MeltDoctor™ HRM Calibration Standard (20X)

We recommend to use 50-250 bp long PCR amplicons. Typically, shorter amplicons can distinguish the genotypes for a SNP better, especially for Type III and Type IV SNPs. This is simply because a single base variation affects the melting behavior of a 100 bp amplicon stronger than of a 500 bp amplicon, for example. In longer fragments, the risk of covering multiple mutations is also increased.

Positive control reactions should contain template DNA with a known sequence. In SNP genotyping experiments, this could be a sample with a known genotype. Positive control(s) for all genotypes should be included where possible to serve as a reference in melting curve comparison and assigning genotypes for test samples. In mutation scanning experiments, a sample with a wild type sequence could serve as a positive control. The controls should preferably have the same DNA concentration as their corresponding test samples. Control DNA should also be eluted and/or diluted in the same buffer as the samples.

A 3-step cycling protocol is recommended for the analysis of complicated (especially Type IV SNP) targets, amplicons longer than 200 bp, and amplicons with a primer annealing temperature that is less than 60 degrees C.

Check that there are no outliers on the plate. You cannot omit any wells on the HRM calibration plate.

There are a few possibilities. First, make sure the HRM Software version is v2.0.1, and the 7900HT Fast Real-Time PCR System software version is v2.3 or above. Second, check that the run method used was as recommended in the HRM protocol; make sure the ramp rate for the dissociation stage is 1%. Then try to open the calibration file from the HRM Software; if it does not open, the calibration file is defective. The defects could be due to a bad calibration plate or instrument uniformity issue.