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

iGEM Marburg - ZSM Karl-von-Frisch-Straße 16, D - 35043 Marburg