MeltDoctor™ HRM Reagent Kit - FAQs

How long should the amplicon be for HRM analysis?

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.

What can be used as positive control reactions in HRM analysis?

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.

In HRM analysis, when is it better to use a 3-step cycling protocol rather than a 2-step protocol?

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.

The HRM software will not accept my calibration file. What is wrong?

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

I have a 7900HT Fast Real-Time PCR System, and cannot open the data file in HRM software. What could be causing this?

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.

My 7500 Fast Real-Time PCR System has been upgraded to v2.0.6, so why can't I analyze my files with HRM Software v2.0.1?

For the 7500 Fast Real-Time PCR System, if the software version is below v2.0.4, the compatible HRM Software is v2.0.1. If the 7500 Fast Real-Time PCR System has been upgraded to software version 2.0.4 or above, then you will need to get HRM Software version 3.0.1.

How can I get positive controls for the different genotypes for my HRM experiment?

You can order a synthetic positive control plasmid using our GeneArt Gene Synthesis services. The final product is sequence verified and provided as 5 µg of lyophilized plasmid. Alternatively, you may be able to find a positive control sample from Coriell.

How do I perform the HRM calibration on my real-time PCR instrument?

The HRM calibration is a 3-step procedure. In some instrument software, the calibration process has been streamlined to a single-step procedure (applies to the ViiA7 Real-Time PCR System and QuantStudio Real-Time PCR Systems).

For the HRM calibration, the first step is a PCR reaction to generate the HRM dye plate. The reaction plate contains a DNA template, a primer pair, and the PCR mix with HRM dye. The second step is dye calibration using the PCR plate from step 1. The last step is a melt curve run, again using the same plate. For details, please refer to the HRM guides for each instrument:

- 7500 Fast Real-Time PCR System (with software version 1.4 or 2.0.4 and below) (https://tools.thermofisher.com/content/sfs/manuals/cms_050347.pdf)
- 7500 Fast Real-Time PCR System (with software version 2.0.5 and above) (https://tools.thermofisher.com/content/sfs/manuals/4457847.pdf)
- 7900HT Real-Time PCR System (https://tools.thermofisher.com/content/sfs/manuals/cms_050347.pdf)
- StepOne and StepOnePlus Real-Time PCR Systems (https://tools.thermofisher.com/content/sfs/manuals/4457847.pdf)
- ViiA 7 Real-Time PCR System (https://tools.thermofisher.com/content/sfs/manuals/4443531.pdf)
- QuantStudio 6 and 7 Real-Time PCR Systems (https://tools.thermofisher.com/content/sfs/manuals/4489823.pdf)
- QuantStudio 6 and 7 Pro Real-Time PCR Systems (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0018981_HighResolutionMelt_DA_SW_UG.pdf)
- QuantStudio 3 and 5 Real Time PCR Systems (https://tools.thermofisher.com/content/sfs/manuals/MAN0014394_HighResMeltExperiment_GSG.pdf)
- QuantStudio 12K Flex Real-Time PCR System (https://tools.thermofisher.com/content/sfs/manuals/4470692A.pdf)

Find additional tips, troubleshooting help, and resources within our High Resolution Melt Support Center.

Do you design primers for HRM?

No. We do not provide primer design service for HRM.

My instrument has been calibrated with MeltDoctor dye. If I now need to use LCGreen Plus for HRM, do I need to recalibrate my instrument?

Yes, the instrument should be calibrated for each HRM dye, and the data analysis should be done with the matching calibration file.

How can I use HRM for genotyping?

You can use HRM to discover novel SNPs over a specific target region, or to quickly screen samples for a specific target SNP. In both cases, you can track the mutation changes by viewing the aligned melt curves.

What can I do if I want to run HRM on my compatible instrument, but in a 96-well standard block?

We do not have any HRM calibration plates for 96-well standard blocks. To perform HRM experiments on such a system, you can instead use our MeltDoctor HRM Calibration Standard to create your own calibration plate.

Do you have any controls for HRM experiments?

Yes, if you are using HRM for genotyping, you can use our MeltDoctor HRM Positive Control Kit to help troulbeshoot your experiment. The kit contains control gDNA for each allele (A, A/G, and G) along with a HRM Primer Mix to match.

What HRM kit should I use for my application?

We have two options for HRM reagents. The MeltDoctor HRM Master Mix contains all components (excluding template and primers) formulated for superior HRM performance across a wide range of genomic targets. It is recommended for most HRM application except methylation-sensitive HRM. The MeltDoctor HRM Reagent Kit provides individually packaged PCR components and the MeltDoctor HRM Dye required for high-resolution melt analysis that is recommended for methylation-sensitive HRM analysis (as it does not contain dUTP).

How do I design primers for HRM applications?

The PCR primers need to be designed for robust performance and must be specific to the region of interest with a minimum of primer-dimers and non-specific products. A variety of software tools are available to help design HRM primers, including Primer Express 3.0 Software, Primer3, Primer3Plus, and Methyl Primer Express Software for methylation-sensitive HRM analysis.

What dye can I use for HRM?

