Luciferase signal - (Jun/04/2010 )

I am having trouble with luciferase assay for last couple of months. I am transfecting PGL.3-TATA vector with different inserts of my interest. I did transfection using Lipofectamine. While I am getting very high signal of Renilla ( between 5000-20,000) , my luciferase signal is incredibly low ( in the range of 50-400). I checked my DNA. It does not have nuclease activity. There is no cell death as well. Any suggestions to overcome the problem?

How about you use less amount of renilla vector than firefly in transfection?

pcrman on Jun 4 2010, 11:03 PM said:

are you suspecting that renilla is quenching firefly signal?

I mean your renilla luc vector is producing strong signal. What renilla vector are you using?

pcrman on Jun 5 2010, 06:38 AM said:

yes, i would like to decrease Renilla signal, but at the same time i would like increased luciferase signal. I think higher renilla signal can quench the signal from firefly luciferase. Is it a possible reason for my low luciferase signal?
I am using pRL vector.

Maybe you could try using the dual luciferase system from Promega