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High firefly luc activity in EMPTY pGL3 Basic vector - (Nov/06/2007 )

Hi,

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! smile.gif

-Snowbird-

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.

-genehunter-1-

One has to normalise the data and look at the data not merely luc reading......

-newarray-