Lipofectamine Transfection of C2C12 Cells

• Cells in a 6 well dish, plated and at ~70% confluence. Typically transfect as myocytes.
• Lipofectamine 2000 (Invitrogen cat# 11668-019 for 1.5mL)
• OptiMEM

• Calculate DNA to transfect. Start with 4 ug per 6 well
• Lipofectamine(in uL) = DNA(in ug) * 2.5
• OptiMEM(in uL, equal volume for DNA and Lipofectamine) = Lipofectamine(in uL) * 25

1. Plate cells and grow to ~70% confluence in 2 mL media without antibiotics (DMEM with 10% FBS, no PSG; use 1 mL for 12 well plate, and use half the amount of DNA)
2. For each well prepare one tube for DNA and one tube for Lipofectamine:
   1. OptiMEM plus required total amount of DNA.
   2. OptiMEM plus required total amount of Lipofectamine.
   3. Incubate ~5 minutes at room temperature.
   4. Combine the equal 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).
6. Examine the cells the next day.

Protocol adapted from Product Manual