( .5) and then added to 1 lite...)Electrotransformation of E. coli With M13 DNA

.5) and then added to 1 liter SOB medium with 10 g/ml tetracycline. Bacteria were grown at 37 C with moderate agitation until they reached an O.D.₆₀₀ of approximately 0.3. Flasks were chilled on ice for 30 minutes, and cells were spun at 3,000 rpm in 250 ml bottles. The supernatants were decanted, and cells were dispersed by gentle inversion in an equal volume of cold 1 mM HEPES, pH 7.0. Cells were washed 3 more times as above with the following: one volume cold water, one-half volume cold water, and 30 ml cold 20% glycerol. The final cell pellet was resuspended in 2 ml cold 10% glycerol. Cells were distributed in 50 l aliquots into chilled Eppendorf tubes and then immediately flash-frozen in a dry ice/ethanol bath. Frozen electroporation competent cells were stored at -70 C.

Electro-transformation
Electroporation competent cell aliquots (above) were thawed on ice and appropriate quantities of DNA in water (see Discussion section) were added in 1 l total volume. Mixtures were pulsed with the Gene Pulser apparatus and Pulse Controller at various voltages with 25 F and 200 Ω in chilled 0.1 cm electroporation cuvettes (Bio-Rad); immediately after pulsing, 200 l of an overnight or late logphase culture of XL1-Blue cells were added to each cuvette and mixed with the pulsed cells. Mixtures were transferred to Falcon 2059 tubes and mixed with 3 ml melted 0.83% L-top agar at 50 C with or without X-Gal (in N, Ndimethylformamide; DMF) and IPTG (in water), then plated immediately on warmed L-agar plates or L-agar plates with 6 mg/ml X-Gal and 9 mg/ml IPTG. Plaques were visible after 6 hours at 37 C.

Competent Cells Prepared by Conventional Methods

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