DNA Engine Opticon System - Bio-Rad · The DNA Engine Opticon system is designed to operate safely under the following conditions: • Indoor use • Altitude up to 2000m • Ambient

DNA Engine Opticon® System

06678 revC.A

For Continuous Fluorescence Detection

PTC-200 DNA Engine® CyclerCFD-3200 Opticon™ Detector

Operations ManualSupports Software Version 1.08

DNA Engine Opticon® SystemFor Continuous Fluorescence Detection

PTC-200 DNA Engine® CyclerCFD-3200 Opticon™ Detector

Operations ManualSupports Software Version 1.08

ii Tech Support: (888) 652-9253 • Sales: (888) 735-8437 • [email protected] • www.mjr.com

Copyright ©2004, Bio-Rad Laboratories, Incorporated. All rights reserved. Reproduction in any form, either print orelectronic, is prohibited without written permission of Bio-Rad Laboratories, Inc.

Chill-out, DNA Engine, DNA Engine Opticon, Hard-Shell, Microseal, MiniCycler, MJ Research and the helix logo,Multiplate, Opticon, Opticon Monitor and PTC-100 are trademarks belonging to Bio-Rad Laboratories, Inc.

Amplifluor is a trademark of Intergen Company. DyNAzyme is a trademark of Finnzymes Oy. Scorpions is a trade-mark of DXS Ltd. SYBR is a trademark of Molecular Probes, Inc. TaqMan is a trademark of Roche Molecular Systems,Inc. Windows is a trademark of Microsoft Corporation.

Practice of the patented polymerase chain reaction (PCR) process requires a license. The DNA Engine Opticon systemincludes an Authorized Thermal Cycler and may be used with PCR licenses available from Applied Biosystems. Its usewith Authorized Reagents also provides a limited PCR license in accordance with the label rights accompanying suchreagents .Some applications may also require licenses from other third parties.

This instrument includes an Authorized Thermal Cycler, Serial No __________________. Its purchase price includes theup-front fee component of a license under United States Patent Nos. 4,683,195, 4,683,202 and 4,965,188, owned byRoche Molecular Systems, Inc., and under corresponding claims in patents outside the United States, owned by F.Hoffmann-LaRoche Ltd, covering the Polymerase Chain Reaction ("PCR") process, to practice the PCR process for internalresearch and development using this instrument. The running royalty component of that license may be purchased fromApplied Biosystems or obtained by purchasing Authorized Reagents. This instrument is also an Authorized ThermalCycler for use with applications licenses available from Applied Biosystems. Its use with Authorized Reagents alsoprovides a limited PCR license in accordance with the label rights accompanying such reagents. Purchase of thisproduct does not itself convey to the purchaser a complete license or right to perform the PCR process. Furtherinformation on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licens-ing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California, 94404, USA.

No rights are conveyed expressly, by implication or estoppel to any patents on real-time PCR.

Applied Biosystems does not guarantee the performance of this instrument.

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Table of Contents

Explanation of Symbols .......................................................................................... ivSafety Warnings .................................................................................................... ivSafe Use Guidelines................................................................................................. vElectromagnetic Interference .................................................................................... vFCC Warning .......................................................................................................... v

1. Introduction ..................................................................................................... 1-12. Layout and Specifications ................................................................................. 2-13. Installation and Operation ................................................................................ 3-14. Compatible Chemistries, Sample Vessels, and Sealing Options ........................... 4-15. Introduction to Opticon Monitor™ Software ...................................................... 5-16. Experimental Setup and Programming .............................................................. 6-17. Run Initiation and Status ................................................................................... 7-18. Data Analysis ................................................................................................... 8-19. Maintenance .................................................................................................... 9-110. Troubleshooting ............................................................................................ 10-1

Appendix A .........................................................................................................A-1Appendix B .......................................................................................................... B-1Appendix C.......................................................................................................... C-1Appendix D .........................................................................................................D-1Appendix E .......................................................................................................... E-1

Index .................................................................................................................. In-1

Declarations of Conformity ...............................................................................DoC-1

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Explanation of Symbols

CAUTION: Risk of Danger! Wherever this symbol appears, always consult notein this manual for further information before proceeding. This symbol identifies com-ponents that pose a risk of personal injury or damage to the instrument if improperlyhandled.

CAUTION: Risk of Electrical Shock! This symbol identifies components that posea risk of electrical shock if improperly handled.

CAUTION: Hot Surface! This symbol identifies components that pose a risk of per-sonal injury due to excessive heat if improperly handled.

Safety Warnings

Warning:Warning:Warning:Warning:Warning: Operating the DNA Engine Opticon system before reading this manual canconstitute a personal injury hazard. Only qualified laboratory personnel trainedin the safe use of electrical equipment should operate this instrument.

Warning:Warning:Warning:Warning:Warning: Do not open or attempt to repair the Opticon tower or base. Doing so willvoid your warranties and can put you at risk for electrical shock. Return theDNA Engine Opticon system to the factory (US customers) or an authorizeddistributor (all other customers) if repairs are needed.

Warning:Warning:Warning:Warning:Warning: The sample block can become hot enough during the course of normal opera-tion to cause burns or cause liquids to boil explosively. Wear safety gogglesor other eye protection at all times during operation.

Warning:Warning:Warning:Warning:Warning: The DNA Engine Opticon system incorporates neutral fusing, which means thatlive power may still be available inside the machines even when a fuse hasblown or been removed. Never open the Opticon base; you could receive aserious electrical shock. Opening the base will also void your warranties.

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Safe Use Guidelines

The DNA Engine Opticon system is designed to operate safely under the following conditions:

• Indoor use

• Altitude up to 2000m

• Ambient temperature 15˚–25˚C

• Maximum relative humidity 80%, noncondensing

• Transient overvoltage per Installation Category II, IEC 664

• Pollution degree 2, in accordance with IEC 664

Electromagnetic InterferenceThis device complies with Part 15 of the FCC Rules. Operation is subject to the followingtwo conditions: (1) this device may not cause harmful interference, and (2) this devicemust accept any interference received, including interference that may cause undesiredoperation.

This device has been tested and found to comply with the EMC standards for emissionsand susceptibility established by the European Union at time of manufacture.

This digital apparatus does not exceed the Class A limits for radio noise emissions fromdigital apparatus set out in the Radio Interference Regulations of the Canadian Depart-ment of Communications.

LE PRESENT APPAREIL NUMERIQUE N'EMET PAS DE BRUITS RADIOELECTRIQUESDEPASSANT LES LIMITES APPLICABLES AUX APPAREILS NUMERIQUES DE CLASS APRESCRITES DANS LE REGLEMENT SUR LE BROUILLAGE RADIOELECTRIQUE EDICTE PARLE MINISTERE DES COMMUNICATIONS DU CANADA.

FCC WarningWarning: Changes or modifications to this unit not expressly approved by the partyresponsible for compliance could void the user’s authority to operate the equipment.

Note: This equipment has been tested and found to comply with the limits for a Class Adigital device, pursuant to Part 15 of the FCC Rules. These limits are designed to providereasonable protection against harmful interference when the equipment is operated in acommercial environment. This equipment generates, uses, and can radiate radiofrequencyenergy and, if not installed and used in accordance with the instruction manual, maycause harmful interference to radio communications. Operation of this equipment in aresidential area is likely to cause harmful interference in which case the user will be re-quired to correct the interference at his own expense.

1-1

1. Introduction

Meet the DNA Engine Opticon System, 1-2Using This Manual, 1-2Important Safety Information, 1-3

1-2 Tech Support: (888) 652-9253 • Sales: (888) 735-8437 • [email protected] • www.mjr.com

Opticon System Operations Manual

Meet the DNA Engine Opticon® SystemThank you for purchasing a DNA Engine Opticon continuous fluorescence detection sys-tem from MJ Research, Incorporated. Designed by a team of molecular biologists andengineers, the Opticon™ system will meet your needs for a sensitive, easy-to-use, and com-pact continuous fluorescence detection system. Some of the DNA Engine Opticon system’smany features include:

• A DNA Engine® Peltier thermal cycler delivers superior thermal accuracy and well-to-well thermal uniformity.

• A 96-well sample block accepts standard consumables (96-well, low-profile micro-plates and low-profile 0.2ml strip tubes).

• An integrated heated lid permits oil-free cycling.

• Long-lived LEDs excite fluorescent dyes in the 450-495nm range.

• Sensitive optics detect fluorophores with emission spectra in the 515-545nm range(SYBR Green, FAM).

• Intuitive Opticon Monitor™ software facilitates experimental setup, run initiation, runstatus, and data analysis.

• Dual modes of temperature control include calculated control for maximum speedand accuracy, or block control for adapting protocols optimized in other cyclers.

• Compact footprint measuring 47cm deep x 34cm wide x 60cm high, allows theOpticon unit to fit comfortably on any lab bench.

• The Opticon detector is available separately as an upgrade for existing DNA Enginethermal cyclers.

Using This ManualThis manual contains instructions for operating your DNA Engine Opticon system safelyand productively:

• Chapter 2 acquaints you with the physical characteristics of the Opticon system.

• Chapter 3 presents the basics of installing and operating the Opticon system.

• Chapter 4 discusses the chemistry and sample vessel compatibilities of theOpticon system.

• Chapters 5-8 step you through the use of the Opticon Monitor software includ-ing how to enter and run protocols, and analyze collected data.

• Chapter 9 explains the proper maintenance of the Opticon system.

• Chapter 10 offers troubleshooting information for the Opticon system.

Tech Support: (888) 652-9253 • Sales: (888) 735-8437 • [email protected] • www.mjr.com 1-3

Introduction

Important Safety InformationSafe operation of the DNA Engine Opticon system begins with a complete understand-ing of how the instrument works. Please read this entire manual before attempting to op-erate the DNA Engine Opticon system. Do not allow anyone who has not read this manualto operate the instrument.

Warning: The DNA Engine Opticon system can generate enough heat to inflictserious burns and could deliver strong electrical shocks if not used ac-cording to the instructions in this manual. Please read the safety warningsand guidelines at the beginning of this manual on pages iv and v, andexercise all precautions outlined in them.

2-1

2. Layout and Specifications

Front View, 2-2Back View, 2-2Specifications, 2-3Gradient Specifications, 2-4Computer Specifications, 2-4



Back View(Figure 2-2)

Front View(Figure 2-1)

Power cord jack(some models)

Power switch(fuses, somemodels)

DAQ (data acquisition)cable port

Serial cable port

Power module,left configuration(standard)

Power cord jack

Power switch(fuses)

Power module,right configuration(some models)

Optical tower

Cycler drawer

Air exhaust vents(also on other side)

Air intake vents(also on other side)

Blue protocol-indicator light

Blue trigger handle(door mechanism)


Layout and Specifications

SpecificationsThermal range: 0˚ to 105˚C, but not more than 30˚C

below ambient temperature

Accuracy: ±0.3˚C of programmed target @ 90˚C,NIST-traceable

Thermal homogeneity: ±0.4˚C well-to-well within 30 seconds ofarrival at 90˚C

Ramping speed: Up to 3.0˚C/sec

Sample capacity: 96-well microplate (low-profile) or96 x 0.2ml strip tubes (low-profile)

Line voltage: 100-240VAC

Frequency: 50-60Hz

Power: 850W maximum

Fuses: Two 6.3A, 250V Type S505, fast acting(user changeable)Two 8.0A, 250V Type S505, fast acting(inaccessible)

Weight: 27kg (excluding computer andmonitor)

Size: 47cm deep x 34cm wide x 60cm high(excluding computer and monitor)

Fluorescence Excitation Range: 450-495nm

Fluorescence Detection Range: 515-545nm



Gradient SpecificationsAccuracy: +0.3°C of target at end columns within

30 seconds (NIST-traceable)

Column uniformity: +0.4°C, in column, well–to–well, within30 seconds of target attainment

Calculator accuracy: +0.4°C of actual column temperature(NIST-traceable)

Lowest programmable 30°Ctemperature:

Highest programmable 105°Ctemperature:

Gradient range: from 1°C up to 24°C(temperature differential)

Computer Specifications(minimum specifications for the computer provided with the Opticon system)

Processor: 2.4GHz processor

Operating System: Windows XP Pro

Display: 15 inch flat-screen monitor

Memory: 256 MB RAM

Storage: 40GB hard drive

Data Acquisition Board: National Instruments PCI-6036E200kS/s (samples per second)

3-1

3. Installation and Operation

Unpacking the Opticon Unit, 3-2Packing Checklist, 3-2Setting Up the DNA Engine Opticon System, 3-3Environmental Requirements, 3-3Power Supply Requirements, 3-4Air Supply Requirements, 3-4

Ensuring an Adequate Air Supply, 3-4Ensuring That Air Is Cool Enough, 3-4Troubleshooting Air Supply Problems, 3-5

Turning the Opticon Unit and Computer On and Off, 3-5Opening and Closing the Cycler Drawer, 3-6Loading Sample Vessels into the Block, 3-6



Unpacking the Opticon™ UnitPlease follow these instructions for unpacking the Opticon unit to reduce the risk of per-sonal injury or damage to the instrument.

Important: DO NOT lift the instrument out through the top of the box.

Important: DO NOT use the blue handle to lift the instrument at any time.

• Cut the band securing the cardboard cover to the support base.

• Open the top of the cardboard cover.

• Remove the top foam insert.

• Remove the accessory box (contents listed below).

• Lift the cardboard cover up and off of the instrument.

• Firmly grasp the sides of the instrument from beneath to support the weight of thecycler and the optical tower. Carefully lift the instrument off of the shipping sup-port. Do not lift the instrument by the blue handle or the cycler drawer.

Packing ChecklistAfter unpacking the DNA Engine Opticon® continuous fluorescence detection system, checkto see that you have received the following:

1. One DNA Engine Opticon unit (Opticon detector with DNA Engine® thermal cycler)

2. One computer with keyboard, mouse, monitor, cables, & installed software (OpticonMonitor and Windows XP pro)

• One serial cable for connecting the Opticon unit’s serial port (figure 2-2) to thecomputer serial port

• One data acquisition cable for connecting the Opticon unit’s DAQ port (figure 2-2) to the data acquisition card in the computer

3. One Opticon accessory pack including:

• One power cord for the Opticon unit

• Two spare fuses

• DNA Engine Opticon® Continuous Fluorescence Detection System OperationsManual (this document)

• Opticon Monitor™ software CD ROM

• Consumables samples including 0.2ml low-profile strip tubes in opaque white(MJ Research catalog no. TLS-0851), optical flat caps for 0.2ml tubes and plates(MJ Research catalog no. TCS-0803), and low-profile Multiplate™ 96-well micro-plates in opaque white (MJ Research catalog no. MLL-9651)


Installation and Operation

If any of these components are missing or damaged, contact MJ Research, Incorporated orthe authorized distributor from whom you purchased the DNA Engine Opticon system toobtain a replacement. Please save the original packing materials in case you need to returnthe DNA Engine Opticon system for service. See Appendix C for shipping instructions.

Setting Up the DNA Engine Opticon SystemThe Opticon system requires a location with three power outlets to accommodate theOpticon unit, the computer, and the monitor. A location with network access (Ethernet10/100BaseT) is recommended if you wish to transfer setup and analysis files betweenthe computer running the Opticon unit and other computers.

The DNA Engine Opticon system requires only minimal assembly. Insert the power cordplug into its jack at the back of the instrument, just below the power switch (see figure 2-2 for the location of the jack). Then, plug the power cord into a standard 110V or 220Velectrical outlet. The Opticon unit will accept 220V automatically, as does the monitor.However, you must set the voltage on the computer. See the “Power Supply Requirements”section below for more information.

Before launching the Opticon Monitor software (see Chapter 5), be sure that the Opticonunit is connected to the computer. There are two cables that connect the Opticon unit tothe computer. Connect the serial cable to the serial cable port on the Opticon unit (seefigure 2-2) and serial port #2 on the computer. Connect the data acquisition cable to theDAQ port on the Opticon unit (see figure 2-2) and the data port on the computer.

Note: The DAQ cable has high-density connectors; take care not to bend any of the pins.

Environmental RequirementsFor reasons of safety and performance, ensure that the area where the DNA EngineOpticon system is installed meets the following conditions:

• Nonexplosive environment

• Normal air pressure (altitude below 2000m)

• Ambient temperature 15˚–31˚C

• Relative humidity above 10% and up to 80%

• Unobstructed access to air that is 31˚C or cooler (see below)

• Protection from excessive heat and accidental spills. (Do not place the DNA EngineOpticon system near such heat sources as radiators, and protect it from danger ofhaving water or other fluids splashed on it, which can cause electrical short circuits.)



