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FAQs
Browse our frequently asked technical questions by selecting a product below.
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.
