Using Sybr green in realtime pcr - (Mar/10/2011 )
I'm new to realtime pcr and I have been given the task of amplifying a 700+ bp cpg island and then determining its melting curve characteristics using sybr green. I have bands when I run my primers in a regular pcr w/no sybr but the moment I put it on the 7900 I get no product..terrible dissolution curves etc. The control (a 100 bp oligo) works fine in this format. I have tried sybr concentration gradients, template gradients, primer gradients, the works. Im wondering if the combination of dmso and sybr could be killing the reaction.
How about the extension time you set on 7900? Longer PCR product need more extension time