06.05.2024

Preparing competent DH5α-cells

Starting culture

• Inoculate 5 mL of LB-medium with DH5α
• Incubate at 37 °C, 160 rpm overnight

Aim: Obtaining competent cells to transform with our plasmid.

07.05.2024

Harvesting of competent DH5α cells

• competent cells where prepared following protocol 1 Preparing Competent Cells with the overnight culture from 06.05.2024
• 50/100 µL Aliquots of the cell suspension where stored in sterile 1,5 mL tubes and frozen at -80 °C

14.05.2024

Testing and transformation of competent DH5α cells

• A tansformation of the competent DH5α cells with pMK1 was carried out following protocol 2 Transformation
• The transformed DHF5α cells where plated on LB agar plates containing ampicilin (amp.; final concentration = 100 µg/mL) as pMK1 contains an amp. resistance incubated at 37 °C over night
• As positive control, cells were plated on LB agar plates without amp., as negative control DH5α cells without the plasmid on LB + amp. plates

Interpretation:

If colonies grow on the positive control, we have living bacteria

If colonies grow on the LB + amp. plates with the non-transformed cells, the antibiotic is not working

If no colonies grow on the LB + amp. plates with the transformed bacteria, the transformation was not successful

If colonies grow on the LP + amp. plates with the transformed DH5α cells, the transformation was successful

15.05.2024

Competent DH5α cells

Colonies were growing on both the positive control and the LB + amp. plates with the transformed cells!

• This means we have successfully produced competent cells and could transform them with a plasmid.

17.05.2024

Top10 and BL21DE3 competent cells for the hyaloronic acid and resilin expression

Starting culture

• 5mL of LB-Medium + Top10 and BL21DE3 were incubated at 37°C, 160rpm over night

18.05.2024

Competent cells

• competent cells were made using the protocol 1. (Competent cells (Zymogen))
• cells were harvested at an OD as shown in the table below

	BL21	Top10
OD	0,03	0,033

27.05.2024

Cloning of RE4 into pMK1 with SacI and NdeI

Cloning of the pMK1 vector with a restriction ligation using enzymes SacI and NdeI to incorporate the fourfold resilin repeat (RE4).
Protocol 3 Digest with Restriction Enzymes was used for the double digestion of pMK1.

Restriction mix contained:

Component	Volume
DNA	1 µg
10X rCutSmart Buffer	5 µL
NdeI	0.5 µL
SacI-HF	0.5 µL
Nuclease-free Water	ad 50 µL

Incubation: 37 °C for 1 h

01.06.2024

T4 Ligation of RE4 into pMK1

• The ligation was perfomed using protocol 4 Ligation (T4 Ligase). with three different insert:vector ratios: 1:5, 1:3 and 1:1

Components	Volume
10X T4 DNA Ligase Buffer	2 µL
Vector (digested pMK1)	0.56 µL
Insert (digested RE4)	0.6 (1:5); 0.32 (1:3); 0.13(1:1)
T4 DNA Ligase	1 µL
Nuclease-free water	ad 20 µL

05.06.2024

Transforming pMK1_RE4 into DHF5α + Amp.

• A Transformation of DHF5α with pMK1_RE4 was made using protocol 2 Transformation

06.06.2024

Over night culture

• Here grew a few colonies on the 1:3 and 1:5 ratio pMK1_RE4 plates
• Making an overnight culture of 4 colonies for each plate for a restriction digestion

Restriction digestion

• Analytic digestion of the 8 pMK1_RE4 mini preps were made with SacI and BsaI using the Protocol 3. (Restriction digestion (NEB)).

expected bonds:

• pMK1:
• pMK1_RE4:

07.06.2024

Mini prep of colony 1:3_3 and 1:3_ containing pMK1_RE4

A miniprep of 9 colonies transformed at the 05.06.2024 was performed following the Miniprep protocol 7.

10.06.2024

Restriction digestion of pMK1_RE4 and gel purifiaction

• Analytic digestion of pMK1_RE4 with SacI and NdeI was performed at 37 °C for 1 h using the Protocol 3 Digestion with Restriction Enzymes with NdeI and SacI.

20.06.2024

Site directed mutagenesis pMK1_RE4 to remove the DpnI restricition side in the ampicillin sequence

• Site directed mutagenesis was done using the protocol 5.1 PCR - Q5 Polymerase from NEB with following primers and templates:

Primer	FW_pMK1_RE4_G->C
	RW_pMK1_RE4_G->C
Template DNA	pMK1_RE4_1:5_2
	pMK1_RE4_1:5_3
	pMK1_RE4_1:3_2
	pMK1_RE4_1:3_4

25.06.2024

Quick change mutagenesis PCR (Q5 Polymerase Protokoll with pMK1_RE4

Templates used:

• (1)pMK1_RE4 5:1_3
• (2)pMK1_RE4 5:1_4
• (3)pMK1_RE4 3:1_3
• (4)pMK1_RE4 3:1_4

Thermocycling conditions:

Step	Temperature [°C]	Time [s]	Cycles
annealing	69	30	30
elongation	72	>100	30

01.07.2024

TOP10 & BL21* Competent cell testing

• Transformation of pMK1 to TOP10 & BL21 competent cells folllowing protocol 2.  
• Neg control: plate with 100µL just bacteria + water

02.07.2024

Competence Test

Results of the competence test: Transformation of pMK1 into TOP10 was successful, but BL21 bacteria did not grow

04.07.2024

Doing a gradient PCR for Quick Change mutagenesis

Repeating the Quick Change mutagenesis using a longer elongation time and a temperature gradient to find the perfect annealing temperature.
template:

Primer	Amount
FW_pMK1_RE4_G->C	0.5 µl
RW_pMK1_RE4_G->C	0.5 µl
Template DNA:
pMK1_RE4_1:3_4	2 ng
Nuclease-Free Water	add to 10 µl

Thermocycling Conditions:

STEP	Temp	Time	Cyles
annealing (gradient)	50°C	30 seconds	30
elongation	72°C	120 seconds	30

For annealing temperature:

lane	1	2	3	4	5	6	7	8	9	10
sample	DNA Ladder	1	2	3	4	5	6	7	8	DNA Ladder
annealing temp.	-	50 °C	53 °C	56 °C	59 °C	63 °C	66 °C	69 °C	72 °C	-

Elongation time was 120 seconds
Results:
The lids popped of, so there was only one PCR to be seen in the gel (lane3).
Problem: The lids were not closed properly
Solution:

• we ordered new primers which should bind better
• we also ordered primes to remove the second BsmBI-Binding sequence behind the poly-A tale
• we would also try a new method to generate repeats called the rolling circle method where circular DNA containing only 4 repeats of resilin is generated. Than a phi29 polymerase will circulate around this circular DNA generating more repeats.

