Internal deletions by PCR

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Tue May 16 11:01:19 EST 2000


Hi Paul,
I also got some problems lately, very weak amplification and no clones
at the end. I re-ordered the primers, and the problem disappeared... 
There is one other thing you could try, adding PCR "enhancers". I had
good success with 2-5% DMSO or 2% formamide. In one case 5% DMSO was the
only way to get the product, and it cloned just fine then. 

Frank


Paul Kowalski wrote:
> 
> 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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