Melting curves are an essential tool to estimate the specifity of a qPCR. You can instantly see if the right target has been amplified, or if unwanted by-products are being produced and detected. We strongly recommend to perform a melting curve analysis to be sure that the desired target has been amplified!

The cycler creates the melting curve by measuring the fluorescence while constantly raising the temperature. As soon as the temperature exceeds the melting temperature of the amplicon, it becomes single stranded resulting in a sudden drop of the fluorescence.

Please be aware that the peak of the melting curve may slightly vary between different master mixes. No reason to worry, as long as there is only the one peak to be expected! If the curve shows several peaks – exception multiplex assay – you cannot really trust the results. Further investigation is necessary to evaluate the reason.

A melting curve analysis requires intercaling dyes like e.g. SYBRGreen oder EvaGreen and is not possible in a probe assay.