Electroporation
DNA Preparation:
1. Cut the required amount of DNA (banded on a CsCl gradient)
with the appropriate restriction enzyme and check for complete digestion
by running 500 ng on a minigel. The DNA concentration should be no
higher than 1 ug/ul in the large-scale digest.
2. Extract the large-scale digest once with an equal volume
of phenol/chloroform and once with an equal volume of chloroform.
Precipitate the DNA with 2.4 volumes of ethanol, spin down and dry in
the Speed-Vac.
3. Resuspend the DNA at the desired concentration (usually 1 ug/ul)
in sterile 0.1X TE (25 ul of DNA per electroporation). Measure
the DNA concentration using the fluorometer.
Cell Preparation:
1. Embryonic stem cells (80% confluent) should be passaged
1:2 the day before electroporation. Cells to be electroporated should
be fed 4 hours before harvesting.
2. Trypsinize cells and resuspend in media (cells from 2 x 10
cm plates can be combined in a total volume of 10 ml in a 15 ml tube).
3. Pellet the cells and aspirate off the supernatant. Resuspend
in 10 ml PBS and determine the total number of cells by counting a 20 ul
aliquot. (Note: The usual yield is 30 x 106 cells per 10 cm plate.)
4. Pellet the cells and aspirate off the supernatant.
Resuspend in PBS at a density of 11 x 106 cells/ml. Count a 20 ul
aliquot to confirm.
Electroporation:
1. Add appropriate amounts of DNA and cells together in a
15 ml tube (25 ul of DNA and 0.9 ml of cells for each electroporation).
2. Allow to sit at room temperature for 5 minutes (this step
may be omitted).
3. Aliquot the cell/DNA mixture into electroporation cuvettes
(0.9 ml per cuvette; the volume is important!). Place the cuvette in
the electroporation holder with the foil electrodes in contact with the
metal holding clips.
4. Set the Biorad GenePulser at 230V, 500 uF (requires the
capacitance extender) and press the two red buttons to electroporate.
The machine will flash "Ch 9" and will beep when electroporation is
complete. (Time constant should read between 5.6 and 7.0.)
5. Leave the cuvette at room temperature for 5 minutes and
then plate the cells at an appropriate density (up to 2 x 107
cells/100 mm plate or 6 x 106 cells/60 mm plate. DO NOT EXCEED THIS
DENSITY, since G418 takes 3-4 days before killing starts and plates
will become over-confluent.). If G418 selection is to be applied, this
will be done 24 hours post-electroporation. (NOTE: G418 concentration
must be titrated for every batch.)
6. Refeed the plate(s) with fresh media + G418 every day
for the first 6-7 days (until colonies are visible and most cell
debris has been removed). If using FIAU (0.2 uM) selection, this
may proceed simultaneously.
7. The typical yield for RV4.0 is up to
104 colonies/107 cells/100 mm plate. Other constructs will deviate
significantly (and unpredictably) from this yield.
8. Colonies may be picked as early as 8 days, are best around
10-11 days, but may be recovered up to 18-21 days after the electroporation.
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