09.07.2024

Trying a new approach Generating a resilin Fragment with four repeats for the rolling circle method.

• Double digestion with BsmBI and BsaI was performed on pMK1_RE4 according to Protocol 3. (Restriction digestion (NEB)).
• Heat inactivation of the resticiton enzyme by heating it to 80 °C for 20 min

10.07.2024

Restriction digestion

• Double digestion with BsmBI and BsaI was performed on pMK1_RE4 according to Protocol 3. (Restriction digestion (NEB))
• an agarose gel was made follwing the 8. protocol (Agarose gel)

expected bonds: 1775 bp, 1382 bp, 252 bp and 180 bp (the fragment with the four resilin repeats)
Problem: 1775 bp and the 1382 bp can be seen in the gel, but not the two small ones.

19.07.2024

Checking the activity of XhoI restriction enzyme

Performing a restriction with XhoI with pMK1 during 1 h at 37 °C
pMK1_RE4 5:1_3 =212 ng/µL
so 2,5 µL are about 0,5 ng

Component	25 µl Reaction
DNA	0.5 µg
10X rCutSmart Buffer	2.5 µl (1X)
XhoI	0.25 µl (10 units)†
Nuclease-free water	to 25 µl = 21.75

24.07.2024

Rolling circle method

Make PCR with new Primers and at the same time try to amplify the RE4 Fragment for the rolling circle method, before cutting it with BsmBI and BsaI.
The PCRs was performed using the 5.1 protocol (Q5 Mastermix from NEB).
The reactions containd the Primers and templates as shown in the Table below and the elontation time was 185 seconds

Reaction	Template	Primer
BsaI	pMK1_RE4_1:3_4	FW_Mut2_BsaI_pMK1_RE4
BsaI	pMK1_RE4_1:3_4	RW_Mut2_BsaI_pMK1_RE4
BsmBI	pMK1-RE4 DNA	FW_Mut2_BsmBI_pMK1_RE4
BsmBI	pMK1-RE4 DNA	RW_Mut2_BsmBI_pMK1_RE4
RE4 Amplification	pMK1-RE4	FW_pMK1_RE4_ColPCR
RE4 Amplification	pMK1-RE4	rev_pMK1_RE4_ColPCR

• The PCR tubes were put in a Thermocycler with a temperature gadient show in the Table below

Temperature [°C]	Probe	Probe	Probe	Probe
65	Bsal (1a)
63.4	Bsal (1b)
60.9	Bsal (1c)
57.1	Bsal (1d)		PCR RE4 Amplification (4a)	PCR RE4 Amplification (4c)
52.6		BsmBI (2a)	PCR RE4 Amplification (4b)	PCR RE4 Amplification (4d)
48.8		BsmBI (2b)
46.4		BsmBI (2c)
45		BsmBI (2d)

28.07.2024

Resilin production with the Reaptigo method

DNA - concentrations
Oligo 1 fw: concentration 1,3876 g/L

• for 4 µg we need 2,883 µL

Oligo 2 rev: concentration 1,3865 g/L

• for 4 µg we need 2,885 µL

Ndel fw - ratios
NdeI fw concentration: 0,6436 g/L

• 32:1 Ratio: 0.0584 µg
  • we take 9.1 µL from the dilution (1:100, c=0.006436 g/L)
• 64:1 Ratio: 0.0292 µg
  • 4.56 µL from the 1:100 dilution
• 124:1 Ratio: 0.0151 µg
  • 2.36 µL from the 1:100 dilution

Ndel fw - ratios
NdeI rev concentration: 1,3308 g/L → dilution 1:100 c=0,01333 g/L

• 32:1 Ratio: 0.1219 µg
  • 9,16 µL from the 1:100 dilution
• 64:1 Ratio: 0.0609 µg
  • 4,75 µL from the 1:100 dilution
• 124:1 Ratio: 0.0315 µg
  • 2,37 µL from the 1:100 dilution

Sac1 fw - ratios
Sac1 fw concentration: 0,72424 g/L 1 :100 dilution c=0,00724 g/L

• 32:1 Ratio: 0.0723 µg
  • 9,98 µL from the 1:100 dilution
• 64:1 Ratio: 0.0361 µg
  • 4,98 µL from the 1:100 dilution
• 124:1 Ratio: 0.0187 µg
  • 2,58 µL from the 1:100 dilution

Sac1 rev- ratios
Sac1 rev concentration:  1,40187 g/L 1:100 dilution c=0,01402 g/L

• 32:1 Ratio: 0.1333 µg
  • 9,51 µL from 1:100 dilution
• 64:1 Ratio: 0.0667 µg
  • 4,76 µL from 1:100 dilution
• 124:1 Ratio: 0.0344 µg
  • 2,46 µL from 1:100 dilution

DNA-Annealing:
1. Prepare Oligos:

• Centrifuge dried DNA
• Dilution of dried DNA oligos to 100µM with Dnase, Rnase free water and vortexed for 20 sec
• After incuation at RT for 30 min centrifuge it for 1 min

2. Annealing:

• Prepare annealing mixture with the desired ratio of the same Oligos
  1. NdeI start fw + NdeI start rev
  2. Oligo 1 fw + Oligo 2 rev
  3. SacI end fw + SacI end rev
• Heat the mixtures to 90 °C for 5 min
• Gradually cool the mixture to room temperature over 12 min in the PCR block

31.07.2024

Resilin production with the Reaptigo method II

Ligation of annealed Oligos: 

• Start & Middle A-Oligos will be ligated first
• 31:1 Start 16,26 µL + Middle 5,76 µL
• 64:1 Start 9,13 µL + Middle 5,76 µL
• 128:1 Start 4,73 µL + Middle 5,76 µL
• Add 2 μl T4 Ligase puffer
• Fill up to 19µL with Rizvee water (Dnase rnase free)
• Add 1µL T4 ligase
• Pipette up & down & centrifuge shortly
• incubate at 16 °C overnight

01.08.2024

Checking and repeating Repeatigo Resilin production

Ligation of annealed Oligos:

• Add end oligo (31:1 = 19,49 µL , 64:1 = 9,74 µL, 128:1 = 5,05 µL)
• Incubate at RT for 1h
• Heat inactivate at 65 °C for 10 min
• put on ice