Power Supply RequirementsThe DNA Engine Opticon unit requires 100-240VAC, 50-60Hz and a grounded outlet.The DNA Engine Opticon unit can use current in the specified range without adjustment,so there is no voltage-setting switch. The monitor can also accept either 110 or 220Vpower without adjustment.

Important! For 220V operation of the computer, the red voltage-settingswitch located on the back of the computer, near the power cordjack, must display 230V rather than 115V.

The Opticon unit is equipped with a power-entry module that accepts cordsets with anIEC 60320-1 type C13 connector (this is the same standard configuration used by manycomputer manufacturers for their equipment). All cordsets used with the Opticon unit mustbe rated to carry at least 10A at 125V or 250V, the latter specification depending uponthe supply voltage used. Additionally, the cordset must meet all other applicable nationalstandards—thus at a minimum, the cordset should carry the mark of a nationally recog-nized testing agency appropriate to your nation.

Note: Do not cut the supplied 120V power cord and attach a different connector. Use a one-piece molded connector of the type specified above.

Air Supply RequirementsThe DNA Engine Opticon unit requires a constant supply of air that is 31˚C or cooler inorder to remove heat from the heat sink. Air is taken in from the lower vents located onthe sides of the instrument and exhausted from the upper vents on both sides (see figure2-1). If the air supply is inadequate or too hot, the instrument can overheat, causing per-formance problems and even automatic shutdowns.

Ensuring an Adequate Air Supply

• Do not block air intake vents (see figure 2-1).

Position the DNA Engine Opticon unit at least 10cm from vertical surfaces and other thermalcyclers or heat-generating equipment (greater distances may be required; see below).

• Do not allow dust or debris to collect in the air intake vents.

Ensuring That Air Is Cool Enough

• Do not position two or more DNA Engine Opticon units (or other instruments) so that hotexhaust air blows directly into the air intake vents.

• Confirm that the DNA Engine Opticon unit receives air that is 31˚C or cooler by measur-ing the temperature of air entering the machine through its air intake vents.


Installation and Operation

Place the DNA Engine Opticon unit where you plan to use it, and turn it on. Try toreproduce what will be typical operating conditions for the machine in that location,particularly any heat-producing factors (e.g., nearby equipment running, window blindsopen, lights on). Run a typical protocol for 30 minutes to warm up the DNA EngineOpticon unit, then measure the air temperature at the air intake vents. If more than onemachine is involved, measure the air temperature for each.

If the air intake temperature of any machine is warmer than 31˚C, consult Table 3-1 forpossible remedies. After implementing possible remedies, verify that the temperature ofthe air entering the air intake vents has been lowered, using the procedure outlined above.

Table 3-1 Troubleshooting Air Supply Problems

Cause Possible Remedies

Air circulation is poor. Provide more space around instrument or adjust roomventilation.

Ambient air temperature Adjust air conditioning to lower ambient airis high. temperature.

Instrument is in warm part Move instrument away from, or protect instrument from,of room. such heat sources as radiators, heaters, other equip-

ment, or bright sunlight.

Instruments are crowded. Arrange machines so that warm exhaust air does notenter intake vents.

Turning the Opticon Unit and Computer On and OffLocate the power switch on the back, left-side of the Opticon unit (back, right-side onsome models) just above the power cord (see figure 2-2). To turn the Opticon unit on,press the switch so that the side marked “1” is depressed. The thermal cycler requiresseveral minutes to warm up after the Opticon unit is powered up. To turn the Opticon unitoff, depress the “0” side of the power switch.

Be sure that the Opticon unit is connected to the computer and turned on prior to launch-ing the Opticon Monitor software. The blue protocol-indicator light on the front of theOpticon unit (see figure 2-1) is illuminated only during a protocol run.

Press the power button on the front of the computer once to turn the computer on. SelectShutdown from the Start menu to turn the computer off. Press the power button on thefront of the monitor once to turn it on, and press it again to turn the monitor off.



Opening and Closing the Cycler DrawerTo gain access to the Opticon unit’s thermal cycling block, first squeeze the blue triggerhandle (1) and allow the spring-loaded door to lift up (2). Then, use the hand hold on thedrawer front to slide the cycler drawer out toward you exposing the 96-well thermal cy-cler block (3).

To return the Opticon unit to the closed position, slide the cycler drawer back into theinstrument and lower the blue handle to secure the cycler drawer. It is not necessary tosqueeze the trigger handle.

Note: Do not open the cycler drawer while the blue protocol-indicator light is illuminated.Opening the door, particularly during a scan of the plate, may interrupt the software’scontrol of the protocol.

Loading Sample Vessels into the BlockImportant! Do not use full height 0.2ml tubes or full height unskirted

microplates. Refer to the “Selecting the Correct Sample Vessel” sectionin Chapter 4 for tube and microplate recommendations.

To ensure uniform heating and cooling of samples, sample vessels must be in completecontact with the block. Adequate contact is ensured by doing the following:

• Ensure that the block is clean before loading samples (see Chapter 9 for cleaninginstructions).

• Firmly press strips of 0.2ml low-profile tubes, or a 96-well, low-profile microplate intothe block wells (see the “Selecting the Correct Sample Vessel” section in Chapter 4).

• MJR strongly recommends that oil not be used to thermally couple sample vessels tothe block.

Tip: Spin down reactions in tubes or microplates prior to loading into the thermal-cyclerblock. Air bubbles in samples or liquid on the plate deck can adversely affect results.

1. 2.

3.

4-1

4. Compatible Chemistries,Sample Vessels, and SealingOptions

Optical System, 4-2Compatible Chemistries, 4-2

SYBR Green I, 4-2Molecular Beacons, 4-3Hydrolysis Probes (TaqMan Probes), 4-3Scorpions Probes, 4-4Amplifluor Universal Detection System, 4-4

Selecting the Correct Sample Vessel, 4-5Vessels Optimized for Fluorescence Detection and Thermal Cycling, 4-5

Sealing Sample Vessels, 4-5Sealing with Optical Caps and the Heated Lid, 4-6Sealing with Chill-out™ 14 Liquid Wax, 4-6

Sample Vessel and Sealing Selection Chart for Optical Assays, 4-7Reaction Volume Recommendations, 4-8



Optical SystemThe Opticon™ detector uses an array of 96 blue LEDs to sequentially illuminate each ofthe 96 wells in the cycler block. The LEDs efficiently excite fluorescent dyes with excita-tion spectra in the 450 to 495nm range. The Opticon detector is optimized to detect dyeswith emission spectra in the 515 to 545nm range, such as SYBR Green and FAM.

The Opticon detector is calibrated at the factory and requires no calibration before use.See Chapter 10, “Troubleshooting” for instructions on testing detector calibration andrecalibrating.

Compatible ChemistriesThe Opticon detector is compatible with popular dye chemistries including SYBR GreenI, molecular beacons, hydrolysis probes (TaqMan probes), Scorpions probes, and theAmplifluor system. In addition to performing real-time quantification and DNA meltingprofiles, the Opticon system is also useful as a temperature-controlled fluorimeter for anumber of applications including ligand binding and protein structure studies. If you havequestions regarding the compatibility of a particular chemistry with the Opticon detector,contact MJ Research technical support at 888-652-9253.

SYBR Green I

SYBR Green I (available from Molecular Probes, Inc. of Eugene, Oregon) is a dsDNAbinding dye thought to bind in the minor groove. The fluorescence of SYBR Green I isgreatly enhanced upon binding dsDNA. This characteristic makes it ideal for detectionof amplification products. The maximum absorbance of SYBR Green I is ~497nm andthe emission maximum is ~520nm*.

SYBR Green I has several advantages for detection of nucleic acids in real time. BecauseSYBR Green I binds to all dsDNA, it does not have to be customized for individual tem-plates thereby providing the advantages of quick protocol adaptation and relatively lowcost. Further, SYBR Green I is very sensitive because multiple dye molecules bind to asingle amplification product. However, because SYBR Green I binds to all dsDNA, falsepositive signals from primer-dimers, secondary structure, or spurious priming can inter-fere with accurate quantification. Measuring fluorescence at elevated temperatures mayhelp reduce the detection of nonspecific products1. Performing a melting curve to ana-lyze product homogeneity can also aid in analyzing quantification results obtained withSYBR Green I.

MJR recommends using buffers containing 5% dimethyl sulfoxide (DMSO) with a concen-tration of 1X or less SYBR Green I with the Opticon detector. For additional informationon optimizing protocols using SYBR Green I with thermostable enzymes available fromMJ Research, contact MJ Research technical support at 888-652-9253.

1Morrison, T.B., J.J. Weis and C.T. Wittwer. 1998. Biotechniques 24:954-962.

*Molecular Probes, Inc.


Compatible Chemistries, Sample Vessels, and Sealing Options

Molecular Beacons

Molecular beacons are dual-labeled oligonucleotide probes designed to form stem-loopstructures in the absence of target. In the hairpin configuration, the fluorophore at oneend of the molecule is brought into close proximity with a quenching moiety at the otherend of the molecule. When the fluorophore is excited in this configuration, it transfersenergy to the quencher rather than emitting that energy as light, in a process known asfluorescence resonance energy transfer (FRET). A “dark” quencher is often used, so theenergy transferred from the fluorophore is emitted in the infrared as opposed to the vis-ible range. If a second fluorophore is used as a quencher, the transferred energy is emit-ted as light at the quenching fluorophore’s characteristic wavelength.

Molecular beacons are designed such that the loop, which is usually 15-30 nucleotides inlength, is complimentary to the target sequence. The arms flanking the loop, which areusually 5–7 nucleotides in length, are designed such that they are complementary andfavor formation of a stem structure. A fluorophore is attached to the end of one arm, and aquencher is attached to the other. Molecular beacons must be carefully designed such thatat the annealing temperature of the reaction hairpins form in the absence of template, butthat in the presence of template, the annealing of the loop sequence to the target is ener-getically favorable. When the loop of a molecular beacon hybridizes to the target sequence,the conformational change of the probe separates the fluorophore and the quencher.When the fluorophore is excited, it now emits light at its characteristic wavelength.

One advantage of molecular beacons is that unlike SYBR Green, molecular beacons spe-cifically detect the target of interest. Great sensitivity, including detection of singlenucleotide polymorphisms (SNPs), is possible with carefully designed molecular beaconsand optimized reaction conditions (temperature, buffer). However, each probe must becarefully and uniquely designed for the detection of a specific target.

Molecular beacons are a technology patented by the Public Health Research Institute ofNew York, NY and are available from a number of licensed suppliers. When designingmolecular beacons for use with the Opticon detector, fluorophores with excitation andemission spectra falling within the Opticon detector’s excitation (450-490nm) and detec-tion (515-545nm) ranges, such as FAM, can be used. Either dark quenchers or a quench-ing fluorophore may be used with the Opticon detector. However, because the Opticondetector is a single-color detection system, light from a quenching fluorophore can not beseparately monitored. Dark quenchers tend to give cleaner signal because there is nooverlapping signal from light emitted by the quenching fluorophore.

Hydrolysis Probes (TaqMan Probes)

TaqMan probes are a patented technology available from a number of licensed suppli-ers. They are oligonucleotide probes whose fluorescence is dependent on the amplifica-tion of a target sequence. TaqMan probes are designed to anneal to the target sequencebetween the forward and reverse primers. A reporter fluorophore is attached to the 5’end of the probe and a quencher to the 3’ end.



When the intact probe anneals to the target sequence, excitation of the reporter is quenchedbecause of its proximity to the 3’ quencher. However, as extension proceeds, the 5’ exonu-clease activity of the polymerase cleaves the probe, separating the reporter from thequencher. TaqMan probes work well with enzymes derived from Thermus species, suchas DyNAzyme™ II DNA polymerase from Thermus brockianus, available from MJ Research,Inc. Liberated reporter molecules accumulate as the number of cycles increases, such thatthe increase in fluorescence is proportional to the amount of amplified product.

One advantage of TaqMan probes, particularly for quantification, is that fluorescence isdependent not only on the presence of a specific target, but also on amplification of thattarget. However, like molecular beacons, TaqMan probes must be individually designedfor specific targets. See the “Molecular Beacons” section above for recommendations onthe use of specific fluorophores and quenchers with the Opticon detection system.

Scorpions Probes

Scorpions probes (available from licensed suppliers) contain both an amplification primerand a target-specific probe separated by an amplification blocker. The probe portion isflanked by complementary sequences favoring formation of a stem structure which bringsa fluorophore and a quencher into close proximity.

During amplification, extension of the target sequence proceeds from the primer portionof the Scorpions probe. As the reaction cools following denaturation, a uni-molecularrearrangement occurs such that the Scorpions probe sequence binds to the amplified tar-get sequence, separating the complementary stem sequences and thus the fluorophoreand quencher. Since the Scorpions probe is integrated into the product, there is a directrelationship between the number of targets generated and the amount of fluorescence.

See the “Molecular Beacons” section above for recommendations on the use of specificfluorophores and quenchers with the Opticon detection system.

Amplifluor Universal Detection System

The Amplifluor system (available from Intergen Company of Purchase, NY) makes use ofa universal primer that emits a fluorescence signal only following incorporation of theprimer into an amplification product. The universal primer consists of a 18 base primertail ("Z sequence") coupled to a hairpin sequence labeled with a fluorophore and aquencher. First, the target is amplified using target-specific primers, one of which has theZ sequence added to its 5' end. In the following round of amplification, the complementto the Z sequence is incorporated into the product. The universal primer then anneals tothe complement of the Z sequence and extension proceeds. In the next cycle, extensionproceeds through the universal primer incorporating it into the amplification product. Inthe process, the hairpin is unfolded separating the fluorophore and quencher and emit-ting a fluorescence signal that is proportional to the amount of amplified product.

See the “Molecular Beacons” section above for recommendations on the use of specificfluorophores and quenchers with the Opticon detection system.



Selecting the Correct Sample Vessel

Important! Do not use full height 0.2ml tubes or full height unskirtedmicroplates. Full height 0.2ml tubes and most unskirted microplates donot provide sufficient clearance between the sample block and lid-heaterassembly. Do not force the cycler drawer closed.

For proper clearance in the Opticon unit, the distance from the bottom of a tube/plate tothe cap rim can not exceed 17.5mm. In general, fully-skirted 96-well microplates, suchas MJ Research Microseal® and Hard-Shell® microplates, provide sufficient clearance whensealed with either domed or flat optical caps (see the "Sample Vessel and Sealing Selec-tion Chart for Optical Assays" below). If unskirted microplates are used, low-profile plates,such as the MJ Research MLL-series Multiplate™ microplates, are required.

Low-profile 0.2ml strip tubes, such as MJ Research TLS-series tubes, are recommendedfor small numbers of samples. Full-height 0.2ml tubes do not provide sufficient clearance.

Vessels Optimized for Fluorescence Detection and ThermalCycling

For optimal sensitivity in fluorescence-detection assays, we recommend thin-walled 0.2mltube strips and microplates with opaque-white wells. MJ Research, Inc. offers microplatesand tubes with opaque white or clear wells designed for fluorescence detection assaysand optimized to ensure a precise fit in the cycler block (see the "Sample Vessel andSealing Selection Chart for Optical Assays" below).

Microplates and tubes with black wells may be useful in applications requiring very lowlevels of background. However, signal strength is significantly reduced when plates andtubes with black wells are used.

Note: In-factory calibration of the Opticon detector is performed with opaque-white plates.If you are using natural (clear) or black plates, refer to Chapter 10 for instructions onperforming a calibration test and recalibrating.

Sealing Sample VesselsSteps must be taken to prevent the evaporation of water from reaction mixtures during ther-mal cycling so as to avoid changing the concentration of reactants. Only a layer of oil orwax, such as Chill-out liquid wax, will completely prevent evaporation from sample ves-sels. However, an adequate degree of protection can be achieved by sealing with opticalcaps, then cycling the samples using the heated lid to prevent condensation/refluxing.



Sealing with Optical Caps and the Heated Lid

The heated inner lid maintains the upper part of sample vessels at a higher temperaturethan the reaction mixture. This prevents condensation of evaporated water vapor ontothe vessel walls, so that solution concentrations are unchanged by thermal cycling. Theheated lid also exerts pressure on the tops of vessels loaded into the block, helping tomaintain a vapor-tight seal and to firmly seat tubes or microplates in the block for themost efficient transfer of heat to and from the samples.

Optical caps must be used along with the heated lid to prevent evaporative losses. Ultra-clear, optical cap strips (available from MJ Research, Inc.) provide high light transmis-sion for fluorescence detection and vapor-tight sealing. Tight-fitting caps do the best jobof preventing vapor loss.

Note: When tubes are cooled to below-ambient temperatures, a ring of condensation mayform in tubes above the liquid level but below the top of the sample block. This is not acause for concern since it occurs only at the final cool-down step when thermal cycling isfinished.

