BIO RAD Tetrad 2, PTC-0240 Operation Manual

DNA Engine Tetrad®2

Peltier Thermal Cycler

Operations Manual

Version 2.0

PTC-0240 DNA Engine Tetrad 2 Cycler

DNA Engine Tetrad®2
Peltier Thermal Cycler

PTC-0240 DNA Engine Tetrad 2 Cycler

Operations manual

Supports software version 4.0

ii

Copyright ©2005, 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.

Alpha, Chill-out, CleanBox, Concord, Disciple Desktop, DNA Engine,
DNA Engine Dyad, DNA Engine Tetrad, Dyad Disciple, Hard-Shell, Hot Bonnet,
iProof, iTaq, Microseal, Moto Alpha, Multiplate, Power Bonnet, Slide Chambers and
Tetrad are trademarks belonging to Bio-Rad Laboratories, Inc.

Teflon is a trademark of E.I. DuPont de Nemours and Company. Windows is a trademark
of Microsoft Corporation.

NOTICE TO PURCHASER

This base unit, Serial No. ____________, in combination with its immediately attached
Bio-Rad sample block module(s), constitutes a thermal cycler whose purchase con-
veys a limited non-transferable immunity from suit for the purchaser’s own internal
research and development and for use in applied fields other than Human In Vitro
Diagnostics under one or more of U.S. Patents Nos. 5,656,493, 5,333,675, 5,475,610
(claims 1, 44, 158, 160-163 and 167 only), and 6,703,236 (claims 1-7 only), or corre-
sponding claims in their non-U.S. counterparts, owned by Applera Corporation. No
right is conveyed expressly, by implication or by estoppel under any other patent
claim, such as claims to apparatus, reagents, kits, or methods such as 5’ nuclease
methods. Further information on purchasing licenses may be obtained by contacting
the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City,
California 94404, USA.

05184 rev E

iii

Table of Contents

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

Safety Warnings and Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .iv

Electromagnetic Interference and FCC Warning . . . . . . . . . . . . . . . . . . . . . . . . . . . .v

Documentation Conventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .vi

Chapter 1: Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-1

Chapter 2: Layout and Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-1

Chapter 3: Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-1

Chapter 4: Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-1

Chapter 5: Creating Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-1

Chapter 6: Managing and Editing Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-1

Chapter 7: Running Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7-1

Chapter 8: Using the Utilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8-1

Chapter 9: Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9-1

Chapter 10: Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10-1

Appendix A: Warranties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A-1

Appendix B: End-User and License Agreement . . . . . . . . . . . . . . . . . . . . . . . . . .B-1

Appendix C: Factory-Installed Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-1

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

Declaration of Conformity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .DoC-1

iv

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 and Guidelines

Warning: When removing an Alpha unit from a DNA Engine Tetrad 2 base, keep

all fingers and foreign objects away from the Alpha unit bays. Keep all
objects clear of the Alpha unit bays until the fan has come to rest.

Warning: Operating the DNA Engine Tetrad 2 cycler before reading this manual

can constitute a personal injury hazard. Only qualified laboratory per-
sonnel trained in the safe use of electrical equipment should operate
these machines.

Warning: Do not open or attempt to repair the DNA Engine Tetrad 2 cycler, any

Alpha unit, or any accessory to the cycler. Doing so will void your war-
ranties and can put you at risk for electrical shock. Return the
DNA Engine Tetrad 2 cycler to the factory (US customers) or an author-
ized distributor (all other customers) if repairs are needed.

Warning: All Alpha unit blocks can become hot enough during the course of

normal operation to cause burns or cause liquids to boil explosively.
Wear safety goggles or other eye protection at all times during operation.

Warning: The DNA Engine Tetrad 2 cycler incorporates neutral fusing, which

means that live power may still be available inside the machine even
when a fuse has blown or been removed. Never open the
DNA Engine Tetrad 2 base; you could receive a serious electrical shock.
Opening the base will also void your warranty.

