Clara™ HRM Mix

This qPCR mix delivers maximum specificity to your high resolution melt (HRM) curve analyses. Accurately detect genetic mutations, quickly identify genotypes based on SNPs, or calculate percent methylation of a target region with HRM analysis.

Clara™ HRM Mix comprises our unique, ultra-pure, PCRBIO Taq DNA Polymerase in a blend containing dNTPs and MgCl2. Powered by our third-generation, DNA-intercalating, SyGreen 2 dye for greatly reduced PCR inhibition, this new generation high-resolution melting qPCR mastermix offers superior performance for accurate SNP discrimination and quantification of methylation differences.

Features

  • Accurate distinction of SNP classes I-IV
  • Quantify methylation of target sequences
  • Super-sensitive product melt curves for distinct allele profiles
  • Compatible with all HRM-suitable real-time instruments
  • Powered by PCRBIO Taq DNA polymerase
CAT No.
Product
Size
Price
QTY
PB20.32-01
Clara™ HRM Mix
100 x 20 μL Reactions
$52.00
PB20.32-05
Clara™ HRM Mix
500 x 20 μL Reactions
$250.00
PB20.32-20
Clara™ HRM Mix
2000 x 20 μL Reactions
$842.00

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More Information

What is HRM?

High Resolution Melt (HRM) curve analysis is a powerful technique used for the analysis of mutations, polymorphisms and epigenetic differences in double stranded DNA samples. This method relies on analysis of the post amplification denaturation of qPCR targets in combination with third generation DNA intercalating dyes with reduced polymerase inhibition (e.g. SyGreen 2, EvaGreen®). HRM analysis exploits the differences in melt curve shapes and DNA melting temperature to discriminate sequence variations between samples.

Usually, in a melt curve analysis run after a standard dye-based qPCR, fluorescence data is acquired every 0.2-0.5 °C, whereas in HRM analysis data is acquired at a much higher density, every 0.1 °C, resulting in far greater resolution of the resulting melt curve. Thus, even small difference (down to single point mutations, SNPs, and base-pair methylation), in target sequences register as a shifted melt curves. HRM thus offers a cheaper effective alternative to probe-based detection for SNP detection, allelic discrimination or target variant detection.

Detectable sequence modifications

Clara™ HRM Mix can distinguish all types of SNPs. These include:

  • Class I: C to T and G to A
  • Class II: C to A and G to T
  • Class III: C to G
  • Class IV: A to T

Additionally, this mix can also be used to quantify CpG methylation in a target sequence. In this case, potentially methylated DNA is subjected to bisulfite treatment prior to running HRM qPCR with primers targeting CpG containing regions. During bisulfite treatment, all unmethylated cytosine residues are deaminated and thus converted to uracil residues, while all methylated cytosine residues remain unaltered. HRM analysis on the bisulfite treated versus a corresponding, untreated, control sample allows the estimation of the target’s cytosine methylation percentage, because methylated sequences will have a higher CG content compared to unmethylated targets following bisulfite treatment.

HRM compatible real-time instruments

Running HRM experiments requires compatible instrumentation. Not all real-time PCR instruments are able to achieve the necessary temperature increment to conduct HRM profiling. We recommend using one the listed qPCR thermocyclers from the following manufacturers:

  • Applied Biosystems: 7900, 7900HT, 7900HT FAST, StepOne™, StepOne™ Plus, 7500, 7500 FAST, Viia7™
  • Bio-Rad®: CFX96™, CFX384™
  • Eppendorf: Mastercycler® ep realplex, Mastercycler® realplex 2S
  • Illumina®: Eco™
  • Qiagen/Corbett: 6000, Q
  • Roche Applied Science: Lightcycler®480, Lightcycler®Nano

Other instruments that are not listed are also suitable for HRM analysis using Clara™ HRM Mix. Please use our qPCR Selection Tool, check your instrument’s product manual, or contact us at [email protected] for help in choosing an appropriate instrument.

Applications

  • SNP genotyping
  • Gene scanning
  • CpG methylation analysis

Specifications

Clara™ HRM Mix

Component

100 reactions

500 reactions

2000 reactions

2x Clara™ HRM Mix

1 x 1 mL

5 x 1 mL

20 x 1 mL

Clara™ HRM Mix

Component

2x Clara™ HRM Mix



100 reactions

1 x 1 mL

500 reactions

5 x 1 mL

2000 reactions

20 x 1 mL

Reaction Volume

Storage

20 μL

On arrival, products should be stored between -30 °C and -15 °C. If stored correctly the kit will retain full activity for 12 months.

Reaction Volume

20 μL

Storage

On arrival, products should be stored between -30 °C and -15 °C. If stored correctly the kit will retain full activity for 12 months.

Instrument Compatibility

This product is compatible with all standard and fast cycling qPCR instruments with High Resolution Melt capability. Use our qPCR Selection Tool to find out which instruments can be used.

Documents

Product Flyers

Product Flyers

Material Safety Data Sheets

Material Safety Data Sheets

FAQs

Can multiplexing be done with Clara™ HRM Mix?

Yes

How can unspecific products be removed in HRM analysis?

