Operator Manual
252 Pages
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Operator Manual
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Smart Cycler Operator Manual
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Copyright 1999-2004 by Cepheid All rights reserved. Printed in USA This manual contains information protected by copyright. No part of this manual may be photocopied or reproduced in any form without prior written consent from Cepheid. Trademark Information Smart Cycler®and I-CORE® are registered trademarks of Cepheid. Smart Cycler® and I-CORE® are not to be used in any type of promotion or advertising without permission from Cepheid. All other trademarks are the property of their respective owners. Cepheid Disclaimer Cepheid makes no warranties, express or implied, including without limitation the implied warranties of merchantability and fitness for a particular purpose, regarding the Smart Cycler® System. Cepheid does not warrant, guarantee or make any representations regarding the use or the results of the use of Smart Cycler® System in terms of its correctness, accuracy, reliability, currentness or otherwise, and the entire risk as to the results and the performance of Smart Cycler® System is assumed by you. The exclusion of implied warranties is not permitted in some states, therefore the above exclusion may not apply to you. In no event will Cepheid, its directors, officers, employees or agents be liable to you for any consequential, incidental or indirect damages (including damages for loss of business profits, business interruption, loss of business information, and the like) arising out of the use or inability to use Smart Cycler® System, even if Cepheid has been advised of the possibility of such damages. Because some states do not allow the exclusion or limitation of liability for consequential or incidental damages, the above may not apply to you. Cepheid's liability to you for actual damages from any cause whatsoever, and regardless of the form of the action (whether in contract, tort [including negligence], product liability or otherwise), will be limited to $50. Notice to Purchaser This real-time thermal cycler is licensed for use in research, diagnostics (except in diagnosis and monitoring of HIV and HCV infections) and all other applied fields under Applera's European Patent No. EP 0 872 562, Japanese Patent No. JP 3136129 and patents pending. No rights are conveyed expressly, by implication or estoppel to any patents on real-time methods, including but not limited to 5' nuclease assays, or to any patent claiming a reagent or kit. Applied Biosystems does not guarantee the performance of this instrument or endorse its use in any application. Authorized Thermal Cycler This instrument, the Smart Cycler® processing block, serial number, _____________________, is an Authorized Thermal Cycler. Its purchase price includes the up-front fee component of a license under United States Patent Nos. 4,683,195, 4,683,202, and 4,965,188, owned by Roche Molecular Systems Inc., and under corresponding claims in patents outside the United States, owned by F. Hoffmann-La Roche Ltd, covering the Polymerase Chain Reaction (“PCR”) process, to practice the PCR process for internal research and development using this instrument. The running royalty
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component of that license may be purchased from Applied Biosystems or obtained by purchasing Authorized Reagents. This instrument is also an Authorized Thermal Cycler for use with applications licenses available from Applied Biosystems. Its use with Authorized Reagents also provides a limited PCR license in accordance with the label rights accompanying such reagents. Purchase of this product does not itself convey to the purchaser a complete license or right to perform the PCR process. Further information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California, 94404. Disclaimer of License No rights for any application, including any in vitro diagnostic application, are conveyed expressly, by implication or estoppel under any patent or patent applications claiming homogenous or real-time detection methods, including patents covering such methods used in conjunction with the PCR process or other amplification processes. The 5´ nuclease detection assay and certain other homogeneous or real-time amplification and detection methods are covered by United States Patent Nos. 5,210,015, 5,487,972, 5,804,375, and 5,994,056, owned by Roche Molecular Systems Inc.; by corresponding patents and patent applications outside the United States, owned by F. Hoffmann-La Roche Ltd; and by United States Patent Nos. 5,538,848 and 6,030,787, and corresponding patents and patent applications outside the United States, owned by PE Corporation. Purchase of this instrument conveys no license or right under the foregoing patents. Use of these and other patented processes in conjunction with the PCR process requires a license. For information on obtaining licenses, contact the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California, 94404, or The Licensing Department, Roche Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, California, 94501. Cepheid Smart Cycler® System Software License Agreement License Grant: Subject to the terms and conditions herein, Cepheid grants Customer a non-exclusive, non-transferable license (the “License”) to use one (1) copy of the Cepheid Smart Cycler System Software program (the “Software”) on the computer provided with the Smart Cycler System and connected to the Smart Cycler processing block(s) and (1) copy on a second computer that is identical to the computer provided with the Smart Cycler System, to be used only for Smart Cycler data analysis and to make one (1) copy for back-up purposes. Other than expressly provided herein, Customer shall not copy, modify, duplicate, translate, disassemble, or decompile the Software without Cepheid's prior written consent. License Type: Single-user Product. Customer shall use the Software only on the computer provided with the Smart Cycler System or a second computer identical to the computer provided with the Smart Cycler System. If the computer is not identical to the one provided with the Smart Cycler System, Cepheid will not guarantee performance and cannot provide technical support for problems arising from the use of a different computer. Customer may not use this single-user product on a network.
