Calculating Tm of PCR Product for RT-qPCR Melting Curve

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Discussion Overview

The discussion revolves around calculating the melting temperature (Tm) of PCR products for use in RT-qPCR melting curve analysis. Participants explore the differences in Tm calculation between primers and PCR products, focusing on the implications for experimental design.

Discussion Character

  • Technical explanation
  • Debate/contested

Main Points Raised

  • One participant inquires about the method for calculating Tm of PCR products, indicating a need for this information for RT-qPCR analysis.
  • Another participant suggests using the same calculation method as for primers, noting that a good calculator should consider the length of the nucleic acid.
  • A third participant provides a link to an online calculator for Tm analysis.
  • One participant emphasizes the distinction between primers (single-stranded) and PCR products (double-stranded), questioning whether Tm should be calculated for the PCR product as if it were single-stranded.
  • Another participant agrees that Tm is calculated for single-stranded DNA when considering the separation of double-stranded DNA during PCR.

Areas of Agreement / Disagreement

Participants express differing views on whether the Tm of PCR products should be calculated similarly to that of primers, highlighting a lack of consensus on the appropriate method for Tm calculation in this context.

Contextual Notes

The discussion does not resolve the differences in approach to Tm calculation, and assumptions regarding the applicability of single-stranded calculations to double-stranded PCR products remain unaddressed.

mountain
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I know how to calculate Tm of a primer but what about of a PCR product?

I want to run the melting curve in RT-qPCR to test for the products and i want to know Tm of the products to be sure.


Thanks.
 
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Why not use the same calculation as the Tm of a primer? A good calculator should takes into account the length of the nucleic acid. How do you calculate it now?
 
Here's an online calculator http://biotools.idtdna.com/analyzer/
 
Last edited by a moderator:
Monique said:
Why not use the same calculation as the Tm of a primer? A good calculator should takes into account the length of the nucleic acid. How do you calculate it now?


The difference between a primer and a PCR product is that the PCR product is a double stranded DNA while the primer is just a singel strand. So should i calculate Tm for the PCR product like as it is a single strand?
 
Although you calculate Tm with a single strand for primer, it is use to separate your double stranded DNA.

So for your PCR product calculate with the single stranded DNA sequence.
 

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