Made a 1% agarose Gel to check the fragment lenghts of resilin see picture following protocol 8. Result

• The longest fragments on the 128:1 ratio sample
• The biggest fragment is about 300bp long
• For a 32 repat fragment one would expect about 1,5kb fragments

Second try Repeatigo resilin production

• We made two different concentration of the middle Oligos using the same volume
  • One with 2 µg and a volume of 10 µL
  • One with 4 µg and a volume of 2,883 µL

DNA - concentrations
Tube 1
Oligo 1 fw: concentration 1,3876 g/L

• For 4 µg we need 2,883µL

Oligo 2 rev: concentration 1,3865 g/L

• For 4 µg we need 2,885µL

NdeI fw concentration: 0,6436 g/L

• Tube 3 64:1 ratio: 0.0292 µg
  • 4,56 µL from the 1:100 dilution
• Tube 4 128:1 ratio: 0.0151 µg
  • 2,36 µL from the 1:100 dilution
• Tube 5 248:1 ratio: 0.0078 µg
  • 1,18 µL from the 1:100 dilution

NdeI rev concentration: 1,3308 g/L → 1 zu 100 verdünnen also c = 0,01333 g/L

• Tube 3 64:1 ratio: 0.0609 µg
  • 4,75 µL from the 1:100 dilution
• Tube 4 128:1 ratio: 0.0315 µg
  • 2,37 µL from the 1:100 dilution
• Tube 5 256:1 ratio: 0.0158 µg
  • 1,19 µL from the 1:100 dilution

Sac1 fw concentration: 0,72424 g/L 1:100 dilution c = 0,00724 g/L

• Tube 6 64:1 Ratio: 0.0361 µg
  • 4,98 µL from the 1:100 dilution
• Tube 7 128:1 Ratio: 0.0187 µg
  • 2,58 µL from the 1:100 dilution
• Tube 8 256:1 Ratio: 0.0935 µg
  • 1,29 µL from the 1:100 dilution

Sac1 rev concentration:  1,40187 g/L 1:100 dilution c = 0,01402 g/L

• Tube 6 64:1 Ratio: 0.0667 µg
  • 4,76 µL from the 1:100 dilution
• Tube 7 128:1 Ratio: 0.0344 µg
  • 2,46 µL from the 1:100 dilution
• Tube 8 256:1 Ratio: 0.0172 µg
  • 1,23 µL  from the 1:100 dilution

02.08.2024

Ligation of annealed Oligos

With 2 µg or 4 µg middle oligos:

• -> Start & middle A-Oligos will be ligated first
• 64:1 start 9,13 µL + middle 5,76 µL → 2,17 H2O
• 128:1 start 4,73 µL + middle 5,76 µL → 6,57 H2O (we only made one of the middle parts, so that we choose the 128:1 ratio since it workt out the best last time)
• 256:1 start 2,36 µL + middle 5,76 µL → 9 H2O

1. Add 2 μl T4 Ligase buffer
2. Fill up to 19 µL with ddH2O
3. Add 1 µL T4 ligase
4. Pipette up and down and centrifuge shortly
5. Incubate at 16 °C overnight

• add end oligo
• 31:1 ends 19,49 µL
• 64:1 ends 9,74 µL
• 128:1 ends 5,05 µL

1. Incubate at RT for 1 h
2. Heat inactivate at 65 °C for 10 minutes
3. Put on ice

Made a 1 % agarose Gel to check the fragment lenghts of resilin see picture following protocol 8. We have the longest fragments in the mixture with the 2 µL of middle oligos. Also the fragments are on average larger than from the first experiment. Gel purification:
Small peaces from the gel of the desired length of the repeating fragements will be cut out of the gel and purified using the protocol 6.1.

05.08.2024

Gradient PCR of purified gel to check the right annealing temperature for oligos

PCR done according to protocol 5 with a temperature range of 48-56 degrees Celsius during annealing.
Tube 1, 2, 3 and 4: DNA concentration of 0,8 ng/µl
Tube 5, 6, 7 and 8: DNA concentration of 1.3 ng/µl

Results:
There are no bonds in the gel. This means that either a pipetting error or the endoligos were involved.
A reason could be that the end Oligos didn't bind because there was not addet additianly T4 Ligase or ATPs, which at this point could be degradet or inactive.
Next step: The next step is to repeat the oligo annealing and ligation step to obtain larger fragments and correctly ligate the end and start oligos.

07.08.2024

Second try Repeatigo resilin production

• We use 4 µg of mid Oligos and up to 20 µL with water, so we have the same concentration as with the experiment that worked out well.
• Then we use two different times for the Oligos to anneals while cooling down in the PCR machine. Since changing the time changed the length of the Fragments from about 300 bp to about 1-2 kb Fragments,
• we will use the old concentration (2 µg in 10 µL → 4µg in 20 µL) and we will use a smaller concentration, as reducing the concentration also has made a big difference:
  • sample 1: 4 µg of mid Oligos and up to 20 µL (previous concentration), shorter time = 38 min
  • sample 2: 4 µg of mid Oligos and up to 20 µL, longer time = 68 min
  • sample 3: 2 µg of mid Oligos and up to 20 µL, longer time = 68 min
  • sample 4: 2 µg of mid oligos and up to 20 µL, shorter time = 38 min
• additionally we will use 3 different ratios of middle oligo to start- and end-oligos:
  • 1:64
  • 1:128
  • 1:256

Protocol:

• Middle-Start and End Oligos where pipetted into PCR tubes as shown in the "Resilin_Oligo_Pipettier_volumina"-Excel document.

