Difference between revisions of "Lipofectamine Transfection of C2C12 Cells"

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(Wrote protocol from ethan's trials)
 
m (changed confluence note)
 
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==Materials==
 
==Materials==
*Cells in a 6 well dish, plated and at >90% confluence
+
*Cells in a 6 well dish, plated and at ~70% confluence. Typically transfect as myocytes.
 
*Lipofectamine 2000 (Invitrogen cat# 11668-019 for 1.5mL)
 
*Lipofectamine 2000 (Invitrogen cat# 11668-019 for 1.5mL)
 
*OptiMEM
 
*OptiMEM

Latest revision as of 16:57, 22 February 2013


Materials

  • 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

Required Amounts

  • 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

Protocol (6 well dish)

  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