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Copyright: The ideas and expressions thereof contained in the Software are confidential and proprietary information and trade secrets of Cepheid that are disclosed to Customer in confidence. Customer shall not cause or permit decompilation, disassembly, or reverse engineering of the Software or disclosure, copying, display, loan, publication, transfer of possession (whether by sales, exchange, gift, operation of law or otherwise) or other dissemination of the Software and related documentation, in whole or part, to any third party without the prior written consent of Cepheid. The Software and its related documentation are owned by Cepheid and protected by United States copyright laws and international treaty provisions. Customer shall not alter or remove any copyright, trade secret, patent, trademark, proprietary and/or other legal notices contained on or in the Software and related documentation. Ownership: Cepheid is the exclusive owner of the Software, related documentation and physical media, and of all copyright, trade secret, patent, trademark and other intellectual or industrial property rights therein. Physical media and copies of the Software, whether in diskette, tape, paper or other forms provided by Cepheid, shall remain the property of Cepheid, and such copies are deemed to be on loan to Customer during the term of the License granted hereby. Customer expressly acknowledges that no title to or ownership of the Software, or any copy or portion thereof, is transferred to Customer hereby. Support: This License entitles Customer to the use of toll free telephone support as provided by Cepheid. Termination: This License is effective until terminated. Cepheid may terminate this License if Customer fails to comply with any of the terms or conditions of this License or of the Cepheid System Agreement. If this License is terminated, Customer must destroy all copies of the Software and its related documentation. Warranty Information: CEPHEID SPECIFICALLY DISCLAIMS ALL OTHER WARRANTIES INCLUDING, BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY AND OF FITNESS FOR A PARTICULAR PURPOSE. Cepheid shall not be liable for incidental or consequential damages resulting from use of the Software. In particular, Cepheid does not warrant that the Software will meet Customer's requirements or that the operation of the Software will be uninterrupted or error free. In the event that the Software media is defective, Cepheid will replace the defective media at no cost to Customer. Government Customers: For Government customer, the Software is commercial computer software subject to restricted rights under FAR 52.227-19 (C) (1, 2). Limitation of Liability: The Software is licensed on an “as is” basis. This means the entire risk as to the quality and performance of the Software is on Customer. IN NO EVENT WILL CEPHEID BE LIABLE FOR ANY LOSS, DAMAGE, OR LIABILITY, INCLUDING, WITHOUT LIMITATION, ANY DIRECT, INDIRECT, SPECIAL, INCIDENTAL OR CONSEQUENTIAL DAMAGES INCURRED BY CUSTOMER OR ANY OTHER PERSON AS A RESULT OF, OR RELATED TO, THE DELIVERY, USE, OR PERFORMANCE OF THE CEPHEID SMART CYCLER SOFTWARE OR ANY BREACH OF THE WARRANTIES CONTAINED HEREIN, WHETHER DUE TO THE NEGLIGENCE OF CEPHEID OR OTHERWISE. Some states do not allow the exclusion or limitation of incidental or consequential damages, in which case the above limitation or exclusion may not apply to Customer.