• for the dimerization the Oligos where heated up to 90 °C and then slowly cooled down to 20 °C in a PCR machine as shown in the table below

Temperature (°C)	90°C	80°C	75°C	70°C	65°C	60°C	50°C	40°C	30°C	20°C
Sample 1 and 4	5 min	5 min	3 min	5 min	3 min	5 min	3 min	3 min	3 min	3 min
Sample 2 and 3	8 min	8 min	5 min	8 min	5 min	8 min	5 min	5 min	5 min	5 min

• the start Oligos where then mixed in differnt ratios with the two different concentrations of Mid Oligos. See "Resilin_Oligo_Pipettier_volumina"-Excel document.
• 2 μl T4 Ligase puffer where addet
• 1 µL of T4 Ligase where addet.
• fill up to 20 µL with DNAse free water
• pipette up and down and centrifuge shortly
• the tubes where incubate at 16 °C overnight

→ this resulted in a total of 12 tubes
The next steps:
add end oligos and run a gel to investiagte the product

08.08.2024

Ligation of end oligos

add the same as shown in the "Resilin_Oligo_Pipettier_volumina":

• incubate at RT for 2 h
• Heat inactivate at 65 °C for 10 minutes
• investigate product on a gel

Results:
The results can be seen in the gel image. We got some bands close to 3 kb. Also, using 2 µg of the middle olgios and using a longer cooling down time in the annealing step resulted in larger fragments on average.
Next steps:
In the next steps, 10 fragments from the gel will be isoalted in the range of 1.5 to 3 kb and the oligos extracted from the gel. Gel:

12.08.2024

Repeatigo

Repetition of the oligo annealing Concentration after gel extraction:

Sample	Concentration [ng/µl]	260/280	260/230	Start/Stop Oligo Ratio
Tube 1, 1.5 kb	8.7	1.7	0.47	1:64
Tube 2, 1.5 kb	9.3	1.59	0.42	1:64
Tube 3, 1.5 kb	9	1.56	0.43	1:128
Tube 4, 1.5 kb	8.6	1.72	0.43	1:128
Tube 5, 1.5 kb	7.8	1.71	0.37	1:256
Tube 6, 1.5 kb	10.09	1.4	0.53	1:256
Tube 1, 2 kb	9	1.43	0.43	1:64
Tube 2, 2 kb	8	1.65	0.42	1:64
Tube 3, 2 kb	7.6	1.58	0.49	1:128
Tube 4, 2 kb	5.5	1.71	0.37	1:128
Tube 5, 2 kb	12.7	1.38	0.56	1:256
Tube 6, 2 kb	7	1.69	0.45	1:256
Tube 1, 3 kb	6.8	1.66	0.47	1:64
Tube 2, 3 kb	6	1.65	0.4	1:64
Tube 3, 3 kb	0	0	0	1:128
Tube 4, 3 kb	7.6	1.63	0.37	1:128
Tube 5, 3 kb	7.1	1.51	0.44	1:256
Tube 6, 3 kb	7	1.71	0.42	1:256

14.08.2024

Agarose gel for the Resilin-Oligo-PCR

1 % agarose gel according to protocol 8.

1	2	3	4	5	6	7	8	9
1kb Plus Ladder	negativ control	tube1	10ng of template for tube 1	tube5	10ng of template for tube 5	tube6	10ng of template for tube 6	1kb Plus ladder

Results:

• the Fragments where exprected at 3 kb.
• as one can see only the primers can bee seen in the gel
• the negativ control cannot be seen, use more than 10 ng

Trouble shooting:

• use more than 10 ng of template next time
• One reason that the PCR did not work could be, that the T4 ligase has not properly ligated start and end oligos to mid fragments, also it could be that the mid Oligos did not ligate properly

Next steps:

• add end and start oligos in ratio 64:1 to the Oligo-Fragments isolated from the PCR-gel and add T4 ligase with ATP buffer.
• test T4 ligase by:
  • resticiton digestion of a Plasmid
  • religation of the fragment using T4 ligase.
  • heat to 95 °C
  • test ligation on a gel

05.09.2024

Re-Ligation of Start & End Oligos to middle oligos extracted from gel over 2 days

• gel extracted oligo samples were used
• 2 and 6 from 1,5 kb
• 1 and 5 from 2 kb
• 4 and 5 from 3 kb

From each tube we transferred 9 µL. The first 2 µL of SacI_ rev 1:100 and 2 µL SacI_fw 1:100 were added and ligated for 1.5 hour at 37 °C. 1 µL T4 Ligase and 2 µL buffer (total volume 20 µL) were added. Then 2µL of NdeI_fw 1:100 and 2µL NdeI_rev 1:100 Oligos were added to the ligation mix. The total ligation lasted for 48 h (total volume now 20 µL).

07.09.2024

PCR with the ligation from 05.09.

All 6 tubes and a positive control prepared.
Mastermix according to protocol 5.1 with 8.75 μl FW_RE_Oligo_amp-Primer and RV_RE_Oligo_amp-Primer each and a total volume of 105 μl. In each PCR tube, 15 μl and 10 μl Template were pipetted.
A positive control was made as follows:

• Q5 High-Fidelity 2X Master Mix: 12,5 μl
• FW_EGFP_SLiCE_PCR: 1,25 μl
• RV_EGFP_SLiCE_PCR: 1,25 μl
• EGFP-Linker: 10 μl

The annealing temperature was 56 °C and the elongation time was 3 min for 30 cycles.
we numbered the PCR tubes from 1-6 and a +

Number	Description
1	1,5 kb Religation from 05.09 for 48h
2	1,5 kb Religation from 05.09 for 48h
3	2 kb Religation from 05.09 for 48h
4	2 kb Religation from 05.09 for 48h
5	3 kb Religation from 05.09 for 48h
6	3 kb Religation from 05.09 for 48h
+	Positive control with EGFP+Linker and the respective primers

The PCR was checked on a 1 % agarose gel (protocol 8).

18.09.2024

DNA-Annealing for Repeatigo

1. Calculate Oligos:

• Oligo 1: ca 70 µg DNA --> 50 μl
• Oligo 2: ca 70 µg DNA --> 50 μl
  • Ansatz An-Oligos: 100 μl mit 140 µg DNA von Oligo 1+2
• NdeI fw: ca 40 µg DNA --> V=m/c = 0.00004 g/ 0.6436 g/ L = 62.1 μl
• NdeI rev: ca 40 µg DNA --> V=m/c = 0.00004 g/ 1.3308 g/ L = 30.06 μl
  • Ansatz An-NdeI: 60 μl fw + 30 μl rev = 90 μl mit ca 80 µg DNA von NdeI fw+rev
• SacI fw: a 40 µg DNA --> V=m/c = 0.00004 g/ 0.72424 g/ L = 55.23 μl
• Sac I rev: a 40 µg DNA --> V=m/c = 0.00004 g/ 1.40187 g/ L = 28.53 μl
  • Ansatz An-SacI: 55 μl fw + 29 μl rev = 84 μl mit ca 80 µg DNA aus SacI fw+rev

2. Annealing:

• Prepare annealing mixture with the desired ratio of the same Oligos (Volume, conc, mass above)

1. An-Oligos: Oligo 1 fw + Oligo 2 rev
2. An-NdeI: NdeI start fw + NdeI start rev
3. An-SacI: SacI ende fw + SacI ende rev

