Abrupt LA-PCR failure?
ladasky at leland.Stanford.EDU
Tue Jun 17 19:50:43 EST 1997
In article <5o6sa8$o9s2 at ncisun1-nf0.ncifcrf.gov>,
Paul N Hengen <pnh at ncifcrf.gov> wrote:
>John Ladasky (ladasky at leland.Stanford.EDU) wrote:
>> 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?
>Don't throw everything out just yet. What you have observed is what
>Wayne Barnes calls the "bad seed" which is probably contaminating
>your pipette barrel. Have a look at the TIBS article on LA-PCR and
>the suggestions he gives. It is linked from my homepage. He suggests
>washing out the pipette with bleach, using filter tips, then starting
>over from your original template. Please let us know if it works for you.
I have always used filter tips. I guess I could also try bleaching
the pipet barrel, but from what you say, it looks like the problem is already
in one of my vials.
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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