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Smart Cycler Processing Block Limited Warranty Cepheid warrants that the Smart Cycler Processing Block (the Instrument) (i) is free from defects in material and workmanship and (ii) conforms to Cepheid's published specifications for the Instrument. This Warranty is for a period of 12 months from the date of shipment to the Purchaser (the Warranty Period). During the Warranty Period, if the Instrument's hardware is found to be defective or if the Instrument fails to meet specifications, Cepheid will repair or replace the Instrument so that it meets specifications, at a site determined by Cepheid at Cepheid's expense. This Warranty is limited and applies only to new products manufactured by Cepheid. Cepheid does not warrant any defects in the Instrument caused by (i) improper installation, removal or testing, (ii) Purchaser’s failure to provide a suitable operating environment for the Instrument, (iii) use of the Instrument for purposes other than that for which they were designed, (iv) unauthorized attachments, (v) unusual physical or electrical stress, (vi) modifications or repairs done by other than Cepheid or a Cepheid authorized service provider, or (vii) any other abuse, misuse, or neglect of the Instrument. Purchaser's exclusive remedy for any defective Instrument is limited to the repair or replacement of any defective Instrument. If Cepheid cannot or does not repair or replace a defective Instrument, Cepheid will remove the Instrument and return the purchase price for the defective Instrument. This warranty extends to Purchaser only, and not to Purchaser's customers or other third parties, except as agreed to in writing by Cepheid. Even if Cepheid cannot or does not repair or replace any defective Instrument and Purchaser's exclusive remedy fails of its essential purpose, Cepheid's entire liability shall in no event exceed the purchase price for any defective Instrument, and Cepheid shall have no liability for general, consequential, incidental or special damages arising from a defect in the Instrument. THERE ARE NO WARRANTIES WHICH EXTEND BEYOND THE FACE HEREOF. CEPHEID DISCLAIMS ALL OTHER REPRESENTATIONS AND WARRANTIES, EXPRESSED OR IMPLIED, REGARDING THE PRODUCTS, INCLUDING ANY IMPLIED WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. CEPHEID SHALL HAVE NO STRICT LIABILITY, PRODUCTS LIABILITY OR NEGLIGENCE, WHETHER ACTIVE OR PASSIVE.
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Table of Contents Preface - Safety Information... 1 Chapter 1 - Theory of Operation... 7 Chapter 2 - Installation... 15 Chapter 3 - The Smart Cycler Software Overview... 27 Chapter 4 - The Smart Cycler Software... 53 Chapter 5 - Tutorial: Running Smart Cycler Assays... 127 Chapter 6 - Maintenance... 163 Chapter 7 -Troubleshooting... 173 Appendix A - User Administration... 199 Appendix B - Optical Calibration... 205 Appendix C - Windows 2000 with the Smart Cycler Software... 223 Appendix D - Smart Cycler Technical Specifications... 227 Index... 229 Smart Cycler Software Release Notes... 239
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Preface - Safety Information Symbols and Conventions Used in This Manual Signal words are as follows: DANGER - indicates an imminently hazardous situation, which, if not avoided, will result in death or serious injury (most extreme). GEFAHR: zeigt eine gefährliche Situation an, die, wenn nicht vermieden, Tod oder die ernste Verletzung ergibt (am extremsten). WARNING - indicates a potentially hazardous situation which, if not avoided, could result in serious injury or death. WARNUNG - zeigt eine möglicherweise gefährliche Situation an, die, wenn Sie nicht vermieden, konnten ernste Verletzung oder Tod ergeben. CAUTION - indicates a potentially hazardous situation which may result in minor or moderate injury to the user or damage to the equipment. VORSICHT - zeigt eine möglicherweise gefährliche Situation an, die kann geringe oder gemäßigte Verletzung zum Benutzer oder zur Beschädigung der Ausrüstung ergeben. Sample hazard symbols are shown below: Beispielgefahrsymbole werden gezeigt unter: WARNING: This heading and symbol are used to indicate that non-compliance with instructions or procedures may lead to injury or even death. WARNUNG: Dieser Kopftext und Symbol werden benutzt, um anzuzeigen, daß Zuwiderhandlung gegen Anweisungen oder Prozeduren zu Verletzung oder sogar Tod führen kann. CAUTION: This heading and symbol are used to indicate that non-compliance with instructions or procedures may cause damage to the instrument. VORSICHT: Dieser Kopftext und Symbol werden benutzt, um anzuzeigen, daß Zuwiderhandlung gegen Anweisungen oder Prozeduren Beschädigung des Instrumentes verursachen kann.
