Hot Start PCR
kim at m44.unm.edu
kim at m44.unm.edu
Fri Oct 2 16:58:10 EST 1992
In article <1992Oct2.122042.15605 at genome.wi.edu>, gsouth at genome.wi.edu (Genome South user) writes:
[Stuff on hot start pcr deleted]
>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.
Hot starts are also done by creating a physical barrier between the primers and
template. This barrier is created by putting a half-reaction mixture (buffer
and primers) into the bottom of the tube and melting wax over the mix. The wax
used can be "PCR Gems" from Perkin-Elmer/Cetus or any number of home-grown
waxes described in this group (e.g. paraffin or Paraplast). After the wax
barrier hardens, put the other half-reaction on top (template, buffer, and
enzyme). Put the tube into the thermocycler and run. As the machine brings
the mix up to denaturation temperature, the wax barrier melts, allowing the two
half reactions to mix only when the temperature has gone above the melting
temperature of the wax. From this point on the PCR cycles proceed normally.
This obviates the need to pre-denature the DNA and watch over the reactions.
In addition, the wax barrier hardens after the cycles are complete and the
temperature of the tube goes to room temp or 4 C. It is fairly to recover the
sample from beneath the barrier with a minimum of contaminating wax.
Perkin-Elmer/Cetus would probably be happy to send literature describing the
Hot Start technique as it pertains to PCR Gems. Several posters (?) described
successful use of alternative waxes to the gems, so there doesn't seem to be
any mystery about them.
KIM at FLOVAX.LANL.GOV
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