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Long real-time Amplicon


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#1 Parc

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Posted 29 October 2009 - 07:18 PM

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)?

#2 fishdoc

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Posted 30 October 2009 - 04:45 AM

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)?




You're better off making new primers to amplify a smaller sequence within that 2 kb amplicon.

#3 Parc

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Posted 30 October 2009 - 06:50 AM

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.




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