Electroporation transfection of mammalian cells

1. Split cells (1:2) in one 150 mm culture dish or 150 cm2 flask. Grow overnight. Cells should be 50-70% confluent next morning.

2. Harvest cells, transfer to sterile tubes. Wash with FCS-free medium, and resuspend the cells in PBS or fresh medium without FCS. Cell concentration should be ~ 1x 10 7 cells/ml.

3. Add cells to the electroporation cuvette, put on ice.

4. Add 10-100 mg DNA. Sit on ice for 10 minutes.

5. Put the cuvette in the eletroporate pulser. Electroporate with a desired setting of voltage and capacitance for your cells. See also Electroporation --- parameters for different cells.

6. Put the cuvette back on ice and allow it to sit for 10 minutes.

7. Remove cells from the cuvette with a pipette and place into 15 ml of culture media with FCS. Plate 2-5x106 cells per 100 mm plate. For establishing stable cell lines, cells should be dispersed into several plates with different folds of dilution.

8. Change to fresh medium next morning.