Note: this heading is used to bring your attention to topics of importance.
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Safety Labels There are two safety labels on the Smart Cycler instrument. The labels contain symbols that indicate the type of potential hazard, as well as signal words accompanied by definitions of the particular hazard. Es gibt zwei Sicherheit Kennsätze auf dem Smart Cycler-Instrument. Die Kennsätze enthalten Symbole, die die Art der möglichen Gefahr anzeigen, sowie die Kennzeichenwörter, die von den Definitionen der bestimmten Gefahr begleitet werden. WARNING: DISCONNECT ALL ELECTRICAL CORDS FROM DEVICE BEFORE PROCEEDING FURTHER. CONSULT USER MANUAL FOR ADDITIONAL INSTRUCTIONS.
This safety label is located inside the Smart Cycler lid. Dieser Sicherheit Kennsatz befindet sich innerhalb der Kappe des Smart Cycler.
¤
C
US
Smart Cycler ONLY MAXIMUM CHAIN OF 3 UNITS
This safety label is located on the back panel of the Smart Cycler instrument. Dieser Sicherheit Kennsatz ist auf der Rückplatte des Smart Cycler.
Smart Cycler Safety Procedures Before using the Smart Cycler system, read the following safety information. Ensure that anyone using the instrument has been instructed in
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general lab safety practices and the specific safety practices for the instrument. Bevor Sie das SmartCycler-System verwenden, lesen Sie die folgenden Sicherheit Informationen. Stellen Sie sicher, daß jedermann, welches das Instrument verwendet, in der allgemeinen Laborsicherheit Praxis und in der Besonderesicherheit Praxis für das Instrument angewiesen worden ist. Moving the Instrument The Smart Cycler weighs 22 pounds (10 kg). Safety training for proper lifting techniques is recommended. Improper lifting can cause painful and perhaps permanent back injury. Das Smart Cycler wiegt 10 Kilogram. Sicherheit Training für korrekte anhebende Techniken wird empfohlen. Das unsachgemäße Anheben kann schmerzliche oder permanente rückseitige Verletzung verursachen. General Laboratory Safety Allgemeine LaborSicherheit Your laboratory should have all equipment ordinarily required for the safety of individuals working with chemicals. A partial list includes fire extinguishers, first-aid equipment, safety shower and eye-wash fountain, and spill-cleanup equipment. Ihr Labor sollte alle Ausrüstung haben, die gewöhnlich für die Sicherheit der Einzelpersonen angefordert wird, die mit Chemikalien arbeiten. Eine teilweise Liste schließt Feuerlöscher, ErsteHilfeausrüstung, Sicherheit Dusche mit Auge-Wäschebrunnen, und Streuungreinigungausrüstung.
Classification Klassifikation The Smart Cycler instrument is classified as: Das Smart Cycler-Instrument wird wie eingestuft: ISM instrument (Industrial Scientific Medical Device), medium sized, for industrial, laboratory and domestic use. ISM-Instrument (industrielle wissenschaftliche medizinische Einheit), mittelgroß, für industrielles, Labor und inländischen Gebrauch. Designed for stationary operation. Entworfen für stationäre Operation. Intended for worldwide use. Beabsichtigt für weltweiten Gebrauch.
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Intended for evaluating preprocessed biological material. Beabsichtigt für vorbearbeitetes biologisches Material Auswertens.
Note on Use with Infectious Material Anmerkung über Gebrauch mit Ansteckendem Material The instrument may not be used to analyze infectious materials, unless additional safety measures to ensure safe sample handling (e.g., placing the instrument in a laminar flow biological safety cabinet) are taken beforehand. Das Instrument darf nicht benutzt werden, um ansteckende Materialien zu analysieren, es sei denn zusätzliche Sicherheitsmaßnahmen die sichere Probebehandlung sicherzustellen (z.B., das Instrument in einen blätterigen Luftstrom biologischen-Sicherheit Schrank legend), vorher genommen werden.
