High Efficient Electrocompetent E. coli Cells

1. Inoculate 2 x 500 ml of LB with 1/100 volume of a fresh overnight culture.
2. Grow cells at 37°C with vigorous shaking to an OD₆₀₀ of 1.0.
3. Transfer the cultures to an ice/water bath and keep or 10 min.
4. Centrifuge at 7000 g for 20 min at 4 °
5. Resuspend pellets in a total of 2 x 250 ml ice-cold MQ water. Centrifuge.
6. Resuspend pellets in a total of 2 x 100 ml ice-cold MQ water. Centrifuge.
7. Resuspend in 20 ml 10% glycerol. Tranfser to a small centrifuge tube. Centrifuge.
8. Resuspend in a small final volume of 10% glycerol.
9. Freeze at - 80°C the suspension in aliquots of 40 μL.

Electro-transformation

1. Thaw the cells on ice for 10 minutes.
2. Add 1-2 μL of DNA. Mix and keep on ice for 1 minute.
3. Set the Gene Pulser apparatus at 25 μF and 2.5 kV. Set the pulse Controller to 400W.
4. Transfer the mixture of cells and DNA to a cold 0.2 cm electroporation cuvette. Place the cuvette in a chilled safety chamber slide, push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber.
5. Pulse once at the above settings. (Time constant should be 4.5 – 5 msec. Field strength will be 12.5 kV/cm).
6. Remove the cuvette from the chamber and immediately add 1 ml cold SOC medium to the cuvette. Mix and transfer to a 10 ml test tube. Incubate at 37°C for 1 hour.
7. Plate on selective media.