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qPCR wide "peak" starting the ramp

qPCR RT-PCR HELP PRIMER peak

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#1 silence_remains

silence_remains

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Posted 03 March 2014 - 01:23 PM

We have been standardizing a primer for pri-miR132 quantitation, using real time two step sybrgreen PCR. We are obtaining a peak that seems to be the product, efficiency of 1.02, Rsq 99. However, the melting analysis shows a wide peak from the beggining of the ramp at 59C and decreases towards 67C, with every sample (see attachment). Could you share your opinion on what could be happening? We fear that the size of the product (50bp) might be ruining things for us. We are using a 9 point efficiency curve, with serial dilutions with a factor of 1/2 up to 1/256.

 

Thanks everyone...

Attached Thumbnails

  • melting primiR132.jpg

Edited by silence_remains, 03 March 2014 - 01:25 PM.






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