qPCRBIO HRM Mix

Developed for reliable and accurate detection of genetic mutations and SNPs using High Resolution Melting (HRM) analysis.

qPCRBIO HRM Mix produces ultra-sensitive melt profiles allowing accurate discrimination of subtle sequence variations including class I to class IV mutations and CpG methylation differences.

Features

  • Fast and accurate detection of subtle sequence differences
  • Ultra-sensitive fluorescence profiles for clear characterisation of samples
  • Third generation saturating dye SyGreen 2
  • Antibody-mediated hot start technology for improved sensitivity
  • Compatible with all real-time instruments with HRM capability
CAT No.
Product
Size
Price
QTY
PB20.31-01
qPCRBIO HRM Mix
100 x 20μL Reactions
$50.00
PB20.31-05
qPCRBIO HRM Mix
500 x 20μL Reactions
$240.00
PB20.31-20
qPCRBIO HRM Mix
2000 x 20μL Reactions
$810.00

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More Information

High Resolution Melt (HRM) is a powerful technique used immediately following PCR for the analysis of mutations, polymorphisms and epigenetic differences in double stranded DNA samples. Samples are characterised based on DNA strand dissociation behavior as temperature is increased in the presence of a fluorescent dye. HRM analysis exploits the differences in melt curve shapes and DNA melting temperature to discriminate sequence variations between samples.

qPCRBIO HRM Mix uses SyGreen 2, a third generation non-PCR inhibiting saturating dye which produces ultra-sensitive melt profiles capable of discriminating class I to IV mutations and CpG methylation differences. Unlike first generation dyes, SyGreen 2 can be used at high concentrations and does not have a different affinity for AT or GC rich sequences.

This 2x mix uses antibody-mediated hot start technology to prevent the formation of primer dimers and non-specific products, leading to improved reaction sensitivity and specificity. Combining the latest advancements in polymerase technology and advanced buffer chemistry we offer fast, reliable and cost effective HRM analysis with minimal or no optimisation required.

Applications

  • SNP genotyping
  • Gene scanning
  • CpG methylation analysis

Specifications

qPCRBIO HRM Mix

Component

100 Reactions

500 Reactions

2000 Reactions

2x qPCRBIO HRM Mix

1 x 1mL

5 x 1mL

20 x 1mL

qPCRBIO HRM Mix

Component

2x qPCRBIO HRM Mix



100 Reactions

1 x 1mL

500 Reactions

5 x 1mL

2000 Reactions

20 x 1mL

Reaction Volume

Storage

20μL

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

Reaction Volume

20μL

Storage

On arrival, products should be stored between -30 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

Material Safety Data Sheets

Material Safety Data Sheets

FAQs

Can multiplexing be done with qPCRBIO 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 PCRBIO 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 qPCRBIO HRM Mix?

qPCRBIO HRM Mixes are ready to use Mastermixes. You only need to add primers, template DNA and PCR grade water during reaction set up.

What is the MgCl2 concentration in qPCRBIO HRM Mix?

All qPCRBIO HRM mixes contain MgCl2 at a concentration of 12 mM. This means the final concentration in the reaction is 6 mM.

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 the qPCRBIO 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 qPCRBIO 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 technical@pcrbio.com and include the following information:

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

More Information

High Resolution Melt (HRM) is a powerful technique used immediately following PCR for the analysis of mutations, polymorphisms and epigenetic differences in double stranded DNA samples. Samples are characterised based on DNA strand dissociation behavior as temperature is increased in the presence of a fluorescent dye. HRM analysis exploits the differences in melt curve shapes and DNA melting temperature to discriminate sequence variations between samples.

qPCRBIO HRM Mix uses SyGreen 2, a third generation non-PCR inhibiting saturating dye which produces ultra-sensitive melt profiles capable of discriminating class I to IV mutations and CpG methylation differences. Unlike first generation dyes, SyGreen 2 can be used at high concentrations and does not have a different affinity for AT or GC rich sequences.

This 2x mix uses antibody-mediated hot start technology to prevent the formation of primer dimers and non-specific products, leading to improved reaction sensitivity and specificity. Combining the latest advancements in polymerase technology and advanced buffer chemistry we offer fast, reliable and cost effective HRM analysis with minimal or no optimisation required.

Applications

  • SNP genotyping
  • Gene scanning
  • CpG methylation analysis

Specifications

qPCRBIO HRM Mix

Component

100 Reactions

500 Reactions

2000 Reactions

2x qPCRBIO HRM Mix

1 x 1mL

5 x 1mL

20 x 1mL

qPCRBIO HRM Mix

Component

2x qPCRBIO HRM Mix



100 Reactions

1 x 1mL

500 Reactions

5 x 1mL

2000 Reactions

20 x 1mL

Reaction Volume

Storage

20μL

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

Reaction Volume

20μL

Storage

On arrival, products should be stored between -30 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

Material Safety Data Sheets

Material Safety Data Sheets

FAQs

Can multiplexing be done with qPCRBIO 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 PCRBIO 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 qPCRBIO HRM Mix?

qPCRBIO HRM Mixes are ready to use Mastermixes. You only need to add primers, template DNA and PCR grade water during reaction set up.

What is the MgCl2 concentration in qPCRBIO HRM Mix?

All qPCRBIO HRM mixes contain MgCl2 at a concentration of 12 mM. This means the final concentration in the reaction is 6 mM.

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 the qPCRBIO 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 qPCRBIO 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 technical@pcrbio.com and include the following information:

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