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Our PCRBIO Hot Start Taq DNA Polymerase is particularly suited for multiplexing due to the presence of our hot-start technology, which blocks the activity of Taq polymerase at low temperatures.
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).
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.
Yes. Use 2 µL blood sample to a 50 µL PCR reaction and follow the general protocol. Please note that blood components may inhibit the PCR reaction. Perform a serial dilution of the sample in order to find the optimal template concentration for the PCR amplification.
No. PCRBIO HS Taq DNA Polymerase has 5’-3’ exonuclease activities, but no 3’-5’ exonuclease (proofreading) activity.
PCR products generated with PCRBIO HS Taq DNA Polymerase are A-tailed and this makes it suitable for cloning into TA vectors. For further reading, refer to this literature1.
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).
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 artefact of running agarose gel electrophoresis with suboptimal conditions. Suboptimal conditions can 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 suggestions 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).
PCRBIO HS Taq DNA Polymerase uses proprietary antibody-mediated hot start technology whereas PCRBIO Taq DNA Polymerase does not have this feature. The interaction of the antibody with the Taq DNA Polymerase leaves the enzyme inactive until the hot start step.
The hot start refers to the initial activation step at 95°C, which subsequently deactivates the antibody bound to the DNA polymerase. Inactivation of the Taq DNA Polymerase below 65°C prevents primer-dimer formation and non-specific amplification. This allows for specific amplification from low copy number target sequences.
The enzyme has an error rate of approximately 1 error per 2.0 x 10⁵ nucleotides incorporated.
For amplicons between 1kb and 5kb, we recommend 15 seconds/kb for amplification from eukaryotic DNA. For shorter amplicons, a 1 second extension is sufficient.