Caution: Never remove an Alpha unit from the DNA Engine Tetrad 2 cycler with

the power turned on and a program running. Doing so can cause elec-
trical arcing that can melt the contacts in the connector joining the
Alpha unit to the cycler.

Caution: Do not attempt to unpack or move the DNA Engine Tetrad 2 cycler

alone. Doing so could results in personal injury. Always enlist the help of
another individual when moving or lifting the DNA Engine Tetrad 2 cycler.

Safe Use Guidelines

The DNA Engine Tetrad 2 cycler is designed to be safe to operate under the following
conditions:

• Indoor use

• Altitude up to 2000 m

• Ambient temperature 5–31°C

• Relative humidity 10–90%, 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 RADIOELEC-
TRIQUES DEPASSANT LES LIMITES APPLICABLES AUX APPAREILS NUMERIQUES
DE CLASS A PRESCRITES DANS LE REGLEMENT SUR LE BROUILLAGE RADIO-
ELECTRIQUE 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.

Shielded cables must be used with this unit to ensure compliance with the Class A
FCC limits.

Note Regarding FCC Compliance: Although this design of instrument has been tested and found to comply with Part
15, Subpart B of the FCC Rules for a Class A digital device, please note that this compliance is voluntary, for the
instrument qualifies as an "Exempted device" under 47 CFR § 15.103(c), in regard to the cited FCC regulations in
effect at the time of manufacture.

v

vi

Documentation Conventions

Before describing the various features of the DNA Engine Tetrad 2 cycler, let’s define
some “common ground” conventions.

• << >> will be used to indicate actual keys on the control panel, such as
<<ENTER>>, <<1>> and <<LEFT>>.

• < > will be used to indicate windowed menu items or buttons, such as <PRO-
GRAMS>, <RUN> and <UTILITIES>.

• Italics will be used to indicate windowed items that are not drop down menu
items or buttons, such as Calculated, Block, and Tracking. Typically, these will be
parameter selection items.

• Select is meant to be synonymous with click on, point-and-click, and any
phraseology implying selection of menu or option items with the mouse. In some
instances, selection is also possible by clicking the <<Enter>> button.

___________________________________________________________________________

This instrument system is labeled, “For Research Use Only.”

Introduction

1

Meet the DNA Engine Tetrad 2 Cycler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-2

Using This Manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-2

Important Safety Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-3

1-1

DNA Engine Tetrad 2 Thermal Cycler Operations Manual

Meet the DNA Engine Tetrad®2 Cycler

Thank you for purchasing an DNA Engine Tetrad 2 cycler. Designed by a team of
molecular biologists and engineers, the DNA Engine Tetrad 2 cycler delivers multi-
block thermal cycling with superior thermal performance. The programmable
DNA Engine Tetrad 2 cycler with its 4-bay chassis is ideal for running multiple proto-
cols and accommodating multiple users.

The DNA Engine Tetrad 2 cycler offers the following features:

• Interchangeable sample blocks—the Alpha™unit family—accommodate a
variety of tubes, microplates, and slides

• Hot Bonnet®heated lid for oil-free cycling or the Moto Alpha™unit for auto-
mated systems

• Intuitive DNA Engine Tetrad 2 system software with user-friendly interface for
programming, editing, file management and much more

• Choice of calculated temperature control for highest speed and accuracy, or
of block or temperature control for compatibility with protocols designed for
a variety of instrument types

• Instant Incubate feature for continuous-temperature incubations

Using This Manual

This manual contains instructions for operating your DNA Engine Tetrad 2 cycler
safely and productively:

• Chapter 2 acquaints you with the physical characteristics of the
DNA Engine Tetrad 2 cycler.

• Chapters 3–4 present the basics of installation and operation for the
DNA Engine Tetrad 2 cycler.

• Chapters 5, 6 and 7 describe the creation, editing, and running of programs.

• Chapter 8 outlines the software utilities.

