Lipofectamine Mediated Knockdown

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  • Cells in a 12 well dish
  • Lipofectamine 2000 (Invitrogen cat# 11668-019 for 1.5mL)

Required Amounts

  • Calculate RNA to transfect (typically 40 pmol per well)
  • Lipofectamine(in uL) = RNA(in nmoles) * 50
  • OptiMEM(in uL, equal for DNA and Lipofectamine) = Lipofectamine(in uL) * 50

Protocol (6 well dish)

  1. Plate cells the day before from a confluent dish diluted to 1:12 into DMEM/FBS with no PSG
  2. For each well prepare, scalin up as required:
    1. 100uL OptiMEM plus 40 pmoles of RNA.
    2. 100uL OptiMEM plus 2 uL of Lipofectamine.
    3. Incubate ~5 minutes at room temperature.
    4. Combine the two volumes of OptiMEM/DNA and OptiMEM/Lipofectamine (should be 200uL/well).
    5. Incubate ~20 min at room temperature.
  3. Add DNA/Lipofectamine complexes (~200 uL) to the 1 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