• Heat the mixtures to 90°C for 5 minutes.
• Gradually cool the mixture to room temperature over 12 minutes in the PCR block

3. Concentrations:
An-Oligos: 139 µg/ 100 μl = 1.39 µg/ μl
An-NdeI: 80 µg/92.16 μl = 0.868µg/ μl
An-SacI: 80 µg/83.76 μl = 0.955µg/ μl
4. Calculate:

• 3 Ansätze:
  • Lig-1: 64:1 --> 6 µg An-Oligos + 0.09 µg Enden jeweils
  • Lig-2: 32:1 --> 4 µg An-Oligos + 0.12 µg Enden jeweils
  • Lig-3: 10:1 --> 4 µg An-Oligos + 0.4 µg Enden jeweils
  • Lig-4: 2:1 --> 4 µg An-Oligos + 2 µg Enden jeweils

5. Dilute:

• An-NdeI 1:10 --> 0.0868 µg/ μl
• An-SacI 1:10 --> 0.0955 µg/ μl
• Ligation 1: Oligos + NdeI jeweils 20 μl Ansätze
  • Lig-1: 4.32 μl von An-Oligos + 1.03 μl von An-NdeI 1:10 + 11.65 μl nuclease-free water 2 μl Ligase buffer + 1 μl Ligase
  • Lig-2: 2.88 µL von An-Oligos + 1.38 µL von An-NdeI 1:10 + 12.74 µL nuclease-free water + 2 µL Ligase buffer + 1 µL Ligase
  • Lig-3: 2.88 µL von An-Oligos + 4.61 µL von An-NdeI 1:10 + 9.51 µL nuclease-free water + 2 µL Ligase buffer + 1 µL Ligase
  • Lig-4: 2.88 µL von An-Oligos + 2.30 µL von An-NdeI + 11.82 µL nuclease-free water + 2 µL Ligase buffer + 1 µL Ligase

Pipette together + mix gently, Centrifuge shortly, incubate at 25 °C for 2 h

• Ligation 2: add An-SacI jeweils zu den 20 µL Erstansätzen
  • Lig-1: add 0.94 µL von An-SacI 1:10
  • Lig-2: add 1.26 µL von An-SacI 1:10
  • Lig-3: add 4.2 µL von An-SacI 1:10
  • Lig-4: add 2.1 µL von An-SacI

Pipette together + mix gently, incubate at RT over night (18.5 h)

1. Heat inactivate at 65 °C for 10 min
2. put on ice

19.09.2024

Gel and Gel Clean-up

Ligation 1 to 4 were loaded into a 1% gel with the NEB 1kb plus ladder.

1	2	3	4	5	6
Ladder 10 μl	Ligation 1 (24 μl)	Ligation 2 (24 μl)	Ligation 3 (24 μl)	Ligation 4 (24 μl)	Ladder 10 μl

Bands were cut out of the gel and cleaned up according to protocol 6.2.

20.09.2024

Amplification of the Oligo Fragments

PCR for amplification of the Oligo fragments that have been cleaned the day before and a checkup with a 1 % agarose gel.
A mastermix (10x) was created (protocol 5.2):
In each PCR Tube 48 μl mastermix and 2 μl Template from each cleanuped tubes were pipetted. For the negative control, 2 μl water were used.
PCR was done with 60 °C annealing temperature and 3 min elongation.
The PCR was checked with a 1 % agarose gel. 20 μl PCR-product were mixed with 4 μl loading dye.
The 10 pockets were loaded as followed:

1	2	3	4	5	6	7	8	9	10
Lig1 3 kb	Lig1 2 kb	Lig1 1,5 kb	Lig1 1 kb	ladder 1kb	Lig2 3 kb	Lig2 2 kb	Lig2 1,5 kb	Lig4 1 kb	negative control

Unfortunately the PCR didn´t work.

25.09.2024

Annealing approach for Resilin via Reapetigo Method

We used the following Oligo reactions:

1. Middle Oligo fw + rev 30 µg
2. Middle Oligo fw + rev 15 µg
3. Middle Oligo fw + rev 0,3 µg
4. Middle Oligo fw + rev & NdeI fw+ rev 30 µg
5. Middle Oligo fw + rev & NdeI fw + rev 15 µg
6. Middle Oligo fw + rev & NedI fw + rev 0,3 µg
7. NdeI fw + rev 1:10. 10X solution
8. SacI fw + rev 1:10. 10X solution

Sample 1-6 were prepared with 40 μl volume:
Sample 1(30 μg):

• Standard Ligation Buffer, 10x conc.: 4 μl
• T4 DNA Ligase: 0.8 μl*
• Oligo_1_repeat_fw (1:10): 10.8 μl
• Oligo_2_repeat_fw (1:10): 10.8 μl
• Nuclease-free water: 13.6 μl

Sample 2 (15 μg):

• Standard Ligation Buffer, 10x conc.: 4 μl
• T4 DNA Ligase: 0.8 μl*
• Oligo_1_repeat_fw (1:10): 5.4 μl
• Oligo_2_repeat_fw (1:10): 5.4 μl
• Nuclease-free water: 23.79 μl

Sample 3 (0.3 μg):

• Standard Ligation Buffer, 10x conc.: 4 μl
• T4 DNA Ligase: 0.8 μl*
• Oligo_1_repeat_fw (1:1000): 10.8 μl
• Oligo_2_repeat_fw (1:1000): 10.8 μl
• Nuclease-free water: 13.6 μl

Sample 4 (30 μg):

• Standard Ligation Buffer, 10x conc.: 4 μl
• T4 DNA Ligase: 0.8 μl*
• Oligo_1_repeat_fw (1:10): 10.8 μl
• Oligo_2_repeat_fw (1:10): 10.8 μl
• NdeI_fw (1:10): 1.7 μl
• NdeI_rev (1:10): 1.67 μl
• Nuclease-free water: 10.23 μl

Sample 5 (15 μg):

• Standard Ligation Buffer, 10x conc.: 4 μl
• T4 DNA Ligase: 0.8 μl*
• Oligo_1_repeat_fw (1:10): 5.4 μl
• Oligo_2_repeat_fw (1:10): 5.4 μl
• NdeI_fw (1:10): 0.85 μl
• NdeI_rev (1:10): 0.84 μl
• Nuclease-free water: 22.1 μl

Sample 6 (0.3 μg):

• Standard Ligation Buffer, 10x conc.: 4 μl
• T4 DNA Ligase: 0.8 μl*
• Oligo_1_repeat_fw (1:1000): 10.8 μl
• Oligo_2_repeat_fw (1:1000): 10.8 μl
• NdeI_fw (1:1000): 1.7 μl
• NdeI_rev (1:1000):1.67 μl
• Nuclease-free water: 10.23 μl
• added after the 55 min annealing time

The samples were put into the thermocycler and heated up to 95 °C. Over 55 min they were cooled down to 20 °C to anneal correctly. After that, the T4 DNA Ligase was added to the samples and incubated overnight at 16 °C.

