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Using Sybr green in realtime pcr


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#1 vswilliamson

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Posted 10 March 2011 - 06:10 AM

Hi all,

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.

Thanks

#2 Harvey

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Posted 13 March 2011 - 06:25 AM

How about the extension time you set on 7900? Longer PCR product need more extension time

Edited by dave.dong, 13 March 2011 - 06:27 AM.





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