• Chapter 9 explains the proper maintenance of the DNA Engine Tetrad 2
cycler.

• Chapter 10 offers troubleshooting information for the DNA Engine Tetrad 2
cycler.

1-2 Tech Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com

Important Safety Information

Safe operation of the DNA Engine Tetrad 2 cycler begins with a complete under-
standing of how the instrument works. Please read this entire manual before
attempting to operate the DNA Engine Tetrad cycler. Do not allow anyone who has
not read this manual to operate the instrument.

Warning: The DNA Engine Tetrad 2 cycler can generate enough heat to

inflict serious burns and can 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.

Warning: Do not block the DNA Engine Tetrad 2 cycler’s air vents (see

figs. 2-1 and 2-4 for locations). Obstructing air vents can lead
to overheating and slightly enhanced risk of electrical shock
and fire.

Introduction

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Layout and

2

DNA Engine Tetrad 2 Cycler Front View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-2

DNA Engine Tetrad 2 Cycler Control Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-2

DNA Engine Tetrad 2 Cycler Back View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-3

DNA Engine Tetrad 2 Cycler Bottom View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-3

Compatible Alpha Units . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-4

DNA Engine Tetrad 2 Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-5

Gradient Specifications (96-Well Alpha unit only) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-5

Specifications

2-1

DNA Engine Tetrad 2 Thermal Cycler Operations Manual

DNA Engine Tetrad®2 Cycler Front View

(Figure 2-1)

™

Alpha

NEXT

BACK

unit bay

Display screen

Numeric keypad

DNA Engine Tetrad 2 Cycler Control Panel

(Figure 2-2)

NEXT

BACK

Air intake vents

1/4 VGA display screen

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Cursor keys

Keypad

DNA Engine Tetrad 2 Cycler Back View

(Figure 2-3)

Power switch

Ethernet port

Layout and Specifications

RS-232 port

Fuses

DNA Engine Tetrad 2 Cycler Bottom View

(Figure 2-4)

Power cord jack

USB A port for mouse

USB B port for
remote command

Air exhaust vent

Air intake vents

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DNA Engine Tetrad 2 cycler rear

DNA Engine Tetrad 2 Thermal Cycler Operations Manual

Compatible Alpha Units

Single-Block Models

60 Single: Holds 60 x 0.5 ml tubes

96 Single: Holds 96 x 0.2 ml tubes or

one 96-well microplate

384 Single: Holds one 384-well

microplate

Flat Block: Holds customer-designed

adapter through four screw-
down points

Dual-Block Models

30/30 Dual: Holds 2 x 30 x 0.5 ml tubes

30/48 Dual: Holds 1 x 30 x 0.5 ml tubes

48/48 Dual: Holds 2 x 48 x 0.2 ml tubes

Slide Block

Slide Chambers
Alpha unit: Holds 2 x 16 standard slides

Moto Alpha Unit

Permits remote control of Alpha unit lid opening;
available in 96, 384, and flat block formats.

and 1 x 48 x 0.2 ml tubes

or half plates

™

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DNA Engine Tetrad 2 Specifications

Thermal range: 0–105°C, but no more than 30°C below ambient

temperature (10–105°C for the Slide Chambers unit)

Layout and Specifications

Temperature accuracy: +

Temperature uniformity: +

Speed of ramping: Up to 3°C/sec for all single- and dual-block Alpha units;

Sample capacity: Varies with installed Alpha unit

Line voltage: 200–240 VAC

Frequency: 50–60 Hz

Power: 1600 W maximum

Fuses: Two 6.3 A, 250 V, 5 x 20 mm

Displays: One 1/4 size VGA screen (320 x 240), 16 colors

Ports: One 9-pin RS-232 serial port

Program Capacity: 1,000 (typical)

Weight: 21.6 kg ( base only)

0.3°C of programmed target at 90°C, NIST-traceable

(+0.4°C for dual Alpha units)

