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Operation ManualPCR SprintThermal CyclerHBSPRMAN Rev. 7 6 January 2004Analyze Detect Measure Control

PCR SPRINT TEMPERATURE CYCLING SYSTEMPCR License RegistrationThis instrument, Serial No. . is anAuthorized Thermal Cycler. Its purchase price includes the up-front feecomponent of a license under United States Patent Nos. 4,683,195,4,683,202 and 4,965,188, owned by Roche Molecular Systems, Inc., andunder corresponding claims in patents outside the United States, owned byF. Hoffman-La Roche Ltd, covering the Polymerase Chain Reaction (PCR)process, to practice the PCR process for internal research and developmentusing this instrument. The running royalty component of that license may bepurchased from Applied Biosystems or obtained by purchasing AuthorizedReagents. This instrument is also an Authorized Thermal Cycler for use withapplications’ licenses available from Applied Biosystems. Its use withAuthorized Reagents also provides a limited PCR license in accordance withthe label rights accompanying such reagents. Purchase of this product doesnot itself convey to the purchaser a complete license or right to perform thePCR process. Further information on purchasing licenses to practice the PCRprocess may be obtained by contacting the Director of Licensing at AppliedBiosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.No rights are conveyed expressly, by implication or estroppel to any patentson real-time methods, including but not limited to 5’ nuclease assays, or toany patent claiming a reagent or kit.Applied Biosystems does not guarantee the performance of this instrument.FS31999HBSPRMAN Rev. 76 January 2004Page 3 of 62

WarrantyThermo Electron Corporation guarantees that the PCR Sprint TemperatureCycling System you have received has been thoroughly tested and meets itspublished specification.This warranty is valid for 12 months* only if this product and functions havebeen used according to the user instruction manual.This warranty period can be extended to a total of 24 months (free ofcharge) by completing the warranty registration card supplied with theinstrument, also available online at www.thermo.com/warrantylog.No liability is accepted for loss or damage arising from the incorrect use ofthe PCR Sprint Thermal Cycler. Thermo's liability is limited to the repair orreplacement of the unit or refund of the purchase price at Thermo's option.Thermo Electron is not liable for any consequential damages.Thermo Electron reserves the right to alter the specification of the PCR Sprintwithout prior notice. This will enable us to implement developments as soonas they arise.The Thermo Electron PCR Sprint is for research use only.Read the Instruction Manual carefully before using the PCR Sprint to ensurethat you obtain the best possible results from the machine.NB: The PCR Sprint should only be used by suitably qualified and trainedpeople. If the PCR Sprint is not used as specified in this Manual, theprotection provided by the equipment may be impaired (see Chapter 2).* Excludes tube thermistor accessory, which carries a warranty for 90 daysonly.Page 4 of 62HBSPRMAN Rev. 76 January 2004

ContentsPCR Sprint Thermal Cycler1. System Description . 72. Installation. 133. Operation . 174. Running a Program. 215. Programming . 316. Comparative Performance Data . 477. Maintenance, Radiochemical and Electrical Safety . 498. Specifications & Ordering Information. 519. APPENDIX 1: Quick Reference Guide . 5310. APPENDIX 2: Peltier Technology . 5511. APPENDIX 3: Pre-Set Programs . 57HBSPRMAN Rev. 76 January 2004Page 5 of 62

Page 6 of 62HBSPRMAN Rev. 76 January 2004

1. System Description1.1OverviewPCR Sprint is designed for fast, accurate, licensed thermal cycling of smallnumbers of samples. PCR Sprint features high-speed sub-ambient blocks toperform oil-free thermal cycling with excellent dynamic uniformity andprecision control of sample temperature.1.2PCR Sprint SystemThe PCR Sprint System consists of a Control Chassis and an InterchangeableBlock Module. Each control chassis can operate one block module, whichcan be changed according to the needs of the sample format. There are twotypes of block modules, which have the following sample formats:The 0.2ml Block Module: holds 24 x 0.2ml individual OmniTubes.The 0.5ml Block Module: holds 20 x standard 0.5ml individual OmniTubes.The temperature cycling blocks for the system is of specially coatedprecision-machined aluminium, ensuring the best possible fit of reactiontubes in the block. This enables rapid and accurate heat transfer from theblock to the samples.Accurate sample temperature control in 0.2ml and 0.5ml block types isachieved by proprietary Active Tube Control software. The tube thermistorprobe monitors the temperature within a dummy sample tube and thisinformation is fed back to precisely control the block temperature to achievethe optimum cycling profile. This feedback loop allows the transition totarget temperature to be accelerated by creating a temperature gradientbetween the tube temperature and the block temperature.HBSPRMAN Rev. 76 January 2004Page 7 of 62

