Team:Manchester-Graz/Project/Protocols
Project Specific Protocols
PCR Mutagenesis of AADC
This protocol was kindly provided to us by Nicholas Weise
1. Prepare 4 x 0.2mL eppendorf tubes, for a total reaction volume of 50μL in each tube.
2. In each tube, prepare each of the following: 1μL Phusion DNA Polymerase, 1μL DNA template, 2μL primer mix (10 pmol/μL stock), 1μL dNTPs, and in tubes 1 and 3, 10μLs of HF Buffer. In tubes 2 and 4, 10μLs of GC Buffer.
In tubes 3 and 4, add 1.5μL of DMSO.
3. Make reaction volumes up to 50μL
4. Run reactions in PCR thermal cycler for 1 cycle of 98°C for 30s, 30 cycles of 98°C for 15s, 58°C for 30s, 98°C for 150s, then 1 cycle of 72°C for 300s.
5. Samples can be stored long-term at 4°C.
Phosphorylation Ligation of AADC Plasmid
1.Run reactions 1-4 on 1% agarose electrophoresis gel
2. Select reaction tube with clearest gel image for phosphorylation-ligation reaction.>
3. Transfer 5μLs of reaction to new 0.2mL eppendorf tube also containing the following: 1μL T4 PNK, 2μL of NEBuffer 4, 2μL T4 ligase buffer, 9μL dH2O.
4. Incubate at 37°C for 1hr without shaking, then add 1μL T4 ligase.
5. Leave for 2hrs at room temperature (or 16°C overnight) and transform immediately.
Transformation of Nissle 1917
Chemically competent Nissle 1917 cells were prepared using the protocol by Penn iGEM 2012
1. Thaw tube on ice for 10 minutes
2. Add 2 μL of plasmid DNA to 100 μL of cells and leave in ice for 30 minutes
3. Heat shock cells at 37°C for 180 seconds
4. Put tubes back on ice for 5 minutes
5. Add 900 μL of SOC and incubate at 37°C shaking for 1 hour
6. Spread varying dilutions of resulting cultures on LB agar plates with appropriate antibiotic added and grow overnight at 37°C
AADC Expression
Transfer 2 mL of inoculum into flask containing 200 mL of sterile LB broth, containing kanamycin
Incubate at 37°c at 180-220 rpm for 2-5 hours.
Once OD600 is at 0.4, add IPTG at final concentration of 1 mM.
Transfer flask to 37°c for 4 hours at 200rpm.
CvATA Expression
Transfer 2mls of inoculum into flask containing 200mls of sterile LB broth, containing kanamycin
Incubate at 37°c at 180-220 rpm for 3-4 hours.
Once OD600 is at 0.6, add IPTG at final concentration of 0.2 mM.
Transfer flask to 20°c while overnight shaking.
