ELECROPORATION OF EUKARYOTIC CELLS

Serum-free electorporation protocol:

Harvest cells (1000rpm, 5 minutes). Wash cells in 1X PBS (calcium, magnesium free) two times to eliminate serum. Resuspend cells in 400m l PBS at a cell density of 5-7 x 10⁶/ml.

Transfer 20m g DNA (supercoiled for transient and linearized for stable transfection) in a volume of up to 100m l PBS to sterile electroporation cuvettes (4mm).

Add 400m l cell suspension to electorporation cuvette and mix by pipeting up and down several times. The final volume within the cuvette should be 500m l.

Incubate the cell/DNA mixture on ice for 10 minutes prior to electroporation..

Dry the cuvette, place in electroporator and pulse at 960m F, 200-260V, and infinite internal resistance. For optimization of the voltage, use 20 volt increments in the range of 200-300V.

Keep the electroporated cells at room temperature for 10 minutes before replating.

Plate the cells in 5-10ml of complete medium by rinsing the cuvette twice with medium.

Harvest cells or begin selection after 14-24 hours.

Alternative protocol:

Harvest cells (1000rpm, 5 minutes). Discard medium and resuspend cells in 400m l complete medium at room temperature to obtain a cell density of 2-5 x 10⁶ cells/ml..

Transfer DNA (supercoiled for transient and linearized for stable transfection) in a volume of up to 40m l to sterile electroporation cuvettes (4mm). For optimization of voltage, use 10m g of pCMVb (Clontech) and 10m g of carrier plasmid DNA. For reporter assays, use 10m g pCMVb and 10m g of reporter plasmid. For stable transfections, use 1-10m g of linear DNA.

Add 400m l of cell suspension to electroporation cuvettes and mix by pipeting up and down several times.

Place the cuvette in electroporator and pulse at 1050m F, 260V, and infinite internal resistance. For optimization of the voltage, use 20 volt increments in the range of 220-300V.

Plate the cells immediately after pulse in 5-10ml of complete medium by rinsing the cuvette twice with medium. Harvest cells or begin selection after 14-24 hours.

Ref: Baum, C., P. Forster, S. Hegewisch-Becker, and K. Harbers. (1994). An optimized electorporation protocol applicable to a wide range of cell lines. Biotechniques 17: 1058-1062.

Jackson's Laboratory | Protocols | Reagents