Kenneth Eum (5/04/2012)
- Prepare a master mix consisting of serum free media (SFM), Novachoice transfection reagent, and the Novachoice booster reagent
- In a 1.5ml tube, add the appropriate amount of pre-warmed SFM
- 100µl of SFM per transfection
- Add the appropriate amount of the Novachoice transfection reagent
- 1µl of Novachoice transfection reagent per transfection
- Add the appropriate amount of the Novachoice booster reagent
- In a new 1.5ml tube, add the appropriate amount of DNA
- 1µg of DNA per transfection
- Add the master mix into the 1.5ml tube containing the DNA
- Incubate for 30 minutes
- 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.
- Add the appropriate master mix + DNA prepared from step 3
- Repeat steps 5 and 6 until all cells of interest have been transfected
- Incubate the cells at 37°C with 5% CO2 for at least 4 hours
- After 4 hours remove the media from the cells and replace with 2ml of the appropriate fresh media
- 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)
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