I have a strange problem.
I use a Firefly plasmid with the promoter and Renilla- TK promoter for normalization.
I want to test the effect of a transcription factor on this promoter (and I do have a positive control as well), when I transfect the cells only with the positive control and the test plasmid, I take a very beautiful curve for my positive control, with fold activation Firefly/renilla ~2. But when I do an experiment where I co transfect cells with the control and the test plasmid, even in the samples that I have as control (only +plasmid) I do not take this fold activation and it is like the negative control with no plasmid at all.
What I have observed by analyzing the graph of Firefly or renilla alone,
In the first experiment renilla graph follow Firefly, as one graph except that firefly it is much higher than renilla (is this normal?)
And in the second one, these two graphs are not so much similar and are very close to each other even in the positive control where I should, at least see the same activation as the first experiment.
Does anyone have an idea what It is wrong, please?
Luciferasse assay question
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