PCR of supercoils

Frank O. Fackelmayer Frank at Fackelmayer.de
Fri Jul 27 05:00:14 EST 2001



"Dr. Duncan Clark" wrote:
> 
> In article <AB72A0E88719D511944A00508BDF89906C1C9A at lscmail1.na.lifetech.
> com>, the eminent Hartley, Jim at BIOSCI/MRC Human Genome Mapping
> Project Resource Centre wrote
> >I would greatly appreciate any observations or suggestions on how to obtain
> >the most product from PCR of supercoiled plasmids.    Any side-by-side
> >comparisons of enzymes or conditions would be most helpful.  Thanks.
> 
> There was quite a long thread on the problem of PCRing supercoiled
> plasmids in the 7700 listserv. Maybe it is worth checking the archives.
> The basic argument, if I remember correctly, was that it should not be
> possible due to the inability to 'unwind' the plasmid.

in theory i would agree. Anyway, it works well in most cases. This does
not say that the yield will be identical in all cases, but at least in
the cases where we have directly compared the results with supercoiled
vs. linearized plasmid we did not find a significant difference.
or, to say it in other words: linearizing a plasmid has never helped us
to get a PCR working that didn´t work with supercoiled plasmid.


> It was argued
> that the reason people can PCR off miniprepped DNA is that there is
> sufficient nicked plasmid in the preps. 

I don´t think so. We often successfully use CsCl purified DNA with no
significant amount of nicked or linear plasmid as a template. Of course
even such preps are never completely free of nicked plasmid, but if
those little amounts were sufficient to allow for an amplification,
noone should ever have problems in amplifying from "supercoiled" DNA, right?


Frank




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