26.09.2024

Adding the end and start oligos to the Ligation from 25.09.2024

The start and end oligos were diluted by the factor 100 to create a 1:1000 dilution.
Tube 9: NdeI fw + rev 1:1000
Tube 10: SacI fw + rev 1:1000
The Start and End Oligos were added to the tubes 1-6 as followed:

tube number	Start Oligo (NdeI fw + rev)	End Oligo (SacI fw + rev)
1	3.37 μl (1:10 dilution (tube 7))	4.1 μl (1:10 dilution (tube 8))
2	1.69 μl (1:10 dilution (tube 7))	2.05 μl (1:10 dilution (tube 8))
3	3.37 μl (1:1000 dilution (tube 9))	4.1 μl (1:1000 dilution (tube 10))
4	-	4.1 μl (1:10 dilution (tube 8))
5	-	2.05 μl (1:10 dilution (tube 8))
6	-	4.1 μl (1:1000 dilution (tube 10))

Then 0.8 μl of Ligase was added again in Tube 1-6 and the tubes were put back into the thermoycycler at 16 degrees for about 5 hours. GEL of the Oligos + PCR of the good Oligo bands Making a 1% Agarose Gels with a total Volume of 75 μl (2 µL of Midori Green pro Gel)
Loading the samples: 20 μl of each sample + 4 μl Loading dye
Ladder: 7 μl

1	2	3	4	5	6	7	8	9
Ladder	Sample 1	Sample 2	Sample 3	Ladder	Sample 4	Sample 5	Sample 6	Ladder

Run the Gel at 120 V Clean up of the three resilin Gel pieces according to protocol 6.2 with the additional ethanol wash step before the elution. The evaporation period of Ethanol is 15-20 min
Weight of the gel pieces and amount of QB buffer.

Resilin 2 3 kb	320 mg	960 μl
Resilin 2 1 kb	347 mg	1041 μl
Resilin 5 1 kb	162 mg	486 μl

Nanodrop measurement:

Resilin 2 3 kb	Dilution to 1 ng/µL	4.55 µL DNA + 15.45 µL water	4.4 ng/µL	2.26	0.24
Resilin 2 1 kb	Dilution to 1 ng/µL	11.1 µL DNA + 18.9 µL water	18.2 ng/µL	1.98	0.47
Resilin 5 1 kb	Dilution to 1 ng/µL	1.7 µL DNA + 18.3 µL water	11.8 ng/µL	1.81	0.58

PCR of the purified Resilin Oligos PCR according to protocol 5.2 with the thermofischer Phusion. Each Resilin sample should get 3 reactions with different amounts of template. (2 ng, 1 ng, 0.5 ng). Therefore 3 x 150 μl were created with 7.5 μl of the Oligo_Amp_fw and Oligo_Amp_rev primers, 3 μl dNTPs, 15 μl Phusion Buffer and 1.5 μl Phusion Polymerase. For each amount of template a different water amount was added:
Mastermix 2 (2 ng): 113.5 μl water
Mastermix 1 (1 ng): 114.5 μl water
Mastermix 0.5 (0.5 ng): 115 μl water
PCR Tube 1

• 48 μl Mastermix 2
• 2 μl 2, 3 kb

PCR Tube 2

• 48 μl Mastermix 2
• 2 μl 2, 1 kb

PCR Tube 3

• 48 μl Mastermix 2
• 2 μl 5, 1 kb

PCR Tube 4

• 49 μl Mastermix 1
• 1 μl 2, 3 kb

PCR Tube 5

• 49 μl Mastermix 1
• 1 μl 2, 1 kb

PCR Tube 6

• 49 μl Mastermix 1
• 1 μl 5, 1 kb

PCR Tube 7

• 5 μl Mastermix 0.5
• 5 μl 2, 3 kb

PCR Tube 8

• 5 μl Mastermix 0.5
• 5 μl 2, 1 kb

PCR Tube 9

• 5 μl Mastermix 0.5
• 5 μl 5, 1 kb

PCR with 60 °C annealing temperature and 20 s elongation time  for 30 cycles.

27.09.2024

Transformation of pET28-c in TOP10

Transformation following protocol 2. Gel of the PCR from 26.09.2024 Make a 1 % 75 mL Agarose gel (according to protocol 8)
NEW STEPS:
Let 20 µL of the Samples 1-9 evaporate in a 1.5 mL Eppi for 10 min
Add 4 μl Loading Dye and let it evaporate for 3 min
Load the samples on the Gel:
1 Lane Ladder
2-10 Lane:   Samples 1-9

• Cut bands of  samples 2, 5,7  out of the gel they are good
• Cut band from sample 1 (Is ok)
• Clean the gel pieces according to protocol 6.2 with the extra ethanol wash step before the elution.

Measurement with Nanodrop:

Sample	ng / μl	260/280	260/230
Sample 1	2.5	2.43	0.12
Sample 2	10.5	1.76	0.21
Sample 5	1.7	2.47	0.07
Sample 7	1.7	2.27	0.10

Repeat the Gel with the rest of the PCR-product repeating the same steps as before
1. Ladder 2. Sample 2 3. Sample 5 3. Sample 7
120 mV
Cut a band from every sample since they were all good.

28.09.2024

Overnight culture of transformed TOP10 with pET28-c

Four colonies from the transformation of pET28-c in TOP10 performed on the 27.09.24 were picked and placed in four tubes with 5 mL of LB-medium.

