Electrotransformation of E. coli

  1. Inoculate 1 liter of LB medium with 5 ml of an overnight culture of bacteria and grow at 37.
  2. When the OD600 reaches 0.5, heat shock the culture for exactly 15 min at 42°C (with agitation) and chill the culture in ice slurry for 10 min. For now on, all procedures are made at 4°C.
  3. Harvest cells at 10.000 rpm for 3 min. Resuspend in 100 ml of ice-cold 10% glycerol and centrifuge again. Repeat this step once again.
  4. Resuspend bacteria to a final volume of 2 ml in ice cold GYT (10% glycerol, 0.125% yeast extract and 0.25% tryptone).
  5. Electroporation is made in a prechilled 0.2 ml cuvette.
  6. Following electroporation, 1 ml of SOC (2% tryptone, 0.5% yeast extract, 10 mM , 10 mM , 10 mM ) is added immediately. Cells are transferred to a tube and incubated at 37°C for 1h to allow expression of the AbR phenotype. Seed cells on the appropriate plates and incubate O.N. at 37°C.