Team:Marburg/Protocols/Protocol2
Competent E.Coli cells (Electroporation)
- Prepare all the media (SOB, 10 % glycerol); prechill a GS3 rotor 4°C; sterilize centrifuge tubes by autoclaving and prechill them!
- Inoculate 30 mL SOB with single colony from a fresh plate and grow them o/n at 37°C.
- Inoculate 1 L SOB (1:100 ie. 10 mL of the starting culture per 1 L SOB) and grow it at 37°C on a shaker with 200 rpm until it reaches an OD600 of 0.4-0.5 (approx. after 4 h). Continue with all working steps on ice or 4°C!
- Centrifuge the cells at 4.000 rpm / 20 min / 4°C.
- Wash with 1 vol. 10 % glycerol.
- Centrifuge at 4.000 rpm / 20 min / 4°C.
- Wash with 0.5 vol. 10 % glycerol.
- Centrifuge at 4.000 rpm / 20 min / 4°C.
- Wash with 0.1 vol. 10 % glycerol.
- Centrifuge at 4.000 rpm / 20 min / 4°C.
- Go to get liquid nitrogen.
- Add 0.5 - 1 mL 10 % glycerol (per liter liquid culture started with) and resuspend pellet and prepare aliquots of 100 µL in 1.5 mL reaction tubes and freeze them immediately in liquid nitrogen.
- Store the electrocompetent cells at -80°C.
SOB-Media -> weight in:
- 20 g Bacto Tryptone
- 5 g Bacto Yeast Extract
- 0.5 g (8,6 mM) NaCl
- 2.5 mL (2,5 mM) KCl (from 1 M stock)
- add 1000 mL of H2O
- test for pH (it should be around pH = ~ 6.8 - 7.0)
- autoclave
- add sterile
- 10 mM MgCl2
- 10 mM MgSO4 f
10 % glycerol-solution:
- get 116 mL of 86 % glycerol
- add 1000 mL of H2O
- autoclave
