store samples on ice or at -20 °C for subsequent transformation. Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases.
• Transform NEB® 5-alpha, NEB Stable or NEB 10-beta Competent E. coli cells (provided in the cloning kit, bundle or purchased separately from NEB) with 2.0 μl (10%) of the chilled assembled product, following the transformation protocol.

Adapted from NEB.

Components	Volume
PCR Sample	20 µL
10x rCutSmart Buffer	2.5 µL
DpnI	0.5 μL
H2O	Ad. 25 µL

• Incubation for at least 2h at 37°C
• Heat inactivation for 30 min at 80°C