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Will GFP interferes with luciferase assay result? - (Mar/03/2008 )


I used an expression vector containing a Transcription Factor and a GFP cDNA. I co-transfected it with pGL3-basic which contains a promoter fragment into HeLa cells. I have included all the necessary controls, but the results were disappointing. The relative luc activity of the empty GFP vector is similar to the GFP expression vector with the TF insert.

Should I use such expression vector? Coz in my lab only have stock for it and i hv no time to prepare new.

Thx in advance.

-hkuspace graduate-

So you have an expression vecotr for the transcription factor fused to EGFP and an reporter in luc, and the co-transfection is not working.
Typically you need a reference plasmid of either beta-Gal or Rluc, how did that treatment work? This could tell you if your transfection was working.
Another thing: what was the ratio you used for the two? You need more reporter than EGFP-tf fusion vector. 2:1 to 5:1 is what I use, depending on the promoter strength.


Thx for suggestion, but should i optimize by adding:

(1) more TF-GFP expression vector & less pGL3-basic-promoter recombinat construct


(2) less TF-GFP expression vector & more pGL3-basic-promoter recombinat construct?


-hkuspace graduate-

Choose option 2, because it has a strong promoter, like CMV, while the promoter that you are studying is less active.