Clara® HRM Mix

Yes

Specificity can be enhanced by raising the annealing temperature or redesigning primers. Ensure no contaminating DNA as this could result in nonspecific signals. When setting up initial reactions, always run a gradient. We also recommend conducting agarose gel analysis for troubleshooting and product identification.

• Ensure excellent DNA sample quality, meaning the DNA should be pure and free from inhibitors as this may affect melting temperatures. There should also be sufficient amounts of DNA in the sample.
• Follow standard precautions when setting up qPCR reactions, e.g., assembly on ice, precise pipetting, proper sealing of qPCR plates ensuring all wells have been sealed after cycling, spinning down samples to eliminate air bubbles and working clean.
• Your amplicon size should be in the range of 80 – 200 bp. A single base change will affect the melting temperature of a short sequence more than a long sequence. Larger amplicons can be analyzed but the resolution will be reduced. Conversely, very short amplicons will have low fluorescence and an unfavorable signal/noise ratio.
• Ensure your positive and negative controls are intact, not contaminated, and treated/purified in the same way as your samples to ensure consistency.
• Ensure your reaction reaches a plateau phase in each case. This will ensure a uniform starting fluorescence across the entire plate. Remember different product concentrations will melt slightly differently.
• Collect enough data points around the melting temperature of your amplicons.
• Examine your amplification plots. Poor amplification plots indicate issues in your qPCR and are sure to result in poor melting behavior.

Clara® HRM Mix is a ready-to-use mix. You only need to add primers, sample DNA, and PCR-grade water during reaction setup.

To achieve efficient amplification under fast cycling conditions, we recommend the amplification product length of 80bp to 200bp. In all manufacturer's mixes, shorter amplicon lengths will allow the reaction to cycle faster. The length of amplification products should not exceed 400bp. Primers should have a predicted melting temperature of approximately 60°C, using default settings of Primer 3 (http://frodo.wi.mit.edu/primer3/).

• 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 can vary significantly in ionic strength and enhancement composition. Cycling conditions may work for one mix but may not be optimal for another; hence a cycling program that works with one mix might not apply to another without optimization. Cycling can be optimized by running a gradient for each amplicon, regardless of predicted or theoretical annealing temperatures.

If all of the above has been applied and results are still unsatisfactory, please email technical@pcrbio.com and include the following information:

• Amplicon size
• Reaction setup
• Cycling conditions
• Screenshots of amplification traces and melting profiles