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FAQs
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Can VeriFi™ Polymerase be used for Multiplex PCR?
VeriFi™ Polymerase doesn’t contain hot-start technology however it does have an intrinsically low activity at low temperatures, which are much lower relative to a Taq Polymerase without the hot start. This means it can be used for some multiplex applications.
When first performing multiplex PCR, we recommend running an annealing temperature gradient from 55 °C to 65 °C. The annealing temperature that results in the best specificity should be used in subsequent experiments. Fast cycling conditions should not be used for multiplex PCR. We recommend a 90 second extension time to begin with and this time may be extended to increase yield.
For multiplex reactions, the reactions should be set up on ice or cooling blocs from start till finish. Primers must be designed carefully to avoid overlapping sequences as much as possible while maintaining diverse amplicon lengths that can be easily analysed with your end-detection method1-3.
1 Markoulatos, P., Siafakas, N. & Moncany, M. Multiplex polymerase chain reaction: a practical approach. J Clin Lab Anal 16, 47-51, doi:10.1002/jcla.2058 (2002).
2 Radhika, M., Saugata, M., Murali, H. S. & Batra, H. V. A novel multiplex PCR for the simultaneous detection of Salmonella enterica and Shigella species. Brazilian Journal of Microbiology 45, 667-676, doi:10.1590/s1517-83822014005000041 (2014).
3 Perez-Perez, F. J. & Hanson, N. D. Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol 40, 2153-2162, doi:10.1128/jcm.40.6.2153-2162.2002 (2002).
