pGL3 problem - HELP!!!! (Jun/15/2008 )

Hello Everyone,

Here is my problem. I successfully cloned my promoter of interest (using restriction sites to MLUI and BGLII) into the pGL3 basic vector, maxiprep etc..., transfected A549 cells, and performed luciferase assay. I did not get any luciferase activity and after talking with the promega rep discovered that I had included too much of exon 1 into my construct. Specifically, I included the ATG start site for my gene and that resulted in no translation of the luciferase protein. I went back and redesigned primers just upstream of translational start site and now I am unable to amplify the promoter from genomic DNA with the new primer. I have varied every condition to no avail. At best, I can get a faint band/smear where I am supposed to but I have tried to digest and ligate this and I get no positive colonies. HELP!!!! please.

Check your primer design with the tools at IDTDNA.com for hairpins and primer/dimers. The problem almost certainly is in the design of your primers. Design several versions and try them all. They are cheap compared to your time and grief. Try automated design tools such as Primer3.

Thanks phage for responding so quickly. I use Primer 3 for designing my primers and I check them for secondary structures. Part of the problem is that i have to start at a specific sequence (+1 in relation to the translational start site) if I am to include all of the 5'UTR. This sequence is less than optimal for designing primers (it blasts to several other genes and there are some secondary structures). My original primer sequence was at +42 and very specific to my gene and worked great, however as I said in my first post, this includes an ATG site that messes up luciferase translation. Do you think I should just forget about the 5'UTR and start further upstream in order to get a better functioning primer? If I do this, will it affect my getting publishable data? thanks again.

if I understand correctly, you want to clone a smaller fragment of the one which you already have, so I would just do the PCR on the pGL3 plasmid you already have available. PCR on genomic DNA, especially in GC regions typical for promoters, can be quite tricky. Using a plasmid gets rid of all the other potential binding sites in the genome.