I am trying to design BSP primers for a region with a high CG content, it is almost impossible to avoid CGs in regular sized (18-22) primers. I have designed 2 primers outside the region of my interest, both primers are CG free but they are 16bp long and there are A and T runs. For the nested primers, I could not excluded CGs (one CG) in forward but reverse is ok but again the problem is forward primer 17bp, reverse primer 16bp long. I was wondering has anyone tried to use BSP primers that short and what do you think whether will it be specific since I am using nested primers.
I also tried to blast my primers but methblast is under maintenance, so I found a web site http://bisearch.enzim.hu/ for blast. But I was expecting my 1st round of PCR to produce a 922bp product, the result gave a lot of smaller products and not located in the region of my interest. Has anyone used bisearch.
I am giving the primers below, it is really important for me I could appreciate any feedback, thank you.
1st round F (5-3) :GTGTAGGTTTATGTGG
1st round R(5-3) :AATAAATCAAAAACCC
2nd round F (5-3): GGAGAGCGTTTTTTGAG
2nd round R (5-3): AAAACCCAACCTCTCC