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Protocols/Notebook
Preparation of chemically competent DH5alpha E. coli cells
Material:
- LB media
- TSS buffer
- DH5alpha E. coli cells (o/n colonies on agar plates)
Method:
- Pick one colony of the plate and transfer into 5 mL of LB media. Grow the culture over night for 16-18 hours at 37 deg. celsius.
- Transfer 1 mL of the overnight culture into a shaking flask with 99 mL of LB media. Measure optical density (OD) at 600 nm and incubate culture at 37 deg. celsius (shaking) to an OD of 0,5.
- Divide the 100 mL into two 50 mL tubes and incubate 10 min on ice.
- Spin the tubes at 3000 rpm for 10 minutes at 4 deg. celsius.
- Resuspend the pellet of competent cells with 10 % TSS buffer (5 mL).
- Aliquot 100 µL of the cell solution into 1.5 mL microtubes (all steps on ice!).
- Store the competent cells at -80 deg. celsius.
Heat-shock Transformation of E. coli cells
Material:
- SOC media
- Agar plates with appropriate antibiotic
Method:
- Thaw chemically competent cells on ice.
- Transfer 50 µL of the cells into a 1.5 mL microtube, add 1 µL of the desired DNA and incubate on ice for 30 minutes.
- Place the tube in a 42 deg. celsius water bath for 60 seconds.
- After heat shock leave cells on ice for 5 minutes.
- Add 950 µL of SOC media and shake cells for 2 hours at 37 deg. celsius.
- Pipet 100 µL of the cells onto an appropriate plate and spread them using sterile glass beads. Incubate overnight at 37 deg. Celsius and hope for colonies in the morning to prove a successful transformation.
Preparation of LB media
Material:
- Tryptone
- NaCl
- Yeast extract
Method:
- Fill a container (bottle) to about 60/70 % its volume with destilled water.
- Add 10 g/L Tryptone, 10 g/L NaCl and 5 g/L of yeast extract.
- Stir properly and fill up the remaining volume with distilled water.
- Treat LB media by autoclave.
Preparation of LB agar
Material:
- Tryptone
- NaCl
- Yeast extract
- Agar
Method:
- Fill a container (bottle) to about 60/70 % its volume with destilled water.
- Add 10 g/L Tryptone, 10 g/L NaCl, 5 g/L of yeast extract and 20 g/L agar.
- Stir properly and fill up the remaining volume with distilled water.
- Treat by autoclave.
Glycerol stocks – storage of bacterial strains
Material:
Method:
- Mix 700 µL of overnight culture with 300 µL glycerol.
- Store at -80 deg. Celsius.
Polymerase chain reaction (PCR)
Material:
- Primer
- Template DNA
- NEB Q5® High-Fidelity 2X Master Mix (dNTPs + Polymerase)
- distilled water
- PCR-Cycler
Method:
- All steps have to be performed on ice.
- 50 µL approach (mix well):
| components | | concentration |
| Q5 Master Mix | 25 µL | 1x |
| 10 µM fw primer | 2,5 µL | 0,5 µM |
| 10 µM rv primer | 2,5 µL | 0,5 µM |
| template DNA | | <1000 ng |
| water | remaining volume to 50 µL |
- PCR-cycler conditions:
| step | cycles | temperature | time |
| Denaturation | 1 | 98°C | 30 sec |
| Annealing | 25-35 | 98°C | 5-10 sec |
| | 72°C | 20-30sec/kb |
| final extension | 1 | 72°C | 2 min |
| hold | | 4-10°C | |
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