Lipofectamine Plasmid Transfection

• 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

1. 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).
2. For each well prepare:
   1. 250uL OptiMEM plus 4 ug of DNA.
   2. 250uL OptiMEM plus 10 uL of Lipofectamine.
   3. Incubate ~5 minutes at room temperature.
   4. Combine the two volumes of OptiMEM/DNA and OptiMEM/Lipofectamine.
   5. Incubate ~20 min at room temperature.
3. Add DNA/Lipofectamine complexes (~500 uL) to the 2 mL of media on the cells
4. Gently rock plate to mix
5. After ~4h aspirate media and re-feed cells with normal media. (It is usually possible to leave overnight).

Protocol adapted from Product Manual