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Protocols: ES Cell Culture - Gene Targeting - Protocols


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.


	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.