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Understanding melt curves for improved SYBR Green assay analysis and troubleshooting

qPCR assays using intercalating dyes, such as SYBR Green dye, are an economical and effective tool for measuring gene expression. To interpret intercalating dye assays, users need to know how to analyze melt curves, and understand the benefits and limitations of such analysis. In this presentation, Nick Downey, PhD, covers melt curve basics and shares examples of multiple peaks due to suboptimal sample prep, primer dimers, and asymmetric GC content of amplicons. He also discusses troubleshooting strategies. Experienced and novice users will benefit from an overview of uMelt software, developed by the Wittwer lab at the University of Utah, that can predict the melt profile of your assay before you run your experiment.

Published on: April 03, 2015