熱循環(huán)儀最重要的要求是溫度的準確性與重現(xiàn)性。然而，由于市售PCR試劑的差異，因此熱循環(huán)儀的溫度性能可能很難確定，除非是采用一款精密的PCR系統(tǒng)。而熱循環(huán)儀糟糕的溫度性能與蒸發(fā)是導(dǎo)致PCR結(jié)果不佳的常見問題。通常，對一個PCR體系進行優(yōu)化是一個費時、費力、費錢的工作。

此外，我們也很難根據(jù)技術(shù)參數(shù)表上的數(shù)據(jù)對溫度性能與蒸發(fā)情況進行評估。本篇應(yīng)用旨在介紹如何使用Eppendorf epMotion 系統(tǒng)進行微量體積的PCR體系構(gòu)建，減少繁瑣的準備工作，提高實驗的準確性。同時，配合具備出色的溫度性能與防蒸發(fā)功能的 Mastercycler X50 PCR 儀，即使在進行微量體積的PCR實驗時，也能獲得可靠的實驗結(jié)果。

Highly reproducible low Volume PCR with Mastercycler^® X50 and epMotion^®
Arora Phang, Tim Schommartz, Eppendorf AG, Hamburg, Germany

Abstract
  Key requirements of a thermal cycler are naturally accuracy and reproducibility in terms of temperature performance. However, due to increasingly robust PCR reagent formulations in the market, temperature performance of a thermal cycler can be difcult to determine until one comes across a delicate PCR system. Optimization of such a system then becomes very tedious, laborious and can be extremely resource-consuming. Poor temperature performance of a thermal cycler and evaporation are two common issues that lead to poor PCR results.
  However, these two factors are difcult to simply assess based on numbers on a technical specifcation sheet. This paper demonstrates how Eppendorf epMotion effortlessly handles low volume PCR set-up, hence cutting down preparation effort while simultaneously increasing accuracy. Furthermore, it presents the robustness of the new Eppendorf Mastercycler X50 even when running low volume PCR, demonstrating its excellent temperature performance and protection against evaporation.
Introduction
  Modern scientifc trends tend towards higher speed, greater convenience and lower volumes. Similarly, scientifc equip ment follows through this line of evolution to push such lim its, giving rise to fancier equipment and techniques. Though easily overshadowed in the wake of such exciting advance ment, performance breakthroughs in simple and established
equipment can also be more useful as they are generally less resource intensive to operate.
  Large PCR setups can be very time-consuming and tedious if done manually. Especially when handling very small volumes; imprecision and pipetting errors caused by the operator can lead to massive variations of the results. This operator-based error carries an even bigger implication when different individuals are involved in the same work flow. A way to overcome this issue is to use an automated liquid handling platform. This allows an increase of sample throughput while at the same time ensuring a reliable handling of small volumes with minimal variations between experimental runs.
  Temperature performance of a thermal cycler is generally measured using an external temperature verifcation device. Such a measurement method can provide a good indication on the performance of the thermal block of a cycler. However, overall PCR performance relies on not just the thermal block but also the additional combination of consumable compatibility and ability to maintain reaction concentration by protecting against evaporation. Thus, the results of an actual PCR run using a temperature sensitive PCR system would present the best direct empirical proof of reliable performance of a thermal cycler. Additionally, a thermal cycler performance in terms of accuracy, homogeneity and robustness can be verifed by performing the same PCR under stricter conditions such as lower reaction volumes, enzyme concentration and template quantity. The results from this Application Note will illustrate the convenience of the epMotion and robustness of the Mastercycler X50 in handling low volume PCR setup and runs.
Materials and Methods
  PCRBio Taq DNA polymerase (NIPPON Genetics) and Human Genomic DNA (Roche®) were used for the following amplifcation. PCR reaction master mix was prepared with 1X reaction buffer and 0.2 µM of each primer, for fnal volumes of 3 µL or 10 µL per reaction respectively. For 10µL reaction, 0.25U of enzyme and 20 ng DNA were used while when amplifying 3 µL reaction volume, the amount of enzyme and DNA was reduced to 0.15U and 10 ng, respectively. The master mix was dispensed into all 96 wells of Eppendorf twin.tec® skirted PCR plates.
  The following primers were used for amplifcation of the human ß-actin gene:
  Forward primer: 5’- ATCGCCGCGCTCGTCGTC-3’
  Reverse primer: 5’- TGGGTCATCTTCTCGCGGTTGG-3’

  Dispensing was carried out by Eppendorf epMotion 5073l equipped with a 0.2–10 µL TS10 single channel dispensing tool using free jet dispensing in pipetting mode. The work table-setup is displayed in Figure 1. Furthermore, this dis pensing protocol uses a device-default template and hence can be easily adapted for other Eppendorf epMotion models equipped with a 0.2–10 uL dispensing tool.

