Applied Biosystems™ MeltDoctor™ HRM Positive Control Kit

Catalog No.	Quantity
44-101-26	1 kit

Catalog No. 44-101-26 Supplier Applied Biosystems™ Supplier No. 4410126

Description

Includes

MeltDoctor™ HRM Allele A DNA (20X), 150μL, MeltDoctor™ HRM Allele G DNA (20X), 150μL, MeltDoctor™ HRM Allele A⁄G DNA (20X), 150μL, MeltDoctor™ HRM Primer Mix (20X), 500μL

Provides nucleic acid controls to demonstrate and troubleshoot High Resolution Melt (HRM) analysis

The kit consists of forward and reverse primers and three DNA templates representing alleles AA, AB, and BB.

Order Info

Shipping Condition: Wet Ice

Recommended Storage

Store at -15° to -25°C.

Specifications

• Concentration: 20X
• Content And Storage: The kit consists of: MeltDoctor™ HRM Allele A DNA (20x), 150 μL, MeltDoctor™ HRM Allele G DNA (20x), 150 μL, MeltDoctor™ HRM Allele A⁄G DNA (20x), 150 μL, MeltDoctor™ HRM Primer Mix (20x), 500 μL
  Store at -15 °C to -25 °C
  
  Guaranteed minimum shelf life is 60 days (exact expiry date printed on product and CofA).
• Control Template: DNA template representing alleles AA, AB, and BB
• Format: Tube(s)
• Gene Symbol: ACTB
• Kit Type: MeltDoctor HRM Positive Control Kit
• Label or Dye: High Resolution Melting (HRM)
• Product Line: MeltDoctor
• Product Type: HRM Positive Control Kit
• Quantity: 1 kit
• Shipping Condition: Wet Ice
• Sufficient For: 450 Reactions
• Target: β-actin
• For Use With (Application): Real Time PCR (qPCR)
• No. of Reactions: 450 Reactions
• Volume: 150 μL, 500 μL

Frequently Asked Questions (FAQs)

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)

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

For Research Use Only. Not for use in diagnostic procedures.