pGL3 transfection and assay - tips for improvement? - improve on low luciferase values?? (Jun/02/2006 )
I am currently trying to optimise transfection of cells (Ht-29 and MCf-7) using Lipofectamine 2000 or plus with pGL3 promoter.
I seed 1million cells in a t25 (small) flask, transfect using protocols that give 30% transfection (by gfp) and assay the luciferase values the next day. However, i have found that i have to use less pGL3 than pGFP (toxicity issues).
I consistently get low (1- 100) luciferase values - any suggestions?? Has anyone got an exisiting protocol that works?? Would it be better to assay after 48 hours??
Measure luciferase atleast after 48 hr, might be better after 72 hrs. It takes like 48 hrs after transfection for protein production to peak.