about nested PCR - (May/16/2006 )
For nested PCR, first I should design some generic sets of primers for the first round of PCR. But I am wondering the word"generic (universal)". Does it mean the primers do not contain CpG "C"s---namely BSP primers?
I got very clear and bright bands using DNA from cell lines in MSP after only one round PCR (not nested PCR). I don't think I need to do nested PCR. Is it right?
But I got very weak PCR bands using human sample DNA, Is it necessary to do nested PCR because the main reason for nested PCR is low concentration of template DNA and weak PCR products?
Thanks for your reply.
In short to your questions:
No, you are right if you see a band it's not necessary to do a secoond round.
Maybe, for MSP you want to detect a band and if you can see it after 1 cycle that is more than enough.
for human samples, maybe the DNA is too little. I have to design BSP primers fist for nested PCR.