Electroporation

11/18/99 ECK
revised 8/6/12

Description

High electric fields transiently rip holes in both membranes and the coat surrounding E.coli, and simultaneously turn molecules of DNA in the medium into electric bullets because of their intense charge density. Some of these bullets penetrate the holes. If such cells are cooled down quickly after the pulse, the holes reseal and the cells are transformed. Efficiency is extremely high, approaching 100% under ideal conditions. Disadvantages are that multiple plasmid copies can enter the same cell (i.e., it can be too efficient!).

We use a BioRad GenePulser and BioRad Pulse Controller to electroporate bacterial cells.

Protocol

Preliminary
  • Check that the GenePulser is connected to the Pulse Controller by two cables (from "output" in pulser to "input" in controller; red = +).
  • The capacitance of the pulser should be set at 250 µFD and the capacitance of the controller at 200 Ohms.
  • Connect the cuvette holder by its cables to the controller at the "output to chamber" receptacles.
  • Turn on the pulser at the rocker switch top left behind the machine.
  • Set the voltage by pressing the "raise" membrane switch until the display reads 250 (the maximum value it can read). This is the only truly obnoxious step!
  • Press the membrane switch "time const.". The display will now read a parameter related to the duration of the electric pulse during electroporation. This will be useful later on.
  • Put cuvettes on ice.
  • Put sufficient L-Broth on ice to dilute each sample to 2 ml.
  • Have Pasteur pipettes and bulb on hand at electroporation station.
  • Label 6-ml plastic plating tubes and have them racked and on hand at station.

  • Preparation of samples
  • Allow frozen electroporation-competent cells to thaw on ice.
  • Add 1 - 5 µL of DNA to cells. Typical plasmid DNA stocks are too concentrated and are commonly diluted 1:100 prior to this step. DNA for linear transformation often comes from a PCR reaction, and should be used full-strength. If the DNA comes from a ligation reaction, the active ligase will interfere with subsequent steps. If so, heat the DNA 15' at 65° or, alternatively, phenol extract, ethanol precipitate and redissolve in deionized H2O.
  • Transfer to cold cuvettes, tapping them on bench to make the thick mixture fall to the bottom. Keep them on ice!

  • Electroporation
  • Place cuvette into cuvette holder. There is only one way to do this, because of a small plastic protrusion on one side of cuvette.
  • Push the cuvette between the electrodes of the cuvette holder using the plastic plunger.
  • Fill a Pasteur pipette with about 1.5 ml cold L-Broth (i.e., one firm bulb press-worth). Hold the pipette in your right hand.
  • Press simultaneously the two red firing buttons top left on the face of the pulser. Display will flash three times, a faint beep will sound and the time constant for the pulse will be displayed. It should be about 4.7 0.2.
  • Immediately withdraw cuvette and, leaving it in its holder, squirt the cold L-Broth into the cuvette, cooling and diluting the cells.
  • Using the same pipette to withdraw the cells and transfer them into a labelled plating tube. These receiving tubes can be left at room temperature.

  • Plating cells
  • Incubate diluted cells 1 hr x 37°.
  • Spread 100 µL onto selective plate. If you expect few transformants, concentrate cells by centrifugation, resuspend them in 100 µL appropriate growth medium and spread them all.

  • Last Update: Thursday, 19-Jun-2014 11:51:56 PDT
    This page has been viewed 354 times.
    Eric Kofoid eckofoid at ucdavis.edu