pGL3 Luciferase assay - need experimental set up help (Aug/20/2007 )
I do a promoter assay using the pGL3 plasmid from Promega. I cloned my potential promoter region into the plasmid and plan on cotransfecting the pGL3 reporter plasmid together with an expression plasmid coding for my transcription factor into HeLa cells. I will do a luciferase assay after transfection.
Now the problem: I got really confuced when I had to make the actual plan for transfection experiments. Especially in terms of controls of transfection efficiencies, normalization of the luciferase, background... I also have the pGL3 control vector, which contains the SV40 promoter and should show luciferase expression. Can this be used to check for transcription efficiency? Should I rather transfect a GFP expression plasmid to check for transfection efficiency? I read that people use ß-Gal. What exactly is that used for? I am pretty new to transfection experiments, so please excuse that I ask some stupid questions.
I would really appreciate some help in this case.
Thanks in advance!
we used to have a gfp plasmid for transfection control and also as a negative control (also as background) for luciferase. We would transfect pGL3-luciferase and also try to knock it down. so the gfp control served for 2 purposes.