Figure 1: epMotion 5073l worktable for PCR setup

  Plates were sealed with Eppendorf Adhesive PCR Film and PCR were carried out on Mastercycler X50s. Cycling conditions are listed in Table 1. The PCR products were detected using GelRedTM (Biotium) following agarose gel electrophoresis and visualized using the Gel Doc XR+ (BioRad®).
 

Table 1: PCR condition with two concurrent gradient setting at denaturation and annealing steps.

Results and Discussion
  The new Eppendorf epMotion TS10 pipetting tool and Mastercycler X50 are a powerful combination in running low volume applications. Not only does an automated pipetting system provide superior accuracy, it is also comparatively more pleasant than setting up PCR reactions manually, especially at high throughput. The error tolerance range is naturally narrower when working with lower liquid volumes. Therefore, automated systems with defned and controlled
force provide more reproducible dispensing of PCR reagents, which in turn result in higher confdence in PCR results. The use of an automated liquid handler also allows the operator to reduce hands-on setting-up time to a bare minimum.
  Additionally, evaporation issues in a thermal cycler are intensifed at lower reaction volumes due to the lower amount of water present in the mastermix. Hence, a change in even a small amount of liquid will lead to a big shift in reagent concentration that will affect PCR amplifcation. As evaporation in each well across the block is uncontrollable, irreproducibility is the most common consequence of evaporation. Therefore, a good thermal cycler is one that has good thermal performance as well as a design that protects against evaporation.

  These evaluation criteria can be easily tested using a temperature sensitive PCR system. The human ß-actin gene used in this study is one such system. Specifc amplifcation will yield 484 bp fragments while sub-optimal condition will give rise to non-specifc amplifcation visible as a 350 bp artefact in the gel. This characteristic can be used to clearly portray how fnely temperature control a cycler has. Figure 2A showed the PCR amplifcation of the ß-actin gene across different temperatures using the gradient function while Figure 2B shows the comparative result of the same system under reduced volume. The results showed that each different annealing temperature gives a different specifc and non-specifc fragment yield, with highest yield at 65.9 °C while highest specifcity at 70.5 °C. Even though the difference in each tested temperature is small, the effect was nonetheless obvious due to the sensitive nature of this PCR system. These differences hence illustrate the fne control and accuracy the Mastercycler X50 has over the temperatures across the block.
  Additionally, these results were highly reproducible at vastly reduced reaction volume (3 µL), showing not only that automated pipetting was very precise, the wells also did not suffer from evaporation problems. At 3 µL, the DNA bands seem to have higher intensity. A possible explanation is that while the amount of enzyme and DNA were reduced accordingly in the 3 µL setup, the overall concentration of enzyme and DNA per total volume is slightly higher, hence resulting in slightly higher yield. Furthermore, it is expected that the effect caused by condensation/evaporation in 3 µL is larger than in 10 µL at the same rate due to the lower amount of water present in the 3 µL setup. This will thus further driveup the concentration of reagents in 3 µL setup and may result in slightly higher fnal yield at the same temperatures. The respective reductions in enzyme consumption and DNA amount required for successful amplifcation at low reaction volumes have two important implications. While many inexpensive PCR reagents exist in the market, DNA polymerases remained one of the most cost-consuming elements in PCR. This is especially true for users with high-throughput or when more expensive reagents are needed to increase efciencies when amplifying difcult targets. Similarly, for users working with precious samples or low concentration targets, the ability of a thermal cycler to reproducibly amplify low amounts of samples can be the determining factor between PCR success or failure.
Conclusion
  The results from this study showed that the Eppendorf Mastercycler X50 is a thermal cycler with excellent tem perature performance and evaporation protection that gives highly reproducible result even for low volume PCR applications or delicate PCR system.
  Combined with the Eppendorf epMotion to handle low volume liquid handling set-up, a PCR can be set up effortlessly and completed with minimal time and effort for maximal accuracy and reproducibility.