FAQs

The 5x VeriFi™ buffer supplied with VeriFi™ Hot Start Polymerase has been developed specifically for this enzyme and we highly recommend using them together. However, VeriFi™ Hot Start Polymerase should be compatible with any PCR buffer developed for use with Pfu polymerase. If you use a customised buffer with VeriFi™ Hot Start Polymerase, keep in mind that reaction parameters such as annealing temperature and concentrations of enzyme, template, dNTPs and MgCl₂, may require optimisation.

Yes. If you’re working from bacterial colonies, use a sterile tip to pick a colony and resuspend into the 50 µL PCR reaction. If working from liquid culture add 5 µL of overnight culture to the final mix. Follow the general protocol and increase the initial denaturation time to 10 min at 95 °C.

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).

If you’re working with GC-rich DNA and finding the yield of your PCR product is too low, try increasing the denaturation temperature to 98-100 °C. VeriFi™ Hot Start Polymerase is capable of withstanding these temperatures and has shown increased yields for DNA with high GC content relative to denaturation at 95 °C. The denaturation time can be varied from 10 seconds up to 30 seconds if required.

Addition of 10x VeriMax Enhancer can also help to amplify GC-rich targets.

VeriFi™ Hot Start Polymerase is available in three formats. One format contains the enzyme (2 U/µL) with a separate 5x VeriFi™ buffer and a separate 10x VeriMax Enhancer tube. The other format contains the enzyme, buffer and enhancer conveniently premixed as a 2x mix. Additionally, the 2x mix is also available with a red dye giving further convenience for direct loading and tracking during agarose gel electrophoresis. All of our formats contain the required reaction components for PCR except for primers, template and PCR grade water.

This non-inhibitory dye, added to enable direct gel loading, runs at a similar rate to 200-300 bp DNA fragments on a 1% agarose gel and at 50-100 bp on a 2% agarose gel. You may notice a shift in this apparent molecular weight when running gels of different agarose content.

VeriFi™ Hot Start Polymerase is the hot start version of VeriFi™ Polymerase, and uses PCR Biosystems’ innovative AptaLock™ technology for increased sensitivity and specificity compared to the two non hot start enzymes. Reactions can also be set up at room temperature when using VeriFi™ Hot Start Polymerase.

Both VeriFi™ Hot Start Polymerase and VeriFi™ Polymerase have an enhanced processivity relative to PCRBIO HiFi Polymerase, resulting in higher yields and the ability to amplify longer (up to 17.5 kb) and more difficult targets. Both enzymes also have a higher fidelity than PCRBIO HiFi Polymerase.

Unlike PCRBIO HiFi polymerase, the VeriFi™ family of enzymes are also available as convenient 2x ready mixes with the option of a red dye for direct gel loading.

VeriFi™ Hot Start Polymerase has a very low error rate, with a fidelity approximately 100 times higher than wild-type Taq DNA polymerase.

PCR products generated by VeriFi™ Hot Start Polymerase have blunt ends, making them suitable for blunt end cloning without requiring any further modification.

If you would like to add A-overhangs, you can use Taq DNA Polymerase or Klenow (exo-) DNA followed by TA cloning¹. Before proceeding with the addition of A-overhangs, ensure the VeriFi™ Hot Start Polymerase has been removed from the reaction. If there is any VeriFi™ Hot Start Polymerase present, the 3’-5’ exonuclease activity of the enzyme can remove A-overhangs.

1  Yao, S., Hart, D. J. & An, Y. Recent advances in universal TA cloning methods for use in function studies. Protein Eng Des Sel, doi:10.1093/protein/gzw047 (2016).

In situations where low yields or no amplification is observed, we recommend adding the 10x VeriMax Enhancer to the reaction mix. This enhancer can improve the performance of VeriFi™ Hot Start Polymerase on some difficult or long templates, for example GC-rich templates or those with complex secondary structures.