Long real-time Amplicon - (Oct/29/2009 )
I have experience using the Power Sybr Green 1-step RNA to Ct kit, but the optimal target length is ~50-150 bp and it is unable to handle long targets >a few hundred bps. Does anyone know of a kit or procedure that will allow me to quantitate much longer amplicons (~2 KB)?
Parc on Oct 29 2009, 10:18 PM said:
You're better off making new primers to amplify a smaller sequence within that 2 kb amplicon.
I'd do that if my goal was to solely quantify the amount of starting product, but I'm actually looking to evaluate primer efficiency to make sure that the primers I am using will adequately amplify a mixed population of viral RNA, a portion of which may have some nucleotide mismatches with the primer pair.