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High Resolution Melt Software v3.0.1 - FAQs

查看更多产品信息 High Resolution Melt Software v3.0.1 - FAQs (4461357)

8 个常见问题解答

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.

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 are the license renewal requirements for the High Resolution Melt (HRM) software?

If you are using the QuantStudio 3/QuantStudio 5 Real-Time PCR Systems, 7500 Real-Time PCR System (with SDS v2.0.5 or later), or StepOne/StepOnePlus Real Time PCR Systems, you can purchase High Resolution Melt (HRM) Software v3.1 (https://www.thermofisher.com/order/catalog/product/A30150?ICID=search-product). Once activated, this software will not require annual renewal. If you have the older HRM software v3.0.1 (for use with StepOne Plus, StepOne or the 7500 Fast (SDS v2.0.5 or later) Real-Time PCR System, it will require license renewal on an annual basis. For use on the 7900HT Fast Real-Time PCR System, please use HRM Software v2.0.1 (https://www.thermofisher.com/order/catalog/product/4397808?ICID=search-product). This software will require license renewal on an annual basis. When using either the ViiA 7, QuantStudio 6/QuantStudio 7 or QuantStudio 12K Flex Real-Time PCR Systems, the HRM software is already included in the instrument's application software, but requires activation of the HRM module within the instrument software by purchase of the license. This license requires an annual renewal.

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