Sealing with Chill-out™ Liquid Wax

Clear Chill-out liquid wax (available from MJ Research, Inc.) may be used to seal samplevessels for optical assays. Clear Chill-out liquid wax is the same easy-to-use alternative tooil as the standard, red-colored Chill-out wax. However, clear Chill-out wax provides ex-cellent light transmission for optimal performance in optical assays. Chill-out liquid waxprovides 100% prevention of condensation and vapor loss. At room temperature andabove, this overlay is transparent and can be applied by pipet. Chill-out liquid wax so-lidifies below 14°C. Use only a small amount of Chill-out liquid wax; 1-3 drops (15-50µl)are usually sufficient. (Include this volume in the total volume when setting up a calcu-lated-control protocol.) Be sure to use the same amount of wax in all samples vessels toensure a uniform thermal profile.



Sample Vessel and Sealing Selection Chart for Optical Assays

The following sample vessels and sealing options are recommended for use with the DNAEngine Opticon® system and are available from MJ Research, Inc. To place an order, call888-729-2165 or fax 888-729-2166.

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sllewetihw/llehsder5169-PSHsllewetihw/llehswolley5269-PSH

sllewetihw/llehseulb5369-PSHsllewetihw/llehsneerg5469-PSH

sllewraelc/llehsetihw1069-PSHsllewraelc/llehskcalb1669-PSH

sllewraelc/llehsder1169-PSHsllewraelc/llehswolley1269-PSH

sllewraelc/llehseulb1369-PSHsllewraelc/llehsneerg1469-PSH

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Reaction Volume RecommendationsReaction volumes of 20-100µl are recommended for most applications. However, it isbeneficial to empirically optimize reagent concentrations and sample volumes with theOpticon detector as the sensitivity of the optical system often allows a cost-saving reduc-tion in reagent concentrations. Volumes as low as 10µl can be used, though sensitivity isslightly reduced.

The maximum recommended sample volume is 100µl. Volumes exceeding 100µl do notmaintain adequate contact with the wells of the sample block resulting in nonuniformheating and cooling within the sample.

The reaction volume is used to calculate the temperature of the samples during a calcu-lated-control run (see the “Temperature Control Method” section in Chapter 6). Therefore,thermal accuracy is optimized when all samples contain identical volumes.

5-1

5. Introduction to OpticonMonitor™ Software

How Opticon Monitor Software Works, 5-2Launching and Navigating Opticon Monitor Software, 5-3Exiting Opticon Monitor Software, 5-5Opticon Monitor File Extensions, 5-6Which Version of Opticon Monitor Software Are You Running?, 5-6Viewing Usage and Message Logs, 5-6



Opticon Monitor software controls all operations on the DNA Engine Opticon® continu-ous fluorescence detection system. This chapter will introduce you to the Opticon Moni-tor software and discuss the basics of launching and navigating the software. Chapter 6describes experimental setup and programming. Chapter 7 discusses run initiation andstatus, and Chapter 8 focuses on data analysis. This manual documents version1.08 of the Opticon Monitor software.

How Opticon Monitor Software WorksThe intuitive Opticon Monitor software is structured such that there are just three phasesfrom protocol creation to analyzed results.

1. Experimental setup and programming. All setup and programming opera-tions are accessed from the master file window. The master file orchestrates the run byspecifying which combination of plate and protocol files to apply to a particular run.Users can create new files or apply existing files, in their current form or after editing.

2. Run initiation and status. After creating a plate and a protocol setup, or select-ing a plate and a protocol file, a run can be initiated. The user has the option to stop therun at any time and to skip protocol steps. The status screen can be used to monitor theprogress and thermal profile of the run. Data collection can be monitored during the runby plotting fluorescence intensity vs. cycle number.

3. Data analysis. Starting copy number can be quantified by using the software to setthe c(t) (cycle threshold) line, view and adjust an automatically-generated standard curve,and apply unknowns to the curve. Products can be identified by melting profile using thesoftware to plot fluorescence vs temperature, and/or the negative first derivative (-dI/dt)of that graph.


Introduction to Opticon Monitor Software

Launching and Navigating Opticon Monitor SoftwareOpticon Monitor software is pre-installed on the computer provided with the Opticon™

unit. The Opticon Monitor software is compatible with Windows 2000 and Windows XPoperating systems. Opticon Monitor software can control the Opticon unit only when run-ning on the computer supplied with the system, which has special hardware required fordata acquisition. Nonetheless, Opticon Monitor software can be installed on any com-puter running Windows 2000 or Windows XP for the purposes of setting up protocols oranalyzing data.

To launch the Opticon Monitor software, choose Programs from the Windows Start menu,and then Opticon Monitor. Upon launching the Opticon Monitor software, the OpticonMonitor window will appear displaying a new master file template. Alternatively, doubleclick on an existing Opticon Monitor master, plate, protocol, or data file to launch Opti-con Monitor and display the chosen file.

4. Log box

1. Toolbar 2. Pull-down menus3. Setup/analysis display window

(master file)



Opticon Monitor window features include:

1. Toolbar: The toolbar contains both menu buttons and run status information. Themenu buttons located on the toolbar are the primary means of navigation betweenthe setup/programming, run/status and analysis windows:

Experimental setup & programming

Run initiation and monitoring

Data analysis


Introduction to Opticon Monitor Software

The run status box on the toolbar indicates if a protocol or infinite incubation step isrunning. It lists the time remaining in the run, the current step and cycle counts, andthe current temperatures.

2. Pull-down menus: The pull-down menus provide access to numerous functionsincluding the ability to print and export data as well as set the default options fordata analysis.

3. Setup/analysis display window: The window displays the selected setup, sta-tus, or analysis screen.

4. Log box: The log box lists the instrument operation log including any errors encoun-tered.

Exiting Opticon Monitor SoftwareExit Opticon Monitor software by selecting Exit from the File menu, or by clicking theclose button in the upper-right corner of the title bar. If a protocol is running, it must bestopped prior to exiting Opticon Monitor software.



Opticon Monitor File ExtensionsWhen saving files, the Opticon Monitor software automatically adds one of the following fileextensions:

.mast Master file: Controls a run by specifying which plate and protocolfiles to apply during the run.

.plate Plate file: Specifies the contents of the 96 wells, any descriptive well labels thatwere assigned, and the amounts of any quantitation standards for use in generat-ing a standard curve.

.prot Protocol file: Specifies the order and parameters of protocol steps includingtemperature incubations, plate reads, temperature gradients, goto steps, andmelting curves.

.tad Data file: Contains the fluorescence and temperature data collected during therun, and any selected options and analysis parameters.

Which Version of Opticon Monitor Software Are YouRunning?

To determine which version of Opticon Monitor software is currently installed on yourcomputer, choose About from the Help menu. The About window will appear displayingthe Opticon Monitor version number. This manual documents Opticon Monitor software,version 1.08.

Select Close to return to the current setup or analysis screen, or click the X in the upper-right corner.

Viewing Usage and Message LogsTo view a record consisting of the dates and times that runs were initiated and success-fully completed, as well as a record of the master, plate, and protocol files applied dur-ing those runs, select Logs from the View pull-down menu and click the Usage Log toggletab. The dates and times that the software was launched and quit will also be displayed.

To view a record of operations performed by the Opticon Monitor software, includingany error messages, select Logs from the View pull-down menu and click the MessageLog toggle tab.

6-1

6. Experimental Setup andProgramming

Creating a Master File, 6-2Specifying a User, 6-3

Adding New Users, 6-3User Password Protection, 6-4Removing Users, 6-4

Assigning New Plate and Protocol Files to a Master File, 6-4Creating a Plate File, 6-4

Assigning Well Contents, 6-5Selecting Wells Using the Plate Diagram, 6-5Selecting Wells Using the Plate Information Table, 6-6Specifying Quantitation Standards, 6-7

Assigning Well Descriptions, 6-8Saving a Plate File, 6-8Creating a Protocol File, 6-9

Choosing a Temperature and a Lid Control Mode, 6-10Temperature Control Method, 6-10Lid Control, 6-11Saving Temperature and Lid Control Settings, 6-12

Designing and Entering a Protocol, 6-12Entering a New Protocol, 6-13

Temperature Step, 6-13Gradient Step, 6-15

Gradient Calculator, 6-17Plate Read Step, 6-17Adding Multiple Temperature Steps, Gradient Steps, or Plate Reads, 6-17Goto Step, 6-18Melting Curve Step, 6-19

Editing a Protocol Step, 6-20Deleting a Protocol Step, 6-20Inserting a Protocol Step Between Existing Steps, 6-20Melting Curve Analysis, 6-21

Saving a Protocol File, 6-23Saving a Master File, 6-23Assigning Existing Plate and Protocol Files to a Master File, 6-23Reusing Master Files, 6-24Using the Quick Load Feature, 6-25



All setup and programming operations can be accessed from the master file window.Before running a protocol on the DNA Engine Opticon® system, a master file specifyingall of the parameters for the run can be created. The master file orchestrates the run byspecifying which plate and protocol files to apply to a run. The first section of this chapterwill describe how to create and assign new plate and protocol files to a master file. Thesecond section will describe how to assign existing plate and protocol files, with or with-out modifications, to a master file. Finally, instructions for reusing and editing existingmaster files will be discussed.

Creating a Master FileUpon launching the Opticon Monitor™ software (see Chapter 5), the Opticon Monitorwindow will appear displaying a new master file template.

New master file

Experimental Setup and Programming


A master file consists of two component files:

1. Plate file: specifies the contents of the 96 wells, any descriptive well labels that wereassigned, and the amounts of any quantitation standards for use in generating astandard curve.

2. Protocol file: specifies the order and parameters of temperature incubations, platereads, temperature gradients, goto steps, and melting curves.

The New, Edit, Open, and Save buttons located in the Plate Setup and Protocol Setupsections of the master file can be used to assign new or existing files to the master file asdescribed below. The Quick Load feature can be used to quickly assign existing plateand protocol files to the master file (see the "Using the Quick Load Feature" section at theend of this chapter).

Specifying a User

The user feature allows users to organize master, plate, protocol and data files by plac-ing them in a Shared folder to which all users have read/write access, or into personalfolders which can be password protected. Files in a password-protected folder cannot beedited or deleted through Opticon Monitor, nor can files be placed in the folder withoutthe password. However, password-protected files can be read by all users. These filescan be edited by any user if a Save as is first performed and the file is assigned to theshared folder or that user's folder. This provides all users access to all files, but ensuresthat the files in an individual user's password-protected folder are only altered by thatuser.

Adding New Users

To add a new user, click the icon in the master file window.



In the Manage users window that appears, select Add. Enter the new user's name in theNew User window that appears and select OK.

User Password Protection

To password protect a user's files, select the name from the Users list in the Manage userswindow and then select Password. To assign a password, enter the password in the NewPassword field and again in the Confirm New Password field. To change an existingpassword, first enter the existing password in the Old Password field, and then enter andconfirm the new password. The user will be prompted to enter their password when theirname is selected from the drop-down User list in the master file window.

Removing Users

To remove a user from the Opticon Monitor software, select the user's name in the Man-age users window and then select Remove. You will be asked to confirm deletion of theuser as all data associated with the user will also be deleted.

Assigning New Plate and Protocol Files to a MasterFile

Creating a Plate File

A plate file functions to identify the contents of the 96 wells as empty (ignored), blank (forbackground subtraction), quantitation standard (for standard-curve generation), or sample(for unknown and control reactions). A plate file may also contain user-specified welldescriptions, and the amounts and units of any quantitation standards.

Click the New button in the Plate Setup section of the master file to create a new platefile.

In the plate file window, begin by entering the volume of your reactions (in µl) in the Reac-tion Volume field. See the "Reaction Volume Recommendations" section in Chapter 4 foradditional information.



Assigning Well Contents

Follow these steps to characterize the contents of wells as empty, blank, quantitation stan-dard, or sample:

1. Select the well or grouping of wells to which a specific content is to be assigned. Youcan select wells by using either the plate diagram or the plate informationtable to the right of the diagram.

Selecting Wells Using the Plate Diagram

Move the cursor over an individual well, row letter, or column number to highlight thewell or wells with a thin outline and darken the corresponding well coordinates (seewell A12 in the diagram above). Clicking on highlighted wells will select them. Se-lected wells appear heavily outlined, and the fields of the corresponding wells arehighlighted in the plate information table (see wells in column 11 in the diagram above).

• Select all wells in the plate by clicking on the * in the upper-left corner of the platediagram.

• Select all wells in a column by clicking on the numbered box at the top of thecolumn. To select multiple columns, hold down the control key and click on thenumbered box at the top of each column to be selected.

Platediagram

Wells with assignedcontents

Selectedwells

Highlightedwell

Plateinformation

table

Plate file window



• Select all wells in a row by clicking on the lettered box at the start of the row. Toselect multiple rows, hold down the control key and click on the lettered box atthe start of each row to be selected.

• Select an individual well by clicking on the well.

• Select multiple wells in an arbitrary pattern by holding down the left mouse but-ton and dragging the cursor over the wells to be selected, or hold down thecontrol key and click on the individual wells you wish to select.

To deselect all wells, click on any blank space in the plate diagram or on anotherwell. To deselect a well, click on the well again.

Selecting Wells Using the Plate Information Table• Select an individual well by clicking on the well’s coordinates (e.g., A1) in the table.

• Select multiple adjacent wells by left clicking on the coordinates of the first well tobe included, holding down the shift key, and left clicking on the coordinates ofthe last well to be included in the group.

• Select multiple, non-adjacent wells by holding down the control key and leftclicking on each well’s coordinates to select it.

2. Assign the appropriate contents to selected wells by clicking on one of the fourcontents buttons:

• Empty (white) – The well is empty. The Opticon™ detector will not measure thefluorescence in the well. Unspecified wells are considered empty.

• Blank (blue) – The well contains a blank reaction (e.g., buffer only). Fluorescenceintensity measurements from blank wells can be used in background subtractioncalculations.

• Quantitation Standard (green)- The well contains a user-specified standard ofknown quantity (see the “Specifying Quantitation Standards” section below).Fluorescence intensity readings from quantitation standards are used to plot astandard curve.

• Sample (red) –The well contains an experimental sample (unknown or control).



The color of assigned wells in the plate diagram should correspond to the color of thecontent specified, and each content assignment should appear in the correspondingContents column of the plate information table.

3. To change the content assignment of a well, select the well as described in step 1,and click on the desired content button. The well’s color and corresponding contentinformation in the plate information table will reflect the content change. If a well isnot designated as empty prior to the run, the content assignment for the well can bechanged post run. Changing a "non-empty" well to "empty" post-run willresult in the irreversible loss of fluorescence data for that well.

Specifying Quantitation Standards

If you are using quantitation standards, click the Specify Quant Standards buttonafter you have designated which wells contain standards. A pop-up window willappear listing all of the wells to which quantitation standards have been assigned.The scroll bar will become active if the number of standards assigned is greater thanthe number that can be displayed in the window.

Enter the quantity of each standard in the Value box. Then, specify the Units of thestandard by choosing either ng, moles, molecules, ge (genome equivalents), or cop-ies from the pull-down menu, or define your own units by selecting the Manage but-ton. Select Add in the Manage Standards window that appears, and type the de-sired unit designation in the Add Item window that appears. To remove unit designa-tions from the Units menu, highlight the designation and click the Remove button.

Select Save to save the standard specifications, or click Cancel to undo any changesto the standard specifications.



A standard curve will be automatically generated using the values supplied duringanalysis of the data. You will have the option to adjust the standard curve by dese-lecting points (see Chapter 8).

Assigning Well Descriptions

To aid in sample identification, you can enter descriptive well labels for individual wellsor groups of wells. Begin by selecting the well(s) using the plate diagram or plate infor-mation table as specified in the “Assigning Well Contents” section above. Then, type adescription in the Well Label field. The well label will be applied to the selected well orwells and appear in the Description column in the plate information table. Alternatively,double click on an individual row in the plate information table and type a well labeldirectly into the well’s Description field. You can also copy and paste a well label fromone Description field in the table to a second Description field by first double-clicking onthe field and then using either (control c) to copy or (control v) to paste. To simultaneouslypaste a well label into the Description fields of multiple wells, select the wells as describedabove and use (control v) to paste into the Well Label field.

Use the Clear Well Label button to delete the well labels for selected wells from theDescription column.

Once you have finished entering plate file parameters, click the OK button in the upper-left corner of the plate file window to return to the master file window. A picture andsummary of the assigned plate contents will appear in the Plate Setup section of the masterfile window.

Alternatively, if you wish to discard the plate file information and return to the master file,click Cancel.

Saving a Plate File

To save the newly created plate file, select the Save button from the Plate Setup section inthe master file window. Enter an appropriate name in the File Name field of the SavePlate File As window.

