PCRBIO Ultra Polymerase & Mixes

The 5x PCRBIO Ultra Buffer supplied with PCRBIO Ultra Polymerase is specifically developed for this enzyme and we strongly recommend using them together. However, PCRBIO Ultra Polymerase should be compatible with any PCR buffer developed for use with wild-type Taq. If you use a custom buffer with PCRBIO Ultra Polymerase, keep in mind that reaction parameters such as annealing temperature and enzyme, template, dNTP, and MgCl₂ concentration may need optimization.

Yes. If you are working from bacterial colonies, use a sterile needle to take a colony and suspend it into the 50µl PCR reaction. If working from liquid culture, add 5µl of overnight culture into the final mixture. Follow the general protocol and increase the initial denaturation time to 10 minutes at 95°C.

Yes. Use 2 µL of blood sample for a 50 µL PCR reaction and follow the general protocol. Please note that components in the blood may inhibit the PCR. Perform a dilution series on the sample to determine the optimal template concentration for PCR amplification.

If smearing is a problem, the best approach is to ensure that it is not an artefact of running an agarose gel under suboptimal conditions. Suboptimal conditions include high voltage or not allowing enough time for the gel to set¹.

You may also need to troubleshoot the PCR reaction and consider the following suggestions².

• Primers should be designed to prevent primer-primer interactions and improve specificity.
• Increase the annealing temperature or perform PCR with an annealing temperature gradient to find the optimal annealing temperature.
• Reduce the amount of template in the reaction. For high-quality DNA, use 1–100 ng of genomic DNA or ≤5 ng of plasmid/lambda DNA per 50 µL reaction.
• Reduce the number of cycles.
• Reduce the amount of enzyme per reaction.
• Decrease the primer concentration, but do not go below 100 nM of each primer.
• Add DMSO into the reaction with a final concentration of 5%–10%.

1  Koontz, L. Agarose gel electrophoresis. Methods in enzymology: DNA. 1st edn, Vol. 529 35-45 (2013).

2  Lorenz, T. C. Polymerase chain reaction: basic protocol and troubleshooting and optimization strategies. J Vis Exp, e3998, doi:10.3791/3998 (2012).

You may want to consider the suggestions below and also consult the literature¹.

• Optimize the annealing temperature for PCR with a gradient of annealing temperatures.
• 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.
• Optimize MgCl₂ concentration.

1  Lorenz, T. C. Polymerase chain reaction: basic protocol and troubleshooting and optimization strategies. J Vis Exp, e3998, doi:10.3791/3998 (2012).

This non-inhibitory dye, added to allow for direct gel loading, migrates 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 with varying agarose content.

This enzyme has an error rate of approximately 1 error per 5.0 x 10⁵ nucleotides incorporated.

For products under 5kb, we recommend using 15 seconds per kb when amplifying from eukaryotic DNA. For longer products, we recommend extension times of 40 to 60 seconds per kb.