MJ Research PTC-200 DNA Engine, CFD-3200 Opticon Operation Manual

DNA Engine Opticon® System

For Continuous Fluorescence Detection

PTC-200 DNA Engine® Cycler

™

CFD-3200 Opticon

Operations Manual

Supports Software Version 1.08

Detector

06678 revC.A

DNA Engine Opticon® System

For Continuous Fluorescence Detection

PTC-200 DNA Engine® Cycler
CFD-3200 Opticon

™

Detector

Operations Manual

Supports Software Version 1.08

ii Tech Support: [phone] • Sales: [phone] • [e-mail] • www.mjr.com

Copyright ©2004, Bio-Rad Laboratories, Incorporated. All rights reserved. Reproduction in any form, either print or
electronic, 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 trade­mark 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 system
includes an Authorized Thermal Cycler and may be used with PCR licenses available from Applied Biosystems. Its use
with Authorized Reagents also provides a limited PCR license in accordance with the label rights accompanying such
reagents .Some applications may also require licenses from other third parties.

This instrument includes an Authorized Thermal Cycler, Serial No __________________. Its purchase price includes the
up-front fee component of a license under United States Patent Nos. 4,683,195, 4,683,202 and 4,965,188, owned by
Roche Molecular Systems, Inc., and under corresponding claims in patents outside the United States, owned by F.
Hoffmann-LaRoche Ltd, covering the Polymerase Chain Reaction ("PCR") process, to practice the PCR process for internal
research and development using this instrument. The running royalty component of that license may be purchased from
Applied Biosystems or obtained by purchasing Authorized Reagents. This instrument is also an Authorized Thermal
Cycler for use with applications licenses available from Applied Biosystems. Its use with Authorized Reagents also
provides a limited PCR license in accordance with the label rights accompanying such reagents. Purchase of this
product does not itself convey to the purchaser a complete license or right to perform the PCR process. Further
information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licens­ing, 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.

06678 revCA

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Table of Contents

Explanation of Symbols .......................................................................................... iv

Safety Warnings .................................................................................................... iv

Safe Use Guidelines................................................................................................. v

Electromagnetic Interference .................................................................................... v

FCC Warning .......................................................................................................... v

1. Introduction ..................................................................................................... 1-1

2. Layout and Specifications ................................................................................. 2-1

3. Installation and Operation ................................................................................ 3-1

4. Compatible Chemistries, Sample Vessels, and Sealing Options ........................... 4-1

5. Introduction to Opticon Monitor™ Software ...................................................... 5-1

6. Experimental Setup and Programming .............................................................. 6-1

7. Run Initiation and Status ................................................................................... 7-1

8. Data Analysis................................................................................................... 8-1

9. Maintenance ....................................................................................................9-1

10. Troubleshooting ............................................................................................ 10-1

Appendix A .........................................................................................................A-1

Appendix B .......................................................................................................... B-1

Appendix C.......................................................................................................... C-1

Appendix D ......................................................................................................... D-1

Appendix E .......................................................................................................... E-1

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

Declarations of Conformity ...............................................................................

DoC

-1

iv Tech Support: [phone] • Sales: [phone] • [e-mail] • www.mjr.com

Explanation of Symbols

CAUTION: Risk of Danger! Wherever this symbol appears, always consult note

in this manual for further information before proceeding. This symbol identifies com­ponents that pose a risk of personal injury or damage to the instrument if improperly
handled.

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

CAUTION: Hot Surface! This symbol identifies components that pose a risk of per­sonal 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 can

constitute a personal injury hazard. Only qualified laboratory personnel trained
in 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 will

void your warranties and can put you at risk for electrical shock. Return the
DNA Engine Opticon system to the factory (US customers) or an authorized
distributor (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 goggles
or other eye protection at all times during operation.

Warning:Warning:

Warning:Warning:

Warning: The DNA Engine Opticon system incorporates neutral fusing, which means that

live power may still be available inside the machines even when a fuse has
blown or been removed. Never open the Opticon base; you could receive a
serious electrical shock. Opening the base will also void your warranties.

Tech Support: [phone] • Sales: [phone] • [e-mail] • www.mjr.com v

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 Interference

This device complies with Part 15 of the FCC Rules. Operation is subject to the following
two conditions: (1) this device may not cause harmful interference, and (2) this device
must accept any interference received, including interference that may cause undesired
operation.

