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Problem of melt curve shape for pikoreal real time PCR

Real-Time PCR

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#1 Nurul

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Posted 03 March 2016 - 11:04 PM

 I have problem with the melt-curve shape. What is the exactly problem present? The primer or DNA used? This is the melt-curve..

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  • melt-curve graph.png


#2 Trof

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Posted 06 March 2016 - 06:23 AM

Well, you say that there is a problem with the melting curve, what exactly do you mean? I see a (thick) curve that is not very narrow, especially on its left side.

Usually this kind of shape means unspecific products that have slightly lower melting point than your product. It is unlikely to be the dimers, as they are usually separated from the main peak with much lower Tm.

 

In some cases however, you can't say if those products are created within the exponential phase (and thus screwing your Ct results with nonspecifities) or this happens in the later cycles only, where it actually don't matter.

You can test that.

 

You didn't mention many details, about, what is this assay, if this happens all the time, or just with one sample or what. And for what reason you do the PCR, what do you want to measure.


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