We offer the MeltDoctor HRM dye for HRM application. Other commonly used dyes for HRM include LCGreen Plus, Eva Green dye, and SYBR Green dye.

What do I need to do to make my real-time PCR instrument ready for HRM?

The instrument will need to be calibrated for HRM. This includes an HRM dye calibration and a melt curve file for data analysis. You can find the appropriate HRM calibration plate for your instrument here (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-reagents/real-time-calibration-kits.html).

Is my real-time PCR instrument compatible with HRM?

Among our real-time PCR instruments, the following are compatible with HRM:

- 7500 Fast Real-Time PCR System
- 7900HT Fast Real-Time PCR System
- StepOne and StepOnePlus Real-Time PCR Systems
- ViiA 7 Real-Time PCR System
- QuantStudio 6 and 7 Pro Real-Time PCR Systems
- QuantStudio 6 and 7 Flex Real-Time PCR Systems
- QuantStudio 12K Flex Real-Time PCR System
- QuantStudio 1 Real-Time PCR System
- QuantStudio 3 Real-Time PCR System
- QuantStudio 5 Real-Time PCR System

What instruments are compatible with High Resolution Melt (HRM)?

HRM can be performed on the 7500 Fast, 7900HT Fast, StepOne/StepOnePlus instruments and requires a separate purchase of HRM software. The following instruments are also compatible with HRM and have a built-in analysis module within the instrument software: ViiA7, QuantStudio 6 and 7 Pro, QuantStudio 6 and 7 Flex, QuantStudio 12K Flex, QuantStudio 1, QuantStudio 3, and QuantStudio 5. To activate the HRM module, you will need to purchase a license.

What reagents do I need to perform an HRM experiment?

To perform an HRM experiment, you will need the reagents to calibrate your instrument for the HRM dye and the software to perform the analysis.

What can I do with HRM?

High resolution melt (HRM) can be used in genotyping, mutation scanning, and methylation studies.

What is the difference between MeltDoctor HRM Master Mix and MeltDoctor HRM Reagent Kit?

The difference between these MeltDoctor products is that the MeltDoctor HRM Reagent Kit provides each of the components separately so you have the flexibility to adjust the concentrations of each component. The MeltDoctor HRM Master Mix comes with all of the components already mixed together in one tube. Please see below for the components of each product:

• MeltDoctor HRM Reagent Kit includes: AmpliTaq Gold 360 DNA Polymerase (2 x 50 µL, each at 5 U/µL), AmpliTaq Gold 360 Buffer, 360 GC Enhancer, GeneAmp dNTP Blend, and MeltDoctor HRM Dye (20x).
  Note: The kit provides sufficient materials for 250 20-µL reactions when using 2 U AmpliTaq Gold 360 DNA Polymerase per reaction.


• MeltDoctor HRM Master Mix includes: A convenient 2X mix containing the following components formulated to obtain optimal HRM results: AmpliTaq Gold 360 DNA Polymerase, MeltDoctor HRM Dye, a dNTP blend including dUTP, Magnesium salts and other buffer components, precisely formulated to obtain optimal HRM results.

How can I create a new variant call group in the HRM app on Thermo Fisher Connect, select a color for the variant, and assign wells to that group?

To create a new variant call group in the HRM app on Thermo Fisher Connect, follow these steps:

1. Select the 'Quality Control & Results' tab.
2. In the 'Quality Control & Results' pane, select your experiment of interest.
3. Select the 'Derivative melt plot'.
4. Click 'Actions', and then select 'Manual Call'.
5. From the 'Manual Call' dialog box, you have two options:
1. To assign the sample to an existing variant call: click 'Select Existing', choose the appropriate call from the 'Group' drop-down menu, and then click 'OK'.
2. To create a new variant call: click 'Create New', enter a name for the new call in the 'Group' field, select a color, and then click 'OK'.
6. Repeat the above steps to assign more manual calls.
7. Click 'Analyze' to reanalyze the data using the manual calls.

What ramp mode should I use for HRM experiments using the QuantStudio 3 and QuantStudio 5 Real-Time PCR Systems?

We recommend using Continuous mode (set to 0.025 degrees C/sec) for HRM experiments. You can also choose “Number of Data Points per Degree”, but if you do so, make sure to collect at least 10 points per degree so that there is enough data for analysis. When in Continuous mode, the instrument uses a fixed ramp rate for the melt step and captures as many data points as possible within the time permitted. When in Data Point Per Degree mode, the instrument adjusts the step and hold for the melt step to achieve the specified number of data points.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.

What calibrate plate can I use for High Resolution Melt (HRM) analysis on the QuantStudio 3 and QuantStudio 5 Real-Time PCR Systems?

We recommend using our MeltDoctor HRM Calibration plates. Use the plate type that matches the block on your QuantStudio 3 or QuantStudio 5 Real-Time PCR System:

0.1 mL, 96-Well: Cat. No. 4425618
384-Well: Cat. No. 4425559
0.2 mL, 96-well: Cat. Nos. 4425562 and 4425557 (Note: We do not have a 0.2 mL calibration plate, but you can make one by combining the MeltDoctor HRM Calibration Standard with the MeltDoctor HRM Reagent Kit)

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Instruments Support Center.