LightCycler Operators Manual Version 3.0 May 1999

Prologue The LightCycler Instrument of Roche Molecular Biochemicals, Mannheim, Germany, is a thermocycler for the rapid analysis of PCR applications. LightCycler technology is the most innovative and rapid possibility for carrying out and simultaneously evaluating PCR experiments. 30 amplification steps including analysis of results can be carried out in less than 30 minutes. Fluorimetric analysis of the PCR products formed is performed as real-time measurement either continuously or at a specifically defined time during each PCR cycle. The analyses can be monitored online by the LightCycler’s user-friendly software, i.e. directly during the reaction.

This Operator’s Manual contains all the necessary technical information required to place the LightCycler instrument in operation and to carry out practical analyses. It contains background information on some of the applications and enables the quick and safe optimization of individual applications.

Symbols and Notes Symbols and Headings

The following symbols and headings are used in this manual: Symbol

Heading WARNING

CAUTION

NOTE

Trademarks and License Disclaimers

Description This heading and symbol are used to indicate that noncompliance with instructions or procedures may lead to injury or even death. This heading and symbol are used to indicate that noncompliance with instructions or procedures may cause damage to the instrument. This heading is used to bring your attention to topics of importance

• The LightCycler technology is licensed from Idaho Technology Inc., Idaho Falls, ID, USA • LightCycler is a trademark of Idaho Technology Inc., Idaho Falls, ID, USA, in the United States and some other countries. • SYBR Green I is a trademark of Molecular Probes Inc., Eugene, OR, USA. • TaqStart is a trademark of Clontech Laboratories, Inc., Palo Alto, CA, USA • Expand is a trademark of a Member of the Roche Group. For License statements, please refer to LightCycler reagent package inserts.

LightCycler Software and License Agreement Software License Agreement

READ THIS LICENSE AGREEMENT BEFORE REMOVING THE PROGRAM CD (HEREINAFTER REFERRED TO AS PROGRAM MEDIA) OR DOCUMENTATION FROM ITS PROTECTIVE COVER. REMOVING THE PROGRAM MEDIA OR DOCUMENTATION WILL CONSTITUTE AGREEMENT TO THE TERMS AND CONDITIONS OF THIS SINGLEUSE, END-USER LICENSE AGREEMENT. IF YOU ARE NOT WILLING TO BE BOUND BY THE TERMS AND CONDITIONS OF THIS LICENSE AGREEMENT, PROMPTLY RETURN THE UNOPENED PACKAGE TO ROCHE DIAGNOSTICS GMBH WITH A COPY OF THE RECEIPT, AND YOUR LICENSE FEE WILL BE REFUNDED.

Program License Agreement

You assume all responsibility and liability for the selection of this software program, hereinafter referred to as Product, to achieve your intended results, and for its installation and subsequent use.

Grant of License

Roche Diagnostics GmbH hereby grants to Licensee a non-exclusive, singleuse license to use the Product upon the terms and conditions contained in this agreement. You may: 1. Use the Product on a single workstation owned, leased or otherwise controlled by you, whether in a network or other configuration. 2. Make one (1) copy of the Product for backup purposes in support of your use of the Product on the single workstation. 3. Transfer the Product and license to another party if the other party agrees to accept the terms and conditions of this Agreement. If you transfer the Product, you must, at the same time, either transfer thel copy of Product to the same party, or destroy any copies not transferred. You must reproduce and include the copyright notice on all copies of the product documentation. Continued on next page

LightCycler Software and License Agreement, Grant of Licence (continued) YOU MAY NOT: 1. use or copy the Product, in whole or in part, except as expressly provided in this Agreement, 2. use the Product on more than one workstation concurrently, 3. copy, rent, distribute, sell, license or sublicense, or otherwise transfer the Product or this license, in whole or in part, to another party, except as specifically set forth in this Agreement, 4. use the Product, or any portion of the Product, to develop, or incorporate into, other software that can be executed without a licensed copy of the Roche Diagnostics Image. The use of the Product, or any portions of the Product, to develop, or incorporate into, other software capable to executing without a licensed copy of Roche Diagnostics Image, is specifically prohibited under the terms of this Agreement. Further, the use of the Product, or any portion of the Product on more than one workstation at one time is specifically prohibited under the terms of this Agreement. For further information, please contact Roche Diagnostics GmbH Roche Molecular Biochemicals Sandhoferstraße 116 D-68298 Mannheim. Germany Terms

