Standard Operating Procedure:

Protocol for DNA transfection

Hazards:

Protection:

Wear gloves and lab coat throughout this procedure.

Waste:

 Bacterial waste should be treated with10% chlorox before disposal.

Spill Clean-Up:

Procedure:

F Cell preparation

If the cells are adherent, use Trypsin/EDTA to get them in solution. Use 0.5 ml Trypsin/EDTA per petri dish. Place at 370C for five minutes and add 9.5 ml of RP10. Take an aliquot to count. Spin 5 minutes at 1000 rpm and resuspend at 10 x 106/ml in OPTI-MEM media. If the cells are not adherent, take an aliquot to count, spin 5 minutes at 1000 rpm and place at 10 x 106/ml in OPTI-MEM media.

F Electroporation

Use 0.4 cm Cuvettes that have been sterilized (60 minutes in the irradiator). Place DNA in cuvettes. I use 25 mg per transfection. If I need to co-transfect with GFP I use 5 mg of GFP per transfection. Add 500 ml of cells (5 x 106). Let sit at room temperature for 10 minutes. Use Shimizu’s electroporator to zap the cells: 320 Volts, 5 mSec pulse length, 2 pulses. Let recover at room temperature. Add complete media to cultures. The cells can either be placed in petri dishes or 6 well plates. GFP is easier to see in 6 well plates. If doing a stable transfection, do not add antibiotic media until two days after electroporation.

F Clean up

Rinse the cuvettes with water and then with ethanol. Then rinse one more time with water and let dry ON. Irradiate the cuvettes as needed.