0.4°C well-to-well within 30 seconds of arrival at

90°C (+0.5°C for dual Alpha units)

Up to 1.2°C/sec for the Slide Chambers Alpha unit

One ethernet port

Size: 47 x 61 x 16 cm (l x w x h, base only)

Projected Life Expectancy: 10 years of normal usage (2 protocol runs/day)

7 years of heavy usage (consistently exceeding
2 protocol runs/day)

Gradient Specifications (96-Well Alpha unit only)

Gradient accuracy: +

Column uniformity: +

Calculator accuracy: +

Lowest temperature 30°C
for gradient:

Highest temperature 105°C
for gradient:

Temperature differential 1–24°C
range:

0.3°C of programmed target at end columns,
30 seconds after the timer starts for the gradient step,
NIST–traceable

0.4°C, well–to–well within column, within 30 seconds
of reaching target temperature

0.4°C of actual well temperature, NIST-traceable

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Installation

3

Unpacking and Moving the DNA Engine Tetrad 2 Cycler . . . . . . . . . . . . . . . . . . . . . . .3-2

Packing Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-2

Setting Up the DNA Engine Tetrad 2 Cycler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-2

External Mouse Device . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-5

Requirements for Robotics Installations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-5

384-Well Microplate Specifics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-6

3-1

DNA Engine Tetrad 2 Thermal Cycler Operations Manual

Unpacking and Moving the DNA Engine Tetrad 2 Cycler

Please read the “Unpacking Instructions for the DNA Engine Tetrad®2 Thermal Cycler”
insert in order to properly and safely remove the instrument from its packaging. Always
enlist the help of another individual when moving or lifting the DNA Engine Tetrad 2 cycler.
In order to lift the instrument, grasp the DNA Engine Tetrad 2 cycler from underneath the
chassis (placing your hands on either side of the DNA Engine Tetrad 2 cycler in between
the feet of the instrument) and lift. Do not insert the Alpha™units until the instrument is
located in its final place.

Packing Checklist

After unpacking the DNA Engine Tetrad 2 cycler, check to see that you have received the
following:

• One DNA Engine Tetrad 2 base

• Four spare fuses

• One power cord

• One external mouse device

• DNA Engine Tetrad 2 Peltier Thermal Cycler Operations Manual (this
document)

• Unpacking Instructions for the DNA Engine Tetrad

If any of these components are missing or damaged, contact Bio-Rad Laboratories to
obtain a replacement. Please save the original packing materials in case you need to
return the DNA Engine Tetrad 2 cycler for service.

®

Setting Up the DNA Engine Tetrad 2 Cycler

The DNA Engine Tetrad 2 cycler requires only minimal assembly, plugging in the power
cord and mounting the Alpha units. Insert the power cord plug into its jack at the back of
the machine (see figure 2-3 for location of jack), then plug the cord into a 220 V electrical
outlet. With the machine switched off, mount the Alpha units (see the “Installing an Alpha unit”
section in Chapter 4).

Caution: Do not insert or remove an Alpha unit with the DNA Engine Tetrad 2
cycler turned on; electrical arcing can result. Read the safety warning at the
beginning of this manual on page iv regarding electrical safety when inserting
or removing an Alpha unit.

2 Thermal Cycler

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External Mouse Device

Included with each shipment of a DNA Engine Tetrad 2 thermal cycler is an externally
attachable mouse, which should be attached prior to power up of the
DNA Engine Tetrad 2 cycler. Usage of the mouse is required to access full function-
ality of the programming software. Underneath the front lip of the DNA Engine Tetrad
2 cycler are two connection ports (see figure 2-4). The mouse should be connected
to the USB A port.To ensure complete compliance with FCC and EMC requirements,
only a mouse with a ferrite core should be used with the DNA Engine Tetrad 2 instru-
ment.

