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Frequently asked questions about High Performance PCR
Q 01: What is High Performance PCR?
It is a three-part solution comprised of Phusion DNA Polymerase, the Piko Thermal Cycler and UTW (ultra-thin walled) PCR vessels, all developed and produced by Finnzymes.
Q 02: What are the benefits of High Performance PCR?
The High Performance PCR system has been specifically engineered to provide the following benefits to the researcher:
- Significant time savings.
- Near error-free amplification.
- Dramatic improvements in yield in every well.
- Robust and specific amplification with a broad range of conditions.
Q 03: How much experimental optimization is required for High Performance PCR?
High Performance PCR is as simple as any PCR, but it follows slightly modified rules for creating the protocol. Most of these changes can be found within the Phusion DNA Polymerases instruction sheets.
The key points to remember include:
- Raising the denaturing temperature to 98°C.
- Raising the annealing temperature by 5-8°C (compared to conventional protocols using Taq).
- Shortening the denaturation, annealing and extension steps.
Q 04: How much time could I save using High Performance PCR?
By simply changing from a standard cycler to the Piko, you can expect to see a 10-15 minute time savings if you use the same protocol. This is derived solely from the faster ramp rates from the Piko Thermal Cycler. High Performance PCR does require a slightly different set of rules to achieve the full benefit; following these rules will give you even faster results.
Q 05: What is the key to maximizing protocol speed with the High Performance PCR?
In High Performance PCR, the polymerase, the thermal cycler and the vessel each offer time savings through different mechanisms. Click here for a chart with a more detailed explanation.
Q 06: Is the model of thermal cycler important to High Performance PCR?
Yes. The Piko Thermal Cycler offers these advantages other PCR machines just can’t match:
- Excellent thermal specifications necessary for very fast protocols [link to technical specifications of Piko]. Accuracy and well-to-well uniformity ensure consistent results with even the fastest protocols.
- Short settling time. Settling time indicates how long it takes the sample to equilibrate at the target temperature of the program. The Piko Thermal Cycler settles in approximately 1 second, allowing hold times to be dramatically shortened. In practice, we recommend hold times of 5 seconds or less for the denaturation and annealing steps; even a 0-second hold time can often provide excellent results.
- Uniform pressure from the heated lid. The computer-controlled lid automatically detects and adjusts lid pressure to ensure even pressure and excellent sealing. Lid temperature is controlled by an optimized heating algorithm that ensures minimal condensation while limiting heat transfer from the lid to the sample.
Q 07: How important to High Performance PCR is the type of vessel used?
Very important. We have finely tuned the samples' thermal profile within UTW vessels by using our proprietary Piko thermal control algorithm. This means that High Performance PCR reactions which are run in conventional thin-walled tubes may give very poor results when compared to UTW vessels. Click here for some examples.
Q 08: Can I use only the Phusion DNA Polymerase to achieve the benefits of High Performance PCR?
Although Phusion DNA Polymerase is a unique enzyme that offers a number of superior features, it requires the complete High Performance PCR solution to take full advantage of its potential. The dramatic time savings and consistent well-to-well yields is only achievable when coupled with the Piko/UTW PCR system. This is a result of the system’s double benefit of fast ramp rates and short settling times, which have been optimized precisely to match the performance of Phusion DNA Polymerases.
Q 09: Other companies advertise polymerases that extend at 1 second/kb. Does High Performance PCR offer a similar advantage?
Yes it does, at least when amplifying smaller amplicons. Phusion DNA Polymerase’s processivity is 10 times that of Pfu, and we recommend extension times of 15 s/kb for Phusion Flash PCR Master Mix. 15 s/kb is a conservative value that we can promise to work with almost any amplicon. In many cases significantly shorter extension times (0-5 s/kb) can be used without compromising the yield. What separates Phusion Flash DNA Polymerase from other fast polymerases is that all steps in the PCR protocol can be shortened, including annealing and denaturation. This results in extremely fast protocols as compared with any other polymerase. For an example, please click here to see the robust yields for a 400 bp amplicon using a 0 second extension step.
Q 10: Can High Performance PCR work well with impure samples or with typical PCR inhibitors present?
High Performance PCR works very robustly under a broad variety of conditions where conventional PCR simply does not work. For example, direct PCR from raw blood gives high product yields even at very high blood concentrations.
