Preparation of the competent cells

1. Inoculate 1l of SOC with 10 ml of a fresh overnight culture
2. grow cells at 37°C with vigorous shaking to an OD600 of 0,5 to 1,0
3. chill the flask on ice for 15 to 30 min, spin in a cold rotor at 4000xg for 5 min (5000rpm, GSA, 10min)
4. discart the supernatant completely, rently resuspend pellets with a 10 ml glass pipette first in a small volume (10ml) of cold water , dilute to 1l, spin as in step 3
5. discard the supernatant, suspend the cells in 500 ml cold water, spin as in step 3
6. discard the supernatant, suspend the cells in 50 ml of cold 10% gycerol, spin as in step 3
7. discard supernatant, suspend cell in final volume of 3 ml 10% glycerol. The concentration should be 1-3x10^10 cells/ml
8. aliquote the suspension, freeze in liq. nitrogen and store at -70°C

Electroporation

1. thaw the cells on ice
2. mix 40 µl of cells with 1-2µl of DNA (TE or water). mix well, let sit on ice for 1 minute or longer
3. set gene pulser apparatus at 25 µF and 2,5 kV. Set the pulse controller to 200 Ohm
4. transfer the DNA/cells to a cold 0,2 cm electropoaration cuvette, knock the suspension to the bottom of the cuvette. 
5. pulse once at the above settings. This should produce a pulse with a time constant of 4-5 ms. 'The field strength will be 15,5 kV/cm.
6. Remove the cuvette from the chamber and immediately add 1 ml of SOC medium to the cuvette and quickly resuspend the cells with a sterile pastuer pipette
7. transfer the cells to a culture tube and incubate at 37°C for upto 1h with shaking
8. plate on selective medium

required materials: