Lipofectamine Mediated Knockdown

Materials

• 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