Marks of Conformity Markierungen der Übereinstimmung The Smart Cycler instrument has been manufactured according to EN 61010-1 (Safety Regulations for Measuring, Control, and Laboratory Instruments); Part 1: General Requirements [IEC 1010-1 + A1: 1992, modified]). The Smart Cycler has been checked in accordance with all relevant safety standards before leaving the factory. Das Smart Cycler-Instrument ist hergestellt worden entsprechend en 61010-1 (Sicherheit Regelungen für Messen, Steuer- und Laborinstrumente); Teil 1: Allgemeine Anforderungen [IEC 1010-1 + A1: 1992, geändert]. Das Smart Cycler ist in Übereinstimmung mit allen relevanten Sicherheit Standards überprüft worden, bevor man die Fabrik verließ.
Electrical Symbols Elektrische Symbole The symbols shown on this page are affixed to the Smart Cycler. Read the explanations and make sure you understand what the symbols mean before you interact with the instrument in any way. Die Symbole, die auf dieser Seite gezeigt werden, werden zum Smart Cycler hinzugefügt. Lesen Sie die Erklärungen und verstehen was die Symbole bedeuten bevor Sie auf das Instrument in jeder Hinsicht einwirken.
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This symbol indicates the on position of the main power switch. Dieses Symbol zeigt die Arbeitsstellung des Betriebsschalters an. This symbol indicates the off position of the main power switch. Dieses Symbol zeigt die Ausschaltstellung des Betriebsschalters an.
This symbol indicates that a protective grounding terminal must be connected to earth ground before any other electrical connections are made to the instrument. Dieses Symbol zeigt an, daß ein Schutzleiteranschluß an Masse Boden angeschlossen werden muß, bevor alle anderen elektrischen Beziehungen zum Instrument hergestellt werden. This symbol indicates that this terminal either receives or delivers alternating current or voltage. Dieses Symbol zeigt an, daß dieses Terminal entweder empfängt oder liefert Wechselstrom oder Spannung.
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Chapter 1 - Theory of Operation
Chapter 1 - Theory of Operation 1.1 Introduction... 9 1.2 Heating/Cooling... 10 1.3 Optical System... 10 1.4 Reaction Tube... 12 1.5 Calibration... 13
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Chapter 1 - Theory of Operation
1.1 Introduction The Cepheid Smart Cycler® System is an integrated DNA/RNA amplification and detection instrument system based on the proprietary microprocessorcontrolled I-CORE® (Intelligent Cooling/Heating Optical Reaction) module. Ease of use is designed into the system through the Smart Cycler Software. Each Smart Cycler processing block contains sixteen independently controlled, programmable I-CORE modules, each with one reaction site. Thermally optimized proprietary reaction tubes combined with the unique design of the I-CORE modules allow very rapid cycling and faster amplification and detection. Up to six Smart Cycler processing blocks can be daisy-chained together, allowing simultaneous custom analysis of 96 discrete samples. The Smart Cycler System is ideally suited to research, such as PCR and RT-PCR, that requires automatic, repeated, rapid heating and cooling cycles for test samples. Specific sequences can be detected using hybridization probes or intercalating dyes. The system has the capacity to store any number of user-generated protocols, limited only by available disk space. All data, including cycling programs and assay results, are stored in a database. Selected data can be exported to spreadsheet programs such as Microsoft Excel. Excitation optical block
Reaction tube
Heater
Fan
Emission detection optical block
I-CORE circuit board
Notch
Figure 1-1: Schematic diagram of the major components of an I-CORE module.
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1.2 Heating/Cooling The disposable reaction tube is inserted into the I-CORE thermal cycling module for amplification. The chamber consists of two heater plates made of a ceramic material that has high thermal conductivity to assure temperature uniformity and rapid heat transfer. Resistive heater elements are deposited on the ceramic plates using thick film technologies and a thermistor attached directly to each plate monitors its temperature. Cooling is accomplished with a high-efficiency fan that moves ambient air across the heater plates. The thermal cycling chamber’s temperature is controlled by the instrument’s firmware. The firmware incorporates a control loop to ensure rapid heating of the plates and to control their temperature overshoot around the desired point, allowing the temperature of the fluid in the reaction tube to be changed rapidly and precisely.
