How to optimize qPCR analysis using a melt curve
Melt curve analysis is a critical quality-control step for qPCR assays using intercalating dyes such as SYBR® Green.
Why use a melt curve?
Intercalating dyes bind to any double-stranded DNA, including nonspecific products. A melt curve helps confirm that amplification is specific to the intended amplicon.
Understanding melting temperature (Tm)
After amplification, temperature is gradually increased. Fluorescence decreases as double-stranded DNA dissociates, producing a melting temperature (Tm) characteristic of the amplicon.
Multiple melt peaks
- Primer dimers or nonspecific products
- GC- and AT-rich regions melting at different temperatures
- Sequence-dependent dissociation patterns
Troubleshooting tips
- Increase annealing temperature
- Reduce primer concentration
- Include no-template and -RT controls
- Validate with agarose gel or prediction software
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