PCR Machines and refridgeration

John Ladasky ladasky at leland.Stanford.EDU
Wed Jun 11 12:36:54 EST 1997


In article <Pine.OSF.3.95.970611120626.26180B-100000 at helix.mgh.harvard.edu>,
Deb Schuback  <schuback at helix.mgh.harvard.edu> wrote:
>The only reason that I prefer a PCR machine to have refridgeration is that
>I can also use it for ligations and the random RE that is at room temp or
>below! I also agree though that keeping PCR reactions at 4 degrees when
>they are finished is a bit ridiculous. I mean hey, they've spent about 20
>minutes at 94 degrees. What could be in there?
>Debs
>
>
>On 10 Jun 1997, Alex Dobrovic wrote:
>
>> I remain unconvinced that it is necessary to bring the samples to 4oC and
>> maintain them at this temperature after the PCR has finished. There is no
[remainder snipped]

	I know that the use of a cold soak at the end of PCR predates the 
use of proofreading thermostable polymerases by several years.  However, a
colleague in my laboratory found that the cold soak was indeed useful when
dealing with Taq/Pfu polymerase mixtures.  If her samples were removed from
the cold block and left at room temperature for later pickup, she had a 
great deal of trouble performing ligations.  But if the samples were kept 
chilled, ligations proceeded normally.  My off-the-cuff explanation for
this is that Pfu's exonuclease activity (perhaps operating aberrantly, 
trimming off more nucleotides than it should) functions at room temperature 
better than any polymerase activity present in the mixture.

-- 
Unique ID : Ladasky, John Joseph Jr.
Title     : BA Biochemistry, U.C. Berkeley, 1989  (Ph.D. perhaps 1998???)
Location  : Stanford University, Dept. of Structural Biology
Keywords  : immunology, music, running, Green



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