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Electroporation Procedure
- Either BTX or BioRad Brand Cuvettes 0.1 cm gap electroporation cuvettes can be used. The latter are available through general stores (Bio Rad Gene Pulser cuvettes, 0.1 cm, pk/50, $128.25, catalog #165-2089)
- Chill cuvettes on ice (10 minutes is OK).
- Remove electrocompetent cells from -70C (When prepared, these are aliquotted in 50 uL per tube, and frozen; one tube of these cells is required for each ligation reaction or DNA to be transformed).
- After the electrocompetent cells are thawed, add 1 ul ligation mix [Note - the limiting factor in setting up the electroporation is the final amount of salt in the cuvette; if there is too much salt, the cuvette will arc when the pulse is applied; Critical factors that determine how much salt is in the cuvette are a) how carefully the electrocompetent cells have been prepared [the procedure is designed to essentially wash away as much salt as possible] and b) how much of the ligation mix is added to the cells. For well-prepared competent cells, we routinely add 1.0 uL of ligation mix without causing the pulse to arc].
- Transfer cells with added ligation mix to a chilled cuvette.
- Set the electroporation voltage, and pulse the cuvette. As the electroporation efficiency is somewhat dependent upon the voltage selected, you need to either experiment or consult the literature for the best values to use. In the literature supplied with the instrument, as well as on the BTX website, there are commonly used values for common E. coli strains used in cloning. The strain that we have used is GI724 (from InVitrogen) and we electroporate at 1.8 kV.
- After pulsing, immediately add 200 uL sterile LB to cuvette.
- Remove sample from cuvette and place in sterile eppendorf tube.
- Grow for 30 min at 37 C with shaking to allow the cells to express their ampicillin resistance gene (need at least 1 hour for cells expressing kanamycin resistance gene).
- Plate 75 ul (using sterile technique) onto LB/AMP plates (or whatever is appropriate for your plasmid) and incubate @ 37C for 12 hrs.
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