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Melting curve is irregular for primer optimization

pcr primer melting curve optimization

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5 replies to this topic

#1 insulin23

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Posted 12 August 2013 - 09:37 AM

Hello,

 

I recently optimized some newly designed primers. I used different concentrations of real-time primers (900, 450, 300, 150 nM). I got Ct values around 24-27, but the dissociation curves were jagged and irregular, and did not follow the usual one peak pattern. When I ran the products on an agarose gel, I observed only one band.

 

What are the possible reasons for this irregular melting curve and how can I resolve this issue?

 

Thanks!



#2 Adrian K

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Posted 12 August 2013 - 09:49 AM

can you show a picture or anything to describe the situation?


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#3 PhalanxBio

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Posted 12 August 2013 - 10:44 AM

i suggest trying the qPCR reaction again to see if this result is reproducible.  if the gel showed only one band, then you should get a clean dissociation curve.  can you also include a positive control reaction that you know gives you a perfect dissociation curve?



#4 insulin23

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Posted 12 August 2013 - 06:37 PM

I actually repeated this experiment twice and  got the same weird melting curves. I ran another real-time pcr experiment right after using different primers, and the melting curves looked normal, so I think it's an issue with the primers that we designed to a specific promoter. I've attached a picture I took of the screen showing my melting curves for all the samples.

Attached Thumbnails

  • melting curve1.png


#5 insulin23

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Posted 13 August 2013 - 06:22 PM

Anyone come across these types of melting curves?



#6 PhalanxBio

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Posted 19 August 2013 - 10:42 AM

No, definitely haven't seen anything like that. Perhaps you should try to design another set of primers.







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