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Melting curve is irregular for primer optimization - (Aug/12/2013 )



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?




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

-Adrian K-

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?


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 Image


Anyone come across these types of melting curves?


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