hot-start PCR

Vladimir Svetlov svetlov at oncology.wisc.edu
Tue Aug 26 09:03:47 EST 1997


In article <M.Champagne-2108971610550001 at ocarina.cellbio.duke.edu>,
M.Champagne at nospam.cellbio.duke.edu (Marc Champagne) wrote:


> I don't know why for sure, but my guess is that by putting your primers and
> template at low temperatures (0-4 C), you give those a chance to anneal more
> than at RT (22-25 C), creating more spurious bands.

Unless I've missed the indication that you are using a ss template this is
not a plausible explanation - most imaginable ds templates would remain ds
and thus unaccessible for primer annealing at 25C as they would at 4C. One
thing to consider is the temperature of the final mix as it goes into the
machine and the temperature of the block - if the block temp is not 95, or
the heat exchange is not fast enough (not a thin-wall tube, bad geometry of
the well etc.), the tube from the ice would take longer to equilibrate with
the block thus creating favorable conditions for extension of primer-dimers
and other odd things. Thus so called "simplified" hot-start (e.g. advocated
by Qiagen) - from ice into the pre-heated block - if not executed properly
might present additional problems rather then eliminate them. 
Regards,
V.



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