Non-specific amplicons, identified by both gel electrophoresis and melting curve analysis, lead to inaccurate real-time PCR results. To avoid this problem, please make sure to perform hot-start PCR and use 60°C annealing temperature. We have noticed that not all hot-start Taq polymerases are equally efficient at suppressing polymerase activity during sample set-up. AmpliTaq Gold® DNA polymerase is highly recommended. If the non-specific amplicon is persistent, you have to choose a different primer pair for the gene of interest. This primer design failure is usually the result of mispriming to a previously unidentified splice isoform (Wang and Seed, 2003a).
Was this article helpful?