ELECTROPORATION OF EUKARYOTIC CELLS
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This protocol uses a Bio-Rad Gene Pulser II apparatus to electroporate DNA into human cell lines. The cells should first be centrifuged, then resuspended in fresh medium without fetal calf serum for electroporation.
- Chill the washed cells and the electroporation cuvette on ice.
- Rinse the cuvette in 70% ethanol, followed by a rinse in PBS with 20 mM HEPES, pH 7.2.
- Add 1 x 10
6 cells in a 0.1 m
l volume, or 5 x 10 6 cells in 0.25 ml to the cuvette.
Add DNA (about 50 µg) to this cell mixture.
Set the Gene Pulser apparatus for 960µF. Set the Pulse Controller to 200 O. With MOLT-4 cells and addition of media immediately following the pulse, the following results were obtained:
Voltage Time constant Notes
0.2 kV
48 Large cell clumps; dead cells.
0.16 kV
46
Many clumps, not as large as those with 0.2 kV.
0.15 kV 45
Lot of fine, particulate clumps of cells.
0.1 kV
45
No clumps of cells
0.1 kV 49
Few small clumps
0.05 kV
63
Very small clumps; cells appear healthy.
These settings must be empirically determined for each cell line. For example, Camille Anderson reports that 250 V works fine for the Jurkat T
cell line. The cuvettes may be recycled by soaking them in bleach for a few minutes, washing extensively with tap water, then in double distilled water, and 70% ethanol. Allow to dry. Do not use acetone or autoclave.
Send comments and updates to
Dr. Bart Frank,
Arthritis and Immunology Program, OMRF
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Copyright 1993, 1996, and 1997 by Mark Barton Frank, Ph.D.
Proper citation for data acquired from this document is: "Frank, M. B. Electroporation of Eukaryotic Cells. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~f
rank/eporeuk.html). 1997. Oklahoma City. Revision Date: October 2, 1997."