PCR: hot start
bsh at MED.PITT.EDU
Thu Apr 21 15:00:15 EST 1994
> Hi there,
> your method is NOT a real hot start, because your polymerase is present when
> the temperature increases from ice-cold to denaturation temperature! To make a
>_hot start_ you must arrange for the polymerase _starting_ to work at above the
>extension temperature (i.e. added in middle of "denaturation" step through oil,
> or separated from main mix during temperature increase by waxy material, or ?
> ... find a trigger for polymerases to start only after an initial heating
> All the best,
I think this method comes as close to hot start as practically
possible. The time it takes for a block to heat up to 90 C is much much more
than the time it takes a sample to go from ice cold to 90 C.
So although technically it is not a hot start, I don't think it makes
much difference in practice.
I have been following this method for quite some time and unless
someone proves to me that this is so much inferior, I am going to stick to it.
bsh at med.pitt.edu
> In article <Pine.3.89.9404131147.A24938-0100000 at unixg.ubc.ca>, brunstei at UNIXG.UBC.CA (John Brunstein) writes:
> > Hello Jiri,
> > There was a thread a few months back where it was suggested that
> > ordinary petroleum jelly (aka Vaseline) would do quite well (sorry, i
> > don't remember who said they had used this). However, our lab has a
> > really simple method for getting hot starts, and I was wondering if
> > perhaps there is some glaringly obvious reason why other labs don't do
> > this as well:
> > we just set up the PCR reaction on ice and KEEP it on ice until
> > the thermocycler blocks hit 90C or so; THEN put the tubes in and go have
> > a coffee. Not only don't you have to buy gems or squish vaseline into
> > tubes but you can mix the reaction mixture yourself instead of hoping
> > convection will do a good enough job for you after the phases come in
> > contact.
> > I would be really interested in hearing other people's opinions
> > on this seemingly simple method. Is there a reason no-one else does it
> > this way?
> > On Wed, 13 Apr 1994, Mgr.Jiri Drabek wrote:
> >> Hello, netters!
> >> Please, does anybody know alternative (cheap, home made) for PERKIN-ELMER
> >> AmpliWax PCR gem (used for hot start)?
> >> Any answer will be appreciated.
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