MeltDoctor™ HRM Master Mix, 50 mL - FAQs

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.

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.

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.

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.

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

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

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.

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.

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.

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).

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.

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.

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).

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

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.

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

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.

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