1.3 Optical System The Smart Cycler optical system uses high intensity light-emitting diodes (LEDs), silicon photodetectors, and appropriate filters for excitation and detection of four different spectral bands. The optical system includes two optical blocks: (1) a four-color excitor module and (2) a four-color detector module. These blocks are positioned within the device such that their apertures mate with the optical windows of the reaction tube, allowing excitation and emission detection of the reaction mixture. Figure 1-2 shows the mechanical layout of the optical blocks.
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Excitation Optical Block
Lens LED
Filters
Lens
Reaction tube
Mirror
Emission Detection Optical Block
Reaction tube
Lens
Detector Mirror Filters Figure 1-2: Schematic diagram of the Smart Cycler optical blocks.
The optical system has four optical channels. The excitation and detection spectral bands for the four channels are as follows: Optical Channel
Excitation
Emission
Calibrated Dye(s)
1
450–495 nm
505–537 nm
FAM
2
495–527 nm
537–565 nm
TET
3
527–555 nm
565–605 nm
TAMRA
4
555–593 nm
605–800 nm
ROX
Table 1–1: Excitation and detection ranges for Smart Cycler channels.
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The excitation and detection spectral bands for the four channels in Smart Cycler II are as follows: Optical Channel
Excitation
Emission
Calibrated Dye(s)
Supported Quenchers
1
450–495 nm
510–527 nm
FAM
BHQ, Eclipse, DABCYL
2
500–550 nm
565–590 nm
Cy3™, TET
BHQ, Eclipse, DABCYL
3
565–590 nm
606–650 nm
Texas Red
BHQ, Eclipse, DABCYL
4
630–650 nm
670–750 nm
Cy5™
BHQ, Eclipse
Table 1–2: Excitation and detection ranges for Smart Cycler II channels.
Although the Smart Cycler System is calibrated using the dyes mentioned in the two tables above, other dyes with excitation and emission wavelengths within these ranges can be used if user-defined optical calibration is performed. By using probes labeled with different fluorescent dyes, up to four dyes and targets can be amplified and detected simultaneously in a single reaction mixture. The optical system always collects data from all four channels. Because the emission spectra of fluorescent dyes can overlap, the presence of a particular dye will result in signals in more than one of the channels. Appropriate calibration and data analysis algorithms are used to separate the signal from each of the dyes.
1.4 Reaction Tube The unique Smart Cycler polypropylene reaction tube (Figure 1-3) is designed for optimal thermal and optical characteristics. The thin reaction tube allows rapid rates of heating and cooling of the reaction mixture, and hence rapid amplification. The reaction tube also includes two optical detection windows at a 90° angle to each other along the bottom edges
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of the tube. These optical windows interface with the I-CORE optical blocks to allow fluorescence excitation and emission detection to occur.
Optical excitation window
Optical detection window
100 µL reaction tube
Optical Optical excitation detection window window 25 µL reaction tube
Figure 1-3: Smart Cycler reaction tubes.
1.5 Calibration The thermistors used to monitor the reaction chamber temperature are calibrated to ±0.50 °C using National Institute of Standards and Technology (NIST)-traceable standards. During the manufacturing process, the temperature of the heating system is measured at two temperatures, 60 °C and 95 °C. Calibration coefficients which correct for small errors in the raw thermistor readings of the heaters are stored in the memory of each I-CORE module. The optical system is calibrated using standard concentrations of the individual unquenched dye-oligos to be detected. For each optical channel, the signal produced by buffer alone (the blank signal) is subtracted from the raw signal produced by the dye-oligo standard to determine the spectral characteristics. Using the individual spectral characteristics of the pure dyeoligos, signals from an unknown mixture of dye-oligos can be resolved into corrected signals for the individual dye-oligos in the mixture.
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