Roche LightCycler User manual

Roche Molecular Biochemicals

LightCycler Oper ator’s 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

Trademarks and License Disclaimers

The following symbols and headings are used in this manual:

Symbol Heading Description

WARNING This heading and symbol are

used to indicate that noncompliance with instructions or procedures may lead to injury or even death.

CAUTION This heading and symbol are

used to indicate that noncompliance with instructions or procedures may cause damage to the instrument.

NOTE 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

Program License Agreement

Grant of License

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.

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.

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 con-

trolled 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

Terms

Roche Diagnostics GmbH Roche Molecular Biochemicals Sandhoferstraße 116 D-68298 Mannheim. Germany

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

Limitations of Remedies

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.

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

A 1

1. Technical Aspects of the LightCycler Instrument

1.1 Table of Contents

1.1

1.2

1.3

1.3.1

1.3.2

1.3.3

1.3.4

1.4

2.1

2.2

3.1

3.2

3.3

3.4

3.5

4. Operation and Maintenance of the LightCycler

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

Topic See Page

A 1 A 2 A 3 A 6 A 6 A 7 A 8

A 9 A 11 A 12 A 12 A 16 A 17 A 17 A 19 A 20 A 21 A 29 A 30

A 2

1.2 LightCycler System

Note

Components of the LightCycler System

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

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

Component Description

System component 1

• LightCycler Instrument

• Sample Carousel (for Ø 1.5 mm capillary)1 pre-

mounted in LightCycler Instrument

System component 2

• LightCycler Capillaries (96 capillaries and stop- pers/box)

• 32 LightCycler Centrifuge Adapters in an alumi- num cooling block

• 1 LightCycler Operator’s Manual

• 1 LightCycler Software Package

• Cable to connect LightCycler Instrument to the computer

• Power Cord (German)

• Power Cord (U.S.)

• Mouse Pad

Hardware

• PC with Pentium processor

• 64 MB SDRAM (minimum configuration)

• 24x CD-ROM drive

• Keyboard and PS/2 mouse

• Internal Iomega ZIP drive

Operating system Monitor Printer

• Windows NT 4.0, including Service Pack III

• 17" monitor

• Hewlett-Packard Color Inkjet Printer

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

Computer workstation configuration may vary from country to country and

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

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

A 3

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

Certified by TÜV Product Service

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

Certified by Underwriters Laboratories Inc.

CUL

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

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

Continued on next page

A 4

1.2 LightCycler System, Continued

Classification

Note on Use with Infectious Material

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.

• The instrument may not be used to analyze infectious materials unless ad-

ditional safety measures to ensure safe sample handling (e.g., placing the instrument in a laminar flow biological safety cabinet) are taken beforehand.

A 5

1.3 Technical Data

1.3.1 Specifications of the LightCycler

General Data

Environmental Parameters

Dimensions Length:

Width:

Height: Weight 19.2 kg Power supply 110–240 V, +/–10%, 47–63 Hz Wattage max. 800 W Maximum current Europe:

US: Noise in accordance with DIN 43635 <60 dBA Heat emission, including PC, monitor and printer Safety symbols CE, GS, UL, CSA or CUL

Temperatures allowed during operation 15° to 35°C Temperatures required to maintain specifications during operation Temperatures allowed during transportation/storage/packaging Relative humidity 20 to 80%, no condensation Altitude/pressure 0 to 2000 m above sea level,

max. 850 W

18° to 30°C

–20° to +60°C

1030 to 850 hp

45 cm 30 cm 40 cm

4 A (220 V) 8 A (110 V)

Samples

Number of samples per run 32 Sample volume 10 to 20 µl

A 6

1.3.2 Specifications for Applications

Temperature for PCR

Temperature range 40° to 98°C Accuracy of “capillary temperature” at thermal equilibrium Accuracy of “displayed temperature” with respect to capillary temperature at thermal equilibrium

Capillary Heating Rates Volume : Heating

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 Volume : Cooling

Cooling rate 95° to 40°C (non-linear)

Cooling rate 95° to 60°C (non-linear)