To connect the mouse, please follow these steps:

1. Verify that the DNA Engine Tetrad 2 cycler is off. Wait for 10 seconds to ensure
that all fans have stopped rotating.

2. Grasping the sides of the DNA Engine Tetrad 2 cycler, tilt the instrument back so
that the underside of the lip is visible.

3. Insert the mouse into the USB A port and push the connector into place.

Installation

4. Tip the DNA Engine Tetrad 2 cycler back down and power up the system.

Environmental Requirements

Ensure that the area where the DNA Engine Tetrad 2 cycler is installed meets the
following conditions, for reasons of safety and performance:

• Nonexplosive environment

• Normal air pressure (altitude below 2000 m)

• Ambient temperature 5–31°C

• Relative humidity of 10–90% (noncondensing)

• 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 Tetrad 2 cycler near such heat sources as radiators, and protect it from
danger of having water or other fluids splashed on it, which can cause shorting of its
electrical circuits.)

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DNA Engine Tetrad 2 Thermal Cycler Operations Manual

Power Supply Requirements

The DNA Engine Tetrad 2 cycler requires 200–240 VAC, 50–60 Hz, and a grounded
outlet on a minimum 20 A line. The DNA Engine Tetrad 2 cycler can use voltage in
the specified range without adjustment, so there is no voltage-setting switch.

Note: Do not cut the supplied power cord and attach a different connector. Use a
one-piece molded connector. If required, additional dedicated power cords may be
purchased through Bio-Rad Laboratories.

Air Supply Requirements

The DNA Engine Tetrad 2 cycler requires a constant supply of air that is 31°C or
cooler in order to remove heat from the Alpha unit’s heat sink. Air is taken in from
vents at the bottom and sides of the machine and exhausted from vents on both
sides (see figures 2-1, 2-3, and 2-4). If the air supply is inadequate or too warm, the
machine can overheat, causing performance problems, software error messages
(particularly “HS Overheating” and “Slow Block Cycling”), and even automatic shut-
downs. Special attention should be paid to airflow and air temperature in robotics
installations of DNA Engine Tetrad 2 cyclers.

Ensuring an Adequate Air Supply

• Do not block the air-intake vents.

Position the DNA Engine Tetrad 2 cycler at least 10 cm from vertical surfaces and
other thermal cyclers (greater distances may be required; see below). Do not put
loose papers, bench paper, or this manual under the instrument; they can be
sucked into the air-intake vents on the bottom.

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

The bottom air vents are particularly liable to collect dust and debris, sometimes
completely clogging up. Check for dust and debris every few months, and clean
the intake vents as needed. Remove light collections of dust with a soft-bristle
brush or damp cloth. Severe collections of dust and debris should be vacuumed
out. Turn the instrument off prior to cleaning or vacuuming air vents.

• Use a solid, non-perforated support material when using the DNA Engine Tetrad 2
cycler on a wire rack.

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Installation

Cause Possible Remedies

Air circulation is poor. Provide more space around machine or adjust room

ventilation.

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

Machine is in warm part of room. Move machine away from, or protect machine from, such

heat sources as radiators, heaters, other equipment, or
bright sunlight.

Machines are crowded. Arrange machines so that warm exhaust air does not enter

intake vents.

Ensuring That Air Is Cool Enough

• Do not position two or more DNA Engine Tetrad 2 cyclers (or other thermal cyclers)
so that the hot exhaust air of one blows directly into the air-intake vents of another.

• Make sure the DNA Engine Tetrad 2 cycler receives air that is 31°C or cooler by measuring
the temperature of air entering the machine through its air-intake vents.

Place the DNA Engine Tetrad 2 cycler 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 Tetrad 2 cycler, then measure the air temperature at the back 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, use Table 3-1 to trou-
bleshoot the problem. Some experimentation may be required to determine the best
solution when more than one cause is involved. After taking steps to solve the
problem, verify that the temperature of the air entering the air-intake vents has been
lowered, using the procedure outlined above.