General Protocols
Preparation of antibiotics stock (1000x)
Ampicillin (100 mg/mL) in H2O, sterile filtered
Kanamycin (50 mg/mL) in H2O, sterile filtered
Chloramphenicol (30 mg/mL) in ethanol
LB medium
10 g/L Trypton
5 g/L Yeastextract
5 g/L NaCl
Autoclave at 121°C for 20 min
SOC medium
Suspend/dissolve the compounds in 1 L of purified water:
Tryptone 20 g
Yeast extract 5 g
NaCl 0.5 g
Dissolve, then add
KCl (250 mM) 10 mL
MgCl2 5 mL
Autoclave, then add
Sterile glucose (1 M) 20 mL
Plasmid preparation
Day before: Preparation of a preculture in LB-medium containing the appropriate antibiotics. The amount of cell culture required for plasmid preparation depends on the copy number of the plasmid (between 5 and 20 mL)
1. Centrifuge the preculture in appropriate tubes for 10 min at 4000 g
2. Carefully remove the supernatant
3. Resuspend pellet in 200 μL resuspension solution
4. Add 200 μL lysis solution and invert the tube gently 1 to 2 times
5. Lysis should not exceed 5 min
6. Add 350 μL neutralization solution and invert the tube 4 to 6 times
7. Centrifuge the suspension for 10 min at 12 000 rcf
8. Prepare the columns by adding 500 μL of column preparation solution and centrifuging it for 1 min at 12 000 rcf. Discard the flow-through
9. Transfer the supernatant of the centrifuged samples onto columns
10. Spin for 1 min at 12 000 rcf and subsequently discard the flow-through
11. Add 750 μL wash solution and spin the loaded column for 1 min at 12 000 rcf. Discard the flow-through
12. Dry the columns by centrifuging them for 1 min at 12 000 rcf
13. Place columns in new collection tubes
14. Elute the plasmid DNA with 50 μL ddH2O to increase the plasmid concentration
The Sigma-Aldrich GenElute™ Plasmid Miniprep Kit was used
Preparation of chemically competent E. coli
Day before: Preparation of a Top10 cells preculture in LB-medium containing streptomycin (25 μg/mL)
1. Addition of 1 mL overnight preculture to 100 mL LB-medium + streptomycin (25 μg/mL)
2. Cultivate culture at 37 °C, 220 rpm until it reaches an OD600 of 0.5
3. Cool culture for 5 min on ice and centrifuge it for 5 min at 4 °C, 4000 g
4. Discard the supernatant and resuspend the cells in cold TFB1 buffer (30 mL, 4 °C)
5. Keep the suspension on ice for 90 min
6. Centrifuge the suspension for 5 min at 4 °C, 4000 g and discard the supernatant
7. Resuspend the cells in 4 mL cold TFB2 buffer
8. Make aliquots of 100 μL and freeze the aliquots in dry ice in ethanol
9. Store aliquots at -80 °C
According to QIAGEN DNA protocols and applications
Preparation of electrocompetent E. coli
1. Grow a 5mL suspension of your cells of interest to OD=0.6
2. Pre-cool your centrifuge to 4°C
3. Pre-cool autoclaved water on ice
4. Cool the cells on ice for 5-10 minutes
5. Centrifuge for 6 minutes
6. Discard supernatant
7. Resuspend pellet with 1mL of chilled water
8. Centrifuge for 6 min
9. Repeat washing step followed by centrifugation 3 times
10. After last centrifugation step, discard supernatant and resuspend pellet in 50uL chilled water.
11. Add plasmid you want to transform
12. Electroporate
13. Add SOC medium and let cells recover at 37°C for 1 hour
14. Streak on LB-Agar plates with the corresponding antibiotic
Transformation of competent E. coli
1. Thaw the competent cells on ice
2. Add 1 μL DNA (0.2 - 200 ng) to 50-100 μL competent cells
3. Leave sample on ice for approximately 20 min
4. Heat shock the cells for 90 s at 42 °C
5. Add 500 μL of SOC to the sample
6. Let the cells recover for 60 min at 37 °C, 220 rpm
7. Plate appropriate amount of cell suspension (50 - 200 μL) on LB-agar-plates containing the appropriate antibiotic
8. Let bacteria grow overnight at 37 °C
Preparation of samples for sequencing at Microsynth
Add 12 μL DNA (60-100 ng/μL) to 3 μL of the corresponding primer (10 μM).