• Additionally, 5 µL of ampicillin (50 mg/mL stock solution) were added
• Finally, the cell suspensions were incubated overnight at 37 °C and 150 rpm on a shaker

Gel clean-up of the gel bands from the 27.09.24
Clean the gel cut out according to protocol 6.2 with the additional ethanol wash step before the elution.
Nanodrop measurements:

Sample	ng / μl	260/280	260/230
Sample 2	1.1	1.88	0.08
Sample 5	1.2	1.79	0.05
Sample 7	1.6	1.35	0.05

Do a PCR to amplify the cleaned repeats again from today (2,5,7) and yesterday (1,2,5,7) because we need higher concentrations for the cloning into the plasmid PCR according to protocol 5.2 with the thermofischer Phusion (50 μl)
25 μl  sample + 5 μl loading dye for the Agarose gel

1	Ladder
2	Sample 1 from gel clean up (27.09.24)
3	Sample 2 from gel clean up (27.09.24)
4	Sample 5 from gel clean up (27.09.24)
5	Sample 7 from gel clean up (27.09.24)
6	Ladder
7	Sample 2 from gel clean up (28.09.24)
8	Sample 5 from gel clean up (28.09.24)
9	Sample 7 from gel clean up (28.09.24)

Cutting bands
The gel pieces were cleaned according to protocol 6.2 with the additional ethanol wash step before the elution.

29.09.2024

Restriction ligation of pET28-c and resilin

Isolation of peT28-c from TOP10 following the Miniprep protocol from Qiagen.

Clone	1.1	1.2	2.1	2.2
Con. (ng/µL)	28.8	24.9	21.8	28.4

The purified resilin samples from the 27.09.24 were pooled (From now on listed as resilin 1) and purified following the Qiagen gel clean up protocol stating at step 5. Before that, the equal amount of sample volume of buffer QB was added. The same steps were repeated with the purified resilin samples from the 28.09.24 (From now on listed as resilin 2).

Sample	Resilin 1	Resilin 2
Con. (ng/µL)	51	46,7

First, the restriction of pET28-c and resilin was carried out with SacI and NdlI for 1 h at 37 °C. Next the samples were heat inactivated for 20 min at 65 °C.

pET28-c	Clone 1.1	Clone 1.2	Clone 2.2	Clone 2.1	Negative control
1 ng template (µL)	35	40	35	45	-
Buffer (µL)	5	5	5	5	-
SacI (µL)	1	1	1	-	-
NdeI (µL)	1	1	1	-	-
ddH2O (µL)	8	3	8	-	-
The samples had a total volume of 50 µL

	Resilin 1	Resilin 2	Resilin 3*	Negative control
Template (µL)	19 (962 ng)	19 (887,3 ng)	10 (109 ng)	7 (76,3 ng)
Buffer (µL)	5	5	1,8	1,2
SacI (µL)	1	1	0,1	-
NdeI (µL)	1	1	0,1	-
ddH2O (µL)	24	24	-	40

• Sample 3 is sample 2 from the 27.09.2024
• The restricted pET28-c plasmid and the resilin sequence were ligated for 1h at RT using the following 50 µL reactions.

	Ligation 1	Ligation 2	Ligation 3	Ligation 4Negative control
Ligase buffer (µL)	4	4	4	4
T4 Ligase (µL)	0,4	0,4	0,4	0
pET28 (µL)	20 (400 ng)	20 (400 ng)	20 (400 ng)	20 (400 ng)
Resilin (µL)	20 (384 ng)	20 (384 ng)	10 (90,8 ng)	20 (N/A)
ddH2O (µL)	5,6	5,6	15,6	5,6

As a control the ligation 1 of was restricted with Sac1.

	Ligation 1
Template (µL)	5
Buffer (µL)	1
SacI (µL)	0,5
ddH2O (µL)	3,5

A 1% agarose gel of 75 mL with 4 µL Midori green was made to verify the restriction ligation of pEt28-c and resilin. 10 µL of each recombination sample were mixed with 2 µL loading dye. Transformation of the recombination samples into DH5α and TOP10 using LB-Kan plates following the transformation protocol 2.

	DH5α	TOP10
1	10 µL	38 µL
2	10 µL	38 µL
3	10 µL	38 µL
1	10 µL	38 µL
3	10 µL	38 µL

Due to too high concentrations of the pET28-c_resilin plasmid no bacteria grew on the plates.

30.09.2024

Repetition of the ligation and transformation of pET28-C_resilin in DH5α

The remaining restriction reactions 1 and 2 from the 29.09.2024 were pooled and ligated for 1 h at RT and then heat inactivated for 10 min at 65 °C

	Ligation
Ligase buffer (µL)	5
T4 Ligase (µL)	0,8
pET28 (µL)	20
Resilin (µL)	20
ddH2O (µL)	4.2

Purification of the ligation reactions
The purified ligation samples from the 29.09.24 were pooled as well as the ligation samples from today and purified following the Qiagen gel clean up protocol stating at step 5. Before that, the equal amount of sample volume of buffer QB was added.

Resilin Ligation Pool	Concentration (ng/µL)
Resilin ligation pool 29.9	27,7 ng/µL
Resilin ligation pool 30.09	87,2 ng/µL

Both ligation pools from the 29.09 and 30.09 were sent for a whole plasmid sequencing Transformation of pET28-c-resilin

• Additionally, a transformation of the two purified pET28-c_resilin plasmid pools was made into DH5α following an optimized version of the transformation protocol 2 using LB-Kan plates.
• Incubate 30 min on ice.
• Incubate for 45 seconds at 42 °C.
• Put on ice for 5 min.
• Add 200 µl prewarmed (37°C) SOC medium to the cells.
• Incubate the cells for 90 min at 37 °C and 500 rpm.
• Plate all on LB-Agar plates with ampicillin.
• Incubate on prewarmed plates at 37 °C overnight.

Ligation reaction		ddH2O	DH5α
Negative control	/	2 µL	33 µL
Resilin ligation pool 29.9	1 µL	/	33 µL
Resilin ligation pool 30.09	0,2 µL	/	33 µL

Colony PCR of pET28-c-resilin in TOP10
A colony PCR was performed from the four colonies that grew on the plates.

	Colonies 1-4	Positive control	Negative control
Taq 2x Master Mix	25 µL	25 µL	25 µL
Oligo_1repeat_fw_primer	1 µL	1 µL	1 µL
Oligo_2repeat_fw_primer	1 µL	1 µL	1 µL
Template	Colony 1, 2, 3 or 4	pET-28-c_resilin 1 µL (1:10)	/
ddH2O	23 µL	22 µL	23 µL

The production of pET28-c_resilin plasmid and it’s transformation into TOP10 was a success, since a band at about a height of 1,3 kbp, representing resilin, can be detected in colony PCR the clones 1-4. Overnight culture of transformed TOP10 with pET28-c_resilin
Four colonies from the transformation of pET28-c_resilin in TOP10 performed on the 29.09.24 that were also picked for the colony PCR were placed in four tubes with 5 mL of LB-medium.