Specificity can be increased by increasing the annealing temperature or redesigning primers. Ensure that there is no contaminating DNA as this could cause the unspecific signal to arise. When performing initial runs, we always recommend running gradients. We also recommend running agarose gel analysis, to fully troubleshoot the reaction and identify products.

What are the general considerations for achieving high-quality results with Clara™ HRM Mix?

  • Ensure that the quality of the DNA sample is exceptional, this mean the DNA should be pure and free of any inhibitors as this could affect the melt. There should also be sufficient amounts of DNA present.
  • Follow regular precautions when preparing qPCR reactions, for example, assemble on ice, precise pipetting, seal the qPCR plate properly and ensure that all wells were sealed post-run, spin the sample down to remove bubbles, and work clean.
  • Your amplicons should be between 80 – 200 bp. A single base variation will affect a melt of a short sequence to a higher degree than a long one. Larger fragments can be analysed but the resolution will be compromised. Conversely, fragments that are too short will have low fluorescence thus unfavourable S/N.
  • Make sure your negative and positive controls are intact, not contaminated and processed/purified in the same way as your sample to ensure consistency.
  • Make sure your reaction reaches plateau in each case. That will ensure similar starting fluorescence intensity for the melt throughout the plate. Keep in mind that different concentrations of products will melt slightly differently.
  • Collect enough data points around the melting temperature of your amplicons.
  • Examine your amplification plots. Poor amplification plots are indicative of qPCR problems and will most certainly result in poor melt behaviour.

What is included in Clara™ HRM Mix?

Clara™ HRM Mix is a ready-to-use mastermix. You only need to add primers, template DNA and PCR grade water during reaction set up.

What should the length of the amplicon be?

For efficient amplification under fast cycling conditions we recommend amplicon lengths between 80bp and 200bp. With all manufacturers master mixes the shorter the amplicon length the faster the reaction can be cycled. Amplicon lengths should not exceed 400bp. Primers should have a predicted melting temperature of around 60°C, using default Primer 3 settings (http://frodo.wi.mit.edu/primer3/).

Which instruments is Clara™ HRM Mix compatible with?

Applied Biosystems: 7900, 7900HT, 7900HT FAST, StepOneTM, StepOneTM Plus, 7500, 7500 FAST, Viia7TM

Bio-Rad®: CFX96TM, CFX384TM

Eppendorf: Mastercycler® ep realplex, Mastercycler® realplex 2S

Illumina®: EcoTM

Qiagen/Corbett: 6000, Q

Roche Applied Science: Lightcycler®480, Lightcycler®Nano

Why are unsatisfactory or different results being observed when comparing Clara™ HRM Mix to a competitor mix?

HRM mixes from different manufacturers vary greatly in their ionic strength and enhancer composition. Cycling conditions that might work for one mix could be suboptimal for another and therefore a cycling program that works on one mix can’t be applied to another without optimisation. The cycling can be optimised by running gradients for each amplicon irrespective of calculated or theoretical annealing temperature.

If all the above has been applied and the results are still unsatisfactory, please email [email protected] and include the following information:

  • Amplicon size
  • Reaction setup
  • Cycling conditions
  • Screen grabs of amplification traces and melting profile

More Information

What is HRM?

High Resolution Melt (HRM) curve analysis is a powerful technique used for the analysis of mutations, polymorphisms and epigenetic differences in double stranded DNA samples. This method relies on analysis of the post amplification denaturation of qPCR targets in combination with third generation DNA intercalating dyes with reduced polymerase inhibition (e.g. SyGreen 2, EvaGreen®). HRM analysis exploits the differences in melt curve shapes and DNA melting temperature to discriminate sequence variations between samples.

Usually, in a melt curve analysis run after a standard dye-based qPCR, fluorescence data is acquired every 0.2-0.5 °C, whereas in HRM analysis data is acquired at a much higher density, every 0.1 °C, resulting in far greater resolution of the resulting melt curve. Thus, even small difference (down to single point mutations, SNPs, and base-pair methylation), in target sequences register as a shifted melt curves. HRM thus offers a cheaper effective alternative to probe-based detection for SNP detection, allelic discrimination or target variant detection.

Detectable sequence modifications

Clara™ HRM Mix can distinguish all types of SNPs. These include:

  • Class I: C to T and G to A
  • Class II: C to A and G to T
  • Class III: C to G
  • Class IV: A to T

Additionally, this mix can also be used to quantify CpG methylation in a target sequence. In this case, potentially methylated DNA is subjected to bisulfite treatment prior to running HRM qPCR with primers targeting CpG containing regions. During bisulfite treatment, all unmethylated cytosine residues are deaminated and thus converted to uracil residues, while all methylated cytosine residues remain unaltered. HRM analysis on the bisulfite treated versus a corresponding, untreated, control sample allows the estimation of the target’s cytosine methylation percentage, because methylated sequences will have a higher CG content compared to unmethylated targets following bisulfite treatment.

