Download ELECTROPORATION OF EUKARYOTIC CELLS

ELECTROPORATION OF EUKARYOTIC CELLS
1) Spin down cells to be transfected, wash once in ice cold PBS (Mg 2+/Ca2+ free) then resuspend at 107
cells/ml and leave on ice for 10 ml (Note A).
- The condition of the cells is very important they must be growing well in log phase for the best
transfection efficiencies -
2) In a sterile hood transfer 0.5ml of the cell suspension to a 0.4cm electroporation cuvette, add the purified
plasmid DNA to a final concentration of approx. 60mg/ml
(Note B). Mix gently and stand on ice for 5 min.
3) Pulse the cells. Settings of voltage and capacitance must be optimized for each cell line. Settings of
25mF and 0.52 - 0.72 Kv have been found to work well with many cell lines and these can be used as a
starting point (Following pulsing of the cells some methods state that leaving the cells on ice for a further
10 min maximises DNA uptake).
4) Gently remove the transfected cells, with a sterile Pasteur pipette, and add to 8ml of RPMI1640/10%
FCS (or relevant growth media) in a small flask prewarmed to room temperature. Gas the cells and put in
the 37oC incubator.
5) After 48 hrs growth, spin down the cells if they grow in solution, and resuspend in RPMI1640/10% FCS
and add selective agent, e.g. G418 at 1 mg/ml or Mycophenolic acid/Xanthine at 25mg/ml and 250mg/ml
respectively (actual concentrations may vary depending on the cell type) If the cells are adherent there is no
need to remove them, just tip off the media and replace with fresh media + selective agent.
6) Change media daily during the first week (until cell death can be seen) then every 2-3 days. Colonies
should appear within 2-3 weeks.
7) Dead cells can be removed either through a Ficoll-Paque gradient or by allowing the clones of interest to
grow through. If single clones are desired the resistant cells can be plated out at a low cell density, e.g. 1-2
cells/well across a 96well microtitre plate.
Notes:
A) It has been reported that electroporation can be performed in 10%FCS/media either at 4 oC or room
temperature.
B) DNA must be sterile. The simplest way to achieve this is by ether treatment. Within the range 30 150mg/ml, the more plasmid DNA used, the higher the transfection efficiency.
Ref: Electroporation for the efficient transfection of mammalian cells with DNA.
Chu et al. (1987). Nucl. Acid Res. 15 (3), p1311-1326.
Taken from http://users.path.ox.ac.uk/~ciu/Methods/DNA/electroporation.html