This is a cached page for the URL ( To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
Go Home
  • Lipofectamine
  • Basal Medium containing 10% fetal bovine serum, 1% glutamine, 1% aa
  • Basal Medium containing 1% glutamine
  • Basal Medium containing 20% fetal bovine serum, 1% glutamine, 1% aa
  • 1. In a six-well or 35 mm tissue culture plate, seed ~2x 105 cells per well in 2 ml Basal Medium containing 10% FBS.

    2. Incubate the cells at 37C in a CO2 incubator until the cells are 70-80% confluent. This will usually take 18-24 h.

    3. Prepare the following solutions in 12 x 75 mm sterile tubes:

    Solution A: For each transfection, dilute 2 µg DNA (plasmid) in 375 µl serum-free basal medium
    Solution B: For each transfection, dilute 12 µl LIPOFECTAMINE Reagent in 375 µl serum-free medium

    4. Combine the two solutions, mix gently, and incubate at room temperature for 15-45 min. The solution may appear cloudy, however this will not impede the transfection.

    5. Wash the cells once with 2 ml serum-free medium.

    6. For each transfection, add 750 µl serum-free medium to each tube containing the lipid-DNA complexes. Do not add antibacterial agents to media during transfection. Mix gently and overlay the diluted complex solution onto the washed cells.

    7. Incubate the cells for 5 h at 37C in a CO2 incubator.

    8. Add 1.5 ml medium with 20% FBS without removing the transfection mixture. If toxicity is a problem, remove the transfection mixture and replace with normal growth medium.

    9. Replace medium at 18-24 h following start of transfection.

    10. Assay cell extracts for gene activity 24-72 h after the start of transfection, depending on cell type and promoter activity or add selective antibiotic for isolating stable transformants.