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The 5x PCRBIO Ultra Buffer supplied with PCRBIO Ultra Polymerase has been developed specifically for this enzyme and we highly 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 customised buffer with PCRBIO Ultra 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.
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.
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 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).
The enzyme has an error rate of approximately 1 error per 5.0 x 105 nucleotides incorporated.
For amplicons below 5kb, we recommend 15 seconds per kb for amplification from eukaryotic DNA. For longer amplicons, we recommend between 40 and 60 seconds per kb.