PCR: hot start

[Helge Weissig]

At 01:00 PM 04/21/94 -0700, Basavaraju Shankarappa wrote:
>> 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
>> step?)
>>  All the best,
>>    Roland
>
>        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 mo=
re
>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.
>Raj Shankarappa
>bsh at med.pitt.edu

I am using this method with success since a while and do agree with Raj.
It is not only the short time the tubes take to heat up that makes this=
 method a very good alternative to a 'pure' hot start! Since your template=
 is most likely double stranded (mine is anyways :)) there will be no=
 annealing of the primers before the template is denatured anyways. So the=
 only thing you need to worry about might be some nuclease activity of=
 'weird' Polymerases like Pfu :)

cheers,
Helge

>
['snip']



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Helge WEISSIG                              e-mail: helgew at ljcrf.edu
La Jolla Cancer Research Foundation        phone: (619) 455 6480 x253
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