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

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

LE PRESENT APPAREIL NUMERIQUE N'EMET PAS DE BRUITS RADIOELECTRIQUES
DEPASSANT LES LIMITES APPLICABLES AUX APPAREILS NUMERIQUES DE CLASS A
PRESCRITES DANS LE REGLEMENT SUR LE BROUILLAGE RADIOELECTRIQUE EDICTE PAR
LE MINISTERE DES COMMUNICATIONS DU CANADA.

FCC Warning

Warning: Changes or modifications to this unit not expressly approved by the party

responsible 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 A
digital device, pursuant to Part 15 of the FCC Rules. These limits are designed to provide
reasonable protection against harmful interference when the equipment is operated in a
commercial environment. This equipment generates, uses, and can radiate radiofrequency
energy and, if not installed and used in accordance with the instruction manual, may
cause harmful interference to radio communications. Operation of this equipment in a
residential area is likely to cause harmful interference in which case the user will be re­quired to correct the interference at his own expense.

1-1

1. Introduction

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

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Opticon System Operations Manual

Meet the DNA Engine Opticon® System

Thank you for purchasing a DNA Engine Opticon continuous fluorescence detection sys­tem from MJ Research, Incorporated. Designed by a team of molecular biologists and
engineers, the Opticon™ system will meet your needs for a sensitive, easy-to-use, and com­pact continuous fluorescence detection system. Some of the DNA Engine Opticon system’s
many 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 micro­plates 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, run

status, and data analysis.

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

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

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

Using This Manual

This manual contains instructions for operating your DNA Engine Opticon system safely
and 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 the
Opticon 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.

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Introduction

Important Safety Information

Safe operation of the DNA Engine Opticon system begins with a complete understand­ing of how the instrument works. Please read this entire manual before attempting to op­erate the DNA Engine Opticon system. Do not allow anyone who has not read this manual
to operate the instrument.

Warning: The DNA Engine Opticon system can generate enough heat to inflict

serious burns and could deliver strong electrical shocks if not used ac­cording to the instructions in this manual. Please read the safety warnings
and guidelines at the beginning of this manual on pages iv and v, and
exercise all precautions outlined in them.

2-1

2. Layout and Specifications

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

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Opticon System Operations Manual

Back View

(Figure 2-2)

Front View

(Figure 2-1)

Power cord jack
(some models)

Power switch
(fuses, some
models)

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 protocol­indicator light

Blue trigger handle
(door mechanism)

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Layout and Specifications

Specifications

Thermal 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 of

arrival at 90˚C

Ramping speed: Up to 3.0˚C/sec

Sample capacity: 96-well microplate (low-profile) or

96 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 and

monitor)

Size: 47cm deep x 34cm wide x 60cm high

(excluding computer and monitor)

Fluorescence Excitation Range: 450-495nm

Fluorescence Detection Range: 515-545nm

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Opticon System Operations Manual

Gradient Specifications

Accuracy: +0.3°C of target at end columns within

30 seconds (NIST-traceable)

Column uniformity:

+0.4°C, in column, well–to–well, within
30 seconds of target attainment

Calculator accuracy:

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

Lowest programmable 30°C
temperature:

Highest programmable 105°C
temperature:

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-6036E

200kS/s (samples per second)

3-1

3. Installation and Operation

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

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

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

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Opticon System Operations Manual

Unpacking the Opticon™ Unit

Please follow these instructions for unpacking the Opticon unit to reduce the risk of per­sonal 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 the
cycler and the optical tower. Carefully lift the instrument off of the shipping sup­port. Do not lift the instrument by the blue handle or the cycler drawer.

Packing Checklist

After unpacking the DNA Engine Opticon® continuous fluorescence detection system, check
to 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 (Opticon
Monitor and Windows XP pro)

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

computer 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 Operations
Manual

(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)

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Installation and Operation

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

Setting Up the DNA Engine Opticon System

The Opticon system requires a location with three power outlets to accommodate the
Opticon unit, the computer, and the monitor. A location with network access (Ethernet
10/100BaseT) is recommended if you wish to transfer setup and analysis files between
the computer running the Opticon unit and other computers.

