Internal deletions by PCR

Paul Kowalski kowalski at stanford.edu
Mon May 15 20:48:39 EST 2000


Ha! And I thought *I* was the only person to use this technique! ;)
It's always worked well for me in the past......except for right now! I've been trying
to "pcr-around-the-plasmid" of a Tet-off construct containing an IRES from EMCV, in
pRC/CMV backbone, and having nothing but trouble. I initially thought it was the
extensive secondary structure in the IRES, but that can be PCRed fine by itself- just
not the whole plasmid (or almost the whole plasmid- I'm doing this to get rid of the
neo marker, to put in puro/hygro) . I've used LTI HiFi DNAP, Qiagen Hotstartaq,
Stratagene Herculase, got long primers with a high Tm (doing 2 step cycling, 68C), have
tried using both circular and linearized (within the to-be-deleted neo ORF) template
DNAs, 10 minute 96C denaturations prior to DNAP addition, and I'm out of ideas. Any
troubleshooting suggestions or controls? Thanks in advance-
pk
--
Paul Kowalski
Postdoctoral Fellow
Genetics - Stanford University School of Medicine
kowalski at stanford.edu


"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". 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,
> Frank
>
> "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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