Then, specify the location to which the plate file should be saved. If a specific user hasbeen designated in the master file window, the plate file may be saved to that user'spersonal folder (Save to Personal Folder), to the shared folder (Save to Shared Folder),or to an alternate location (Specify Other Save Location). If the designated user is Shared,



only the last two options are available. If the Specify Other Save Location option is cho-sen, select the Browse button to access a standard Windows browse screen, and selectthe location to which you wish to save the file.

Creating a Protocol File

A protocol file contains a program that controls the thermal-cycling parameters of a runand specifies when during the run the Opticon detector will measure the fluorescence inthe wells designated as samples, quantitation standards, and blanks. Protocol steps canbe entered and edited in the protocol file window and a listing and graphical represen-tation of the protocol is displayed for easy review.

Click the New button in the Protocol Setup section of the master file to create a new pro-tocol file.

Protocol file window

Insertprotocol

steps

Select methodsof temperatureand lid control

Protocollisting

Protocolgraphical

representation

Editprotocol

steps



Choosing a Temperature and a Lid Control Mode

Click the Edit button in the Temperature and Lid Mode panel to specify the temperatureControl Method and the Lid Control method to be used in the run. The Protocol Optionswindow will be displayed.

Temperature Control Method

The DNA Engine Opticon system can control block temperature in two different ways,each of which has implications for the speed and accuracy of sample heating.

1. Calculated Control is the default method of temperature control. Calculatedcontrol is the method of choice for most protocols, yielding consistent, reliable,and fast programs. When using calculated control, the DNA Engine Opticonsystem maintains a running estimate of sample temperatures based on the block’sthermal profile, the rate of heat transfer through the sample tube, and the samplevolume. Since this estimate is based on known quantities and the laws of thermo-dynamics, sample temperatures are controlled much more accurately than withblock temperature control.

Hold times can be shortened significantly when protocols are run under calcu-lated control. In addition to the simple convenience of spending less time runningreactions, shorter protocols also help preserve enzyme activity and minimizefalse priming. Cycling denaturations run under calculated control are usuallyoptimal at five to 30 seconds, though optimization of denaturation time may bebeneficial for quantitative protocols. Annealing/extension steps can also be short-ened—but the periods for these will be reaction specific.

Calculated control provides for shorter protocols in three ways:



• Brief and precise block-temperature overshoots are used to bring samplesto temperature rapidly.

• Incubation periods are timed according to how long the samples, not theblock, reside at the target temperature.

• The instrument automatically compensates for vessel type and reactionvolume.

2. When using Block Control, the DNA Engine Opticon system adjusts the block’stemperature to maintain the block at programmed temperatures independent ofsample temperature. Block control provides less precision in control of actualsample temperature than calculated control provides. Under block control, thetemperature of samples always lags behind the temperature of the block. Thelength of the time lag depends on the vessel type and sample volume but istypically between 10 and 30 seconds. Block control is used chiefly to run proto-cols developed for other thermal cyclers that use block control including the PTC-100® cycler and the MiniCycler® personal cycler from MJ Research.

Lid Control

When a sample is heated, condensation on the tube cap or the plate cover can oc-cur. This changes the volume of the sample, the concentration of components and thusthe kinetics of the enzymatic reaction. Use of a heated lid minimizes condensation bykeeping the upper surface of the reaction vessel at a temperature slightly greater thanthat of the sample itself.

The DNA Engine Opticon system can control lid temperature in three possible ways:Constant, Tracking, or Off.

• Constant: Keeps the inner lid at a specified temperature (˚C). This is the defaultmethod of control. To use constant lid-temperature control, select Constant andenter a Lid Temperature between 30°C and 110°C or use the arrows to scroll tothe desired temperature. A temperature of 5°C to 15°C above the highest tem-perature in a protocol is recommended. You can also specify a sample-blocktemperature below which the heated lid will turn off. Enter a Lid Shutoff Tempera-ture between 1°C and 50°C or use the arrows to scroll to the desired tempera-ture.

• Tracking: Offsets the temperature of the heated inner lid a minimum specifiednumber of degrees Celsius in comparison to the temperature of the sample block.Tracking is useful for protocols with long incubations in the range of 30-70°C,where it may be undesirable to keep the lid at a very high temperature. An offsetof 5°C above block temperature is adequate for most protocols. To use trackinglid-temperature control, select Tracking and enter the number of degrees, from1°C to 45°C, the lid temperature should be maintained above the block tempera-ture, using the format Lid Temperature = Block Temperature +. You can also usethe arrows to scroll to the desired temperature. To specify a sample-block tem-perature below which the heated lid will turn off, enter a Lid Shutoff Temperaturebetween 1°C and 50°C or use the arrows to scroll to the desired temperature.



Note: Because there is no active cooling of the lid, a decrease in the lid tem-perature may not be observed during rapid cycling. In addition, the lid heats moreslowly than the sample block as a result of its additional thermal mass.

• Off: No power is applied to the heated lid. In this mode, condensation will occurat a rate consistent with the incubation temperature and the type of tube or platesealant being used. This option is recommended only when using an oil or waxoverlay.

Saving Temperature and Lid Control Settings

Click the OK button to apply your temperature and lid control settings to the protocol,or choose Cancel to close the window without changing the settings applied to theprotocol. The settings should appear in the Temperature Control and Lid Settings fieldsabove the protocol listing and graphical display.

Designing and Entering a Protocol

Programming the DNA Engine Opticon system consists of entering a series of steps en-coding a protocol. This section will present a sample protocol and describe how to enterthe protocol steps. Additional protocol options will also be described.

Consider the following example protocol:

1. Incubate at 94°C for 30 seconds

2. Optimize annealing temperature by incubating at a range of 55°C to 65°C acrossthe 12 columns of the sample block

3. Read the fluorescence intensity of the Blank, Quantitation Standard, and Samplewells

4. Incubate at 72°C for 1 minute

5. Sequentially repeat steps 1-4, 24 more times, then proceed to step 6

6. Identify and determine the purity of reaction products by melting profile—raise thetemperature from 55°C to 90°C, and read the fluorescence 10 seconds after every1°C increase in temperature

7. End program



Entering a New Protocol

As you insert steps into a protocol, descriptions of the steps will appear in the upper proto-col display window and a graphical representation of each step’s temperature and timeperiod (in minutes) will appear in the lower protocol display window. Use the HorizontalZoom slider and the scroll bar to clearly view the graphical representation of the protocol.

Before beginning to enter a new protocol, note that the END step is highlighted in theupper display window. Opticon Monitor software adds new steps before the step that ishighlighted. Protocols are limited to a maximum of 99 steps.

Temperature Step

A temperature step specifies incubation temperature and duration. The DNA EngineOpticon system ramps the sample to this temperature at its maximum rate—unlessramp modifying instructions are added to the program (see the “Manual Ramp Rate”description near the end of this section).

Click the Temperature button to enter a temperature incubation step (e.g., step 1 orstep 4 from our example) into a protocol.

Plate read Melting curve



Enter the desired temperature, from 0°C to 105°C, into the Set Temperature to fieldor use the arrows to scroll to the desired temperature—which is 94°C in step 1 of ourexample.

Enter the desired incubation time, to a maximum of 18 hours, in the Maintain TempFor field. Click in the hour: minute: or second field and enter a time period, or use thearrows to scroll to the desired time—00:00:30 in step 1 of our example. Alterna-tively, you can select Forever to maintain the desired temperature for an infinite pe-riod of time. A forever incubation step at the end of a protocol can be useful for hold-ing reaction products at a sub-ambient temperature (we recommend 10°C) until theycan be processed. In the graphical representation of the protocol, a forever incuba-tion is indicated with an .

Click the Insert button to add the temperature step to the protocol without further modi-fications. The temperature step should appear as step 1 in the upper protocol displaywindow, and a graphical representation of the temperature and duration should ap-pear in the lower window. Note that the END step is again highlighted indicatingthat the next step will be added above the END step and therefore after step 1.

You can also choose to modify a temperature step before inserting it into the protocolby adding options. Available options include:

1. Manual Ramp Rate: Set a slower-than-maximum rate of heating or cooling.A slower-than-maximum ramp rate ranging from 0.1°C to 2.5°C per second canbe specified. Fast thermal ramping between incubation steps can often help re-duce overall reaction times by 10% to 30% and may help reduce production ofnon-specific products.



2. Increment Temperature: Modifies a temperature step to allow a “per cycle”increase or decrease of temperature (0.1°C to 10.0°C per cycle) each time thestep is executed. This feature is useful when annealing stringency is a consider-ation such as in a touchdown program.

In a touchdown program, the annealing temperature begins higher than the cal-culated temperature, and incrementally decreases each cycle, first reaching, andeventually falling below the calculated annealing temperature. With the reactionbeginning at a temperature favoring high stringency in hybridization andincrementing to lower stringency, the higher stringency favors the desired prod-uct by creating a high proportion of signal relative to noise in the early amplifica-tion cycles.

3. Extend Time: Modifies a temperature step to allow a “per cycle” lengtheningor shortening of a temperature step hold (by 1–60 sec/cycle) each time a step isexecuted.

This capability is useful for slowly increasing (typically by 2 to 5 seconds percycle) the hold time during an extension step. The number of bases that a poly-merase must synthesize during the extension step increases in later cycles be-cause there are more template molecules, because there are fewer active poly-merase molecules, or both. The extra time can allow synthesis to be completed.

4. Beep When Completed: Modifies a temperature step so the instrument willbeep when the target temperature is reached.

Gradient Step

The temperature gradient feature allows you to optimize denaturing or annealingconditions by incubating at several different temperatures simultaneously. For example,determining the best denaturation temperature may be especially important in opti-mizing the efficiency of amplification reactions used for quantification. With the MJgradient feature, such optimization can be performed in a single experiment. Therange of temperatures that can be achieved from left to right across the 96-well sampleblock can be as small as 1°C or as great as 24°C. The maximum programmabletemperature is 105°C; the minimum programmable temperature is 30°C.

Click the Gradient button to insert a gradient step into a protocol.



The minimum gradient temperature is assigned to the far left column (column #1) ofthe sample block and can range from 30°C to 104°C. In the Set Temperature To fieldin our example, L=55. The maximum gradient temperature is assigned to the far rightcolumn (column #12) and can range from 31°C to 105°C. In the Set Temperature Tofield in our example, R=65. The minimum temperature differential between the farleft and far right columns is 1°C and the maximum differential is 24°C.

After entering the range of temperatures for the gradient, enter the desired incuba-tion time in the Maintain temperature For field by clicking in the hour: minute: or sec-ond field and entering a time period, or use the arrows to scroll to the desired time—00:00:30 in step 2 of our example. Alternatively, you can select Forever to maintainthe desired temperature for an infinite period of time.

Click Insert to add the gradient step to the protocol. The gradient step should ap-pear as step 2 in the protocol display window. Note that the END step is againhighlighted indicating that the next step will be added above the END step andtherefore after step 2.



Gradient Calculator

To accurately predict the temperature of each of the twelve columns across theblock during a gradient incubation, select Gradient Calculator from the Tools menu.

Use the arrows to scroll to the desired left and right column temperatures. Pleasenote that the gradient temperature distribution is not linear, with a broader spreadin temperature between the center columns of wells. This is a consequence of thegeometry of the Peltier-Joule heaters that underlie the block and is normal. Restassured that the temperatures displayed are quite accurate for each well in thatcolumn (± 0.4°C of actual column temperature). The predicted temperature forthe column that yields the best results can then be accurately transferred to a tem-perature step in a non-gradient protocol.

Plate Read Step

Insertion of a plate read step directs the Opticon detector to measure the fluorescenceof the wells designated as samples, quantitation standards, and blanks. The plateread begins immediately after the programmed end of the previous incubation step,step 2 in our example. The Opticon detector performs the plate read at the currentincubation temperature, and then initiates the next step, step 4 in our example.

To insert a plate read step, click the Plate Read button. A plate read step will be in-serted into the protocol. The plate read step should appear as step 3 in the upperprotocol display window in our example. A plate read appears as an eye icon in thegraphical protocol display. Protocols cannot contain a plate read as a first step.

Adding Multiple Temperature Steps, Gradient Steps, or PlateReads

To add additional temperature steps, gradient steps, or plate reads to your proto-col, click the appropriate button and follow the directions for the specific step asoutlined above.

Following our example, step 4 is a temperature incubation step of 72°C for a dura-tion of 00:01:00 with no additional options.



Goto Step

The goto step allows a protocol of many repeating steps to be shortened. When theprotocol encounters a goto step, it returns to a user-specified step, repeats that stepand all steps that follow back to the goto step. When the protocol has cycled back tothe goto step a specified number of times, the protocol moves on to the step that fol-lows the goto step. Keep in mind that the maximum length of a protocol is 99 steps; agoto step (regardless of the number of times that a protocol loop will be performed)counts as only one step.

Step 5 of our example protocol indicates that the protocol should return to step 1,repeat steps 1-4 24 additional times, for a total of 25 cycles, and then proceed tostep 6. This can be accomplished by including a single goto step.

Click the Goto button, and enter the line number of the step to which the protocolshould return. In our example, enter 1 in the Goto Line field, and 24 in the How ManyMore Times? field. Step 5 of our protocol will then direct the protocol to repeat steps1,2,3, and 4 a total of 24 times before continuing on to step 6.

Click the Insert button to add the goto step to the protocol.



Melting Curve Step

In some instances, such as to verify product identity, you may want to perform a melt-ing curve following a cycling protocol. Melting curve profiles are influenced by sev-eral factors including the number and concentration of discreet fragments produced,the length and G+C content of each fragment, and other factors that influence themelting temperature of nucleic acids such as buffer conditions.

To add a melting curve step following a cycling protocol, click the Melting Curve button.

Enter a Starting Temperature (0.0°C to 99.0°C), and an Ending Temperature (1.0°Cto 100.0°C).

Next, specify when during the melting curve step the Opticon detector should mea-sure fluorescence. Designate a Temperature Increment Between Reads of 0.1°C to10°C and a Hold Time Between Reads (1 second to 1 hour) corresponding to theduration for which the temperature increment should be maintained before the fluo-rescence is read. A temperature Increment of 0.2°C and a hold time of 1 second isrecommended for many protocols.



Melting Curve Analysis Options can be specified at the time the melting curve step iscreated, or analysis options can be altered during analysis of the melting curve data.See the "Defining Default Data Analysis Options" and "Display" sections in Chapter8 for an explanation of the available analysis options and information on specifyingdefault analysis options and altering options during data analysis.

Click the Insert button to add a melting curve step to the protocol. Data from only onemelting curve step per protocol can be analyzed.

Editing a Protocol Step

To edit a protocol step, first click on the step to highlight it in the protocol display window.Then, click the Edit Step button. The parameters for the step as it is currently entered willappear. After making the desired changes, click the Replace button to enter the editedstep into the protocol, or click Cancel to leave the step unedited.

Deleting a Protocol Step

To delete a protocol step, click on the step to highlight it in the protocol display window.Then, click the Delete Step button to remove the step from the protocol. The remainingprotocol steps will automatically renumber.

Inserting a Protocol Step Between Existing Steps

To insert a protocol step between existing steps, highlight the step in the protocol displaywindow that will follow immediately after the newly inserted step. All protocol steps areadded immediately before the step that is highlighted in the protocol display window.Then, click the button corresponding to the type of step you would like to add.

Once you have finished entering protocol file parameters, click the OK button in the up-per-left corner of the protocol file window to return to the master file window. A graphicalrepresentation of the protocol and a summary of the total number of plate reads, meltingcurves, and the estimated run duration will appear in the Protocol Setup section of themaster file window.

Alternatively, if you wish to discard the protocol file information and return to the masterfile, click Cancel.



Melting Curve Analysis

A melting curve can be performed to identify specific fragments and/or assess the homo-geneity of a sample. Melting curve profiles are influenced by several factors includingthe number and concentration of discreet fragments produced, the length and G+C con-tent of each fragment, and other factors that influence the melting temperature of nucleicacids such as buffer conditions.

The Opticon apparatus can be programmed to run a melting curve independent of acycling protocol. This analysis can be useful in a variety of applications including homo-thermic assays, sizing fragments relative to ladders, and utilizing the 96-well capacity ofthe Opticon apparatus to perform endpoint assays to increase throughput.

Melting curves are often useful in verifying the identity of amplification products, as wellas distinguishing positive internal controls from amplified products. In these cases, simplyspecify a melting curve after a cycling run, and the instrument will perform both proce-dures automatically.

To program a melting curve independent of cycling, click the Melting Curve button.



Enter a Starting Temperature (0.0°C to 99.0°C), and an Ending Temperature (1.0°C to100.0°C).

