Materials:       

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.