Sack: Cell Transfection (Novachoice)

Kenneth Eum (5/04/2012)

1. Prepare a master mix consisting of serum free media (SFM), Novachoice transfection reagent, and the Novachoice booster reagent
   1. In a 1.5ml tube, add the appropriate amount of pre-warmed SFM
      • 100µl of SFM per transfection
   2. Add the appropriate amount of the Novachoice transfection reagent
      • 1µl of Novachoice transfection reagent per transfection
   3. Add the appropriate amount of the Novachoice booster reagent
      • 0.5µl per transfection
2. In a new 1.5ml tube, add the appropriate amount of DNA
   • 1µg of DNA per transfection
3. Add the master mix into the 1.5ml tube containing the DNA
4. Incubate for 30 minutes
5. In the 35mm dishes where the cells are growing, remove 1.1ml of media leaving only 0.9mL of media in the dish with the cells.
6. Add the appropriate master mix + DNA prepared from step 3
7. Repeat steps 5 and 6 until all cells of interest have been transfected
8. Incubate the cells at 37°C with 5% CO2 for at least 4 hours
9. After 4 hours remove the media from the cells and replace with 2ml of the appropriate fresh media
10. After 24 hours, begin to select with selection agents
    • If using blasticidin, use 10µg/ml (50µl blasticidin in 50ml of media)
    • If using zeocin, use 250µg/ml (125µl of zeocin to 50ml of media)
    • If using geneticin, use 1000µg/ml (1000µl of geneticin to 50ml of media)