PCR Problems! Please help!

Christopher T. Cole colect at caa.mrs.umn.edu
Wed May 29 11:23:30 EST 1996


Holly--

I would second John Brennand's reply.  The devil is in the details.  We ran 
into a problem that superficially sounds very similar but could arise from 
totally different causes (e.g. Taq lot?).  Turned out our problem came from 
"position effects", differences in cooling & heating rates at different 
positions in the thermocycler.  Whether or not this made a difference 
depended not only on the number of samples (i.e. place) used, but also on 
the profile: more difference when the spread between denaturing and 
annealing temps was greater.  Solution was easy after we'd figured that 
out, but embarassing how long it took to understand.  Also, the 
denaturing temp sounds risky, high enough to be ruining the polymerase, 
especially if the temp varies (higher) in different positions, or with lots 
of cycles, etc.....

Best wishes.

-Chris Cole

On Thu, 23 May 1996 14:50:02 -0800, 
Holly West  <hwest at fred.fhcrc.org> wrote:

>I have been trying to do a scale-up of a rather difficult bcl-2 PCR.  
>I thought I had found the correct parameters--strong, bright bands on 
>preliminaries--but then when I did a master mix and alliquotted into 
>12 or 15 sample tubes, only 3 or 4 of the samples worked.  It is the 
>same story when I add reagents to each tube individually.  Does anyone 
>have any ideas.  I'm using 4% DMSO, and denaturing at 99.  Thanks!
>




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