Abrupt LA-PCR failure?

Gail Otulakowski gotulak at sickkids.on.ca
Tue Jun 17 13:30:49 EST 1997


John Ladasky wrote:
> 
> Hi, all,
> 
>         Lately everything I touch turns to dust.  I have designed a
> 6 kB PCR that uses a mixture of Taq and Pfu polymerase.  It has worked
> beautifully for months.  Suddenly, I'm getting high-molecular weight
> smears (3 - 30 kB).  I have re-run the reaction using the same genomic
> DNA templates that I previously used successfully, and they, too, smear.
> I have confirmed that no one has messed with my thermal cycling proto-
> col.  I have taken all of my reagents from the same vials I used before,
> even including the distilled water.  Any ideas?  Can primers fail in a
> single freeze-thaw cycle?  The problem with just throwing out all of
> my reagents at this stage is that *everything* is costly -- the Taq,
> the Pfu, even the primers.  They're HPLC purified with 3' phosphorothi-
> oate ends.  Mucho dinero.
> 
> --
> 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


I have sometimes been able to "rescue" primers which suddenly stopped 
working by heating them to 95C for 5 min and then snap freezing on dry 
ice.  I assume this treatment removes secondary structures that have 
accumulated during previous freeze-thaws, perhaps better than the initial 
denaturation step in the PCR cycle program.  Doesn't always work but 
sometimes it is quite dramatic.  Good Luck

Gail Otulakowski
Hospital for Sick Children
Toronto, Canada



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