Lipofectamine Plasmid Transfection

• Cells in a 6 well dish, plated and at >90% confluence
• Lipofectamine 2000 (Invitrogen cat# 11668-019 for 1.5mL)

1. Plate cells the day before so that they are at 90-95% confluence in 2 mL media without antibiotics, or alternatively change media before transfection to OptiMEM
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 re-feed cells with normal media.

Protocol adapted from Product Manual