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The 5x PCRBIO VeriFi buffer supplied with PCRBIO VeriFi Polymerase has been developed specifically for this enzyme and we highly recommend using them together. However, PCRBIO VeriFi Polymerase should be compatible with any PCR buffer developed for use with Pfu polymerase. If you use a customised buffer with PCRBIO VeriFi Polymerase, keep in mind reaction parameters such as annealing temperature and concentrations of the 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 re-suspend into the 50µl PCR reaction. If working from liquid culture add 5µl of overnight culture to the final mix. Follow the general protocol and increase the initial denaturation time to 10 min at 95°C.
PCRBIO 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).
My results contain a high background of non-specific amplicons or smears. What trouble-shooting suggestions do you have?
If smears are a concern, it’s good practice to ensure they are not an artifact of running agarose gel electrophoresis with sub optimal conditions. Sub optimal conditions can include high voltage or not allowing enough time for the gel to set etc)1.
You may also need to troubleshoot the PCR reaction. If this is the case, consider the suggestions and literature below2.
- Primers should be designed to prevent primer-primer interactions and improve specificity.
- Increase the annealing temperature or conducting an annealing temperature gradient PCR to determine the optimal annealing temperature.
- Reduce the amount of template in the reaction. For high quality DNA, use 1–100 ng genomic DNA or ≤5 ng plasmid/lambda DNA per 50 µL reaction.
- Reduce the number of cycles.
- Reduce the amount of enzyme per reaction.
- Reduce the primer concentration, but not lower than 100 nM of each primer.
- Include DMSO in the reaction to a final concentration of 5%–10%.
1 Koontz, L. Agarose Gel Electrophoresis. Laboratory methods in enzymology : DNA. First edition. edn, Vol. 529 35-45 (2013).
2 Lorenz, T. C. Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies. J Vis Exp, e3998, doi:10.3791/3998 (2012).
You may want to consider the suggestions below and also refer to the literature1.
- Optimise the annealing temperature in an annealing temperature gradient PCR.
- Increase the amount of template in the reaction.
- Increase the number of cycles.
- Increase the amount of enzyme per reaction.
- Increase the primer concentration, but do not exceed 1 µM of each primer.
- Try a fresh dNTP solution.
- Optimise the MgCl2
1 Lorenz, T. C. Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies. J Vis Exp, e3998, doi:10.3791/3998 (2012).
The high GC content in my DNA is causing low yields of PCR product. What is the optimal denaturation temperature and time for PCRBIO VeriFi Polymerase in this case?
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. PCRBIO VeriFi 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.
PCRBIO VeriFi Polymerase is available in two formats. One format contains the enzyme (2units/µl) with a separate 5x VeriFi buffer and the other format contains the enzyme and buffer 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, the template and PCR grade water.
PCRBIO VeriFi Polymerase has an enhanced processivity relative to PCRBIO HiFi Polymerase, resulting in shorter extension times (10-30s/kb), higher yields and the ability to amplify longer (up to 17.5kb) and more difficult targets. PCRBIO VeriFi Polymerase also has a higher fidelity than PCRBIO HiFi Polymerase. Unlike PCRBIO HiFi polymerase, PCRBIO VeriFi Polymerase is available as a convenient 2x ready mix with the option of a red dye for direct gel loading, saving time during reaction setup and analysis.
PCRBIO VeriFi Polymerase has a very low error rate, with a fidelity approximately 100 times higher than wild-type Taq DNA polymerase.
30 seconds per kilobase (kb) is recommended for amplification from eukaryotic DNA. Shorter extensions times are possible provided there isn’t too much template DNA being loaded. If there are non-specific bands present after amplification the amount of template DNA should be decreased. 2-step protocols which combine annealing and extension are also possible. Optimisation of the cycling conditions and template concentration may be required if you deviate from the conditions and concentration recommended in the manual.
What type of cloning strategy should I use with a fragment generated using PCRBIO VeriFi polymerase?
PCR products generated by PCRBIO VeriFi 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 PCRBIO VeriFi polymerase has been removed from the reaction. If there is any PCRBIO VeriFi 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 PCRBIO VeriFi™ Polymerase on some difficult or long templates, for example GC-rich templates or those with complex secondary structures.