Abrupt LA-PCR failure?
ladasky at leland.Stanford.EDU
Tue Jun 17 06:01:40 EST 1997
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
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