5 replies to this topic

[blue_lotus]

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?

[pcrman]

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

[blue_lotus]

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

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

are you suspecting that renilla is quenching firefly signal?

[pcrman]

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

[blue_lotus]

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

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

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.

[zodiac1505]

Maybe you could try using the dual luciferase system from Promega