cDNA Transfection using FuGENE 6 (Boehringer Mannheim)

1. The day before transfection seed cells in 6 well plates to achieve approx 50% confluency: 1ml RPMI + 10% FCS per well (antibiotics can be used here)
2. Make a master mix of TopFlash or FopFlash DNA (500 ng/transfection) and TKRenilla internal control DNA (100 ng/transfection). Aliquot the Top or FopFlash /TKRenilla DNA mix to separate sterile eppendorf tubes (1200ng total per tube for each duplicate transfection)
3. Dilute 12 : l FuGENE reagent directly in 188 : l serum-free RPMI (approx 6:1 ratio of FuGENE6: DNA) for each duplicate transfection, mix by gentle tapping and incubate for 5 minutes at room temperature
4. Add the 200 : l FuGENE mix dropwise onto the DNA in each tube, tap gently to mix and leave at room temperature for 15 minutes
5. Add 100 : l of the mix to each of the duplicate wells and swirl to distribute
6. 24-48 hours after transfection assay for Luciferase and Renilla activity as described above.