Next, specify when during the melting curve step the Opticon detector should measure fluo-rescence. Designate a Temperature Increment Between Reads of 0.1°C to 10°C and a HoldTime Between Reads (1 second to 1 hour) corresponding to the duration for which the tem-perature increment should be maintained before the fluorescence is read. A temperatureIncrement of 0.2°C and a hold time of 1 second is recommended for many protocols.

Melting Curve Analysis Options can be specified at the time the melting curve step iscreated, or analysis options can be altered during analysis of the melting curve data. Seethe "Defining Default Data Analysis Options" and "Display" sections in Chapter 8 for anexplanation of the available analysis options and information on specifying default analysisoptions and altering options during data analysis.

Click the Insert button to add a melting curve step to the protocol. Data from only onemelting curve step per protocol can be analyzed.



Saving a Protocol File

To save the newly created protocol file, select the Save button from the Protocol Setupsection in the master file window. Enter an appropriate name in the File Name field of theSave Protocol File As window. Then, specify the location to which the protocol file shouldbe saved. If a specific user has been designated in the master file window, the protocolfile may be saved to that user's personal folder (Save to Personal Folder), to the sharedfolder (Save to Shared Folder), or to an alternate location (Specify Other Save Location).If the designated user is Shared, only the last two options are available. If the SpecifyOther Save Location option is chosen, select the Browse button to access a standardWindows browse screen, and select the location to which you wish to save the file.

Saving a Master FileTo save a master file, click the Save button under Master File. Enter an appropriate namein the File Name field of the Save Master File As window. Then, specify the location towhich the master file should be saved (see the "Saving a Protocol file" section above foran explanation of location options).

You can also choose to not save this collection of component files and proceed directly tothe run (see Chapter 7 for information on initiating a run).

Assigning Existing Plate and Protocol Files to a Mas-ter File

To assign existing plate and protocol files to a new or existing master file, either click theOpen button in the section of the master file corresponding to the type of file you wish toassign, or use the Quick Load feature to rapidly assign existing plate/protocol files to amaster file (see the "Using the Quick Load Feature" section below).

Selecting Open will display all of the plate/protocol files in either the Shared folder or aspecific user's folder, if a user has been assigned to the master file. Select the desired fileor use the Windows browse screen to locate the file if it has been saved to an alternatelocation, and then click Open. The plate/protocol file will be applied to the master fileand a corresponding summary will appear in the master file window.

To view the newly assigned plate or protocol file and/or make any necessary modifica-tions, click the Edit button in the appropriate section of the master file window. The plateor protocol file window will open allowing you to modify the file parameters. Select OKto retain any modifications and return to the master file window, or select Cancel to re-turn to the master file without modifying the plate/protocol file. Select Save to save anychanges to the plate/protocol file under the same or a newly assigned file name. See the"Saving a Plate/Protocol File" sections in this chapter for additional information on sav-ing plate/protocol files.

Click the Save button in the master file section to save any changes to the master file.



Reusing Master FilesA new master file need not be created for every run. Existing master files may be reusedwithout modifying the plate or protocol files, or the master file may be edited to accom-modate changes such as a different arrangement of samples in the plate.

If a run has just completed, or a data file for a previously completed run has just beendisplayed, and you wish to use the same master file, select the Repeat This Run button. Toaccess a new master file template, select the Prepare New Run button. To display an ex-isting master file, click the Open button near the top of the master file window, or use theQuick Load feature to rapidly assign an existing master file to a master file template (seethe "Using the Quick Load Feature" section below).

Selecting Open will display all of the master files in either the Shared folder, or a specificuser's folder, if a user has been assigned to the master file template. Select the desiredfile or use the Windows browse screen to locate the file if it has been saved to an alter-nate location, and then click Open. The master file will be applied to the master file tem-plate and the corresponding plate and protocol file summaries will appear in the masterfile window.

To use the master file without any changes, proceed to Chapter 7, Run Initiation and Status.

You can also modify the master file before initiating a run, by editing the assigned plateor protocol files (Edit button), by substituting files (Open button or Quick Load), or bycreating new component files (New button).

Note: If a component plate or protocol file is edited and saved under the same name,the edited file will replace the original file in all master files to which that file has beenassigned. Therefore, if you save a master file to your password-protected folder, be surethat the component plate and protocol files are also saved in your folder. If the compo-nent plate and protocol files are not password protected, another user could modify thesefiles and inadvertently modify your password-protected master file.

Click the Save button to save any changes to the master file.



Using the Quick Load FeatureThe Quick Load feature can be employed to rapidly access any existing plate, protocol,master, or data files. If the Plates/Protocols/Masters option is selected, all of the avail-able plate, protocol, or master files that have been saved to the Shared folder or indi-vidual user folders are displayed in the drop-down menu. The files are listed along withtheir associated user as shown below.

Scroll to locate the desired file in the drop-down menu, and select the file. If the Data Filesoption is selected, all of the data files are listed in the drop-down menu. Selecting a datafile will apply the plate, protocol, or master file that was used to generate that data file tothe current master file template.

7-1

7. Run Initiation and Status

Running a Protocol, 7-2Monitoring Run Status, 7-2

Protocol Information On the Toolbar, 7-2The Status Window, 7-3

Thermal Cycler Status, 7-3Optical Read Status, 7-4



Running a ProtocolBefore initiating a run, check that the appropriate master file is displayed (see Chapter 6for instructions on how to create and edit master files).

Initiate the run by clicking the Run button on the toolbar. A windows browse screen willappear asking you to name the file to which the data will be saved. Click Save to acceptthe default filename consisting of the year, month, day, and six-digit run identificationnumber (e.g., 20010618_110812), or enter an appropriate filename and then click theSave button. The data file will be saved as a .tad (acquired data) file.

Click the Stop button on the toolbar to halt the run at any time. The Skip button can beused to skip to the next step in the protocol file.

Monitoring Run Status

Protocol Information On the Toolbar

A summary of run information is displayed at the bottom of the toolbar. This run summaryincludes:

• Protocol information: indicates if a protocol or a forever incubation is currentlyrunning.

• Time Remaining: displays an estimate of the time remaining for the run.

• Program Counter: displays the current step and cycle number.

• Temperature: displays the current sample, block, and lid temperatures.

Run Initiation and Status


The Status Window

Use the status window to monitor run progress using the thermal cycler status screen, ormonitor real-time data collection using the optical read status screen. The status windowdisplaying the thermal cycler status screen is automatically displayed when the run is ini-tiated. To access the status window after the run has completed, click the Status button onthe toolbar. The tabs in the lower-left corner of the status window toggle the display be-tween Thermal Cycler Status and Optical Read Status.

Thermal Cycler Status

The run status is graphically displayed in the top portion of the thermal cycler status win-dow. Select the appropriate boxes to display a graph of the sample, block, and/or lidtemperatures over time. The protocol is listed in the bottom portion of the window withthe current step highlighted. The END step is highlighted if the run has finished.

Time sliderGraph selection

bar

Protocolwindow

Toggle tabs Thermal cycler status window



Optical Read Status

Click the Optical Read Status tab to monitor real-time data collection.

A graph of Fluorescence versus Cycle number can be displayed for selected wells. Eitherraw or normalized data can be displayed depending on the data processing optionsspecified in the options file. Use the Plate diagram to select the wells to be included in theData and Standards graph. See the “Selecting Wells Using the Plate Diagram” section inChapter 6 for additional information.

Selected wells will appear outlined in color. The color outlining the well corresponds tothe color of the well coordinates in the Graphed Samples list and to the color of the fluo-rescence intensity trace in the Data and Standards Graph. The interior color of the wellcorresponds to the contents (empty-white, blank-blue, quantitation standard-green, sample-red) assigned to the well in the plate file.

Deselect all wells by clicking on any blank area between the wells. To deselect a well orsubset of wells, hold down the control key, and click on the well(s) you wish to deselect.The well(s) will no longer appear outlined in color, and the corresponding fluorescenceintensity trace will be removed form the graph.

Optical read status window

Run Initiation and Status


To highlight the results for a particular sample in the Plate diagram, Data and Standardsgraph, and the Graphed Samples list:

• Move the cursor over a well on the Plate diagram. The column number and row lettercoordinates of the well will be highlighted in the Plate diagram, and a well label willappear displaying the assigned contents and well description. The well will also behighlighted in the Graphed Samples list, and the trace corresponding to the well willbe thickened in the graph.

• Move the cursor over a particular trace on the graph to thicken the trace and high-light the corresponding well coordinates in both the Plate diagram and GraphedSamples list. The x and y coordinates, cycle number and fluorescence intensity, cor-responding to the position of the cursor on the trace will also be displayed.

• Select a well from the Graphed Samples list to highlight the well coordinates in thePlate diagram and thicken the corresponding trace in the graph.

Use the Parameters box to display the signal intensity data for a particular Step, if a plateread is included in more than one step of the protocol. Use the Cycle and Read boxes tohighlight the trace for a particular cycle or read number in the Data and Standards graphwith a dotted-vertical line.

8-1

8. Data Analysis

Quantitation, 8-2Graphs, 8-3

Using the Plate Diagram to View Fluorescence Intensity, 8-3Data Graph, 8-4

Adjusting Data Analysis Options, 8-6Adjusting the Cycle Threshold Line, 8-6

Standards Graph, 8-7Adjusting the Standard Curve, 8-8Changing the Values of Quantitation Standards, 8-8Quantity Calculations, 8-9Printing and Exporting Quantitation Data, 8-10Saving the Quantitation Analysis, 8-10

Melting Curve, 8-10Display, 8-11Calculations, 8-14Printing and Exporting Melting Curve Data, 8-15Saving the Melting Curve Analysis, 8-15

Exporting and Printing Data, 8-15Exporting Data, 8-15

Copying Data to the Clipboard, 8-16Printing Data, 8-16

Saving a Data File, 8-17Defining Default Data Analysis Options, 8-18



QuantitationContinuous, real-time, fluorescence detection of amplification products allows you to ac-curately calculate the quantity of template initially present in a sample. During the expo-nential phase of amplification, there is a highly reproducible relationship between theinitial amount of template present in the reaction, and the number of cycles required be-fore a significant increase in fluorescence signal is observed. The larger the initial tem-plate number, the fewer cycles required before significant fluorescence signal is detected.

This relationship between starting copy number and the number of cycles preceding de-tection can be used to calculate the initial quantity of template in a sample. First, the po-sition of the cycle threshold or C(t) line must be defined on a graph of Fluorescence vs.Cycle number. The C(t) line is often positioned on a graph of baseline-subtracted data(see the “Defining Default Data Analysis Options” section in this chapter) at a point wherethe signals surpass background noise and begin to increase. The threshold cycle for anindividual sample is then defined as the cycle at which the sample’s fluorescence tracecrosses the C(t) line. By including quantitation standards with varying initial amounts oftemplate in the run, a standard curve of Log Quantity vs. C(T) Cycle can be plotted. Thequantity of initial template in unknown samples can then be calculated by applying thesample’s threshold cycle to the standard curve. Initially, or for applications requiring ahigh degree of precision, including replicate quantitation standards in the run can aid inpositioning the C(t) line. The use of replicates allows you to determine which options forsetting the C(t) line parameters, described below, provide the tightest fit of the replicatesonto the standard curve.

If a run has just completed, or a previously generated data file has been opened by select-ing Open data file from the File menu, click the Quantitation button on the toolbar to ana-lyze Data and Standards graphs, adjust the data analysis options, position the cycle-thresh-old line, adjust the automatically generated standard curve, and calculate the quantity ofsample initially present in a reaction. The Graphs screen is the default quantitation screen.

Data Analysis


GraphsA Data graph of Fluorescence (or Log Fluorescence) versus Cycle number, a Standardsgraph of Log Quantity versus C(T) Cycle, or Both can be displayed by clicking on theappropriate tab at the bottom-left side of the quantitation window.

Using the Plate Diagram to View Fluorescence Intensity

The Plate diagram can be useful for selecting wells to include in the Data graph and foranalyzing end-point fluorescence intensity. The interior color of a well in the Plate dia-gram correlates with the signal intensity measured in the well for the specified Step, Cycle,and Read number. Dark grey or black wells indicate no or weak signal while white orlight grey wells indicate strong signal.



To display the fluorescence data for a particular step, if a plate read is included in morethan one step of the protocol, enter the step number in the Step field or use the arrows toscroll to the desired step. Use the Cycle and Read boxes to view the signal intensity mea-sured during a specific cycle, or a specific plate read if more than one plate read is in-cluded per cycle. You can also use the Cycle and Read boxes to highlight a particularcycle or read number in the graph display with a dotted-vertical line.

Data Graph

Use the Plate diagram to select the wells to be included in the Data graph. See the “Se-lecting Wells Using the Plate Diagram” section in Chapter 6 for additional information.

Selected wells will appear outlined in color. The color outlining the well corresponds tothe color of the well coordinates in the Graphed Samples list and to the color of the fluo-rescence intensity trace in the Data graph.

Deselect all wells by clicking on any blank area between the wells. To deselect a well orsubset of wells, hold down the control key, and click on the well(s) you wish to deselect.The well(s) will no longer appear outlined in color, and the corresponding fluorescencetrace will be removed form the graph.

Click the Data tab to display a large graph of Fluorescence versus Cycle number. The de-fault data analysis options are used to generate the initial data graph (see the “DefiningDefault Data Analysis Options” section for information on setting default analysis options).

Data Analysis


To display the log of fluorescence vs. cycle number, select the Log option in the lower-leftcorner of the graph.

To clearly view regions of the graph, use the X and Y sliders to zoom along the x and yaxes of the graph. The scroll bar can be used to position the region of interest in thedisplay window. Alternatively, right click and drag the box that appears around the areaof the graph that you wish to magnify.

C(t) line



Moving the cursor over the data trace for a well will thicken the trace and display the C(t)value, along with the x and y coordinates corresponding to the current position of thecursor over the trace. Moving the cursor over a well in the Plate diagram or selecting awell in the Graphed Samples list will thicken the trace.

Adjusting Data Analysis Options

After viewing a graph of the fluorescence data versus cycle number, you may wish toadjust some of the data analysis options. You can adjust the following data analysis op-tions in the quantitation window without altering the default analysis options (see the“Defining Default Data Analysis Options” section near the end of this chapter for a de-scription of the analysis options and information on altering the default analysis options):

• Subtract Blanks

• Subtract Baseline

• Threshold Cycle (see “Adjusting the Cycle Threshold Line” immediately below)

Adjusting the Cycle Threshold Line

The C(t) (cycle threshold) line appears as a dotted horizontal line on the Data graph at aposition specified in the default data analysis options. To readjust the position of the C(t)line, select one of the Threshold options, or click and drag the C(t) line on the graph tothe desired position. The C(t) line is often positioned such that the C(t) line intersects thefluorescence traces, on a graph of baseline-subtracted data, at a point where the signalssurpass background noise and begin to increase. If no C(t) line appears on the Datagraph, select Manual from the Threshold options and enter a value for the C(t) line lessthan the maximum fluorescence value displayed on the y-axis of the graph. Then, clickand drag the C(t) line to the desired position.

Options for setting the C(t) line include:

• Manual: The C(t) line can be set manually by entering a threshold value for fluores-cence intensity between 0-10, or by dragging the C(t) line to the desired position onthe graph.

C(t) line

Data Analysis


• Standard Deviation over Cycle Range: The C(t) line can be automatically set toa multiple of standard deviations above the mean where the multiple and the cyclerange are specified by the user. Enter the desired cycle range in the Cycle Rangeboxes, and enter the multiple in the Standard Deviation over Cycle Range X box. TheCycle Range settings are applied to both the Threshold and Subtract Baseline func-tions.

It is often useful to display both the Data and the Standards graphs when establishing theposition of the C(t) line. The best option for setting the C(t) line can often be determinedby observing the effects of each option on the fit of the quantitation standards to a linearstandard curve. The effect of the position of the C(t) line on the standard curve can easilybe visualized by displaying both graphs and dragging the C(t) line up and down.

In establishing the position of the C(t) line, it may also be helpful to display the log offluorescence vs. cycle number by selecting the Log option in the lower-left corner of thegraph window. Often, the C(t) line can be set at a lower position upon examination ofthe log graph.

Standards Graph

Click the Standards tab to display a large Standards graph.



A standard curve is automatically generated using the information that was provided inthe Specify Quantitation Standards window during creation of the plate file. The Stan-dards Graph displays the base-10 logarithm of initial quantity (ng, moles, molecules,genome equivalents, copies, or user-defined units) versus the C(T) Cycle, the cycle num-ber at which the intensity trace intersects the C(t) line. The equation describing the linearstandard curve is displayed in the form y = mx + b where m = the slope of the line and b= the y-intercept. The R-square (R^2) value indicates how well the fit of the standard curvedescribes the variation in the data. The value of R-square can vary between 0 and 1,with values closer to 1 signifying a good fit. An R-square value of 0.999 indicates thatthe fit of the linear standard curve explains 99.9% of the variation in the data.

