The new peqSTAR 2X − two independently useable 48-well thermocycler in one unit.

PCR results in only 9 minutes!News from our application lab – 'Fast PCR' with the peqSTAR 2X

Hold timesBesides heating and cooling rates, the hold time (plateau time) for each temperature also has a big impact on the overall run time. To reduce hold times, we performed our next test using a commercially-available Fast DNA polymerase. With such a polymerase, a synthesis speed of around 1 kb/s is achieved without loss of sensitivity or yield. Using the protocol as recommended by the manu-facturer, we were able to reduce the run time down to only 32 minutes. We then further optimised the protocol, cutting the number of temperature steps from 3 to 2, and further reducing the hold times. In this way, the complete run time was reduced to just 18 minutes.

IntroductionNowadays PCR is an essential standard method for every molecular biology lab. With the technique so well established, one of the main challenges today is to try to get valid results in ever shorter process times. Scientists and engineers worldwide are working on the development of 'Fast' and even 'Ultra Fast' PCR technologies, presenting their results under headlines like 'Under-three minute PCR' (DOI: 10.1039/C1AN15365J) and point out their spectacular time savings compared to traditional 90 minute run times. Although these advances are due in part to the advent of 'Fast' PCR reagents, we often see upon closer inspection that these innovative new systems do not support conventional thermocycler platforms and/or cannot be used with stan-dard PCR plastics.

Fast PCR with the peqSTAR 2X..?Considering the above, we wanted to know to what extent it might be possible to reduce the run time of a standard PCR by using a conventio-nal, but modern, thermocycler like the peqSTAR 2X, along with standard PCR plastics and commercially-available reagents. For a standard PCR amplification of a 200bp Lambda DNA fragment, we experimented with modifying a number of different parameters to produce a step by step reduction of run times. Heating and cooling rateAs starting point we performed the standard protocol, using standard PCR reagents on a Primus 96 advanced® thermocycler, and noted a run time of 95 min. We then ran the same protocol using the modern peqSTAR 2X thermocycler, and observed a run time of 67 min. The time saving of 28 minutes was due not only to the faster temperature ramping capabili-ties of the peqSTAR 2X, but also to its superior control circuitry and ultra-precise temperature regulation. This eliminates delays caused by oscillation around the target temperature (overshoot/undershoot) that is a feature of many other thermocycler makes and models.

Agarose gel electrophoresis of PCR products after the stepwise protocol optimization. With only a marginally reduced yield it was possible to reduce the run time by 95% down to only 9 min. We used the thermocyclers Primus 96 advanced® (1) and peqSTAR 2X (2 to 6) with standard PCR plastic. 1: standard protocol (95 °C 2 min; 30 x [95 °C 30 sec, 55 °C 30 sec, 72 °C 30 sec]; 72 °C 5 min), Primus 96 advanced® and Standard PCR reagents 2: Like (1), but on the peqSTAR 2X 3: Fast

Polymerase manufacturer’s standard protocol (95 °C 2 min; 30 x [95 °C 15 sec, 55 °C 15 sec, 72 °C 1 sec]; 72 °C 30 sec), peqSTAR 2X and Fast DNA polymerase. 4: Like (3), but further optimized 2-step protocol (95 °C 30 sec; 30 x [95 °C 5 sec, 55 °C 1 sec]). 5: Like (4), but only 20 cycles. 6: Like (4), but only 15 cycles. M: peqGOLD Ultra Low Range DNA ladder II.

able to reduce the complete run time from 95 min down to only 9 min – a reduction of 95%.

Summary: how to minimise PCR reaction times•Heating and cooling rate Thermocycler with high ramp rates•Hold times of the individual steps Use special Fast PCR reagents•Number of steps and cycles 2-step protocols/fewer cycles•Reducing ramp times further by reducing the temperature differential Use primer with high Tm

Number of cyclesFinally we wanted to find out how many cycles are really necessary to get detectable and reproducible PCR results. With our template concentration at 0.2 ng/µl, we reduced the number of cycles stepwise from 30 down to 15. In this way we were able to reduce the run time down to only 9 minutes.

Conclusion'Fast PCR' with the peqSTAR 2X is a reality. We have shown that, due to the high heating and cooling rates and superior temperature control capabilities of the peqSTAR 2X, and by using a Fast DNA polymerase with appropriate protocol optimization, it is possible to run Fast PCR reactions on a conventional platform using standard PCR plastics. In our example we were

Application Note