Problems in my Multiplex PCR - (May/23/2002 )
i had a problem about multiplex PCR my result showed nonspecific bands . My work had five primers in one single tube and amplified for only one band but it show non specific bands, so I need to knew additive or optimized condition for PCR for specific product oh,my primers have varies Tm. Now I use annealing temperature is 55 C. Thanks for answer.
Are you sure you haven't contaminations?
If not, try to add 10% DMSO and i also do the "hot start" before the reaction: 5' at 80°C with all reagents except taq and template, then you'll add template and taq
you check your primer concentration and template amount my multiplex rect. 5pmol primer and 50ng template amount best result
Try increasing the annealing temperature.
Be sure that your primers target a very conserved areas of the sequence of your interest. Do a Blast with your primers in order to see where else they could anneal.
Second, try gradient PCRs in order to find the most appropriate annealing temp for your primers. Try to avoid the lower temps, since they favor non specific binding of any primer.
Third, try any hot start enzyme (e.g. HotStarTaq from QIAGEN), since adding the taq and template after the incubation of 80C may promote ideal conditions for contamination.
I dont even tried this before, but why you dont try a touchdown PCR approach?