Abrupt LA-PCR failure?
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
Hospital for Sick Children
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