Select the Show selected wells option to apply the Graphed Samples to the standard curve.The samples will appear as gray dots while the standards appear as black dots.

If a Standards graph is not automatically displayed, check that the C(t) line has beenappropriately set on the Data graph, and that the quantitation standards have been de-fined in the Specify Quant Standards window (see the “Changing the Values of QuantitationStandards” section below).

Adjusting the Standard Curve

If desired, you can adjust the standard curve by deselecting outlying points. Moving thecursor over a data point will increase the size of the point and highlight the correspond-ing well in the plate diagram, Graphed Samples list, and Data graph. To exclude a pointfrom the standard curve, click on the point and it will turn red indicating that it is no longerbeing used in the calculation of the curve. The standard curve will be automatically re-plotted to exclude the deselected point.

If multiple sets of standards have been included in a single run, points may be excludedsuch that the standard curves of interest are serially displayed. Recall that only the black(selected) standards are used in quantity calculations and only these standards will ap-pear when the graph is printed.

Changing the Values of Quantitation Standards

If, during creation of the plate file, a mistake was made in entering the values of quanti-tation standards, or quantitation standards were not specified, it is possible to change oradd quantitation standards during the data analysis phase. To access the Specify QuantStandards window, select Plate Setup from the View menu, and then click the SpecifyQuant Standards button. Enter the value and units of each standard and then click theOK button. Save the changes to the plate file, and then click the Quantitation button onthe toolbar to continue analyzing data with the modified standards.

Data Analysis


Quantity Calculations

Click the Quantity Calculations tab to display the quantity calculations window.

The Quantity Calculations table lists:• Well coordinates

• The Type of well contents

• A descriptive well Label (if specified)

• The C(T) value, the cycle number at which the fluorescence intensity trace for a wellintersects the C(t) line on the Data graph.

• The initial quantity of template calculated to be present in the reaction. This can beexpressed in several different units including ng, ge (genome equivalents), moles,molecules, copies, or user-defined units.

To view the quantity calculations for only those wells selected in the plate diagram, choosethe Selected Wells option in the Data Source box. To view the quantity calculations for allnon-empty wells, choose the All Wells option in the Data Source box.

To display the quantity calculations in order by rows (A1-A12, B1-B12, etc.), choose theRows option in the Order by box. To display the quantity calculations in order by col-umns (A1, B1, C1, D1, etc.), choose the Columns option in the Order by box.



Printing and Exporting Quantitation Data

For information on printing and exporting quantitation data, refer to the “Printing andExporting Data” section near the end of this chapter.

Saving the Quantitation Analysis

See the “Saving a Data File” section below for information on saving a data file with theapplied analysis options.

Melting CurvePerforming a melting curve analysis following amplification can aid in product identifi-cation and determination of product homogeneity, often eliminating the need for time-consuming electrophoresis. If a chemistry’s fluorescence is dependent on annealing, adecrease in fluorescence is observed as melting progresses. Because the melting tem-perature of nucleic acids is affected by length, G+C content and the presence of basemismatches among other factors, products can often be distinguished by their meltingcharacteristics.

If a run has just completed, or a previously generated data file has been opened by se-lecting Open data file from the File menu, click the Melting Curve button on the toolbarto analyze melting curve data.

Data Analysis


Display

The Display screen is the default screen in the melting curve analysis window.

A graph of Fluorescence versus Temperature, -dI/dT versus Temperature, or both can bedisplayed for selected wells.

1. Use the Plate diagram to select the wells to be included in the graph. See the “SelectingWells Using the Plate Diagram” section in Chapter 6 for additional information.

Deselect all wells by clicking on any blank area between the wells. To deselect a well orsubset of wells, hold down the control key, and click on the well(s) you wish to deselect.The well(s) will no longer appear outlined in color, and the corresponding trace(s) will beremoved form the graph.

Selected wells appear outlined in color. The color outlining the well corresponds to thecolor of the well coordinates in the Graphed Samples list and to the color of the trace inthe melting curve graph.

Tm (-dI/dT max)

dI/dTFWHM

Intensity

-dI/dT



2. Use the Display panel to choose how the melting curve data is graphed.

• Select Intensity to graph Fluorescence intensity versus Temperature.

• Select -dI/dT to graph the negative first derivative of the fluorescence intensity versusTemperature. The dotted vertical line, drawn in the same color as the correspondingtrace, marks the maximum -dI/dT value, the temperature at which the rate of changein fluorescence is the greatest. This corresponds to the melting temperature (Tm) ofthe product. The dotted horizontal line indicates the sharpness of the -dI/dT curve asthe number of degrees Celsius over which the curve spans (i.e., the curve width) athalf of the maximum -dI/dT value calculated for the well.

• Select both Intensity and -dI/dT to simultaneously display the Intensity and the -dI/dTgraphs.

• Select Show Relative Intensities to display the relative intensities of the signals for theselected wells. Deselect this option to autoscale each trace on the graph.

3. After viewing a graph of the Fluorescence data versus Temperature, you may wish toadjust some of the data analysis options initially specified in the creation of the meltingcurve step or in the default analysis options. You can adjust the following data analysisoptions in the melting curve window without altering the default analysis options (see the“Defining Default Data Analysis Options” section for a description of the analysis optionsand information on altering the default analysis options):

• Subtract Blanks

• Subtract Baseline

Data Analysis


Note: If Minimum over Temp. Range or Average over Temp. Range is selectedas the Subtract Baseline option, and the Temp. Range values are altered, selectthe reload button to redraw the melting curve graph applying the new baselinetemperatures.

4. The Temperature Cursor controls can be used to highlight the results associated witha particular temperature by drawing a dotted vertical line on the graph at the designatedtemperature. Either use the scroll bar to scroll to the desired temperature, or enter thedesired temperature in the box located to the right of the scroll bar.

5. The Peak Location Boundaries box allows you to limit the area in which -dI/dTpeaks are found and used to calculate melting temperatures. This can be particularlyuseful for excluding unwanted peaks, or determining the melting temperature, Tm (-dI/dTmaximum), for a second, smaller peak (e.g., genotyping heterozygotes). You can set leftand right peak location boundaries by entering the temperature or by using the arrows toscroll to the desired temperature. Alternatively, drag the peak location boundary guidesto the desired location on the graph.

6. The Point Smooth slider allows you to change the number of points that are includedin calculating the smoothing of the melting curve graph. This can be particularly usefulfor resolving peaks when many reads have been collected over small intervals in tem-perature resulting in a choppy graph. The default for well-resolved data is a setting of 3.

Without peak location boundaries

Note that the Tm (-dI/dT max)is now calculated forthe smaller peak

With peak location boundaries

reload



Calculations

Click on the Calculations tab at the bottom of the melting curve window to display themelting curve calculations screen.

The Calculations table lists:

• Well coordinates.

• The Type of well contents.

• A descriptive well Label, if entered during creation of the plate file.

• Tm (-dI/dT Max): The melting temperature (Tm), the temperature at which -dI/dT is atthe maximum value calculated for the well.

• dI/dT FWHM (Full Width Half Maximum): Describes the sharpness of the -dI/dTcurve as the number of degrees Celsius over which the curve spans, i.e., the curvewidth, at half of the maximum -dI/dT value calculated for the well.

To view the melting curve calculations for only those wells selected in the plate diagram,choose the Selected Wells option in the Data Source box. To view the calculations for allnon-empty wells, choose the All Wells option in the Data Source box.

Data Analysis


To display the calculations in order by rows (A1-A12, B1-B12, etc.), choose the Rowsoption in the Order by box. To display the calculations in order by columns (A1,B1, C1,D1, etc.), choose the Columns option in the Order by box.

Printing and Exporting Melting Curve Data

For information on printing and exporting melting curve data, refer to the “Printing andExporting Data” section below.

Saving the Melting Curve Analysis

See the “Saving a Data File” section below for information on saving a data file with theapplied analysis options.

Exporting and Printing Data

Exporting Data

For customized data analysis, Opticon Monitor™ software provides the option to writethe fluorescence data collected during the run, along with the protocol and run param-eters, to either an Excel or CSV (comma separated values) file. Select Export from eitherthe Quantitation or Melting Curve pull-down menu, and select Excel or CSV.

If the Excel option is chosen, the processed Data, either quantitation or melting curvedata, for the currently displayed step, along with the Analysis Options, Protocol and Platesummaries will be written to the Excel compatible file. The Excel export options can notbe customized. In the Export Options window, select OK and type an appropriate filename in the Windows browse screen that appears, if the default name, data filename_platereads is not acceptable. The Excel compatible file will be saved as an .xls filein the Opticon Monitor data folder unless an alternate file location is specified.

Note: Excel 2000 must be installed on the computer in order to use the Export Exceloption.

If the CSV option is chosen, the export options can be customized. Select Data to exportthe fluorescence values measured in either all steps of the protocol (All Run Data), or thevalues from the Currently Displayed Step Only. Then, specify if the Raw, Normalized, orProcessed fluorescence values should be exported. The Raw option will export the fluo-rescence values measured by the Opticon™ detector with no data processing. The Nor-malized option will export fluorescence values normalized to account for any variation insignal measurement between wells by applying a normalization constant to the data col-lected in each well. These constants are calculated by subtracting the signals obtainedwhen no plate is present in the instrument from signals obtained with a plate of uniformfluorescence. The Processed option exports fluorescence values from which blanks and/or a baseline value have been subtracted from the normalized data.



Select Analysis Options to include a summary of the analysis options applied to the dataincluding whether blanks were subtracted and the method of baseline subtraction, if any.Select Protocol to include a summary of the protocol used to generate the data. SelectPlate to include a summary of the plate file information including the well contents andany well descriptions.

After choosing the desired export options for the CSV file, click OK to display a Win-dows browse screen allowing you to enter a file name and specify a location in which tosave the file.

Copying Data to the Clipboard

From the quantitation window, you have the option to copy the Data Graph, StandardsGraph, and Quantity Calculations to the clipboard for pasting into word processing or spreadsheet programs. From the melting curve window, the Data Graph and Calculations can becopied to the clipboard. Select Copy to Clipboard from either the Quantitation or MeltingCurve pull-down menu, and then select the desired graph or calculations option.

Printing Data

To print quantitation or melting curve analysis graphs and calculations, select Print fromthe File menu while in the appropriate data analysis window. Select Print Preview fromthe File menu to view the data in the form in which it will be printed.

Data Analysis


Saving a Data FileTo save analyzed data, which could include the samples graphed, the analysis optionsettings, the position of the C(t) line, a standards graph including any deselected points,and/or a melting curve graph including peak location boundaries and display options,select Save data file or Save data file as from the File pull-down menu.



Defining Default Data Analysis OptionsTo define the default options for data analysis, select Options from the Tools pull-downmenu.

The Analysis Defaults are set separately for Quantitation and Melting Curve data. Dataanalysis options include:

• Subtract Blanks: If this option is selected, the fluorescence measured in all wellsdesignated as blanks (blue) will be averaged and subtracted, as background, fromthe fluorescence measured in all wells designated as samples (red) or quantitationstandards (green).

• Subtract Baseline: If this option is selected, the baseline signal, an absolute fluo-rescence value, will be subtracted from the fluorescence data collected in each well.This value is calculated based on the signals measured in each well and thus willvary from well to well.

There are three options for defining the baseline signal value for a well:

• Minimum over all Data: The baseline signal is defined as the weakest fluores-cence signal measured in the well. This value will be set to zero.

• Average over Cycle Range (Temp Range): The baseline signal value is defined asthe average of the measured fluorescence calculated from a specified range ofcycles (quantitation data) or temperatures (melting curve data).

• Minimum over Cycle Range (Temp Range): The baseline signal value is definedas the minimum fluorescence value measured in a specified range of cycles (quan-titation data) or temperatures (melting curve data).

Data Analysis


• Threshold Cycle (Quantitation only): This quantitation option positions theC(t) (cycle threshold) line for use in quantitation of starting copy number. The C(t)line is often positioned on a graph of baseline-subtracted data at a point wherethe signals surpass background noise and begin to increase (see the “Quantita-tion” theory on page 8-2).

There are two options for setting the cycle threshold line:

• Manual: The C(t) (cycle threshold) line can be set manually by entering athreshold value for fluorescence intensity between 0-10 on the y-axis.

• Standard Deviation over Cycle Range: The C(t) line can be automaticallyset to a multiple of standard deviations above the mean where the mul-tiple and the cycle range are specified by the user. Enter the desired cyclerange in the Cycle Range boxes, and enter the multiple in the StandardDeviation over Cycle Range X box. The Cycle Range settings are appliedto both the Threshold and Subtract Baseline functions.

Setting the C(t) position in the default options prior to a run is particularly usefulwhen using an established set of reaction conditions. When using new chemis-tries or changing reaction conditions, it is often desirable to reposition the C(t)line in the Quantitation window by clicking and dragging the C(t) line on theData and Standards graph or by using the Threshold options in the quantitationwindow (see the “Adjusting the Cycle Threshold Line” section in this chapter).

Note: Refer to the “Running a Protocol” section in Chapter 7 for information onsetting the Collection Variables.

After you have completed defining the Analysis Defaults, select OK to save yourchanges and return to the previously displayed setup or analysis screen.

9-1

9. Maintenance

Cleaning the DNA Engine Opticon, 9-2Cleaning the Chassis and Block, 9-2Cleaning the Air Vents, 9-2Cleaning the Optics, 9-2

Changing the Fuses, 9-3



Cleaning the DNA Engine Opticon® Unit

Cleaning the Chassis and Block

Clean the outside of the DNA Engine Opticon unit with a damp, soft cloth or tissue when-ever something has been spilled on it, or when the chassis is dusty. A mild soap solutionmay be used if needed.

Clean block wells with swabs moistened with water, 95% ethanol, or a 1:100 dilution ofbleach in water. If using bleach, swab wells with water afterward to remove all traces ofbleach. Clean spilled liquids out of the block as soon as possible; dried fluids can be diffi-cult to remove. Do not clean the block with caustic or strongly alkaline solutions (e.g., strongsoaps, ammonia, or bleach at a higher concentration than specified above). These willdamage the block’s protective anodized coating, and possibly lead to electrical shorting.

Caution: Do not pour any cleaning solution into the block’s wells and thenheat the block, in an attempt to clean it. Severe damage to theblock, the heated lid, and the chassis can result.

Cleaning the Air Vents

With the Opticon™ unit turned off, clean the air intake and exhaust vents with a soft-bristlebrush, a damp cloth, or a vacuum cleaner whenever dust is visible (see figure 2-1). Ifthese vents become clogged with dust and debris, airflow to the heat sink is hampered,causing performance problems related to overheating.

Tip: To prevent problems with overheating, check regularly for dust buildup.

Cleaning the Optics

The optical components of the detector should not be cleaned by the user. Disassembly ofthe optical tower will void your warranty.

Should you suspect difficulty with the optics, please contact the customer service staff ofMJ Research, Inc. or one of its distributors.

Maintenance


Changing the FusesThe circuits in the DNA Engine Opticon unit are protected by four fuses, two external,user-changeable 6.3A, 250V Type S505, fast acting, 5 x 20mm fuses and two user-inac-cessible 8A fuses. When a fuse blows, an error message will appear in the OpticonMonitor™ software indicating that it can not communicate with the instrument.

Warning: The DNA Engine Opticon unit incorporates neutral fusing, whichmeans that live power may still be available inside the unit evenwhen a fuse has blown or been removed. Never disassemble thethermal cycler base. You could receive a serious electrical shock.Disassembling the base will also void your warranty.

To change the 6.3A fuses:

1. Disconnect the power cord from the back of the instrument. Move the power switch tothe “0” (off) position.

2. Turn the computer off, and disconnect the serial and DAQ cables from the Opticonunit. The Opticon unit also draws power from the computer.

3. Insert one corner of a small flat-head screwdriver just under the fuse plug, locatedimmediately below the power switch and just above the power cord jack, and gentlypry the plug loose. Pull the plug straight out as far as it will go, then push it down-ward to expose the 6.3A fuses.

4. Remove both fuses and replace with new ones (it is impossible to visually determinewhich fuse is blown). You may also test the fuses with an ohmmeter to determinewhich is defective and replace just that one.

5. Gently press the fuse cover back in place, and reconnect the power cord and thecomputer.

6. Once the fuse has been replaced and the power restored, the DNA Engine Opticonunit will resume the run, but will not communicate with the Opticon Monitor software.Restart the software to halt the run as no data will be collected.

