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clear bands on agarose gel but problems in melting curve after qPCR


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#1 cansy

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Posted 04 August 2011 - 05:19 AM

Dear all,

I am trying to optimize a primer pair in qPCR but I've problems in melting curves. I have run the products on agarose gel and they seem clean but there some weird peaks in melting curve. I am attaching the images of agarose gel and melt curve. Here I have used 3 different cDNA dilutions and a no template control.
What could be the reason? Any suggestions to solve my problem?

Thanks :)

Attached Thumbnails

  • agarose gel.jpg
  • melt curve.jpg


#2 Telomerase

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Posted 04 August 2011 - 08:49 AM

Looks like a primer dimer problem. The unspecific stuff has lower melting temperature than your product, so it should be smaller. You run the gel too low though. To check what's happening, I'd run agarose gel again, only this time actually leave some space under your band.
Anyway, you might not even see it on the gel if it's not much.
As for PCR, I'd dig for higher temp. of annealing and/or less magnesium. If it does not work, primers are crap.

Edited by Telomerase, 04 August 2011 - 08:51 AM.

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#3 cansy

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Posted 04 August 2011 - 11:25 PM

Looks like a primer dimer problem. The unspecific stuff has lower melting temperature than your product, so it should be smaller. You run the gel too low though. To check what's happening, I'd run agarose gel again, only this time actually leave some space under your band.
Anyway, you might not even see it on the gel if it's not much.
As for PCR, I'd dig for higher temp. of annealing and/or less magnesium. If it does not work, primers are crap.



There was another gel photo in which bands are not close to the bottom , but still no extra band was observed. It seem as a sharp clear PCR product. I will try higher temperature but I cannot decrease the magnesium concentration because the Sybr mix that I'm using has a fix magnesium concentration.

Thnaks:)




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