"R. Jayakumar" wrote:
> I have a problem. I got a 1kb insert cloned into a pGEM-T vector. The
> insert contains two BamHI sites which need to be removed. Can anyone
> suggest a suitable strategy for this. The insert does not encode a protein
> and is actually an intron.
> I have already tried 1) Klenow endfilling of BamHI cohesive ends 2) Iam
> planning to remove these sites by PCR invitro-mutagenesis. Since this is a
> bit costly, It would be nice if I could get some economical means of doing
> it. Any adapters for this?
> thanking everyone in advance.
There are several possible ways to remove the sites. The choice is up to you
and depends on what you want to do with the fragment later. If I understand you
right, you don´t care so much about the actual sequence at the sites, but just
want to remove them. In that case, you can trim the ends by a nuclease before
religating them. This approach may be a problem in your particular case with
TWO sites, because the 3 fragments will religate randomly, with only a small
fraction in the right configuration. The same is true for religation after
fill-in. In a alternative approach, you can ligate the fragments in presence of
a adaptor that destroys the sites (and BamHI to cut religated fragments without
inserted adaptor). As Bam cuts GGATCC, a possible adaptor could be:
Again, however, you will have the problem of random rearrangement of the
The simplest and fastest approach is certainly in vitro mutagenesis by PCR,
using e.g. the quickchange kit from stratagene (no affiliation).