I am having some trouble cloning and wondered if anyone on this forum would be able to offer me some advice.
My aim is to clone a conserved enhancer element into a yeast 1-hybrid vector - I need three identical copies in the same orientation. I have achieved this in the past by blunting in a PCR product, checking insert size by PCR and then sequencing; with an unmutated enhancer element, this worked on the first or second attempt and all inserts were in the same orientation.
However, here's the problem bit - I now want to mutate a putative transcription factor binding site and re-make the construct. I can't do site-directed mutagenesis on the construct described above, since the primers will bind to each insert, so I've tried to do it by mutagenizing the enhancer element and blunting in as before. Myself and a colleague have tried this quite a few times now and we cannot ligate in the mutated enhancer. So we have been looking at another strategy - that of adding enzyme sites on the pcr products using tagged primers. So that we can be sure we've digested out the insert we want, we topo-cloned it, amplified using M13F/R primers (to get a really long piece of DNA outside of the restriction sites), phenol/chloroform extracted/ethanol precipitated this DNA and digested using xbaI/nheI. Cut DNA was then gel-purified and ligated into an XbaI site in our vector (which was cut, gel purified and antarctic phosphatase-treated). The rationale behind this is that the xbaI site will be preserved, but the nheI site not, so we can re-cut the 1-insert vector and add another insert and so on. Positive clones can be identified by PCR to assess the direction of the inserts using one oligo inside the insert and one in the vector.
Problems however; I can get one copy in no problem, but we just don't seem to be able to get a second or third. Does anyone have a good way to trouble shoot this? Or, alternatively, is there a good method anyone knows for building a concatamer that might be more appropriate?
Any help gratefully received!
Cloning a concatamer into yeast vector
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