10-1

10. Troubleshooting

Calibration, 10-2Testing Calibration, 10-2Recalibrating, 10-2

Software Error Messages, 10-3



CalibrationThe Opticon™ detector is calibrated at the factory and this calibration will accommodatemost applications. If you wish to reload the factory default calibration after manuallyrecalibrating the instrument, select Restore Factory Calibration from the Tools menu.

Testing Calibration

An accurately calibrated instrument will detect the same fluorescence intensity in everywell when reading samples of uniform composition.

To determine if the Opticon detector is accurately calibrated:

1. Prepare a test plate by accurately pipetting 50µl of a carefully-prepared 500nMfluorescein solution into each of the 96 wells of a microplate.

2. Program the following protocol:

• Temperature step of 30°C for 30 seconds

• Plate Read step

• Goto line 1 for a total of 2 more times

• End.

3. Analyze the fluorescence data for all 96 wells using the Optical Read Status screen.If the instrument is accurately calibrated, the fluorescence data should appear asrelatively straight lines that are tightly clustered—within the accuracy of pipetting.Several test plates should be measured to determine the error resulting from pipetting.

4. If the fluorescence data do not appear as tightly-clustered straight lines, follow theinstructions below for recalibrating the instrument.

Recalibrating

1. Select Calibrate Instrument from the Tools menu.

2. Remove the plate from the cycler block, if present, when instructed by the software todo so.

3. Insert a calibration plate when instructed. (Prepare a calibration plate by accuratelypipetting 50µl of a carefully-prepared 500nM fluorescein solution into each of the96 wells of a microplate.)

4. Rotate the plate 180° when instructed.

The instrument is now calibrated.

Troubleshooting


Software Error Messages

The following tables list software error messages along with their probable causes andsuggested resolutions. For help resolving software problems or for additional informa-tion, contact MJ Research technical support at 888-652-9253 (in the US or Canada) orcontact your local distributor (outside the US or Canada).

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Troubleshooting



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Tech Support: (888) 652-9253 • Sales: (888) 735-8437 • [email protected] • www.mjr.com A-1

The functional heart of every DNA Engine® thermal cycler is a high-performance Peltier-effect heatpump (also known as a “thermoelectric module”). The “MJ” module version is a solid-state devicemanufactured to withstand the thermal stresses associated with rapidly cycling temperatures.

A thermoelectric module consists of numerous pairs ofcrystalline semiconductor blocks precisely sandwichedbetween two layers of ceramic substrate (fig. A-1). Theblocks are of two varieties: “N-type,” which has a sur-plus of electrons in its crystalline structure, and “P-type,”which has a deficit of electrons. The two types are posi-tioned in alternating pairs within the innermost layer ofthe sandwich.

The two types of blocks are wired together in alternat-ing pairs. When electrical current is passed through theblocks, electrons in the N-type blocks and the “holes,”or empty electron spaces, in the P-type blocks are excitedat one conductor-semiconductor interface, which absorbsa small amount of heat. The electrons and holes flowthrough the crystalline blocks and return to a low-energystate at the other conductor-semiconductor interface, with the release of the previously absorbedheat. A thermal gradient of up to 70°C can be generated across the blocks in this manner.

The direction of heat pumping is reversed by reversing the polarity of current flow through thethermoelectric module, and the amount of heat pumped is changed by changing the amount ofcurrent passed. Both direction and amount of current flow are dictated by a microprocessor, al-lowing precise control of thermal cycling in the Alpha™ unit block.

How a Peltier Heat Pump Works

Appendix A

Electron

HoleN-type bismuth telluride

N-type bismuth telluride

Metal conductor

P-typebismuthtelluride

Ceramic substrate

Power input

Power input

Figure A-1 A thermoelectric module.

Tech Support: (888) 652-9253 • Sales: (888) 735-8437 • [email protected] • www.mjr.com B-1

Almost all solid-state electronic devices, including the DNA Engine® thermoelectric modules, re-quire direct current (DC) for operation. However, electric utilities supply low-Hertz alternatingcurrent (AC), which varies in voltage and frequency from nation to nation. The DNA EngineOpticon™ system uses switching transistors, combined with high-frequency, resonant transform-ers, to convert the incoming AC to DC.

The power supply first chops the AC power into small bursts of energy (over 100,000 per sec-ond) with the aid of high-current switching transistors called MOSFETs (metal-oxide semiconduc-tor field-effect transistors). The energy bursts are channeled into a high-frequency transformer. Bychanging the duration of the bursts that charge the transformer’s magnetic core (pulse-widthmodulation), a specific voltage output can be maintained even when the incoming voltage varies(between 100 and 240 volts in the case of the DNA Engine Opticon unit). Because the incomingpower is being chopped so rapidly, the incoming frequency is unimportant; it can even be DC.Spikes and surges in the incoming power no longer pose a problem since they are choppednearly to oblivion. The addition of resonance to the transformer design gives it extraordinaryefficiency. These design innovations have made the DNA Engine Opticon unit’s power supplysmall in size, universal in input, and resistant to noise.

Appendix B

How a Switching Power Supply Works

Tech Support: (888) 652-9253 • Sales: (888) 735-8437 • [email protected] • www.mjr.com C-1

Shipping Instructions for US ResidentsUsers residing in the United States should follow these instructions for shipping a machine to MJResearch for factory repair or an upgrade. Users outside of the United States should send ma-chines to their distributor, in accordance with shipping instructions obtained from the distributor.

1. Call MJ Research (888-652-9253) to obtain a return materials authorization (RMA) num-ber. Machines returned without an RMA will be refused by the Receiving Department.

2. Thoroughly clean the machine, removing excess oil and radioactive and otherbiohazardous substances. To protect the health of our employees, MJ Research willnot repair or upgrade any machine that is excessively oily or that emits ionizingradiation upon arrival at our factory. PLEASE ELIMINATE ALL BIOHAZARDSAND RADIATION!

3. Pack the machine in its original packaging. If this has been misplaced or discarded, callMJ Research to request shipment of packaging materials. You can also request a loanermachine, which will be provided if available (a rental fee may apply). You can use theloaner’s packaging to return the machine needing repair.

4. Write the RMA number on the outside of the box.

5. Ship the machine (freight prepaid) to the following address. We recommend you pur-chase insurance from your shipper.

Ship to: Repair DepartmentMJ Research, Incorporated590 Lincoln StreetWaltham, MA 02451

Appendix C

Tech Support: (888) 652-9253 • Sales (888) 735-8437 • [email protected] • www.mjr.com D-1

Warranties

The DNA Engine Opticon® system (CFD 3200 & PTC-200) is warranted against defects in mate-rials and workmanship. For specific Warranty information, contact your local Bio-Rad office. Ifany defects should occur during the warranty period, Bio-Rad will replace the defective partswithout charge. However, the following defects are specifically excluded:

1. Defects caused by improper operation or by improper packaging of returned goods.

2. Repair or modifications done by anyone other than Bio-Rad Laboratories.

3. Use with tubes, plates, or sealing materials not specified by Bio-Rad Laboratories for use with the DNA Engine Opticon system.

4. Deliberate or accidental misuse.

5. Damage caused by disaster.

6. Damage due to use of improper solvent or sample.

The warranty does not apply to fuses.

For inquiry or request for repair service, contact Bio-Rad Laboratories after confirming the modeland serial number of your instrument.

For Technical Service call your local distributor or in the U.S. call 1-888-652-9253, or visit ourwebsite at www.mjr.com.

Appendix D

Tech Support: (888) 652-9253 • Sales: (888) 735-8437 • [email protected] • www.mjr.com E-1

END-USER AGREEMENT AND LICENSE

FOR

MJ RESEARCH OPTICON MONITORTM SOFTWARE

(ALL VERSIONS)

IMPORTANT! PLEASE READ THE FOLLOWING LICENSE AGREEMENT.

YOU HAVE PURCHASED THE MJ RESEARCH DNA ENGINE OPTICON®

CONTINUOUS FLUO-

RESCENCE DETECTION SYSTEM WHICH INCLUDES CERTAIN EQUIPMENT, DEVICES AND IN-

STRUMENTATION AS WELL AS A LICENSE TO COMPUTER SOFTWARE.

This End-User License Agreement (‘AGREEMENT’) is a legal agreement between you (either an individual or a

single legal entity) (hereinafter ‘LICENSEE’) and MJ Research, Incorporated (‘MJR’) for the MJ Research

DNA Engine Opticon Continuous Fluorescence Detection System (the ‘SYSTEM’) which includes certain equip-

ment, devices, or instrumentation, MJR proprietary computer software (‘SOFTWARE PRODUCT’), and may

include “online” or electronic documentation, associated media, and printed materials, including an operator’s

manual (‘MANUAL’). The SYSTEM also contains software owned and produced by third parties (‘THIRD

PARTY SOFTWARE’) which is licensed for use on the equipment, devices and instrumentation of the SYSTEM

only, and the terms of the SOFTWARE PRODUCT license below apply.

BY OPERATING THE SYSTEM OR INSTALLING, COPYING, OR OTHERWISE USING THE SOFT-

WARE PRODUCT, OR ANY UPDATE THERETO, YOU AGREE TO BE BOUND BY THE TERMS OF

THIS AGREEMENT AND ANY ACCOMPANYING AGREEMENTS. IF YOU DO NOT AGREE TO

THE TERMS OF THIS AGREEMENT OR ANY ACCOMPANYING AGREEMENTS, DO NOT OPER-

ATE THE SYSTEM OR INSTALL, COPY, OR OTHERWISE USE THE SOFTWARE PRODUCT. IF

YOU DO NOT AGREE TO THE TERMS OF THIS AGREEMENT, YOU MAY RETURN THE SYSTEM

AND SOFTWARE PRODUCT TO MJR OR THE AUTHORIZED SELLER FROM WHICH YOU PUR-

CHASED THE SYSTEM AND SOFTWARE PRODUCT, BEFORE OPERATING THE SYSTEM, IN-

STALLING, COPYING, OR OTHERWISE USING THE SOFTWARE PRODUCT, FOR A FULL RE-

FUND OF THE PURCHASE PRICE THEREOF. IN ADDITION, BY INSTALLING, COPYING, OR

OTHERWISE USING ANY UPDATES OR OTHER COMPONENTS OF THE SOFTWARE PRODUCT

THAT YOU RECEIVE SEPARATELY AS PART OF THE SOFTWARE PRODUCT, YOU AGREE TO BE

BOUND BY THIS AGREEMENT AND BY ANY ADDITIONAL LICENSING TERMS THAT ACCOM-

PANY SUCH UPDATES. IF YOU DO NOT AGREE TO THE ADDITIONAL LICENSE TERMS THAT

ACCOMPANY SUCH UPDATES, YOU MAY NOT INSTALL, COPY, OR OTHERWISE USE SUCH

UPDATES, BUT MAY RETURN SUCH UPDATES TO MJR FOR A FULL REFUND OF THE PURCHASE

PRICE THEREOF, IF ANY.

Appendix E

E-2 Tech Support: (888) 652-9253 • Sales: (888) 735-8437 • [email protected] • www.mjr.com


1. SCOPE OF AGREEMENT

1.1 No License to Trademarks. No license is granted hereunder in connection with any trademarks or service

marks of MJR or its suppliers.

1.2 This AGREEMENT does not effect any transfer of title in the SOFTWARE PRODUCT.

1.3 The equipment, devices, and instrumentation included in the SYSTEM are not specifically warranted under

this AGREEMENT and any warranties related to the equipment, devices, and instrumentation are provided

solely by the manufacturers of the equipment, devices, and instrumentation provided in the SYSTEM.

2. SOFTWARE PRODUCT LICENSE GRANT

2.1 MJR hereby grants LICENSEE, a limited, non-exclusive, license to:

(a) Use the SOFTWARE PRODUCT on a number of computer central processing units at any one

time, but expressly provided that LICENSEE may use THIRD PARTY SOFTWARE only with

the SYSTEM.

(b) Use the MANUAL and other documentation in support of LICENSEE’s use of the SOFTWARE

PRODUCT.

(c) Install the SOFTWARE PRODUCT into memory on any number of computers and make one (1)

copy of the SOFTWARE PRODUCT for backup purposes only, provided that such backup copy is

a complete copy containing all copyright and trademark notices and any other restrictive property

legends of MJR that appear on and in the SOFTWARE PRODUCT as originally provided to LIC-

ENSEE by MJR.

(d) Make a one-time permanent transfer of the license granted herein and copies of the SOFTWARE

PRODUCT and THIRD PARTY SOFTWARE directly to a third party end user in connection with

the sale of the SYSTEM to such third party end user provided that (i) the SOFTWARE PRODUCT

and THIRD PARTY SOFTWARE are transferred in their entirety to the third party, including all

component parts, along with all associated media and printed materials, including this AGREE-

MENT, as originally received by LICENSEE, (ii) LICENSEE does not retain any copies of the

SOFTWARE PRODUCT or THIRD PARTY SOFTWARE, or any portion thereof, after transfer

thereof to the third party, and (iii) the third party agrees in writing to comply with all of the terms and

conditions contained in this AGREEMENT, including the obligation not to further transfer this

AGREEMENT and SOFTWARE PRODUCT. Such transfer may not be by way of consignment

or any other indirect transfer.

2.2 Restrictions. LICENSEE agrees that LICENSEE shall not:

(a) Separate the components of the SOFTWARE PRODUCT for use by more than one user.

(b) Copy the SOFTWARE PRODUCT, THIRD PARTY SOFTWARE or the MANUAL except and

to the extent provided in Paragraph 2.1(c).

(c) Sublicense, distribute, disclose or transfer the SOFTWARE PRODUCT or THIRD PARTY SOFT-

WARE in whole or in part, to any third party, except to the extent as provided in Paragraph 2.1(d)

and 2.2(d).


End User Agreement and License

(d) Sublicense the SOFTWARE PRODUCT or the THIRD PARTY SOFTWARE, except in connec-

tion with the rental or leasing of the entire SYSTEM to a single end user in a bona fide commercial

rental or leasing transaction, and only if said end user agrees in writing to be bound by the terms of

this Agreement.

(e) De-compile, reverse-compile, reverse-engineer, disassemble, or modify the SOFTWARE PROD-

UCT or THIRD PARTY SOFTWARE, or any portion thereof, in any way.

(f) Use the SOFTWARE PRODUCT or THIRD PARTY SOFTWARE, or any portion thereof, for

development of any infringing or derivative works.

2.3 Notice. The SOFTWARE PRODUCT and THIRD PARTY SOFTWARE are protected by copyright laws

and international copyright treaties, as well as other intellectual property laws and treaties. The SOFT-

WARE PRODUCT and THIRD PARTY SOFTWARE are licensed to LICENSEE, not sold.

2.4 No License to Methods of Use. No license is granted hereunder to any process, method or use for which the

SOFTWARE PRODUCT may be used either by itself or in combination with an analytical instrument

except to the extent such a process is described in the MANUAL and MJR has the right to grant a license to

such a process hereunder.

3. SOFTWARE PRODUCT LIMITED WARRANTY

3.1 Limited Warranty. MJR warrants that the SOFTWARE PRODUCT will perform substantially as described

in the MANUAL, if used in the manner described therein, for a period of ninety (90) days from the date of

receipt. MJR also warrants that the media on which the software is distributed is free from defects in

materials and workmanship. To the extent allowed by applicable law, implied warranties on the SOFT-

WARE PRODUCT, if any, are limited to the same ninety (90)days.

3.2 Customer Remedies. MJR’s entire liability and your exclusive remedy shall be, at MJR’s sole option, either

(a) return of the purchase price paid by LICENSEE, if any, for the SOFTWARE PRODUCT that does not

meet MJR’s limited warranty or (b) repair of the SOFTWARE PRODUCT that does not meet MJR’s

limited warranty, or (c) replacement of the SOFTWARE PRODUCT that does not meet MJR’s limited

warranty, provided that under (a), (b), or (c) above the SOFTWARE PRODUCT that does not meet MJR’s

limited warranty is returned in its entirety to MJR or the authorized seller from which the SOFTWARE

PRODUCT was purchased, complete with a dated proof of payment, within 90 days of the date of delivery.

Any repaired or replaced SOFTWARE PRODUCT will be warranted as described in Paragraph 3.1 for the

remainder of the original warranty period or thirty (30) days, whichever is longer.

