1. When I did normal PCR with them at 58 degree annealing, there was a clear band in no-template-control, the same size (160bp) as bands in my Chip sample and input. It must not be problems about other reagents such as water or PCR mix because there were not bands in no template controls of other pairs of primers. So I thought the primers might be contaminated and I reordered them. But when I did PCR with the new primers, the band was still there. The more interesting thing is, when I increased annealing temperature to 60, the band in no-template-control disappeared while my Chip sample and input bands still remained. I can't understand.
2. When I did qPCR with these two primers, the issue became simple. Whether I chose 58 or 60 annealing, the no template control always gave a Ct around 28-32 and a single dissociating peak at the same site as those of my Chip sample and input.
What happens in hell on my primers?! They made me go insane. I do hope somebody can give me an explanation. I appreciate it very much!
Edited by yg_eagle, 09 June 2009 - 09:27 PM.