Team:Aix-Marseille/Protocols
DH5α competent cells preparation
- The day before the preparation, make overnight starters with 100 µL of bacterial cells diluted in 5 mL of LB in a 50 mL Erlenmeyer.
- Prepare buffers 1 and 2 and put them on ice. Put Eppendorf tubes and 50 mL Falcon tubes, and p1000 tips at -80°C for future use.
- The next day, recover your starters and seed 1/60 bacterial cells in 200 mL of LB, then incubate at 37°C and 180 rpm.
- Keep measuring the OD600. When it's between 0,4 and 0,6, recover your cells an pool them in cold 50 mL Falcon tubes.
- Centrifuge tubes at 4000 rpm and 4°C for 10 minutes.
- Discard supernatant and resuspend each Falcon tube in 20 mL of buffer 1. Afterward, centrifuge at 3500 rpm and 4°C for 5 minutes.
- Discard supernatant and resuspend each Falcon tube in 8 mL of buffer 2.
- Take 100 µL aliquots in Eppendorf tubes and stock them at -80°C.
Buffer 1 preparation
| Product
|
For 40 mL
|
| KAc (1M)
|
1.2 mL
|
| MnCl2 (0.5M)
|
4 mL
|
| KCl (1M)
|
4 mL
|
| CaCl2 (0.1M)
|
4 mL
|
| Glycerol (80%)
|
7.5 mL
|
| H2O
|
19.3 mL
|
Buffer 2 preparation
| Product
|
For 8 mL
|
| KCl (1M)
|
800 µL
|
| MOPS (1M)
|
400 µL
|
| CaCl2 (0.1M)
|
6 mL
|
| Glycerol (80%)
|
1.5 mL
|
| H2O
|
500 µL
|
Transformation
- Recover the needed number of competent cells tubes, adding an additional negative control tube.
- Add DNA (1µL) into the competent cells tubes.
- Incubate tubes on ice for an hour.
- Incubate tubes at 42°C for 2 minutes for thermal choc.
- Leave tubes on ice for 5 minutes.
- Leave tubes at room temperature for 5 minutes.
- Add 1 mL of LB per tube, then incubate for 1 hour at 37°C and 180 rpm.
- Spread cells on Petri dishes and incubate overnight at 37°C.
