Currently, I encountered problems with optimizing my pyrosequencing PCR primers. I tried different annealing temperatures, number of cycles, Mg concentrations, and all with or without Q-solution... nothing works and I do not get any bands on the gel.
First, I ordered the same primers without biotin label, since they do not have the best score according to the Qiagen design software (score:62, which means ) and to minimize costs. In a first gradient PCR I did obtain bands at 520C and 45 cycles, so I ordered one of the primer with biotin label. However with the biotin labelled primer I get nothing! How can that be? Could it be that the primer forms loops, so that it cannot bind to the DNA?
I found a protocol online, in which the authors suggests to add a NN-tail to the 5' end of the not-biotin labelled primer in order to minimize template loops. Does anyone here have experience with it? Or do you have other suggestions?
Thanks a lot in advance!!