HRM compatible real-time instruments

Running HRM experiments requires compatible instrumentation. Not all real-time PCR instruments are able to achieve the necessary temperature increment to conduct HRM profiling. We recommend using one the listed qPCR thermocyclers from the following manufacturers:

  • Applied Biosystems: 7900, 7900HT, 7900HT FAST, StepOne™, StepOne™ Plus, 7500, 7500 FAST, Viia7™
  • Bio-Rad®: CFX96™, CFX384™
  • Eppendorf: Mastercycler® ep realplex, Mastercycler® realplex 2S
  • Illumina®: Eco™
  • Qiagen/Corbett: 6000, Q
  • Roche Applied Science: Lightcycler®480, Lightcycler®Nano

Other instruments that are not listed are also suitable for HRM analysis using Clara™ HRM Mix. Please use our qPCR Selection Tool, check your instrument’s product manual, or contact us at [email protected] for help in choosing an appropriate instrument.

Applications

  • SNP genotyping
  • Gene scanning
  • CpG methylation analysis

Specifications

Clara™ HRM Mix

Component

100 reactions

500 reactions

2000 reactions

2x Clara™ HRM Mix

1 x 1 mL

5 x 1 mL

20 x 1 mL

Clara™ HRM Mix

Component

2x Clara™ HRM Mix



100 reactions

1 x 1 mL

500 reactions

5 x 1 mL

2000 reactions

20 x 1 mL

Reaction Volume

Storage

20 μL

On arrival, products should be stored between -30 °C and -15 °C. If stored correctly the kit will retain full activity for 12 months.

Reaction Volume

20 μL

Storage

On arrival, products should be stored between -30 °C and -15 °C. If stored correctly the kit will retain full activity for 12 months.

Instrument Compatibility

This product is compatible with all standard and fast cycling qPCR instruments with High Resolution Melt capability. Use our qPCR Selection Tool to find out which instruments can be used.

Documents

Product Flyers

Product Flyers

Material Safety Data Sheets

Material Safety Data Sheets

FAQs

Can multiplexing be done with Clara™ HRM Mix?

Yes

How can unspecific products be removed in HRM analysis?

Specificity can be increased by increasing the annealing temperature or redesigning primers. Ensure that there is no contaminating DNA as this could cause the unspecific signal to arise. When performing initial runs, we always recommend running gradients. We also recommend running agarose gel analysis, to fully troubleshoot the reaction and identify products.

What are the general considerations for achieving high-quality results with Clara™ HRM Mix?

  • Ensure that the quality of the DNA sample is exceptional, this mean the DNA should be pure and free of any inhibitors as this could affect the melt. There should also be sufficient amounts of DNA present.
  • Follow regular precautions when preparing qPCR reactions, for example, assemble on ice, precise pipetting, seal the qPCR plate properly and ensure that all wells were sealed post-run, spin the sample down to remove bubbles, and work clean.
  • Your amplicons should be between 80 – 200 bp. A single base variation will affect a melt of a short sequence to a higher degree than a long one. Larger fragments can be analysed but the resolution will be compromised. Conversely, fragments that are too short will have low fluorescence thus unfavourable S/N.
  • Make sure your negative and positive controls are intact, not contaminated and processed/purified in the same way as your sample to ensure consistency.
  • Make sure your reaction reaches plateau in each case. That will ensure similar starting fluorescence intensity for the melt throughout the plate. Keep in mind that different concentrations of products will melt slightly differently.
  • Collect enough data points around the melting temperature of your amplicons.
  • Examine your amplification plots. Poor amplification plots are indicative of qPCR problems and will most certainly result in poor melt behaviour.

What is included in Clara™ HRM Mix?

Clara™ HRM Mix is a ready-to-use mastermix. You only need to add primers, template DNA and PCR grade water during reaction set up.

What should the length of the amplicon be?

For efficient amplification under fast cycling conditions we recommend amplicon lengths between 80bp and 200bp. With all manufacturers master mixes the shorter the amplicon length the faster the reaction can be cycled. Amplicon lengths should not exceed 400bp. Primers should have a predicted melting temperature of around 60°C, using default Primer 3 settings (http://frodo.wi.mit.edu/primer3/).

Which instruments is Clara™ HRM Mix compatible with?

Applied Biosystems: 7900, 7900HT, 7900HT FAST, StepOneTM, StepOneTM Plus, 7500, 7500 FAST, Viia7TM

Bio-Rad®: CFX96TM, CFX384TM

Eppendorf: Mastercycler® ep realplex, Mastercycler® realplex 2S

Illumina®: EcoTM

Qiagen/Corbett: 6000, Q

Roche Applied Science: Lightcycler®480, Lightcycler®Nano

Why are unsatisfactory or different results being observed when comparing Clara™ HRM Mix to a competitor mix?

HRM mixes from different manufacturers vary greatly in their ionic strength and enhancer composition. Cycling conditions that might work for one mix could be suboptimal for another and therefore a cycling program that works on one mix can’t be applied to another without optimisation. The cycling can be optimised by running gradients for each amplicon irrespective of calculated or theoretical annealing temperature.

If all the above has been applied and the results are still unsatisfactory, please email [email protected] and include the following information:

  • Amplicon size
  • Reaction setup
  • Cycling conditions
  • Screen grabs of amplification traces and melting profile