Hot Start PCR

Genome South user gsouth at genome.wi.edu
Fri Oct 2 07:20:42 EST 1992


"Hot-start" PCR is a method that generally produces cleaner PCR
products.  Take your template DNA, denature it, and, leaving it
well above the annealing temp of your primers, add your buffer,
dNTPs, primers, and Taq.  The results tend to be cleaner because
the primers don't have a chance to anneal non-specifically at
a lower temperature.

This method is really a pain to do (you pretty much have to use
a 100C heat block as your work surface), so I do what I call
"hemi-demi-semi Hot-start";  denature your template DNA, snap
cool on ice, and LEAVE it on ice while you add the PCR mix
(primers, buffer, dNTPs, Taq).  Then put it on the PCR machine
(excuse me _thermocycler_) which is _already)_ at the denaturing
temperature.  This means that the primers never get a chance
to anneal at the wrong temperature.

Joyce
jcmill at eagle.mit.edu



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