Nanodrop question - Spectrophotometrical measurment of bisulfite treated DNA (Nov/30/2006 )
sorry, I just saw that I have written 65°C as annealing temperature - that surely was wrong, I ment 65°C as elongation temperature - annealing should be like Nick has said. Sorry for that...
Nick, I generally agree with you on primer design, but in this case, I use the same primers and have never seen a C outside a CpG in the sequence... This should indicate full conversion, shouldn't it?
Should I set elongation temperature to 65 C just for 2 round of PCR (nested PCR)? How about first round? Should it be 72 C?
PS (to moderator): I apologise because discussion has gone outside of initial topic (nanodrop)
I tried it for both rounds, but it seemed necessary only for the second round? Doesn't make too much sense, I know, but it works well for me. For the reelin primer; I use 55°C as annealing and 72°C as elongation for the first round PCR, cycle conditions following Nick's protocol. The second round is slightly different due to the modified primers.
Thanks for the tips Krümel.
if you are seeing full conversion with your primers then it should be okay. I wouldn't change anything.
I am pretty relaxed here, doesn't matter to me if you go off topic.