Requirements for Robotics Installations

Robotics installations require special attention to airflow and air temperature. Typically in
these installations, DNA Engine Tetrad 2 cyclers and other thermal cyclers are restricted to
a small area, along with other heat-generating equipment. Overheating can quickly occur
when many of these instruments are operating at once, unless preventive measures are
taken.

Follow the procedures described above to ensure adequate airflow and an air-intake
temperature of 31°C or cooler. Air-intake temperature must be verified by measurement.

Do not use oil or glycerin to thermally couple sample vessels to the blocks of machines in
a robotics installation. This can make plates difficult to remove.

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DNA Engine Tetrad 2 Thermal Cycler Operations Manual

384-Well Microplate Specifics

Some users find that a 384-well microplate can be difficult to remove from the 384-well
block after completing their thermal cycling protocol. The plate fits very snugly in the block,
and the 384 points of contact can provide a significant amount of friction. Fortunately, it is
relatively simple to ameliorate this problem if it occurs in your application.

In our experience, a very thin coating of a Teflon-based dry lubricant sprayed onto the
block will solve the sticking problem very effectively. The coating eventually wears off so
the block should be re-coated as needed, probably about once every 10 to 20 runs. Your
experience will be the best guide in establishing the frequency for re-coating. As you will
see, a very thin coat is sufficient to eliminate any sticking.

TFE (tetra-fluoroethylene) dry lubricant is available from many sources. One source in the
United States is:

Miller-Stephenson Chemical Co., Inc.
in Danbury, CT: 203-743-4447
in Morton Grove, IL: 847-966-2022
in Sylmar, CA: 818-896-4714

TFE Dry Lubricant/Release Agent
Cat.# MS-122DF (aerosol, 10 oz can) approx. $10.50/can

Here are some guidelines for applying the TFE lubricant:

1. Cool the block and lid to room temperature (below 38°C).

2. Cover the lid and any other areas that you don’t want to get slippery.

3. Shake the can well.

4. Spray for about 1 second onto the block.

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Operation

4

Turning the DNA Engine Tetrad 2 Cycler On . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-2

Using the Control Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-3

Display Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-3

Operation Keys . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-3

Block Status Lights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-4

Using the Data Ports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-4

Operating Alpha Units . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-4

Installing an Alpha Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-4

Removing an Alpha Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-5

Opening an Alpha Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6

Closing an Alpha Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6

Selecting the Correct Sample Vessel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6

0.5 ml Tubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-7

Thin-Walled vs. Thick-Walled Tubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-7

0.2 ml Tubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-7

Microplates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-7

Sealing Sample Vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-8

Sealing with Oil or Wax . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-8

Sealing with the Hot Bonnet Lid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-8

Adjusting the Hot Bonnet Lid’s Pressure . . . . . . . . . . . . . . . . . . . . . . . . . . 4-9

Loading Sample Vessels into the Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-10

Using Oil to Thermally Couple Sample Vessels to the Block . . . . . . . . . . . . . . . 4-10

Appendix 4-A: Tube, Microplate, and Sealing System Selection Chart . . . . . . . . . . 4-12

Appendix 4-B: Safety Warning Regarding Use of 35S Nucleotides . . . . . . . . . . . . . 4-13

4-1

DNA Engine Tetrad 2 Thermal Cycler Operations Manual

Turning the DNA Engine Tetrad®2 Cycler On

Caution: Do not insert or remove an Alpha™unit with the DNA Engine Tetrad 2

cycler turned on; electrical arcing can result. Read the safety warning
regarding electrical safety on page iv of this manual before inserting or
removing an Alpha unit or operating the DNA Engine Tetrad 2 cycler.

The Alpha units must be installed prior to powering up the DNA Engine Tetrad 2
cycler (see the "Operating Alpha Units" section below for installation instructions).
The power switch is located at the back of the instrument (see figure 2-3). Turn the
power switch on. The fan will turn on, the display screen will illuminate, and the
microprocessor will implement a boot-up protocol lasting about 1 minute, 30 sec-
onds. During the boot sequence, the user is presented with several options including:

1. Selftest — Choose number 1 on the keypad to instruct the DNA Engine Tetrad 2
cycler to perform a diagnostic system test and report any errors.

