Type-it HRM PCR Kit (2000)

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	DNA quantification prior to performing the HRM procedure is a necessary step to avoid introducing error due to modifications in the shape of the melt curves

Protocol tips
DNA quantification prior to performing the HRM procedure is a necessary step to avoid introducing error due to modifications in the shape of the melt curves

Publication protocol

Singleplex PCR amplifications: Singleplex endpoint PCRs of ST131 DNA template and individual MLST primer sets were performed using the Qiagen Type-it HRM PCR kit in a final volume of 25 μl with 10 ng of DNA template and 12.5 pMol each primer (30). DNA quantification prior to performing the HRM procedure is a necessary step to avoid introducing error due to modifications in the shape of the melt curves. The cycling conditions consisted of a 5-min denaturation at 95°C, followed by 30 cycles of a 10-s denaturation at 95°C and 30 s of annealing at 51°C and then by 10 s of extension at 72°C. Amplicons were analyzed by HRM at a 0.1°C resolution from 50 to 95°C to determine the absolute melt curve of each amplicon. The derivative melt curves of MLST amplicons were compared to the melt curves predicted by uMelt software, developed at the University of Utah, to confirm sequence identity. PCR amplicons were also separated on a 1% agarose gel and visualized with ethidium bromide to confirm that only a single product of the expected size was amplified.

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