Team:UNICAMP-Brazil/Protocols/Electroporation

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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

Thermal transformation

1. Keep the E. coli on ice for 30 minutes

2. Add 10μl ligation reaction

3. Keep the E. coli on ice for more 30 minutes

4. 42ºC for 1`30``

5. Put in the ice immediately for 5 minutes.

6. Add 500μl LB media

7. 37ºC for 1 hour

8. Plate aliquots of the mixture on LB plates supplemented with the appropriate antibiotics. Incubate plates at 37°C.