Electroporation of Mammalian cells
Dean Danner Lab
djdlab at bimcore.emory.edu
Wed Mar 30 12:13:03 EST 1994
In article 3eu at mercury.hgmp.mrc.ac.uk, jslingsb at crc.ac.uk (Mr. J.H. Slingsby) writes:
>We are interested in electroporating HepG2 cells. Has anyone successfully
>transfected constructs into these cells? If so any guidance regarding
>preparation of cells for electroporating, as well as which parameters
>(voltage, cpacitance, resistance) might be used will be appreciated.
>There appears to be no reference in the literature to successful electro-
>poration of HepG2. If there are any refs. then this will suffice.
>Mr. J Slingsby and Dr. A K Vaishnaw
>E-mail jslingsb at uk.ac.mrc.hgmp
I have successfully electroporated HepG2s in a BioRad Gene Pulser.
- Isolate cells. Two confluent T150s will provide enough
cells for about 12 transfections. Rinse twice
in PBS, Trypsinize, wash with media (w/ 15% FBS) to
inactivate trypsin, resuspend in .2ml X (# of rxns +1).
- Set up reactions. add 200 ul of cell suspension to cuvette
containing 10 ug circular plasmid. (I use 1 ug of
B-gal control, 2 ug of plasmid of interest and 7 ug
of carrier plasmid)
- Zap. My best conditions are: .25kV, 960uF. Let sit in
cuvette for 15 min, add to 8ml fresh media (I use
60mm dishes for this). This yields time constants from
high 40's to mid 50's.
- Harvest after 36-48 hours
Notes: all of this is done at room temperature.
I have only used this for transient transfections with
CAT plasmids, but I don't see why it wouldn't
work for stables.
Good luck. If you need more details, e-mail me!
Department of Genetics and Molecular Medicine
djdlab at bimcore.emory.edu or
tsitler at gmm.gen.emory.edu
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