The license is effective until terminated. You may terminate this Agreement at any time by destroying the Product together with the copy and documentation in any form. It will also terminate automatically and without notice from Roche Diagnostics GmbH if you fail to comply with any term or condition of this Agreement. You agree to destroy the Product and the copy ,if any, of the Product upon termination of this Agreement. Continued on next page

LightCycler Software and License Agreement, Limited Warranty

The Product is provided as is without warranty of any kind, either expressed or implied, including, but not limited to the implied warranties of merchantability and fitness for a particular purpose. The entire risk as to the quality and performance of the Product is with you as Licensee, should the Product prove to be defective. You assume the entire costs of all necessary servicing, repair, or correction. However, Roche Diagnostics GmbH warrants that the program media on which the software is furnished is free from defects in materials and workmanship under normal use for a period of ninety (90) days from the date of delivery as evidenced by a copy of your receipt. ROCHE DIAGNOSTICS GMBH MAKES NO FURTHER WARRANTIES OR GUARANTEES NOR EXPLICIT NOR IMPLIED.

Limitations of Remedies

Roche Diagnostics GmbH’s sole liability and your sole remedy shall be: 1. the replacement of the program media not meeting Roche Diagnostics GmbH’ s limited warranty and which is returned to Roche Diagnostics GmbH with a copy of your receipt; 2. if Roche Diagnostics GmbH is unable to deliver replacement program media which is free of defects in workmanship, you may terminate this Agreement by returning the Product and a copy of your receipt to Roche Diagnostics GmbH, and your money will be refunded. In no event will Roche Diagnostics GmbH be liable to you for any damages, including any lost profits, lost savings, or other indirect, special, exemplary, incidental or consequential damages, claims or actions, arising out of the use or inability to use the Product, even if Roche Diagnostics GmbH has been advised of the possibility of such damages, claims or actions. Further, in no event will Roche Diagnostics GmbH be liable for any claim by any other party arising out of your use of the Product. Continued on next page

LightCycler Software and License Agreement, General Information

You may not sublicence, assign or transfer the license or the Product, in whole or in part, except as expressly provided in this Agreement. Any attempt otherwise to sublicense, assign or transfer any of the rights, duties or obligations hereunder is void. • This Agreement will be governed by the laws of Germany. • Should any part of this agreement be declared void or unenforceable by a court of competent jurisdiction, the remaining terms shall remain in full force and effect. • Failure of Roche Diagnostics GmbH to enforce any of its rights in this Agreement shall not be considered a waiver of its rights, including but not limited to its rights to respond to subsequent breaches. By opening and using this software you acknowledge that you have read this Agreement, understand it, and agree to be bound by its terms and conditions. You further agree that this agreement is the complete and exclusive statement of the Agreement between you and Roche Diagnostics GmbH and supersedes any proposal or prior agreement, oral or written, any other communications between you and Roche Diagnostics GmbH relating to the subject matter of this Agreement.

Chapter A Technical Aspects of the LightCycler Instrument

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1. Technical Aspects of the LightCycler Instrument 1.1 Table of Contents 1. 1.1 1.2 1.3 1.3.1 1.3.2 1.3.3 1.3.4 1.4 2. 2.1 2.2 3. 3.1 3.2 3.3 3.4 3.5 4.

Topic Technical Aspects of the LightCycler Instrument Table of Contents LightCycler System Technical Data Specifications of the LightCycler Specifications for Applications Specifications for the Detectors Temperature Control Safety Precautions for the LightCycler System Description The LightCycler PC Configuration Installation Installation Requirements Installation of the LightCycler Installation of the Computer Installation of LightCycler Software Version 3 Dismantling the LightCycler System Operation and Maintenance of the LightCycler

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See Page A1 A2 A3 A6 A6 A7 A8 A9 A 11 A 12 A 12 A 16 A 17 A 17 A 19 A 20 A 21 A 29 A 30

1.2 LightCycler System Note

Using the system improperly may compromise the integrity of the results, result in poor performance, or even permanently damage the equipment.

