Team:UNICAMP-EMSE Brazil/protocols/Transforming bacteria electro

Transforming bacteria: Electroporation

Electroporation

Before starting the protocol, esterilize cuvettes under UV light for at least 20 minutes. After that, place them in ice untill they cool. Place the eletrocompetent cells on ice too untill they thaw.

1. Set the electroporation apparatus to 2.5 kV.

2. Add 2-7 µl plasmid DNA to tubes containing 40 µl of thawed cells. 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 between 700µL and 1 ml LB medium and transfer the cells to a sterile polypropylene culture tube.

6. Incubate cultures for 60 minutes at 37°C

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

See how to make competent cells