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