Abrupt LA-PCR failure?

John Ladasky ladasky at leland.Stanford.EDU
Tue Jun 17 06:01:40 EST 1997


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



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