2. Send Files — Choose number 2 on the keypad to prepare the cycler to transfer
stored program files to another DNA Engine Tetrad 2 cycler (see Chapter 8 for
instructions on transferring program files).

3. Receive Files — Choose number 3 on the keypad to prepare the cycler to receive
stored program files from another DNA Engine Tetrad 2 cycler (see Chapter 8 for
instructions on transferring program files).

If no option is selected, the boot sequence will automatically exit after approximately
six seconds.

Following boot-up, the DNA Engine Dyad/Tetrad 2 logo screen is briefly displayed.
The DNA Engine Tetrad 2 cycler’s status window will then be visible. The cycler is
now ready to accept, edit, and execute programs.

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Using the Control Panel

The control panel (see figure 2-2) includes: a display screen, cursor keys, a numeric
keypad, and enter key.

Display Screen

• The display screen is a 1/4 size VGA screen for displaying thermal cycler condi-
tions and programs.

Operation Keys

• Navigation keys (left and right arrows, next, back) — Use to move around
within the display screen.

• Numeric keypad — Use to enter numeric values.

• Enter key — Use to accept specific programming additions and modifications.

Operation

Note: The external mouse is used to execute selected commands, access menus,
and to toggle through selection options in a list.

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DNA Engine Tetrad 2 Thermal Cycler Operations Manual

Block Status Lights

• When illuminated, these blue lights indicate whether the left and/or right Alpha
units are in use.

Using the Data Ports

The DNA Engine Tetrad 2 cycler has two data ports located at the rear of the machine —
an RS-232 port and an Ethernet port. See Chapter 8 for information on using these ports.

Operating Alpha Units

Note: Operation of the Slide Chambers™Alpha unit will not be discussed, owing to the

many differences between this type of Alpha unit and the others. Please see the
Slide Chambers Alpha Unit Operations Manual for operating instructions.

Note: Moto Alpha units are installed and removed as described below. See the Moto Alpha

Unit User’s Manual and "The Moto Alpha Unit" section in Chapter 5 for information on
opening and closing Moto Alpha units.

Installing an Alpha Unit

Caution: Do not insert or remove an Alpha unit with the DNA Engine Tetrad 2

cycler turned on; electrical arcing can result. Read the safety warning at
the beginning of this manual on page iv regarding electrical safety when
inserting or removing an Alpha unit.

1. Turn off the DNA Engine Tetrad 2 cycler (see the Caution above).

2. Hold the Alpha unit at its front and back edges.

3. Lower the Alpha unit into the DNA Engine Tetrad 2 base, leaving at least 3 cm
between the front edge of the Alpha unit and the center of the DNA Engine Tetrad
2 cycler base.

4. Raise the handle at the back of the Alpha unit, and slide the block toward the
center of the base as far as it will go (see figure 4-1A).

5. Push the handle down until it is completely vertical (see figure 4-1B); firm pres-
sure may be required, but do not force the handle into position. A definite click
signals that the Alpha unit’s connectors have mated with the DNA Engine Tetrad 2
cycler’s connectors.

™

When the handle is in the down position, the Alpha unit is locked into place.

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Operation

Removing an Alpha Unit

Caution: Do not insert or remove an Alpha unit with the DNA Engine Tetrad 2

cycler turned on; electrical arcing can result. Read the safety warning at
the beginning of this manual on page iv regarding electrical safety when
inserting or removing an Alpha unit.

1. Turn off the DNA Engine Tetrad 2 cycler (see the Caution above).

2. Pull upward on the handle. When the lock releases, you will hear a click, and the
Alpha unit will slide a little outward from the center of the cycler. The electrical
connectors of the Alpha unit and the DNA Engine Tetrad 2 cycler are now disen-
gaged, and there is little danger of electrical shock.

