Internal deletions by PCR

Philip Carl plc at med.unc.edu
Wed May 17 15:19:10 EST 2000


Hi Paul,

	I am just about to try this technique for a class I teach on cloning
methods so I'm afraid I'm in no position to give you advice.   Actually,
I was wondering if you could give me some.   I've read a lot of
techniques papers on Inverse PCR, and think I know what to try first,
but if you had a detailed protocol of what worked in the past or any
hints that you found important, I'd sure like to learn of them.  In
particular I'd like to use the technique for creating a substitution
mutation rather than a deletion.  I was planning on putting the base to
be altered about midway in a 26-mer primer.   Does that sound about
right?  I note that Frank Fackelmayer who replied to your request had a
brief note on the technique in June of 98 that I had somehow saved.  In
that note he recommende using 50 ng of template.   Now he says no more
than 1.   I don't know what caused him to change his advice, but I'll
try to find out.  

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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