The DNA Engine Opticon system requires only minimal assembly. Insert the power cord
plug into its jack at the back of the instrument, just below the power switch (see figure 2­2 for the location of the jack). Then, plug the power cord into a standard 110V or 220V
electrical 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 Opticon
unit is connected to the computer. There are two cables that connect the Opticon unit to
the computer. Connect the serial cable to the serial cable port on the Opticon unit (see
figure 2-2) and serial port #2 on the computer. Connect the data acquisition cable to the
DAQ 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 Requirements

For reasons of safety and performance, ensure that the area where the DNA Engine
Opticon 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 Engine
Opticon system near such heat sources as radiators, and protect it from danger of
having water or other fluids splashed on it, which can cause electrical short circuits.)

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Opticon System Operations Manual

Power Supply Requirements

The 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 220V
power without adjustment.

Important! For 220V operation of the computer, the red voltage-setting

switch located on the back of the computer, near the power cord
jack, must display 230V rather than 115V.

The Opticon unit is equipped with a power-entry module that accepts cordsets with an
IEC 60320-1 type C13 connector (this is the same standard configuration used by many
computer manufacturers for their equipment). All cordsets used with the Opticon unit must
be rated to carry at least 10A at 125V or 250V, the latter specification depending upon
the supply voltage used. Additionally, the cordset must meet all other applicable national
standards—thus at a minimum, the cordset should carry the mark of a nationally recog­nized 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 Requirements

The DNA Engine Opticon unit requires a constant supply of air that is 31˚C or cooler in
order to remove heat from the heat sink. Air is taken in from the lower vents located on
the sides of the instrument and exhausted from the upper vents on both sides (see figure
2-1). If the air supply is inadequate or too hot, the instrument can overheat, causing per­formance 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 thermal
cyclers 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 hot
exhaust air blows directly into the air intake vents.

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

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Installation and Operation

Place the DNA Engine Opticon unit where you plan to use it, and turn it on. Try to
reproduce what will be typical operating conditions for the machine in that location,
particularly any heat-producing factors (e.g., nearby equipment running, window blinds
open, lights on). Run a typical protocol for 30 minutes to warm up the DNA Engine
Opticon unit, then measure the air temperature at the air intake vents. If more than one
machine 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 for
possible remedies. After implementing possible remedies, verify that the temperature of
the 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 room

ventilation.

Ambient air temperature Adjust air conditioning to lower ambient air
is 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 not

enter intake vents.

Turning the Opticon Unit and Computer On and Off

Locate the power switch on the back, left-side of the Opticon unit (back, right-side on
some 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 requires
several minutes to warm up after the Opticon unit is powered up. To turn the Opticon unit
off, depress the “0” side of the power switch.

Be sure that the Opticon unit is connected to the computer and turned on prior to launch­ing the Opticon Monitor software. The blue protocol-indicator light on the front of the
Opticon 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. Select

Shutdown

from the

Start

menu to turn the computer off. Press the power button on the

front of the monitor once to turn it on, and press it again to turn the monitor off.

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Opticon System Operations Manual

Opening and Closing the Cycler Drawer

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

To return the Opticon unit to the closed position, slide the cycler drawer back into the
instrument and lower the blue handle to secure the cycler drawer. It is not necessary to
squeeze 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’s
control of the protocol.

Loading Sample Vessels into the Block

Important! Do not use full height 0.2ml tubes or full height unskirted

microplates. Refer to the “Selecting the Correct Sample Vessel” section

in Chapter 4 for tube and microplate recommendations.

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

• Ensure that the block is clean before loading samples (see Chapter 9 for cleaning

instructions).

• Firmly press strips of 0.2ml low-profile tubes, or a 96-well, low-profile microplate into

the 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 to

the block.

Tip: Spin down reactions in tubes or microplates prior to loading into the thermal-cycler

block. 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 Sealing
Options

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

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

Selecting the Correct Sample Vessel, 4-5

Vessels Optimized for Fluorescence Detection and Thermal Cycling, 4-5

Sealing Sample Vessels, 4-5

Sealing with Optical Caps and the Heated Lid, 4-6

Sealing with Chill-out™ 14 Liquid Wax, 4-6
Sample Vessel and Sealing Selection Chart for Optical Assays, 4-7
Reaction Volume Recommendations, 4-8

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Opticon System Operations Manual

Optical System

The Opticon™ detector uses an array of 96 blue LEDs to sequentially illuminate each of
the 96 wells in the cycler block. The LEDs efficiently excite fluorescent dyes with excita­tion spectra in the 450 to 495nm range. The Opticon detector is optimized to detect dyes
with 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 and
recalibrating.

