FAQs

Yes

• 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.

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.

• Applied Biosystems: 7900, 7900HT, 7900HT FAST, StepOne, StepOne Plus, 7500, 7500 FAST, Viia7
• Bio-Rad: CFX96, CFX384
• BMS: Mic
• Eppendorf: Mastercycler ep realplex, Mastercycler realplex 2S
• Illumina: Eco
• 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