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ApplicationsNote 244 | December 2011Excellent temperature homogeneitycontributes to high PCR performance ofthe Mastercycler ® nexusNils Gerke 1, Henner Tasch 2 , Kirsten Schicke 2 , Andrea Hellberg 21 Eppendorf AG, Hamburg, Germany, 2 Eppendorf Instrumente GmbH, Hamburg, GermanyAbstractTemperature measurements for the purpose of evaluating block temperature homogeneity were performed onthe Mastercycler nexus and on two other thermal cyclers by competing manufacturers. The results illustratethe characteristics of the selectable temperature control modes and demonstrate the excellent homogeneoustemperature control of the Mastercycler nexus. Furthermore, reliable PCR performance is shown by using acommercially available PCR test kit.IntroductionMany factors influence PCR – apart from PCRcomponents such as primers and quality of template DNAand reagents, the technical parameters influencing PCRplay a major role. For example:- Thermal cycler (performance of the thermoblock)- PCR setup (e.g. liquid handling)- Quality of the reaction vesselsThe cyclical approach of the different temperature levels isa core function of the thermal cycler during the course ofa PCR reaction [1]. All block positions are to be controlledas homogeneously as possible to the set temperature.If this condition is fulfilled, a major contribution to theminimization of technical variability has been achieved.As a consequence, the certainty that the PCR productsobtained may be interpreted as actual biological results,rather than the result of technical artifacts, increases. Suchartifacts may manifest as non-specific products, falsepositive results or, in the worst case scenario, as falsenegative results.A high quality thermal cycler with a high level oftemperature accuracy and homogeneity in all positionsfulfills the basic technical requirements for minimization oftechnical variability, thus playing a major part in obtainingaccurate and reproducible results.A further aspect is highlighted by the fact that thermalcyclers made by different manufacturers may differconsiderably when it comes to temperature control.Consequently, a PCR system which has been optimizedand established on one thermal cycler may not deliver acomparable PCR product, despite employing the samePCR program, when a thermal cycler by a differentmanufacturer is involved.In this Application Note, the homogeneous and accuratetemperature control of the Mastercycler nexus isdemonstrated by temperature measurements and by theperformance of a commercially available PCR test kit forthe purpose of quality control.

Application Note 244 | page 3Mastercycler nexusfast modeCompetitor Afast-analog mode1001009090Temperature [°C]80706050Temperature [°C]80706050404030340 440 540 64030zoom-in: 95 °C stepTemperature [°C]9695.59594.59493.59392.592360 365 370 375 380 385 390 395 400 405 410 415 420 425Temperature [°C]9695.59594.59493.59392.592660 665 670 675 680 685 690 695 700 705 710 715 720 725Mastercycler nexusstandard modeCompetitor Bstandard-analog mode, setting: 50 µLTemperature [°C]11010090807060504030Temperature [°C]11010090807060504030315325335345355365375385395405415425435445455465475485495505515525535545555565575585595605615625635645655455465475485495505515525535545555565575585595605615625635645655665675685695705715725735745755765775785795805815zoom-in: 95 °C step107107Temperature [°C]1041019895Temperature [°C]104101989592330 335 340 345 350 355 360 365 370 375 380 385 390 395 40092480 485 490 495 500 505 510 515 520 525 530 535 540 545 550Figure 2: Recorded temperature profiles of the Mastercycler nexus and two other thermal cyclers. In both modes, the 95 °C stepwas selected for a zoom-in view.

Application Note 244 | page 4Temperature Spread [°C]1,51,41,31,21,110,90,80,70,60,50,40,30,20,10MC nexus(fastmode)MC nexus(standardmode)95 °C 72 °C 60 °CA(fast-analogmode)B(standard-analogmode)A1C1B12540 bp90 bpD12Figure 3: Thermoblock homogeneity: Temperature spreadbetween the lowest and the highest recorded temperaturevalue at the three different temperature steps.E1F12For each of the three tested temperature steps theMastercycler nexus showed the best block homogeneity(Figure 2 and 3). In particular, the 95 °C step revealed asignificantly smaller temperature spread than the two otherthermal cyclers.G1H122) Quality control of PCR performanceEach of the 96 well positions of a Cyclercheck platecontains two primer pairs that amplify two fragments of540 and 90 bp, respectively. According to the manufacturerboth primer pairs are designed to work optimally underthe same conditions in all individual positions of theplate (further information is provided by the Cyclercheckmanufacturer Protans). The separation of the PCR productson an agarose gel revealed a reliable amplification ofthe expected two PCR products at all 96 well positionsof the Mastercycler nexus (Figure 4). Thus, the hightechnical performance of the Mastercycler nexus shownby the temperature measurements (see above) could beconfirmed using this quality control PCR application.Figure 4: Performance of the Cyclercheck kit (Protrans) in all96 well positions of the Mastercycler nexus (Length standardin the first position of every row on the gel: GeneRuler 50bpDNA Ladder, Thermo Fisher Scientific).ConclusionThe Mastercycler nexus is characterized by high technicalperformance which contributes to minimizing technicalvariability in PCR sample processing. The results presentedhere demonstrate the outstandingly homogeneous andaccurate temperature control of the Mastercycler nexus.