Dear Cintia

Some fragments of foreign DNA can be unclonable in E coli vectors for vaguely understood reasons (hairpins, incompatibility with plasmid origins, whatever)  One thing to test is whether the behaviour is plasmid specific - can you clone the same promoter fragment in pUC or pBR322 or some other vector?  Can you switch to a lower copy plasmid vector and rebuild the promoter luciferase construct? 

Cheers

Hi Malcolm, nice hear from you! We did not have problem for cloning our promoters in TOPO-TA

Malcolm, It seems I had a problem with my original answer to you. I am sending it again.

Hi Malcolm, nice hear from you! Thank you for your return.  We did not have problem for cloning our promoters in TOPO-TA. I believe the problem is plasmid specific. The idea is transfect mammalian cells with different haplotypes of the same promoter to measure the effect of some SNPs on luciferase expression. I liked your idea to switch for a low copy plasmid vector, but I need plasmid mass to transfect the cells. After so many trials and controls I truly believe that E. coli strains simply destroy human control regions DNA cloned upstream the luciferase, or  maybe the E. coli over express the protein, and it is toxic to the bacteria. Because we do not have problem with the control plasmid. I do not understand. I had the same problem with promoters from more than one gene. I do not want to give up, but pGL3 vector is giving us a hard time. Science as usual! 

Cheers,