No product/primer dimers after BSP - (Aug/16/2005 )
I am attempting to replicate the work done by a previous lab member and am using her protocol (which is similar to all those I've read on this site), and her primers, all of which worked beautifully for her in the past. However, when I run the PCR, I get very strong primer dimers and no detectable product, nested or single-round. I have used the Chemicon kit in addition to her protocol and the MethPrimer program to design new primers, and still only see dimers after PCR. The only thing I can think of is that my template is the problem- I extracted genomic DNA using Qiagen's genomic extraction kit, and performed diagnostic digests that seemed normal. I did, however, keep the DNA at 4 degrees for about a month, but still have a respectable OD reading. According to a primer-dimer prediction program, the dimers are only about 1.1kcal. Any ideas about the problem?
buckie....more often than not I really think it is the primer design, methprimer doesn't pick optimal BSP primers.
do the primer dimers also appear in the negative water control? if so the primers aren't optimal for the annealing temperature set and you may need to increase the Tm.
can you post your sequence and primers to the board?