The amounts below are given for a 12-well plate format. Use Table 2 to adjust the reagent volumes for other plate sizes.
- Plate cells so they reach a confluence of 60-90% at the time of transfection.
- For each transfection well, add 1.5 µg of plasmid DNA into 0.6 ml serum-free high glucose DMEM in a microcentrifuge tube.
- Gently mix TransPass D2 Transfection Reagent tube before pipetting. Do not vortex.
- Add 1.5-3 µl TransPass D2 Transfection Reagent per tube and mix well by flicking the tube. Incubate at room temperature for 20-30 minutes to form the transfection complexes.
- Wash cells once with serum-free medium.
- Aspirate the culture medium from the cells and immediately replace with the transfection mixture. Rock the plate gently in order to evenly disperse the complex mixture.
- Return the plate to the incubator and incubate cells for 2-3 hours.
- Replace transfection medium with complete growth medium containing serum and incubate for 24-72 hours before assaying.
- Replace medium 24 hours post-transfection or as needed to maintain healthy cells.