PCR and qPCR
Support and Educational Content

Melt curve analysis for improved intercalating dye qPCR


Performing qPCR using intercalating dyes, such as SYBR® dyes, involves use of just 2 amplification primers to provide assay specificity. Because fluorescence in intercalating dye assays is based on the production of any double-stranded DNA, off-target amplification can generate false-positive signals, compromising assay data. To address this problem, researchers use melt-curve analysis to assess whether their assays have produced single, specific products. 

In April 2015, IDT hosted a webinar, Understanding Melt Curves for Improved SYBR® Green Assay Analysis and Troubleshooting, that covered several aspects of melt curve analysis. Now available as a recording, this webinar features our resident expert, Nick Downey, PhD, demonstrating some of the benefits and limitations of melt-curve analysis through clear examples. The presentation also provides an overview of the uMeltSM software for guiding assay design. You can access the recording of this webinar through the following video link:

Product focus—qPCR primer assays for use with intercalating dyes

PrimeTime® Predesigned qPCR Primer Assays

These predesigned qPCR assays are designed to detect genes in the human, mouse, and rat transcriptomes. The primer sets are ideal for use with SYBR® Green, EvaGreen®, and other intercalating dyes.

PrimeTime qPCR Primer Assays:

  • Fully plan and analyze your experiments with disclosed primer and probe sequences.
  • Easily  transition from primer-only assays to probe-based PrimeTime qPCR 5' Nuclease Assays if your experimental needs change or increased specificity is needed.
  • Choose the delivery format of your forward and reverse primers, delivered in single tube or plate configurations (96-well plates or matrix plates are available).
  • Begin your project sooner with 2–3 day product shipping.

Additional reading

Interpreting melt curves: An indicator, not a diagnosis—Performing intercalating dye PCR/qPCR assays? Review examples of PCR melt curve data with our scientists to determine what it can/cannot tell us about resulting PCR amplicons.

qPCR with intercalating dyes: PrimeTime® qPCR Primers—Product spotlight: Need qPCR assays to use with intercalating dyes? PrimeTime qPCR Primers are now available to detect genes in human, mouse, and rat transcriptomes with intercalating dyes such as SYBR® Green and EvaGreen®.

SYBR® to 5’ nuclease assays—Product spotlight: Researchers often use intercalating dyes, like SYBR® Green I, for qPCR experiments for the advantage they bring in lowered price and reduced turnaround time. However, the disadvantages to using these reagents are significant. Read how easy it is to convert to 5’ nuclease assays and the advantages the addition of a hydrolysis probe will provide.

Review other DECODED Online newsletter articles on PCR and qPCR applications.

You can also browse our DECODED Online newsletter for additional application reviews, lab tips, and citation summaries to facilitate your research.

Author: Hans Packer, PhD, is a scientific writer at IDT.

© 2015, 2016 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.

Predesigned qPCR Assays

Probe-based qPCR assays for quantification of human, mouse, and rat gene expression. Order in plates or tubes.

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