Yes.

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.

• 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.

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

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

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/).

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

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