Hi, I am doing luciferase assay. I am using HCT116 and transfected by using Lipofectamine. I checked luciferase assay with only pGL3 Basic (2ug) and then performed luciferase assay according to protocol but I am having high luminance values more than 300. I checked PLB , cells without transfection and another luciferase reagent too. Thank you.
If you are talkning about relative luciferase units then 300 is not really high. It could very well be the background. You need to measure difference between signal and background and, if it is not significant, you may need to optimize transfection.
Even though pGL3-basic is supposed to give you no signal, it will show some background signal. I agree with answers from upstairs. You can reduce integration time or reduce amount of transfected plasmids.
I think you should reduce the concentration of your plasmid may be around 500 ng.
In case of HCT116, I recommend using Effectine.
And also you should increase of your luc vector amount.
Good luck!
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