3. Slide the Alpha unit from the center of the DNA Engine Tetrad 2 base, about 3 cm.

4. Grasp the front and back edges of the Alpha unit, and lift it out of the machine.

Figure 4-1 Installing an Alpha unit (as shown on a DNA Engine Dyad®thermal cycler).

A

B

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DNA Engine Tetrad 2 Thermal Cycler Operations Manual

Opening an Alpha Unit

Grip the front edge of the top lever of the Hot Bonnet®lid as shown in figure 4-2A,
and pull upward firmly. The top lever will pop open to reveal the entire thumbwheel
(see figure 4-2B). Continue pulling upward to open the lid. The Hot Bonnet lid will tip
backward, revealing the entire block.

Caution: Do not pull on the thumbwheel to open the unit. This can damage the

Hot Bonnet lid’s mechanism.

Closing an Alpha Unit

Press down on the top lever. The lever will close over the thumbwheel as the lid
closes over the sample block. A click signifies that the Hot Bonnet lid’s latch has
engaged.

Selecting the Correct Sample Vessel

The DNA Engine Tetrad 2 cycler’s wide variety of interchangeable Alpha units affords great
scope in choosing sample vessels. Keep in mind that differences in tube and plate com-
position and wall thickness among the many brands available can affect reaction
performance. Protocols may require some adjustment to ensure optimum performance
when using a new vessel type. Bio-Rad offers a full range of tubes and microplates, man-
ufactured to the specifications of each type of Alpha unit to ensure a precise fit. See
Appendix 4-A of this chapter for a complete list.

Figure 4-2 Opening an Alpha unit (as shown on a DNA Engine®thermal cycler).

A

B

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Operation

0.5 ml Tubes

Thick-walled 0.5 ml tubes may not fit tightly in thermal cycler wells and typically provide
poor thermal transfer, since these tubes were originally designed for centrifuges. For
best results, we recommend using thin-walled 0.5 ml tubes specifically designed for
thermal cycling. The higher quality brands provide a good and consistent fit. Bio-Rad
thin-walled 0.5 ml tubes are designed for precise block fit and tight sealing of reactions
down to 10 µl.

Thin-Walled vs. Thick-Walled Tubes

The thickness of sample tubes directly affects the speed of sample heating and thus
the amount of time required for incubations. Thick-walled tubes delay sample
heating, since heat transfers more slowly through the tubes’ walls. For the earliest
types of thermal cyclers, this delay mattered little. These machines’ ramping rates
were so slow (below 1°C/sec) that there was plenty of time for heat to transfer
through the tube wall to the sample, during a given incubation.

Modern thermal cyclers have much faster ramping rates (up to 2–3°C/second), so the
faster heat transfer provided by thin-walled tubes allows protocols to be significantly
shortened.

0.2 ml Tubes

All types of thin-walled 0.2 ml tubes may be used. Bio-Rad offers high-quality 0.2 ml
tubes in a number of styles, including individual and strip tubes.

Microplates

A variety of polycarbonate or polypropylene microplates can be used in Alpha units
as long as they fit the wells snugly. Polypropylene microplates are usually preferred
because they exhibit very low protein binding and, unlike polycarbonate microplates,
do not lose water vapor through the vessel walls. This allows smaller sample volumes
to be used—as little as 5–10 µl.

Several varieties of microplates are available from Bio-Rad (see the "Tube, Microplate,
and Sealing Selection Chart"), including Hard-Shell®thin-wall microplates. Hard-
Shell microplates feature a skirt and deck molded from a rigid, thermostable polymer
that completely resists the warping and shrinkage experienced with traditional one-
component plates. The rigid skirt improves robotic handling such that stackers and
robotic arms can grip and move Hard-Shell plates securely and reliably. In a separate
step, thin-wall wells are molded of virgin polypropylene selected for low DNA binding
and optimized for thermal cycling.

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