PCR...help

[JOHN NASH]

On 26 January '90, Johan de Boer wrote:

>Recently we are experiencing problems with our PCR reactions.
>One day the reactions work fine, the next day, if we repeat the same
>reactions (everything the same) they just don't work. It does not
>depend on the person who does it. We use a Perkin Elmer machine and Cetus
>or Pharmacia Taq and amplify an approx. 2000 bp piece. Does any one
>have ideas about the reproducebility problems more people must be
>experiencing?

We had similar problems trying to amplify a 1200 bp piece of DNA, and
the following things helped:

1.      Of great benefit (most of the time) was to heat everything,
including the overlaying oil, at 94 degrees for 5 min.

2.      We had a lot of trouble when we used NON-Cetus Taq polymerase (from
two different sources).

3.      We improved our yields by doing a step-type reaction:
        5 cycles at (94 deg/1 min; 45 deg/1 min; 55 deg/2 min);
        30 cycles at (94 deg/1 min; 55 deg/1 min; 65 deg/2 min) etc..

We had to do this because we were using two "guessimers" (23 bp with 128
mismatches), and determined the lower temp. set of conditions by doing a
Tm experiment with labelled primers.

Hope this helps,

John Nash <NUM208JN at NRCCAD.NRC.CA>