Internal deletions by PCR
jmac_66 at hotmail.com
Fri May 12 10:59:44 EST 2000
"Frank O. Fackelmayer" wrote:
> Hi Ulf,
> We do that quite routinely, and the method that works best is what I
> call "PCR around the plasmid".
we always called this "backasswards PCR"--glad to see someone else uses it (it works,
by the way)
> The idea is to design two primers that
> flank the region to be deleted, but point in "the wrong direction" i.e.
> the other way round than those that would amplify the region in PCR.
> Perform PCR with a good proofreading enzyme (we use Pfu) and long
> extension times. You´ll get a (blunt ended) fragment of "plasmid minus
> deleted region"-size. Gel-isolate the fragment, then ligate.
> Important details:
> * primers must be 5´phosphorylated and good quality
> * use no more than 1ng of template
> * it is often useful to destroy the original template after ligation by
> digestion with DpnI (which won´t harm the PCR product because it is not methylated)
> In most cases, greater than 80% of clones are ok.
> Hope this helps,
> "Ulf S. Andersson" wrote:
> > Hi,
> > I wonder if anybody could tip me on a good and fast way of making
> > internal deletions in a plasmid by PCR. I have previously done it by
> > introducing new restriction sites by site directed mutagenesis (and then
> > digesting and re-ligating) which takes quite a long time... there must
> > be a smarter way.
> > /Ulf.
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