PCRBIO Classic Taq

The PCRBIO 10x reaction buffer supplied with PCRBIO Classic Taq has been specifically formulated for this enzyme and we highly recommend using them together. However, PCRBIO Classic Taq should be compatible with any PCR buffer designed for use with a wild-type Taq. If you use a custom buffer with PCRBIO Classic Taq, please bear in mind that reaction parameters such as annealing temperature and concentrations of enzyme, template, dNTPs, and MgCl₂ may need optimization.

Yes. If working from bacterial colonies, use a sterile pipette tip to pick a colony into a 50µl PCR reaction. If working from a liquid culture, add 5µl of overnight culture to the final mix. Follow the standard procedure and increase the initial denaturation step to 10 minutes at 95°C.

Yes. Use 2 µL blood sample per 50 µL PCR reaction and follow the standard procedure. Be aware that components of blood may inhibit the PCR reaction. Perform a serial dilution of the sample to find the optimal sample concentration for efficient PCR amplification.

No. PCRBIO Classic Taq has a 5’-3’ exonuclease activity, but it does not have a 3’-5’ exonuclease (proofreading) activity.

PCR products generated by PCRBIO Classic Taq are A-tailed, making them suitable for TA cloning. For more information, please refer to the document¹.

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

If smearing is a concern, good practice is to ensure that it is not an artifact caused by running an agarose gel with suboptimal conditions. Suboptimal conditions may include high voltage or not letting the gel solidify sufficiently¹.

You may also want to reevaluate the PCR reaction and consider the following suggestions².

• Primers should be designed to prevent primer-dimer formation and improve specificity.
• Increase the annealing temperature or perform a 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 below 100 nM of each primer.
• Add DMSO to the reaction at a final concentration of 5%–10%.

1  Koontz, L. Agarose gel electrophoresis. Methods in enzymology: DNA. First Edition, Volume 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 following suggestions and also refer to the document¹.

• Optimize the annealing temperature in a 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 new dNTP solution.
• Optimize MgCl₂

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

Upon receipt, the kit should be stored at -20°C. Avoid prolonged exposure to light. If stored properly, the kit will maintain full activity for 12 months. The kit can be stored at 4°C for 1 month.

The PCRBIO Classic Taq enzyme is supplied with 10x PCRBIO Classic Buffers, with the buffer containing 30 mM MgCl₂. dNTPs are sold separately and must be added to the final reaction. The PCRBIO Taq DNA Polymerase is supplied with 5x PCRBIO Reaction Buffer, with the buffer containing both MgCl₂ and dNTPs. Additionally, PCRBIO Taq DNA Polymerase is also available as a 2x Mix for added convenience, with and without a red dye for direct loading onto agarose gels.

The enzyme has an error rate of approximately 1 error per 2.0 x 10⁵ nucleotides incorporated.

15 seconds per kilobase (kb) for amplification from eukaryotic DNA with amplicons between 1kb and 6kb. For shorter amplicons, a 1-second extension is sufficient.