Transformation by electroporation (Dower et al. 1988)

Grow cell stocks on M9 minimal medium.

Day 1:
Inoculare 4 X 4 ml LB; incubate overnight

Day 2:
Inoculate 4 X 250 ml of LB medium, pre-incubated at 37°C with the overnight cultures.

Incubate with vigorous shaking at 37°C (up to an O.D.₆₀₀ » 0.6)

Collect cells by centrifugation

Wash cell pellet twice with 25 ml ice-cold sterile water

Wash cell pellet once with 25 ml ice-cold glycerol (10% w/v)

Concentrate cells in 3 ml of the glycerol solution (up to a final cell density of approximately 3.10¹⁰ cells/ml).

At this stage, competent cells can be stored at -70°C.

Mix approximately 1 ng of DNA in a solution of low salt concentrations with 40 ml electro-competent cells.

Transfer this mixture to a pre-chilled electroporation cuvette (0.1 cm electrode gap; Eurogentec, Seraing, Belgium)

Apply a high voltage pulse of a Gene Pulserä apparatus (Bio-Rad Laboratories, Hercules, CA) set to 1.7 kV, 25 mF, and pulse controller to 200 W .

Immediately after electroporation, add 1 ml SOC medium supplemented with 20 ml/ml of a 1 M glucose solution,

Incubate at 37°C for 30 to 60 minutes

plate onto selective media

Remarks:
Growth of E. coli at low temperature dramatically increases the transformation frequency by electroporation (Chuang et al. (1995) Nucl. Acids Res. 23, 1641).

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