Team:UNICAMP-Brazil/Protocols/Electroporation

Electroporation

1. Set the electroporation apparatus to 2.5 kV.

2. Add 2 µl plasmid DNA to tubes containing 40 µl fresh or thawed cells on ice. Mix by swirling with pipette tip.

3. Transfer the DNA and cells to a pre-chilled electroporation cuvette (0.2 cm electrode gap) using a narrow pipette tip. Wipe any ice or water from sides of cuvette using a Kimwipe. Place the cuvette into the sample chamber.

4. Energize the electroporation apparatus and deliver the pulse by pushing in both charging buttons simultaneously and holding until a short beep is heard. Note the time constant of the pulse and the actual voltage delivered.

5. Remove the cuvette from the sample chamber. Immediately add 1 ml LB medium and transfer the cells to a sterile polypropylene culture tube using a Pasture pipette. (Failure to immediately add SOC to the electroporated cells can significantly reduce cell viability and decrease transformation efficiency.)

6. Incubate cultures for 60 to 180 minutes at 37°C on a roller or with moderate shaking to allow for plasmid expression.

7. Plate aliquots of the electroporation mixture on L-agar plates supplemented with the appropriate antibiotics. Incubate plates at 37°C.

Protocol adapted from: http://wheat.pw.usda.gov/~lazo/methods/goldberg/electro.html