Components of the LightCycler System

The components of the LightCycler System are listed in the following table. Please note that these components may vary from country to country. Component System component 1 • • System component 2 • • • • •

Hardware

Operating system Monitor Printer

• • • • • • • • • • •

Description LightCycler Instrument Sample Carousel (for Ø 1.5 mm capillary)1 premounted in LightCycler Instrument LightCycler Capillaries (96 capillaries and stoppers/box)1 32 LightCycler Centrifuge Adapters in an aluminum cooling block1 1 LightCycler Operator’s Manual 1 LightCycler Software Package1 Cable to connect LightCycler Instrument to the computer Power Cord (German) Power Cord (U.S.) Mouse Pad PC with Pentium processor2 64 MB SDRAM (minimum configuration) 24x CD-ROM drive Keyboard and PS/2 mouse Internal Iomega ZIP drive Windows NT 4.0, including Service Pack III 17" monitor2 Hewlett-Packard Color Inkjet Printer2

1

Also available as single components: LightCycler Sample Carousel LightCycler Capillaries LightCycler Centrifuge Adapters LightCycler Software Package

Cat. no. 1 909 282 (1 piece) Cat. no. 1 909 339 (1 set, 768 capillaries) Cat. no. 1 909 312 (1 Set) Cat. no. 1 909 304

2

Computer workstation configuration may vary from country to country and may change due to component availability and technical advances. To confirm exact computer configuration, please contact your local Roche Molecular Biochemicals representative. Continued on next page

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1.2 LightCycler System, Continued

Marks of Conformity

The LightCycler 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]) and has been checked in accordance with all relevant safety standards prior to leaving the factory. The instrument has been approved for use by recognized testing institutions. This is confirmed by the following test/conformity symbols: Acronym

Test Symbol

Testing Institution

GS

Certified by TÜV Product Service

CE

The instrument conforms to current directives as issued by the European Union

UL

Certified by Underwriters Laboratories Inc.

CUL

Certified by Underwriters Laboratories for Canada – a testing facility recognized by the Standards Council of Canada (SSC)

Equipment to be connected must fulfill the standards set by IEC 950 (Information security in technical equipment, including electronic business machines). Continued on next page

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1.2 LightCycler System, Continued Classification

The LightCycler Instrument is classified as: • ISM instrument (Industrial Scientific Medical Device), medium-sized, for industrial, laboratory and domestic use. • Designed for stationary operation. • Intended for worldwide use. • Intended for evaluating preprocessed biological material.

Note on Use with Infectious 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.

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1.3 Technical Data 1.3.1 Specifications of the LightCycler General Data Dimensions Weight Power supply Wattage Maximum current Noise in accordance with DIN 43635 Heat emission, including PC, monitor and printer Safety symbols

Length: 45 cm Width: 30 cm Height: 40 cm 19.2 kg 110–240 V, +/–10%, 47–63 Hz max. 800 W Europe: 4 A (220 V) US: 8 A (110 V) <60 dBA max. 850 W CE, GS, UL, CSA or CUL

Environmental Parameters Temperatures allowed during operation Temperatures required to maintain specifications during operation Temperatures allowed during transportation/storage/packaging Relative humidity Altitude/pressure

15° to 35°C 18° to 30°C

Number of samples per run Sample volume

32 10 to 20 µl

–20° to +60°C 20 to 80%, no condensation 0 to 2000 m above sea level, 1030 to 850 hp

Samples

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1.3.2 Specifications for Applications Temperature for PCR Temperature range Accuracy of “capillary temperature” at thermal equilibrium Accuracy of “displayed temperature” with respect to capillary temperature at thermal equilibrium Capillary Heating Rates Heating rate 40° to 95°C (non-linear) Heating rate 50° to 72°C (non-linear) Heating rate 72° to 95°C (non-linear) Capillary Cooling Times Cooling rate 95° to 40°C (non-linear) Cooling rate 95° to 60°C (non-linear)

40° to 98°C ±0.4°C ±0.4°C Volume : Heating Time Required 10 µl: ≤13 s 20 µl: ≤15 s 10 µl: ≤6 s 20 µl: ≤8 s 10 µl: ≤6 s 20 µl: ≤8 s Volume : Cooling Time Required 10 µl: ≤23 s 20 µl: ≤24 s 10 µl: ≤7 s 20 µl: ≤7 s

Temperature Tolerances, Short Term Precision of capillary temperature over all capillary positions when measured for 30 s at 95°C Precision of capillary temperature over all capillary positions when measured for 30 s at 70°C Precision of capillary temperature over all capillary positions when measured for 30 s at 45°C Temperature Difference, Melting Curves Systematic difference between sensor temperature and capillary temperatures when chamber is heated from 50°C to 95°C at a rate of 0.2°C/s

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±1.5°C ±1.0°C ±0.5°C <0.1°C

1.3.3 Specifications for the Detectors Excitation Type Median wavelength Wattage at 470 to 490 nm

LED 470 nm 0.1 mW

Filter Detector 1 Detector 2 Detector 3

Bandpass 530 nm, HBW 20 nm, dichroic Bandpass 640 nm, HBW 20 nm, dichroic Bandpass 710 nm, HBW 40 nm, dichroic

Detector Type Sensitivity at 530 nm, 20 µl sample volume Resolution Range of detection sensitivity

Photohybrid 10 fM fluorescein 12 bit Adjustable by a factor of 1 to 256

Signal acquisition time for 32 capillaries

≈ 5.0 s

Typical Time

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1.3.4 Temperature Control Visual Display of Temperature Profiles

The LightCycler software graphically displays the current temperature in the thermal chamber. These graphs appear under Temperature History on the Running Screen (see below). The graphs show “overshoots” and “undershoots” for each temperature transition (see arrows below).