Compatible Chemistries

The Opticon detector is compatible with popular dye chemistries including SYBR Green
I, molecular beacons, hydrolysis probes (TaqMan probes), Scorpions probes, and the
Amplifluor system. In addition to performing real-time quantification and DNA melting
profiles, the Opticon system is also useful as a temperature-controlled fluorimeter for a
number of applications including ligand binding and protein structure studies. If you have
questions 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 dsDNA
binding dye thought to bind in the minor groove. The fluorescence of SYBR Green I is
greatly enhanced upon binding dsDNA. This characteristic makes it ideal for detection
of amplification products. The maximum absorbance of SYBR Green I is ~497nm and
the emission maximum is ~520nm*.

SYBR Green I has several advantages for detection of nucleic acids in real time. Because
SYBR Green I binds to all dsDNA, it does not have to be customized for individual tem­plates thereby providing the advantages of quick protocol adaptation and relatively low
cost. Further, SYBR Green I is very sensitive because multiple dye molecules bind to a
single amplification product. However, because SYBR Green I binds to all dsDNA, false
positive signals from primer-dimers, secondary structure, or spurious priming can inter­fere with accurate quantification. Measuring fluorescence at elevated temperatures may
help reduce the detection of nonspecific products

1

. Performing a melting curve to ana­lyze product homogeneity can also aid in analyzing quantification results obtained with
SYBR Green I.

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

1

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

*Molecular Probes, Inc.

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Compatible Chemistries, Sample Vessels, and Sealing Options

Molecular Beacons

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

Molecular beacons are designed such that the loop, which is usually 15-30 nucleotides in
length, is complimentary to the target sequence. The arms flanking the loop, which are
usually 5–7 nucleotides in length, are designed such that they are complementary and
favor formation of a stem structure. A fluorophore is attached to the end of one arm, and a
quencher is attached to the other. Molecular beacons must be carefully designed such that
at the annealing temperature of the reaction hairpins form in the absence of template, but
that in the presence of template, the annealing of the loop sequence to the target is ener­getically 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 spe­cifically detect the target of interest. Great sensitivity, including detection of single
nucleotide polymorphisms (SNPs), is possible with carefully designed molecular beacons
and optimized reaction conditions (temperature, buffer). However, each probe must be
carefully and uniquely designed for the detection of a specific target.

Molecular beacons are a technology patented by the Public Health Research Institute of
New York, NY and are available from a number of licensed suppliers. When designing
molecular beacons for use with the Opticon detector, fluorophores with excitation and
emission spectra falling within the Opticon detector’s excitation (450-490nm) and detec­tion (515-545nm) ranges, such as FAM, can be used. Either dark quenchers or a quench­ing fluorophore may be used with the Opticon detector. However, because the Opticon
detector is a single-color detection system, light from a quenching fluorophore can not be
separately monitored. Dark quenchers tend to give cleaner signal because there is no
overlapping signal from light emitted by the quenching fluorophore.

Hydrolysis Probes (TaqMan Probes)

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

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Opticon System Operations Manual

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

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

Scorpions Probes

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

During amplification, extension of the target sequence proceeds from the primer portion
of the Scorpions probe. As the reaction cools following denaturation, a uni-molecular
rearrangement occurs such that the Scorpions probe sequence binds to the amplified tar­get sequence, separating the complementary stem sequences and thus the fluorophore
and quencher. Since the Scorpions probe is integrated into the product, there is a direct
relationship between the number of targets generated and the amount of fluorescence.

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

Amplifluor Universal Detection System

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

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

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Compatible Chemistries, Sample Vessels, and Sealing Options

Selecting the Correct Sample Vessel

Important! Do not use full height 0.2ml tubes or full height unskirted

microplates. Full height 0.2ml tubes and most unskirted microplates do

not provide sufficient clearance between the sample block and lid-heater
assembly. Do not force the cycler drawer closed.

For proper clearance in the Opticon unit, the distance from the bottom of a tube/plate to
the cap rim can not exceed 17.5mm. In general, fully-skirted 96-well microplates, such
as MJ Research Microseal

®

and Hard-Shell® microplates, provide sufficient clearance when
sealed with either domed or flat optical caps (see the "Sample Vessel and Sealing Selec­tion 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 recommended
for small numbers of samples. Full-height 0.2ml tubes do not provide sufficient clearance.

