NUM208JN@NRCCAD.NRC.CA (JOHN NASH) (02/24/90)
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@NRCCAD.NRC.CA>