• Additionally, 5 µL of ampicillin (50 mg/mL stock solution) were added to generate.
• Finally, the cell suspensions were incubated overnight at 37 °C and 150 rpm on a shaker

03.10.2024

Overnight cultures for Sequencing and long term storage

• Discard overnight culture of DH5a with pET28(c)+_resilin from tuesday
• Make 4 new overnight cultures from each clones picked on 01.10.24 and 03.10.24
• 5 mL LB-medium and Kanamycin

04.10.2024

Sequencing and long term storage of DH5a with pET28(c)+_resilin

• Send clones 1 and 2 from the 01.10.24 overnight culture and clones 3 and 4 from the 03.10.24 overnight culture to sequencing with e.coli nightseq
• Made glycerol stocks with the overnight cultures for storage with 500 µL of the overnight culture and 500µL of 50% glycerol
• • Overnight cultures were stored in the fridge at 4 °C after incubation

10.10.2024

Miniprep pET28c(+)

• • Miniprep following protocol 7.1 with the overnight cultures from the 04.10.2024 from the clones 1-4.

Clone	Con. (ng/µL)	260/280	260/230
1.10 1	73.7	1.83	1.55
1.10 2	42.2	1.9	2.58
1.10 3	81.2	1.92	2.47
1.10 4	54.4	1.85	1.61
3.10 1	51.5	1.92	2.57
3.10 2	64	1.82	1.26
3.10 3	69	1.84	1.30
3.10 4	44.7	1.8	1.28

Restriction of pET28c(+)_resilin
Made a Restriction reaction with a total volume of 0.5 µg.

• 1µL XhoI
• 1µL NdhI
• 5µL 10× rcutsmart buffer
• 0.5 µg template
• add to 50 µL ddH2O

Each of the 8 plasmids from above were cutted with the reaction from above. Additionally, there was a reaction with just pET28c(+) as a template.
They were incubated for 1h at 37 °C.
Afterwards they were checked with a 1% agarose gel. For this 20 µl restriction reaction was mixed with 5 µl loading dye and loaded onto the gel, as well as 8 µl of the ladder.

1	Ladder
2	pET28(c)+ digested
3	pET28(c)+_resilin 3.10 1 digested
4	pET28(c)+_resilin 3.10 2 digested
5	pET28(c)+_resilin 3.10 3 digested
6	pET28(c)+_resilin 3.10 4 digested
7	pET28(c)+_resilin 1.10 1 digested
8	pET28(c)+_resilin 1.10 2 digested
9	pET28(c)+_resilin 1.10 3 digested
10	pET28(c)+_resilin 1.10 4 digested
11	pET28(c) undigested

Unfortunately no resilin insertion was observed in the plasmid from the DH5α bacteria.

16.10.2024

Competent cells JM109

The E. coli strain JM109 was made competent following the protocol 1. 50 mL LB-Medium with 2 mL overnight culture of JM109 was put into a 500 mL culture flask and put into an incubator at 37 °C. The OD600 was measured and as the OD600 = 0,570 was reached the whole media was transferred into a 50 mL falcon.
Afterwards 100 µl of cell culture was put into 1,5 mL tubes and freezed at -80 °C.

17.10.2024

Transformation of pET28c(+)_resilin into JM109

Transforming pET28c(+)_resilin into JM109 bacteria following protocol 2. The bacteria were plated on LB-agar plates spiked with kanamycin. As a negative control JM109 bacteria were transformed with only pET28c(+). As a second negative control JM109 were plated out without being transformed.
We transformed the JM109 bacteria cells with:

• 5 µl pET28c(+)_resilin2 (1)
• 5 µl pET28c(+)_resilin2 (1)
• 5 µL of pET28(c)+_resilin1, 2, 3
• 5 µl pET28c(+)

18.10.2024

Making overnight cultures from the transformed bacteria from the 17.10.24

Add one clone in 5 mL LB-Media with 10 µL Kanamycin (Stock: 50 mg/mL). Incubate them on a shaker at 37 °C and 180 rpm overnight.

• 2 overnight cultures with pET28(c)+
• 3 overnight cultures with pET28(c)+_resilin from ligation 2
• 3 overnight cultures with pET28(c)+_resilin from ligation pool1+2+3

19.10.2024

IPTG induction of resilin expression

6 asseys from transformation (see 17.10.2024) with:

• 50 μl kanamycin
• 1000 μl from
  • "pET28(c)+_resilin1, 2, 3" → three assays
  • "pET28(c)+_resilin2" → two assays
  • "pET28(c)+" without resilin → one assay (negative control)
• 49 ml LB media each
• incubated at 37 ° C and RPM 200 until OD 0.5 was reached

Induction of 6 asseys with IPTG:

• added 30 μl IPTG to each assay to induce resilin expression
• sample of each taken immediately afterwards and frozen at -20 ° C → "Ex0" (negative control)
• incubated rest of IPTG assays over night at 37 ° C and RPM 200 for expression of resilin

20.10.2024

SDS-PAGE for resilin detection

21 hours after the IPTG induction the cells were harvested. 20 mL of each culture was transferred into two 50 mL falcon tubes and centrifuged for 15 min at 3400g. The cell pellet of one half of the culture was resuspended with 3 mL lysis buffer and the other half was resuspended in LB-Medium.

• pET28(c)+
• pET28(c)+_resilin ligation 1+2+3 1
• pET28(c)+_resilin ligation 1+2+3 2
• pET28(c)+_resilin ligation 1+2+3 3
• pET28(c)+_resilin ligation 2.1
• pET28(c)+_resilin ligation 2.2

Then two different lysis approaches were performed. First we did Heat Lysis with each of the 6 expression cultures in Lysis buffer and LB-Medium. This totals 12 eppis, each with 1 mL culture. They were heated for 10 min at 90 °C and then centrifuged for 25 min at 16.000g.
1 mL of each of the 6 expression cultures were used for sonification lysis. They were sonified for 10 s with a 30 s break for a total of 5 repeats. After that they were centrifuged for 25 min at 16.000g.
50 µl of the supernatant of the heated and sonified samples were mixed with 50 µl reducing sample buffer.
The samples without lysis were also mixed with 50 µl reducing sample buffer.
Three 10% SDS-Gels were prepared following the protocol 9. On Gel 1 the samples before and after the induction were put onto. Gel 2 contained the heat lysis samples and Gel 3 the sonified samples of all six expression cultures.
The gels were stained over night with Coomassie.

21.10.2024

SDS-PAGE for resilin detection- destaining

Destaining with water for 3 hours. Waterchange and again destaining for 4 hours. After that the pictures were taken.