Alternatively, Simulated Tube Control may be used for reactions when it isnot appropriate to use the tube thermistor probe (for example in very small( 20µl) reactions). The temperature control algorithm is similar to activetube control, but is based on calculated values for the sample temperaturerather than values fed back by the tube thermistor.Heating and CoolingThe PCR Sprint sub-ambient blocks are built to proven designs, providing anaccurate, reliable and durable thermal cycling system.The sub-ambient aluminium block is heated and cooled by the latest in Peltiertechnology. The block modules excel in performing applications such asRAPD and Differential Display, which require cycling temperatures close toambient. It mirrors the temperature control characteristics of larger Thermoblocks in thermal cycling applications, allowing protocols to be directlytransferred. The block modules will control the temperature of the samplesfrom 20 C to 99 C for cycling reactions in all reaction formats. In addition,static incubation steps may be performed down to 4 C.Programming and OperationCycling programs are simple to perform, using the combination of userfriendly screens and operating keys. During programmed operation, thedisplay screens provide comprehensive information for the block includingsample temperature, number of cycles completed, estimated time forcompletion, etc.The PCR Sprint can be programmed to perform all types of temperaturecontrolled reactions, from simple one step incubations, to complex multi-steptemperature cycling protocols. Three additional temperature control softwareoptions are available to the user in the Advanced Edit Menu if required:Temperature Ramping, Time Increment/Decrement and TemperatureIncrement/Decrement.The unit has program space for up to 60 full cycling protocols including thepreset protocols. Programs can be saved in one of five user directories, eachof which can be identified by user or protocol type.Preset ProgramsThe PCR Sprint is supplied with 10 non-editable preset protocols stored asprograms 51-60 in the F:THERMO directory. These protocols cover the mostcommon thermal cycling techniques and can be used to create customizedprotocols by editing (see section 5). Full details of the cycling parameters ofeach can be found in Appendix III.Page 8 of 62HBSPRMAN Rev. 76 January 2004

Advanced Edit - Temperature RampingThe temperature ramping (Ramp Rate) enables the rate of change of sampletemperature (degrees centigrade per second) to be slowed down.Under normal circumstances the temperature cycling times are very rapid,which minimizes non-specific reactions. The rate of sample temperaturechange during thermal cycling is controlled so that it is as fast as possiblewithout affecting the block uniformity and accuracy. In some instances,however, it may be advantageous to limit the rate of change of temperature,for example, to allow limited extension of short or degenerate primersbetween primer annealing and DNA synthesis steps to stabilize the primer/template duplex.Advanced Edit - Time/Temperature IncrementThese features enable the time interval and/or the temperature of a specifiedprogrammed step to be increased or decreased with successive temperaturecycles. For example, it may be advantageous to increment the extension timeinterval to compensate for deterioration of enzyme activity in later cycles.Alternatively, temperature decrements can be used to perform TouchDownPCR in which it is required to decrease the annealing temperature oversuccessive cycles.Temperature ControlTemperature accuracy and uniformity across the block, particularly duringrapid temperature cycling operations, have been of paramount importanceduring the design and development of the PCR Sprint. Every machine iscalibrated using a number of thermistor probes, located in tubes, which areplaced at several block positions to ensure that the required temperaturesand incubation times achieved are identical in all samples.1.3The Heated LidThe unit can be operated with or without the lid being activated. The heatedlid enables PCR Sprint users to run temperature cycling protocols without theneed for paraffin or mineral oil overlays. In a unit without a heated lid suchvapour barriers are required to prevent evaporation of reaction constituentsbefore thermal cycling is complete.The heated lid operates by positioning a heated plate in intimate contact withthe top of the reaction tubes or wells. The heating plate then raises the airtemperature at the top of each tube to a temperature that is permanentlyhigher than the sample temperature. The elevated air temperature, relativeto the sample temperature, minimizes evaporation as the reaction mixture isrepeatedly heated and cooled.The lid is opened by pressing the black catch. On closing, the height of theheating plate inside the heated lid is automatically adjusted on light pressuresprings. The heated lid is designed to be compatible with both reactionsample formats, i.e. 0.5ml tubes and 0.2ml tubes. In most cases, anexperimental protocol is largely unchanged when switching from using oiloverlay to an oil-free system.HBSPRMAN Rev. 76 January 2004Page 9 of 62

!WARNINGThe tops of the reaction vessels and the surfaces of the HeatedLid assembly (in particular the inner surfaces) can become veryhot during normal operation. Touching the surfaces can causeburns. Do not touch the heated plate.The temperature of the heating plate is set at 115 C (max. surfacetemperature).The heating plate inside the lid is switched on and off from within theprogram. The lid operates once a program is activated, with a heating timeof typically 3 minutes before cycling commences.1.4SafetyThe PCR Sprint has been designed for safe operation. The following symbolsappearing on the unit and their meanings should be noted.I Indicates the ON position of the main power switch.O Indicates the OFF position of the main power switch.Consult the manual for further information.Consulter les documents d’accompagnement.!WARNINGIndicates a heating hazard.Proceed with caution to avoid burn injury.ATTENTION: Surface chaude.!WARNINGSAFETY NOTE: This symbol indicates high voltage. Risk ofelectric shock.AVERTISSEMENT: Risqué de choc electrique.Page 10 of 62HBSPRMAN Rev. 76 January 2004