High firefly luc activity in EMPTY pGL3 Basic vector - (Nov/06/2007 )
I really need some input on my ongoing luciferase assay... Right now I'm trying to optimize it, but the results are very strange.
I'm transfecting melanocytes using Lipofectamine 2000 (DNA:Lipf ratio 1:2), with pGL3 Basic as my experimental vector and phRG-TK as my control vector. When I transfect the cells with an EMPTY (it's empty, I've sequenced it...) pGL3 Basic vector and phRG-TK as control, I get very high firefly luciferase activity, between 25000-30000 RLU. As far as I've understood, the activity of empty pGL3 Basic should be really low, since there´s no promoter or enhancer in it.
What could be the problem? Has anyone similar experiences?
I'm so greatful for every input anyone can give!
I think it is the ratio of luc counts between an empty vector and a testing vector that matter the most, not the absolute value. The basic vector will have some background transcription level, although low, 100-500 typically, and that is related to how much cell lysate that you loaded to an assay.
One has to normalise the data and look at the data not merely luc reading......