3.3 Warranty Void. The limited warranty of Paragraph 3.1 and remedies of Paragraph 3.2 are void if:

(a) failure of the SOFTWARE PRODUCT has resulted from accident, abuse, or misapplication of the

SYSTEM or SOFTWARE PRODUCT;

(b) the SOFTWARE PRODUCT is installed in any computer or is used with any operating system

other than the computer and operating system included in the SYSTEM;

(c) the SOFTWARE PRODUCT is used for the analysis of data generated by any analytical instrument

other than the MJR analytical instruments with which the SOFTWARE PRODUCT was designed

to be employed and which are specifically listed in the MANUAL;

E-4 Tech Support: (888) 652-9253 • Sales: (888) 735-8437 • [email protected] • www.mjr.com


(d) failure of the SOFTWARE PRODUCT has resulted from anyone other than MJR or its authorized

representative installing or running any software on the computer running the SOFTWARE PROD-

UCT, other than the SOFTWARE PRODUCT itself; or

(e) for failure to comply with any of the provisions of this License Agreement.

3.4 NO OTHER WARRANTIES. TO THE MAXIMUM EXTENT PERMITTED BY APPLICABLE LAW,

MJR DISCLAIMS ALL OTHER WARRANTIES AND CONDITIONS, EITHER EXPRESS OR IM-

PLIED, INCLUDING, BUT NOT LIMITED TO, IMPLIED WARRANTIES OR CONDITIONS OF

MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, TITLE AND NON-INFRINGE-

MENT, WITH REGARD TO THE SOFTWARE PRODUCT.

4.0 LIMITATION OF LIABILITY

4.1 LIMITATION OF LIABILITY. TO THE MAXIMUM EXTENT PERMITTED BY APPLICABLE

LAW, IN NO EVENT SHALL MJR OR ITS SUPPLIERS OR EMPLOYEES BE LIABLE FOR ANY

SPECIAL, INCIDENTAL, INDIRECT, OR CONSEQUENTIAL DAMAGES WHATSOEVER (IN-

CLUDING, WITHOUT LIMITATION, DAMAGES FOR LOSS OF BUSINESS PROFITS, BUSI-

NESS INTERRUPTION, LOSS OF BUSINESS INFORMATION, OR ANY OTHER PECUNIARY

LOSS) ARISING OUT OF THE USE OF OR INABILITY TO USE THE SYSTEM OR SOFTWARE

PRODUCT OR THE FAILURE TO PROVIDE SUPPORT SERVICES, EVEN IF MJR HAS BEEN

ADVISED OF THE POSSIBILITY OF SUCH DAMAGES. IN ANY CASE, MJR’s ENTIRE LI-

ABILITY UNDER ANY PROVISION OF THIS AGREEMENT SHALL BE LIMITED TO THE PUR-

CHASE PRICE PAID BY LICENSEE FOR THE SYSTEM AND SOFTWARE PRODUCT.

4.2 The SYSTEM, SOFTWARE PRODUCT and THIRD PARTY SOFTWARE are not intended for HIGH

RISK ACTIVITIES (as defined below). In particular, the SYSTEM, SOFTWARE PRODUCT and THIRD

PARTY SOFTWARE are not fault-tolerant and are not designed, manufactured or intended for use or resale

as on-line control equipment in hazardous environments requiring fail-safe performance, or any other appli-

cations or situations in which the failure of the SYSTEM or SOFTWARE PRODUCT could lead directly

to death, personal injury, or severe physical or environmental damage (“HIGH RISK ACTIVITIES”).

Accordingly, MJR specifically disclaims any express or implied warranty of fitness for HIGH RISK AC-

TIVITIES. LICENSEE agrees that MJR will not be liable for any claims or damages arising from the use

of the SYSTEM or SOFTWARE PRODUCT in such applications or situations.

5. INDEMNIFICATION

5.1 LICENSEE agrees to indemnify and hold harmless MJR (including its officers, directors, employees, and

agents) and suppliers from and against any claims or lawsuits, including attorney fees, that arise or result

from any negligent, unlawful, or unauthorized use, transfer, or distribution of the SOFTWARE PRODUCT

or THIRD PARTY SOFTWARE by LICENSEE.

6. TERM AND TERMINATION

6.1 Term. The term of this AGREEMENT and the license granted hereunder shall commence upon first use of

the SYSTEM or first copying of the SOFTWARE PRODUCT by LICENSEE and shall terminate upon

LICENSEE’s discontinued use of the SOFTWARE PRODUCT or the transfer of the SOFTWARE PROD-

UCT as provided in Paragraph 2.1(d), whichever occurs earlier; provided, however, that this AGREEMENT

and the license granted hereunder shall survive and continue as to the transferee.


End User Agreement and License

6.2 Termination. This AGREEMENT and the license granted hereunder may be terminated by MJR, without

prejudice to any other rights, by written notice to LICENSEE only in the event that LICENSEE is in breach

of any material provision of this AGREEMENT, which breach is not cured after reasonable notice. In the

event of termination by MJR, LICENSEE shall destroy all copies of the SOFTWARE PRODUCT in

LICENSEE’s possession, and all of its component parts. LICENSEE may terminate any license granted

hereunder by returning the SOFTWARE PRODUCT and any documentation, including the MANUAL, to

MJR.

7. GENERAL PROVISIONS

7.1 Entire Agreement. This AGREEMENT is the entire agreement and understanding of the parties hereto with

respect to the SOFTWARE PRODUCT, and supersedes all prior oral, written, or other representations and

agreements. This AGREEMENT may only be amended in writing by an authorized agent of MJR.

7.2 If any provision of this AGREEMENT is held invalid, the offending clause will be modified so as to be

enforceable and, as modified, shall be fully enforced, and the remainder of the AGREEMENT will continue

in full force and effect.

7.3 Intellectual Property Rights. All title and intellectual property rights, including copyrights, in and to the

SOFTWARE PRODUCT, the THIRD PARTY SOFTWARE, the accompanying printed materials, in-

cluding the MANUAL, and any copies of the SOFTWARE PRODUCT, are owned by MJR or its suppli-

ers. All rights not expressly granted are reserved by MJR.

7.4 U.S. Government Restricted Rights. The SOFTWARE PRODUCT and any documentation are provided

with RESTRICTED RIGHTS. Use, duplication, or disclosure by the Government is subject to restrictions as

set forth in subparagraph (c)(1)(ii) of the Rights in Technical Data and Computer Software clause at DFARS

252.227-7013 or subparagraphs (c)(1) and (2) of the Commercial Computer Software- Restricted Rights

clause at 48 CFR 52.227-19, as applicable. Manufacturer is MJ Research, Incorporated, 590 Lincoln Street,

Waltham, MA 02451.

7.5 Export Restrictions. The SOFTWARE PRODUCT, the THIRD PARTY SOFTWARE, any part thereof,

and any accompanying documentation, including the MANUAL, shall not be exported or re-exported out-

side of the United States without MJR’s prior written permission, and, if MJR gives such permission, the

SOFTWARE PRODUCT, THIRD PARTY SOFTWARE, any part thereof, or accompanying documenta-

tion shall not be exported or re-exported to any country, person, entity or end user subject to U.S. export

restrictions.

7.6 Applicable Law. This AGREEMENT shall be governed by the laws of the Commonwealth of

Massachusetts.

7.7 Survival. Sections 2.2, 2.3, 2.4, 3, 4, 5, and 7 shall survive termination, cancellation or completion of thisAGREEMENT.

BY USING THE SYSTEM OR BY USING OR COPYING THE SOFTWARE PRODUCT YOU ACKNOWLEDGE

THAT YOU HAVE READ AND UNDERSTOOD THE FOREGOING AND AGREE TO BE BOUND THEREBY AS

LICENSEE OF THE SOFTWARE PRODUCT

Tech Support: (888) 652-9253 • Sales: (888) 735-8437 • [email protected] • www.mjr.com index-1

IndexAAbout window 5-6Adding users 6-3–6-4Air exhaust vents, location of 2-2Air intake vents, location of 2-2Air supply

ensuring adequate 3-4ensuring cool enough 3-4requirements 3-4troubleshooting problems with 3-5

Amplifluor system 4-4Analysis options 8-18

subtract baseline signal 8-18subtract blanks 8-18

Assigning well contentschanging contents 6-7content options 6-6selecting wells 6-5

Assigning well descriptions 6-8

BBeep when completed 6-15Blank wells 6-6Block control 6-11Blue protocol-indicator light, location of 2-2Blue trigger handle, location of 2-2

CC(t) cycle 8-8C(t) line. See Cycle threshold lineCalculated control 6-10Calculations

melting curve 8-14quantitation 8-9

during the run 7-6Calibration

recalibrating 10-2testing calibration 10-2

Capsoptical 4-6

Chemistries 4-2–4-4Chill-out 14 liquid wax 4-6

index-2 Tech Support: (888) 652-9253 • Sales: (888) 735-8437 • [email protected] • www.mjr.com


Cleaningair vents 9-2chassis and block 9-2optical components 9-2

Collection variablesintegration time 7-2warm-up time 7-2

Condensation in tubes following holds 4-6Copying data to the clipboard 8-16CSV 8-15Cycle threshold line

adjusting 8-6–8-7options for adjusting

manually set 8-6standard deviation over cycle range 8-7, 8-19

Cycler drawer, location of 2-2Cycler drawer, opening & closing 3-6

DData analysis options

subtract baseline signal 8-18subtract blanks 8-18threshold 8-19

Data filesaving 8-17

Data graph 8-4–8-7Declarations of Conformity DoC-1–DoC-2Default data analysis options 8-18Deleting a protocol step 6-20Dyes

compatible w/ Opticon detector 4-2

EEditing a protocol step 6-20Electromagnetic interference vEmission range 4-2Empty wells 6-6Environmental requirements 3-3Error messages 10-3Excel files, writing 8-15–8-19Excitation range 4-2Exporting data 8-15–8-16

copying to clipboard 8-16CSV 8-15Excel 8-15

Extend time 6-15


Index

FFCC warning vFile extensions 5-6First derivative 8-12Fluorescence detection range 2-3Fluorescence excitation range 2-3Fluorescent dyes

compatible w/ Opticon detector 4-2FRET 4-3Fuses

changing 9-3

GGoto step 6-18. See also ProgrammingGradient calculator 6-17Gradient step 6-15–6-17. See also Programming

adding multiple 6-17Gradient, temperature range 2-4

HHeated lid 4-6. See also Sample vessels: sealingHydrolysis probes 4-3

IIncrement temperature 6-15Inserting a protocol step 6-20Integration time 7-2

LLaunching

Opticon Monitor software 5-3Layout

back view 2-2front view 2-2

Lid temperature control 6-11–6-12constant 6-11off 6-12tracking 6-11

Loading sample vessels 3-6Log box 5-5Log option

data graph 8-5Logs

message 5-6usage 5-6



MManual ramp rate 6-14Master file

assigning existing plate or protocol files 6-23assigning new files to 6-4–6-23component files 6-3–6-4creating 6-2–6-4reusing 6-24saving 6-23

Melting curve 8-10–8-19calculations 8-14–8-19display 8-11–8-13first derivative 8-12fluorescence intensity 8-12peak locations boundaries 8-13point smooth 8-13relative intensities 8-12reload 8-13temperature cursor 8-13theory 8-10

Melting curve step 6-19–6-20, 6-21–6-22. See also Programmingindependent of cycling 6-21

Message log 5-6Microplates

recommended for optical assays 4-7required clearance 4-5

Molecular beacons 4-3

NNavigating

Opticon Monitor software 5-3–5-5

OOpening/closing cycler drawer 3-6Optical read status 7-3Opticon Monitor software

exiting 5-5file extensions 5-6how it works 5-2launching 5-3navigating 5-3version documented 5-2version number 5-6

Optionsadjusting during analysis 8-6


Index

PPacking checklist 3-2Password protection 6-4Peak location boundaries 8-13Peltier effect A-1Plate diagram

viewing fluorescence intensity 8-3Plate file

assigning well descriptions 6-8creating 6-4reaction volume 6-4saving 6-8specify quantitation standards 6-7

Plate read step 6-17. See also Programmingadding multiple 6-17

PortsDAQ cable port, location of 2-2serial cable port, location of 2-2

Power cord jack, location of 2-2Power supply requirements 3-4Power switch, location of 2-2Prepare new run 6-24, 7-2Printing

analysis graphs and calculations 8-16Programming

deleting a step 6-20editing a step 6-20entering a protocol 6-12–6-22

goto step 6-18gradient step 6-15–6-17inserting a step 6-20melting curve step 6-21–6-25plate read step 6-17–6-25temperature step 6-13–6-15

Protocol filecreating 6-9–6-22saving 6-23–6-25

QQuantitation

calculations 8-9–8-19data graph 8-4–8-19standards graph 8-7–8-19theory 8-2–8-3

Quantitation during the run 7-6



Quantitation optionssubtract baseline 8-18subtract blanks 8-18threshold 8-19

Quantitation standard wells 6-6Quantitation standards

changing values of 8-8managing 6-7specifying 6-7

Quantity calculations 8-9Quenchers 4-3Quick load 6-25

RR-square value 8-8Ramp rate, slower than maximum 6-14Reaction volume 4-8, 6-4Reload, melting curve 8-13Repeat this run 6-24, 7-2Run

initiating 7-2monitoring the status of 7-2–7-6skipping steps 7-2stopping 7-2

Run status 5-5, 7-2–7-6

SSafety

explanation of symbols ivgeneral instructions 1-3guideline for safe use vwarnings iv

Sample vesselsoptimized for fluorescence detection 4-7

clear 4-5opaque white 4-5

required clearance 4-5sealing 4-5

Chill-out wax 4-6optical caps 4-6

selecting 4-5, 4-7Sample wells 6-6


Index

Save to personal folder 6-8Save to Shared folder 6-8Saving

data file 8-17master file 6-23plate file 6-8protocol file 6-23temperature and lid control settings 6-12

Scorpions probes 4-4Selecting wells 6-5Setting up, Opticon system 3-3Setup/Analysis display window 5-5Shared folder 6-3Shipping instructions C-1Skip 7-2Slope 8-8Specifications

computer 2-4detection range 4-2excitation range 4-2gradient 2-4Instrument 2-3

Specify other save location 6-8Standard curve 8-7–8-8Standard deviation over cycle range 8-7Standards

specifying 6-7Standards graph 8-7–8-19

applying samples 8-8deselecting points 8-8R-square value 8-8slope 8-8

Status 5-5, 7-2–7-6optical read status 7-3–7-6thermal cycler status 7-3

Stop 7-2Subtract baseline signal 8-18Subtract blanks 8-18Switching power supply B-1SYBR Green I 4-2

recommended concentration 4-2

TTaqMan probes 4-3–4-4Temperature control mode 6-10–6-11

block control 6-11calculated control 6-10



Temperature cursor 8-13Temperature step 6-13–6-15. See also Programming

adding multiple 6-17options 6-14–6-15

beep when completed 6-15extend time 6-15increment temperature 6-15manual ramp rate 6-14

Thermal cycler status 7-3Thermoelectric unit A-1Threshold cycle 8-8Threshold options 8-19Toolbar 5-4–5-5

functionality 5-4status information 5-5

Tubes, 0.2ml low-profile 4-5Turning computer on/off 3-5Turning Opticon system on/off 3-5

UUnpacking instructions 3-2Usage log 5-6Users

adding new 6-3–6-4password protection 6-4removing 6-4specifying 6-3–6-4

Using oil 3-6

WWarm-up time 7-2Warranties D-1Well descriptions 6-8

Declarations of Conformity DoC-1

Declaration of Conformity

Bio-Rad Laboratories, Inc., 1000 Alfred Nobel Drive, Hercules, California,94547, U.S.A., declares that the product

PTC-200, The DNA Engine® Thermal Cycler

to which this declaration relates, is in conformity to the following standards ornormative documents.

IEC61010-1EN61326: CLASS A

following the provisions of the 73/23/EEC, 89/336/EEC & 93/68/EEC Directive.

Test Data to verify this conformity are available for inspection at our EuropeanTTRepresentative Office at Literbuen 10B, 2740 Skovlunde, Denmark.ff

13 September, 2004

date of issue

Brad Crutchfield

Vice President

11142 rev A.A

Declarations of Conformity DoC-2

Declaration of Conformity

Bio-Rad Laboratories, Inc., 1000 Alfred Nobel Drive, Hercules, California,94547, U.S.A., declares that the product

CFD-3200 & CFD-3220, The DNA Engine Opticon® System and The DNAEngine Opticon 2 System

to which this declaration relates, is in conformity to the following standardsor normative documents.

IEC61010-1EN61326: CLASS A

following the provisions of the 73/23/EEC, 89/336/EEC & 93/68/EEC Directive.

Test Data to verify this conformity are available for inspection at our EuropeanTTRepresentative Office at Literbuen 10B, 2740 Skovlunde, Denmark.ff

13 September, 2004

date of issue

Brad Crutchfield

Vice President

11148 rev A.A

Documents

DNA Engine Opticon System - Bio-Rad · The DNA Engine Opticon system is designed to operate safely under the following conditions: • Indoor use • Altitude up to 2000m • Ambient