Bacterial Electroporation

Procedure

  1. Allow the electrodes to warm up for at least a half hour.

  2. Keep everything very cold throughout the process.

  3. Only use prechilled electrode cuvettes, tips, solutions, etc.

  4. Very gently add 50 μL of the electrocompetent cells into the tube with the resuspended DNA from the ethanol precipitation.

  5. Allow to sit for 30 seconds.

  6. Very gently transfer the electrocompetent cells and DNA into the prechilled electrode cuvette.

  7. Gently tap the cuvette on a benchtop to get the cells the bottom and eliminate bubbles.

  8. Allow the cells to incubate with DNA for 5 minutes on ice within electrode cuvette.

  9. Insert electrode cuvette into the electroporation equipment, and press the “pulse” buttons simultaneously.

  10. Immediately after electroporating the cells, add prewarmed 450 μL of prewarmed SOC to the cuvette.

  11. Gently transfer the 500 μL of SOC/cell mixture to a sterile culture tube.

  12. Rinse the electrode cuvette 3x with 500 μL of SOC, bringing the total volume up to 2 mL.

  13. Place in shaker for 1 hour at 37 Celsius.

  14. Plate a dilution of the transformation (10-3, 10-4), and back-calculate the total number of colonies.

  15. Dilute remainder of culture to 10 mL.

  16. Add appropriate antibiotic.

  17. Place in shaker overnight.

  18. The following day, proceed to purify DNA with a mini-prep, inoculate a larger culture for a midi-prep, or create glycerol stock of bacteria.

Notes and Cautions

  • Settings are 2 kV, 200 Ω, and 25 μF.

  • The smaller the volume of DNA added to the cells, the higher the competency.

  • It will flash, and then beep.