Two round MSP with the same primer - (Nov/13/2005 )
As we know that after the bisulfite reaction,DNA is degraded by about 80%!Two rounds of PCR is ideal .Is it OK to run two rounds of pcr using the same MSP primers only when you cannot find the right universial primers(no CpG "Cs" in primer?If not ,why?Please help me!Thank you very much!
like with conventional PCR, performing two rounds of PCR at 30 cycles each with the same primer could lead to disaster!
if your primer set misprimes in the first round, the second round will also misprime and amplify spirious amplicons.
hemi nested PCR is the next best option, I have tried this for bisulfite PCR and seqeuncing I am not too sure the same can be said for MSP.
It would be better to have a universal set of primers that ampliufy your region of interest which you can then use in the second round for both your M and U primer sets. I would not fully rely on primer picking programs as they can be too stringent, try picking some by eye.