High electric fields transiently rip holes in both membranes and the coat
E.coli, and simultaneously turn molecules of DNA in the medium into
bullets because of their intense charge density. Some of these bullets
the holes. If such cells are cooled down quickly after the pulse, the
reseal and the cells are transformed. Efficiency is extremely high,
100% under ideal conditions. Disadvantages are that multiple plasmid
can enter the same cell (i.e., it can be too efficient!).
We use a BioRad GenePulser and BioRad Pulse Controller to electroporate
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
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
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
Label 6-ml plastic plating tubes and have them racked and on hand at
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!
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
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
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