Lipofectamine Plasmid Transfection

Revision as of 14:45, 13 August 2009 by Davebridges (Talk | contribs) (Protocol (6 well dish))

Revision as of 14:45, 13 August 2009 by Davebridges (Talk | contribs) (Protocol (6 well dish))


Materials

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

Protocol (6 well dish)

  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 re-feed cells with normal media.

Protocol adapted from Product Manual