Vessels Optimized for Fluorescence Detection and Thermal
Cycling

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

Microplates and tubes with black wells may be useful in applications requiring very low
levels of background. However, signal strength is significantly reduced when plates and
tubes 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 on
performing a calibration test and recalibrating.

Sealing Sample Vessels

Steps must be taken to prevent the evaporation of water from reaction mixtures during ther­mal cycling so as to avoid changing the concentration of reactants. Only a layer of oil or
wax, such as Chill-out liquid wax, will completely prevent evaporation from sample ves­sels. However, an adequate degree of protection can be achieved by sealing with optical
caps, then cycling the samples using the heated lid to prevent condensation/refluxing.

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Opticon System Operations Manual

Sealing with Optical Caps and the Heated Lid

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

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

Note: When tubes are cooled to below-ambient temperatures, a ring of condensation may

form in tubes above the liquid level but below the top of the sample block. This is not a
cause for concern since it occurs only at the final cool-down step when thermal cycling is
finished.

Sealing with Chill-out™ Liquid Wax

Clear Chill-out liquid wax (available from MJ Research, Inc.) may be used to seal sample
vessels for optical assays. Clear Chill-out liquid wax is the same easy-to-use alternative to
oil as the standard, red-colored Chill-out wax. However, clear Chill-out wax provides ex­cellent light transmission for optimal performance in optical assays. Chill-out liquid wax
provides 100% prevention of condensation and vapor loss. At room temperature and
above, this overlay is transparent and can be applied by pipet. Chill-out liquid wax so­lidifies 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 calcu­lated-control protocol.) Be sure to use the same amount of wax in all samples vessels to
ensure a uniform thermal profile.

Tech Support: [phone] • Sales: [phone] • [e-mail] • www.mjr.com 4-7

Compatible Chemistries, Sample Vessels, and Sealing Options

Sample Vessel and Sealing Selection Chart for Optical Assays

The following sample vessels and sealing options are recommended for use with the DNA
Engine Opticon® system and are available from MJ Research, Inc. To place an order, call
888-729-2165 or fax [phone].

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golataChcraeseRJM

rebmuN

sthgilhgihtcudorP

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detriksnu

llew-69

setalporcim

)etihweuqapo(1569-LLM

)raelc(1069-LLM

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langis

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llew-69®laesorciM

s

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)etihweuqapo(1569-PSM

)raelc(1069-PSM

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gniweivelpmasdnalangis

llew-69®llehS-draH

setalporcimdetriks

sllewetihw/llehsetihw5569-PSH
sllewetih

w/llehskcalb5669-PSH

sllewetihw/llehsder5169-PSH

sllewetihw/llehswolley5269-PSH

sllewetihw/llehseulb5369

-PSH
sllewetihw/llehsneerg5469-PSH

sllewraelc/llehsetihw1069-PSH
sllewraelc/llehskcalb1669-PSH

sllewraelc

/llehsder1169-PSH

sllewraelc/llehswolley1269-PSH

sllewraelc/llehseulb1369-PSH

sllewraelc/llehsneerg1469-

PSH

selpmas69roflaedI•

talfyletulosbasniameretalP•

rof,gnilcyclamrehtgnirud

noitcellocthgilmrofinu

rofdoog

erasllehsderoloC•

gnidoc-roloc

snoitpOgnilaeS

.spactalflacitpO

8fospirts

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thgilhgihrofraelc-artlU•

noissimsnart

lµ5>semulovgnilcyclamrehT•

diuqiL

™tuO-llihC

edarg-lacitpo,xaW

1141-OHC

yalrevoliolarenimsecalpeR•

noissimsnartthgilhgiH•

lµ2>semulovgnilcyc

-lamrehT•

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Opticon System Operations Manual

Reaction Volume Recommendations

Reaction volumes of 20-100µl are recommended for most applications. However, it is
beneficial to empirically optimize reagent concentrations and sample volumes with the
Opticon detector as the sensitivity of the optical system often allows a cost-saving reduc­tion in reagent concentrations. Volumes as low as 10µl can be used, though sensitivity is
slightly reduced.

The maximum recommended sample volume is 100µl. Volumes exceeding 100µl do not
maintain adequate contact with the wells of the sample block resulting in nonuniform
heating and cooling within the sample.

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