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

±0.4°C ±0.4°C

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

Time Required

10 µl: ≤23 s 20 µl: ≤24 s

10 µl: ≤7 s 20 µl: ≤7 s

±1.5°C ±1.0°C ±0.5°C

<0.1°C

A 7

1.3.3 Specifications for the Detectors

Excitation

Type LED Median wavelength 470 nm Wattage at 470 to 490 nm 0.1 mW

Filter

Detector 1 Bandpass 530 nm, HBW 20 nm, dichroic Detector 2 Bandpass 640 nm, HBW 20 nm, dichroic Detector 3 Bandpass 710 nm, HBW 40 nm, dichroic

Detector

Type Photohybrid Sensitivity at 530 nm, 20 µl sample volume 10 fM fluorescein Resolution 12 bit Range of detection sensitivity Adjustable by a factor of 1 to

256

Typical Time

Signal acquisition time for 32 capillaries

≈ 5.0 s

A 8

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).

A 9

Continued on next page

1.3.4 Temperature Control, Continued

100

Temperature [ C]

200

Fluid temperature in capillary

Ambient temperature in thermal chamber

Autocorrection

Effectiveness of 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.

The graph below shows the changes in temperature in both the thermal 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.

0 50 100 150

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.

A 10

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 in- struments.

• 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 op-

erating. 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.

A 11

2.1. The LightCycler

Schematic of the LightCycler

2. System Description

A 12

Continued on next page

2.1 The LightCycler, Continued

Description of the Instrument

Communication between LightCycler and PC

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.

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 move-

ment 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

A 13

2.1 The LightCycler, Continued

Temperature Setting of the Thermal Chamber

Fluorimeter

Temperature is controlled with hot air and air at ambient temperature. Varying the voltage supplied to the heating coil regulates the temperature. A sensor provides reference values for control purposes. During the heating phase, the fan in the thermal chamber operates at low speeds to ensure homogenous distribution of temperature. During the cooling phase, the fan operates at higher speeds so that the capillaries and the heating coil can be cooled efficiently. Two sensors are integrated into the LightCycler to prevent unduly high temperatures:

• Sensor I is located in the thermal chamber and switches off the heat when a

temperature of 125°C has been reached.

• Sensor II monitors the temperature on the aluminum base plate, and

switches off the entire LightCycler to protect the electronics when the temperature exceeds 55°C.

A three-channel fluorimeter is used for detection purposes. A blue diode (LED) with maximum emission of 470 nm serves as the energy source for sample excitation. Screens are used to diffuse the light emitted by the LED to ensure uniformity. The exhaust and ventilation channels have been designed to prevent ambient light from entering directly into the thermal chamber. Fluorescence is detected at 530 nm, 640 nm, and 710 nm with the aid of photohybrids. Note: Acquisition time for fluorescence acquisition time is 20 msec (16.6 msec in the U.S.).

Measurement of Samples

The thermal chamber and the fluorimeter are placed optimally for sample measurement. Only the sample carousel, which holds the capillary, rotates during measurement of the various samples.

Continued on next page

A 14

2.1 The LightCycler, Continued

LightCycler Diodes

Position of

Diode

Top Power on Green On Instrument is switched on

Middle Cycling Red On Instrument is running

Bottom Run Yellow On PCR run is completed

Three diodes are located at the front of the LightCycler. All of the diodes come on when the instrument is switched on. In this way, the instrument tests for proper functioning of the diodes. During instrument operation, the diodes function as described in the table below:

Label Color of Diode Function Indication

Off

Flashing Instrument is defective

completed Off Instrument is running

• No power

• Instrument is defective

A 15

2.2 PC Configuration

General

The computer workstation contains the following components:

Hardware

• PC with a Pentium processor

• 64 MB SDRAM (minimum configuration)

• 24x CD-ROM drive

• Keyboard and PS/2 mouse

• Internal Iomega ZIP drive

Operating system Monitor Printer

Computer workstation configuration may vary from country to country and

• Windows NT 4.0, including Service Pack III

• 17" monitor

• Hewlett-Packard Color Inkjet Printer

may change due to component availability and technical advances. To confirm exact computer configuration, please contact your local Roche Molecular Biochemicals representative.

A 16

3. Installation

3.1 Installation Requirements

Note

Space and Power Requirements

• Do not place the LightCycler next to instruments that cause electromag- netic interference or have high inductance (e.g., centrifuges or mixers).

• Peripheral instruments connected to the LightCycler System must meet the IEC 950 (UL 1950) standard.

• All plugs used in the LightCycler System (PC, printer, monitor) should have the same phasing in order to prevent switch-on peaks and electronic noise generated by other instruments or by the power supply itself. We recommend using a dedicated multiple-outlet surge protector for the LightCycler System.

