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  • TransPass R2: siRNA Transfection Protocol

    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.

    Protocol

    1. 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.
    2. 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.
    3. 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.
    4. Wash cells once with serum-free medium.
    5. Aspirate the culture medium from the cells and immediately replace with the transfection mixture. Evenly disperse the siRNA complexes by gently rocking the plate.
    6. Incubate the cells for 2-4 hours. Add 1 ml of complete medium containing serum per well and incubate at 37°C overnight.
    7. Replace the culture medium and incubate 24-72 hours before assaying target gene inhibition.