PCR protocol for phusion DNA polymerase
| Components | 20 μL reaction volume | 50 μL reaction volume |
|---|---|---|
| 5x Phusion HF buffer | 4 μL | 10 μL |
| 10 mM dNTPs | 2 μL | 5 μL |
| Forward Primer (10 μM) | 1 μL | 2.5 μL |
| Reverse Primer (10 μM) | 1 μL | 2.5 μL |
| DMSO | 0.6 μL | 1.5 μL |
| Phusion DNA polymerase | 0.2 μL | 0.5 μL |
| DNA | 40-200 ng | 40-200 ng |
| H2O | add to a volume of 20 μL | add to a volume of 50 μL |
Restriction Endonuclease Reaction (double digestion)
1-2.5 μL restriction endonuclease 1
1-2.5 μL restriction endonuclease 2
5 μL Cut Smart Buffer
1-3 μg template DNA
add H2O to reach a total volume of 50 μL
Enzymes, buffers and protocol are from New England BioLabs
Agarose gel electrophoresis
1. For a 5 cm x 6 cm gel add 0.25 g Agarose to 25 mL TAE (0.5x) and heat up in microwave to dissolve it
2. Let the solution cool down to approximately 50 °C
3. Add nucleic acid dye (e.g. EtBr) and mix
4. Pour the solution in a tray using an appropriate comb
5. Add loading dye (e.g. NEB purple loading dye) to the samples
6. Fill the samples and ladder in the wells
7. Run the gel at 135 V
Site-directed mutagenesis
14 μL H2O
2 μL HF buffer
1.6 μL dNTPs
0.5 μL of primer 1
0.5 μL of primer 2
0.4 μL Phusion polymerase
1 μL template DNA (2-20 ng)
1. Run PCR
2. Digestion of template DNA: Addition of 1 μL DpnI, 1h at 37 °C
3. Heat inactivation of DpnI: 20 min at 80 °C
Protocol based on QuikChange Site-Directed Mutagenesis
Gibson Assembly
One-step isothermal DNA assembly protocol: the exonuclease amount is ideal for the assembly of DNA molecules with 20–150 bp overlaps
1. Mix the backbone and PCR fragments in 5 µL total volume in equimolar amounts
2. Thaw the Gibson assembly reaction mixture on ice
3. Add DNA mixture (5 µL) to the reaction mixture (15 µL)
4. Run the reaction for 30-60 min at 50 °C
5. Subsequently the reaction mixture (5 uL are enough) can be used directly to transform competent cells (75 uL)
SDS-PAGE Gel preparation
First prepare the separation gel (12,5%) For the preparation of 2 thick (1mm) gels:
- 3.77 mL H2O dest.
- 2.81 mL TrisHCl-Buffer (1.5 M, pH 8.8)
- 4.5 mL Acrylamid/Bis(30%) Toxic
- 112.5 µL SDS (10% w/v)
After adding SDS carefully mixing -> foam
- Ammonium persulfate (APS 10% w/v) 56.25 µL
- TEMED 11.25 µL
Pour the gel (leave some space for the stacking gel) and cover it with n-butanol, wait for aprox. 1-2 hours until it gets solid. Remove the n-butanol (with filter papers) and prepare the stacking gel (4%) (can be prepared after pouring the separation gel; Attention: adding of persulfate and TEMED shortly before pouring the stacking gel)
Preparation of two thick (1mm) stacking gels:
- 4.58 mL H2O dest.
- 1.88 mL TrisHCl-Buffer (0.5 M, pH 6.8)
- 0.98 mL Acrylamid/Bis(30%) Toxic
- 75 µL SDS (10% w/v)
- 37.5 µL Ammonium persulfate (APS 10% w/v)
- 7.5 µL TEMED
Use the combs (mind that there are different ones for 0.75 mm and 1 mm) and wait for aprox. 30 min – 1 h until it gets solid.
Running an SDS-PAGE
1. Add SDS-Loading Buffer (consists of reducing agent and loading dye) to your samples (total sample volume per slot should not exceed 15 )µL
2. Prepare 250 mL / 2 gels 1x Running Buffer (use 25 mL of 10x Running Buffer + 225 mL dest. H2O)
3. Build up the SDS-Running chamber, fix your gels and add the 1x Running Buffer
4. Load your samples
5. Close the lid (mind the correct arrangement of the electrodes)
6. Run your gels at 125 V. Duration is about 50 min but depends on the size of your proteins.
Stain your gels with coomassie blue for 10 min, then wash with 10 % acetic acid over night.
