PCR melt curve predictor
This tool predicts what a SYBR Green melt curve will probably look like for a qPCR product produced by a specified pair of primers. You give it a gene, an organism, and your known forward and reverse sequences. It then pulls the RefSeq mRNA sequence from NCBI, finds where those primers bind, cuts out the amplicon and sketches a melt curve plus Tm. This is handy when you want a rough estimate of what your melt curve should look like before you load a plate or when the melt plot on the instrument looks wrong and you are wondering what you should have seen.
WHAT IT’S FOR
Melt analysis after SYBR qPCR tells you whether you have amplified one thing or several in your reaction. A single sharp peak is what you should get from a clean, specific reaction. Extra peaks often mean primer dimers, off-target products, or mixed isoforms, all of which are quantified together with any specific product you may have made. This obviously messes up the quantification of your specific target amplicon. See our melt curve analysis guide for reading real instrument data.
This predictor certainly does NOT replace the post-run check. It answers a different question: given these primers and this gene, what melt peak should I expect? Typical uses for us are sanity-checking primers from an old notebook or a paper before ordering a new assay, comparing a predicted Tm to what QuantStudio software reports, and spotting cases where the amplicon sequence might melt at an unexpected temperature (long GC-rich products, splice isoforms, that sort of thing).
If you still need primers designed from scratch, use the qPCR primer designer first and come back here once you have a pair to check.
HOW IT WORKS
When you click Predict melt curve, the tool looks up your gene and organism in NCBI and
fetches a RefSeq mRNA (NM_ by default). It searches that template for your forward and
reverse primer sequences (5′→3′, spaces and line breaks stripped) and extracts the DNA
between them, which represents your amplicon.
If the primers were designed on genomic DNA or span an intron, paste a template accession
(NG_ or a specific NM_) in the optional field so the match is on the right
sequence.
Melt prediction uses the same nearest-neighbour thermodynamics as our qPCR primer designer and uMelt-style tools: SantaLucia & Hicks (2004) parameters with SL (2004) salt correction. Default buffer settings are 50 mM [Mono+] and 3 mM free Mg2+ — change those if your master mix differs.
The plot shows a schematic derivative melt curve. We report a primary melt peak (the main peak SYBR software would pick) and a separate thermodynamic Tm; the thermodynamic value is usually a few degrees higher than the peak you see on the instrument. That gap is normal. Real runs also depend on ramp rate, dye, and how the software finds the peak.
Primer binding is found by plain sequence match on the downloaded template. It does not model mis-priming, SNPs, or alternative splice forms unless you point it at the right accession. Treat the output as a guide, not a guarantee.
HOW TO USE IT
- Enter the gene symbol (e.g.
ACTB,TP53) and organism (e.g. Homo sapiens). - Paste forward and reverse primer sequences in 5′→3′ orientation.
- Adjust buffer [Mono+] and Mg2+ if needed; add a template accession only when mRNA lookup is not enough.
- Click Predict melt curve and compare the amplicon sequence and predicted peak to your assay.