Continued on next page

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1.3.4 Temperature Control, Continued Autocorrection Autocorrection compensates for physical differences in heat capacity in air and water. This specific autocorrection ensures that every programmed temperature is adjusted within the sample capillaries during thermal cycling. Effectiveness of The graph below shows the changes in temperature in both the thermal Autocorrection chamber and a sample capillary during thermal cycling. Differences at any given time between the temperature in the two locations are shown as spaces between the two temperature curves. 100

Temperature [o C]

90 80 70 60 50

Ambient temperature in thermal chamber Fluid temperature in capillary

40 0

50

100

150

200

Time [sec] Conclusion: This graph demonstrates that: • The temperature profile shown under Temperature History on the Running Screen exactly corresponds to the capillary temperature, and • The fluid temperature in the capillaries coincides with the programmed profile.

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1.4 Safety Precautions for the LightCycler General Safety Precautions

Always observe these precautions when using the LightCycler Instrument: • Follow all safety instructions printed on, or attached to, the analytical instruments. • Observe all general safety precautions which apply to electrical instruments. • Never touch switches or power cord with wet hands. • Do not open the housing of the LightCycler. Note: Only authorized service personnel should perform service or repairs required for this unit. • Do not open the LightCycler thermal chamber during operation. • The chamber lid and the sample rotor are hot while the instrument is operating. Note: The corresponding symbol is attached to the front side of the LightCycler. • Always wear safety goggles and gloves when dealing with toxic, caustic, or infectious materials . • When analyzing infectious materials, use the LightCycler only in specially equipped rooms with controlled exhaust equipment, e.g., biological safety cabinets.

Safety Precautions for Centrifuge Adapters

Loading capillaries to perform fluorescence measurement with the LightCycler requires a centrifugation step in a tabletop centrifuge with specially designed adapters. A centrifuge adapter with capillary weighs approximately 6.2 grams. Always observe the following precautions during the centrifugation step: • Always distribute the adapters in the rotor so the weight in the rotor is balanced, and • Adjust the speed of the centrifuge so the centrifugal force does not exceed 700 x g. Note: For details on the maximum weight that can be processed in your centrifuge, refer to the product specification sheet for that centrifuge.

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2. System Description 2.1. The LightCycler Schematic of the LightCycler

Continued on next page

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2.1 The LightCycler, Continued Description of the Instrument

The LightCycler basically consists of an upper unit and a lower unit. The upper unit contains the heating coil. The lower unit contains the thermal chamber, fluorimeter, drive units, electronic boards and power supply. The various elements are mounted on a 10-mm cast aluminum base plate. This guarantees stability, especially for the thermal chamber and fluorimeter. Hot or ambient temperature air, introduced into the thermal chamber, regulates the temperature of the sample capillaries. A heating coil heats the air, which is then fed into the chamber by a fan. The fan ensures efficient air circulation and temperature homogeneity during the heating cycle. During the cooling cycle, the fan operates at a higher speed to ensure adequate cooling. During measurements, a stepper motor rotates the sample carousel to position the capillary tip precisely at the focal point of the fluorimeter optics. The fluorimeter itself is positioned radially to the maximum signal to compensate for any radial deviation of the capillary tip.

Communication between LightCycler and PC

For online display, data are transmitted to and from the PC via a serial interface. Control is made possible by three microprocessors that are integrated into the instrument: • Processor 1 is for communication, • Processor 2 regulates the temperature, and • Processor 3 controls the measurement procedures as well as the movement of the rotor and fluorimeter. Users enter data on samples (number of samples, name, concentration, etc.) and on the experimental protocol into the PC; the PC transmits the data to the LightCycler. The PC also monitors the temperatures and fluorescence signals during the PCR run and operates all the analytical programs.

Power Supply Unit

All high-voltage components are located in the power-supply unit. Note: Electrical safety information and test symbols apply to this unit. To minimize interference to the detector electronics, the electronics that control the drive units are located on a separate circuit board. Continued on next page

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