BamHI/XhoI ligation - (Jan/10/2009 )

Hi! I'm having problems with a ligation using BamHI and XhoI.
I'm using a plasmid that has an insert between BamHI and XhoI sites, I cut the vector sequencially with both enzymes and I see this insert is liberated. Then I gel purified the vector. I cut a pcr product with both enzymes and I ligate it to that vector. All the cuts are checked and gel purified and they seem to be ok.
The problem is that I have low efficiency in the ligation procedure. It seems like the vector is religating in the sites where it is cut by both enzymes, I've seen it in the sequence. The sequence is OK up to the middle of the BamHI site. Then, I can see the region of the sequence that comes after the XhoI site, but the XhoI site is not there. I know BamhI has star activity but that doesnt explain at all what is happening. Has anybody seen something like this before?

Hi

I have had problems cloning PCR products that are dircetly digested and used for ligation. In these cases, I just phosphorylate the PCR product and perform a blunt ligation in Eco RV digested pBS or Stu I/ Sna BI digested pLitmus28. Then I pick up the insert from these clones and ligate in teh desired vector. For soem reason it works very well.

Best
TC

Thank you for your answer. I've already tried cloning the pcr product in a p-GEMTeasy vector, but I didn't get better results. I think that the problem is how the enzymes are cutting the vector, or maybe the sticky ends are damaged during the process of purification, but I don't know if that is possible.

Hi

Get yr p-GEMTeasy clones sequenced. Maybe there is a problem in oligo synthesis. Before that, just digest yr p-GEMTeasy clone with both the enzymes individually. If you see d fragment release in any of the sinle enzyme digestions, tht means that one of the sites is not present due to bad oligo synthesis and u see the fragment release due to another site in vector on the opp site of the gene cloned. Like if u hav a Nde I site at bp, there is another Nde I after yr gene and in teh vector. I have faced this problem and had to order fresh oligos to get a clone.

Best

TC