Abrupt LA-PCR failure?

Dr. Duncan Clark duncan at genesys.demon.co.uk
Fri Jun 20 03:12:34 EST 1997


In article <5o5qqk$2hv at cardinal1.Stanford.EDU>, John Ladasky
<ladasky at leland.Stanford.EDU> writes
>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. 


What we found when developing a long PCR mix was that low pH led to
smears and to high a pH no product. Magnesium is also verycritical for
really long PCR ie 15kb plus with lambda or 6kb+ for human genomic. A
shift of just 0.1mM from the optimum has a huge effect on product yield.
For short PCR shifting by 0.5mM was not too much of a problem. Finally,
we still have primer sets that work one day and not the next but worked
again say a week later. We never found what caused this after running
hundreds of test PCRs. It is still an enigma.

Duncan   
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
TEl/FAX 01252376288
http://www.dnamp.com
http://www.genesys.demon.co.uk



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