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2 peaks for identical sequences in HRM

HRM melting curve Tm melting peaks melt-curve

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#1 itsron

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Posted 08 April 2015 - 10:11 PM

Hello,

 

I have 2 100% identical sequences but when doing an HRM, I am getting 2 different peaks! How is this even possible?

 

Any ideas?

 

Thanks!



#2 dpo

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Posted 09 April 2015 - 02:19 AM

many possible reasons: a different plateau level at the end of the PCR, different buffer composition (eg salt concentration is crucial in HRM), presence of certain chemicals in one of your samples, bad calibration of your machine  ...

 

To minimize this difference, you can dilute your DNA before your PCR or you can perform a second PCR on the first with the same primers (using only a tiny amount of your first PCR product of course)



#3 itsron

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Posted 09 April 2015 - 03:01 AM

Thanks, will try this!







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