• Use only the supplied power lines and RS 232 connector.

Place the instrument in a site that can support the following instrument requirements:

Dimensions The LightCycler is 30 cm wide, 45 cm long, and 40 cm

high. . Weight Approximately 20 kg Voltage Requirements

The LightCycler operates between 120 and 240 V (50 to 60

Hz).

Note: The LightCycler adjusts automatically to the avail-

able voltage when the instrument is plugged in. The user

does not have to set the instrument to the correct voltage

manually.

Power Consumption

Do not open the LightCycler housing.

The LightCycler uses 800 W max.

PC and printer consume an additional 500 W (approx.).

Continued on next page

A 17

3.1 Installation Requirements, Continued

Environmental Parameters

Storage Conditions

The instrument requires the following environment for accurate operation:

Ambient temperature 15° to 35°C Ambient temperature required to main specifications during operation Humidity 20 to 80%, no condensation Altitude Sea level to 2000 m Excess voltage Category II Degree of contamination 2

The LightCycler can be stored under the following conditions:

Ambient temperature –20° to +60°C Humidity 20 to 80%, no condensation

18° to 30°C

A 18

3.2 Installation of the LightCycler

Installation of the LightCycler Instrument

Your Roche Molecular Biochemicals representative will normally install the LightCycler Instrument at your site. Should this not be possible, follow these steps to install the instrument successfully:

Step Action

1 Unpack the instrument. 2 Position the instrument on the workbench. Allow 10-cm spaces to

the left, right and behind the instrument to ensure sufficient cooling of the electronic components. Note: The workbench should not be covered with paper.

3 Make the following electrical connections:

• Connect the LightCycler to the PC using the RS 232 cable (serial interface) provided with the system.

• Connect the LightCycler, PC, monitor, and printer to the same multiple-outlet distributor plug.

Ensure that PC, monitor, and printer have been set to the correct voltage.

A 19

3.3 Installation of the Computer

Installation of the PC

To install the PC, do the following:

Step Action

1 Connect mouse, keyboard, and monitor to the computer. 2 Connect the LightCycler to the computer with the RS 232 cable

(serial interface) provided with the system.

3 Connect the computer, monitor, and LightCycler to the same

multiple-outlet distributor plug. The computer is now ready for operation.

A 20

3.4 Installation of LightCycler Software Version 3

Logging In

Installation of LightCycler Software

A prerequisite for the software installation is that the operating system WINNT 4.0 (Service Pack 3) has been installed. For the installation of the software and the setting up of users it is recommended that you log in as administrator. All instructions are based on the English WIN NT 4.0 version. The operating system programs and folders in the German versions have different names.

Insert the CD with the LightCycler Software version 3 into the CD-ROM drive of your computer. The autorun function will automatically start the Setup program. If the autorun function is not available, the CD must be opened from the Explorer, and the 'setup' program started manually by doubleclicking on it. The Setup program displays the following start screen:

Click the “Next” button to continue with the installation. Click “Cancel” to abort the program.

Continued on next page

A 21

3.4 Installation of LightCycler Software Version 3, Continued

License Agreement Information of LightCycler Software

Here, the customer is provided with a display of the license agreement. Scroll bars can be used to view the entire text. Choose “Yes” to accept the license agreement. If you choose “No”, the program will be exited. Clicking on “Back” will redisplay the start screen.

Installation Directory of LightCycler Software

Continued on next page

A 22

3.4 Installation of LightCycler Software Version 3, Continued

Installation Directory of LightCycler Software,

Continued

The software will be installed to the recommended directory “LightCycler3”. The disk drive corresponds to the drive to which WIN NT was installed, here: drive “D”.

If “Next” is clicked on, the installation will be continued. If “Cancel” is chosen, the installation will be exited, and “Back” will redisplay the previous screen.

If you wish to change the disk drive or the directory, click the “Browse” button. A popup window will display permitting the desired path and directory to be entered.

The installation to the “LightCycler3” directory will have no consequence for any previous installations of version V1.22 since that version was installed in the directory named “LightCycler”. It is therefore recommended that directory “LightCycler3” be used so as to avoid any conflict with respect to future versions and to be able to quickly locate the relevant versions.

Continued on next page

A 23

+ 316 hidden pages