FAQs

VeriFi™ Hot Start Polymerase contains the innovative AptaLock™ technology which minimises unwanted reactivity at room temperature and makes this product suitable for multiplexing.

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.

The optimal extension time for multiplex reactions will be dependent on the complexity of template, the length of amplicons, and the number of targets. We recommend starting with the extension time of the longest fragment, and then increasing in increments of between 10 and 30 seconds if necessary. For example, for the multiplex reaction shown on the website (10-plex with fragments from 139 bp and 962 bp), a 90 second extension time was used.

Fast cycling conditions should not be used for multiplex PCR.

Thanks to AptaLock™ hot start technology, there is no need to set up reactions on ice or cooling blocks 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 method^1-3.

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).