Calculation of Tm of amplicon when using SYBR Green (View forum version)



Arzeecue

Posted 17 May 2010 - 04:00 PM

Hi everyone,

When using aspecific detection of PCR amplicons by SYBR Green, a melting curve analysis is done after the PCR to check if the expected PCR product has been made and to check for the absence or presence of additional peaks, caused by primer dimers or aspecific primer annealing etc.

But suppose I find one, unique peak in this analysis? How can I be sure that this peak belongs to the amplicon I want to produce and not to primer dimers or a wrong amplicon? Can the Tm of an amplicon be calculated? If so, how accurate is this calculation and can the software of the qPCR machine do this calculation?

I found e.g this calculator: http://www.mycalcula...jmm/biot16.html , but is calculation of the Tm of amplicons done routinely or do most people just check if there is one, unique, relatively sharp peak?

Thanx,
Peter

ivanbio

Posted 17 May 2010 - 04:12 PM

Calculating a Tm using software works fine, but if you want to be 100% sure that you are getting the amplicon you expect you need to sequence the amplicon. Just run it on a gel, extract the band, and send it for sequencing. It is more work than most people do when running qPCR assays, but it is better to make sure you are looking at the right thing instead of finding out many experiments later that you were studying a primer dimer.

Good luck!

criscastells

Posted 18 May 2010 - 12:42 AM

To be sure it is best not only use bioinformatic tools. Run a gel could be enought but if you need to be completely sure that it was your amplicon it will be needed to sequence it.

Prep!

Posted 18 May 2010 - 12:54 AM

Calculating a Tm using software works fine, but if you want to be 100% sure that you are getting the amplicon you expect you need to sequence the amplicon. Just run it on a gel, extract the band, and send it for sequencing. It is more work than most people do when running qPCR assays, but it is better to make sure you are looking at the right thing instead of finding out many experiments later that you were studying a primer dimer.

Good luck!


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