The following protocol is given with amounts for a 12-well plate format. Use Table I to adjust the reagent volumes for other size plates.
- Plate cells in complete growth medium containing 10% serum and no antibiotics/antimycotics at an appropriate density, so that they will reach 40-60% cell density at time of transfection.
- Add 1.25 µl TransPass R2 solution A to 0.6 ml serum free medium (DMEM high glucose medium) and mix thoroughly, add 2.5 µl TransPass R2 solution B and mix thoroughly.
- Add 0.1-1 µl siRNA1 to the diluted transfection reagent mix gently the tube and incubate for 20 minutes at room temperature to form the transfection complexes.
1 For a stock of siRNA at 10 µM, the final concentration of siRNA will be 2.5-25 nM.
- Wash cells once with serum-free medium.
- Aspirate the culture medium from the cells and immediately replace with the transfection mixture. Evenly disperse the siRNA complexes by gently rocking the plate.
- Incubate the cells for 2-4 hours. Add 1 ml of complete medium containing serum per well and incubate at 37°C overnight.
- Replace the culture medium and incubate 24-72 hours before assaying target gene inhibition.