Bacterial Electrocompetent Cells and Electroporation

Electrocompetent Cells

For 2 ml final volume of competent cells:

  1. Inoculate 1 fresh colony into 5 ml SOB. Grow at 37oC with rotation for 2.5 hours.

  2. Inoculate 500 ml SOB with the 5 ml culture. Grow at 37oC on shaker to O.D.(600) 0.75. Takes ~2.5 hours.

  3. Transfer cells into two chilled 250 ml centrifuge bottles. Pellet cells at 2600 g at 4oC for 15 minutes. Discard supernatant and remove residual liquid by pipette.

  4. Resuspend the cells in an equal volume of chilled 10% glycerol by repeated pipetting while keeping the cells on ice. Pellet cells at 2600 g at 4oC for 15 minutes. Immediately decant the supernatant.

  5. Repeat step 5.

  6. Resuspend the cells in 200 ul of 10% glycerol, with the total volume equal to about 2 ml.

  7. Dilute a 10 ul aliquot to 3.0 ml with 10% glycerol. Measure the O.D.(550).

    1. Example: If O.D.(550) of 1/300 dilution = 0.75, the O.D.(550) of original suspension = 225.

    2. The O.D.(550> of the original suspension should be 200-250 units/ml, if not then dilute it with chilled 10% glycerol to achieve that range.

  8. Aliquot in desired volumes and freeze at -70 to -80oC. Cells will last a long time at these temperatures.

  9. Use 40 ul aliquots for transformations.

Transformation

  1. Gently thaw cells. Don't let them get too warm. Place on ice when thawed.

  2. Chill cuvettes on ice for 5 minutes.

  3. Add 0.5-1.0 ul ligation or DNA to 40 ul cells and add to cold cuvette.

  4. Set the electroporator to 2.5 kV, 25 uF, and 400 ohms.

  5. Pulse, and record the actual voltage and time constant. If the pulse sets of a spark, the cells are pretty much dead.

  6. Immediately add 1 ml SOC media to cells and transfer to a sterile culture tube.

  7. Incubate 30-60 minutes at 37oC.

  8. Plate cells on selective media.