Lipofectamine Plasmid Transfection
From Bridges Lab Protocols
- Cells in a 6 well dish, plated and at >90% confluence
- Lipofectamine 2000 (Invitrogen cat# 11668-019 for 1.5mL)
- Calculate DNA to transfect
- Lipofectamine(in uL) = DNA(in ug) * 2.5
- OptiMEM(in uL, equal for DNA and Lipofectamine) = Lipofectamine(in uL) * 25
Protocol (6 well dish)
- Plate cells the day before so that they are at 90-95% confluence in 2 mL media without antibiotics (DMEM with 10% FBS, no PSG).
- For each well prepare:
- 250uL OptiMEM plus 4 ug of DNA.
- 250uL OptiMEM plus 10 uL of Lipofectamine.
- Incubate ~5 minutes at room temperature.
- Combine the two volumes of OptiMEM/DNA and OptiMEM/Lipofectamine.
- Incubate ~20 min at room temperature.
- Add DNA/Lipofectamine complexes (~500 uL) to the 2 mL of media on the cells
- Gently rock plate to mix
- After ~4h aspirate media and re-feed cells with normal media. (It is usually possible to leave overnight).
Protocol adapted from Product Manual