BIO RAD CFX96, CFX384 Instruction Manual
CFX96™ and CFX384™ Real-Time
PCR Detection Systems
Instruction Manual
Catalog # 184-5384
# 185-5384
# 184-5096
# 185-5096
Copyright ©2008 Bio-Rad Laboratories, Inc. Reproduction in any form, either print or electronic, is
prohibited without written permission of Bio-Rad Laboratories, Inc.
Excel, Microsoft, PowerPoint, Windows, and Windows Vista are trademarks of Microsoft Corporation.
Adobe and Reader are trademarks of Adobe Systems Incorporated. Cy is a trademark of GE Healthcare
group companies. Fluor and Quasar are trademarks of Biosearch Technologies, Inc. SYBR and Texas
Red are trademarks of Invitrogen Corporation. Bio-Rad Laboratories, Inc. is licensed by Invitrogen
Corporation to sell reagents containing SYBR Green I for use in real-time PCR, for research purposes
only. Other brands or product names are trademarks of their respective holders.
LICENSE NOTICE TO PURCHASER
Bio-Rad Real-time thermal cycler CFX96 or CFX384 is licensed real-time thermal cycler(s) under Applera's United
States Patent No. 6,814,934 B1 for use in research and for all other fields except the fields of human diagnostics and
veterinary diagnostics.
This CFX96 or CFX384 detection module, when combined with a C1000 thermal cycler for which the applicable realtime thermal cycler royalty fee has been paid, constitutes a real-time thermal cycler licensed under U.S. Patent No.
6,814,934 and corresponding claims in any Canadian counterpart patent thereof owned by Applera Corporation, for
use solely in research and all applied fields except human and veterinary in vitro diagnostics. These license rights
are effective only if this detection module is combined with a Bio-Rad thermal cycler for which the applicable realtime thermal cycler royalty fee has been paid and not with any other thermal cycler. No rights are conveyed
expressly, by implication or estoppel to any patents on real-time methods, including but not limited to 5' nuclease
assays, or to any patent claiming a reagent or kit. For further information on purchasing license rights, contact the
Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California, 94404, USA.
This product is covered by one or more of the following U.S. patents, their foreign counterparts, or their foreign
patents pending, owned by Eppendorf AG: US Patent Nos. 6,767, 512 and 7,074,367. No rights are conveyed
expressly, by implication or estoppel to any patents on PCR methods, or to any patent claiming a reagent or kit
Bio-Rad’s Hard-Shell® plates are covered by one or more of the following U.S. patents, patents pending, or their
foreign counterparts, owned by Eppendorf AG: US Patent Nos. 7,347,977, 6,340,589, 6,528,302 and US application
2007246858, 20080084004, 20030180192 and 20060120927
Bio-Rad Resources
Bio-Rad provides many resources for scientists. The following websites contain useful information about
running PCR and real-time PCR experiments:
• Gene Expression Gateway (www.bio-rad.com/genomics/)
This site provides rich technical resources on a wide variety of methods and applications related to
PCR, real-time PCR, and gene expression
• Life Science Research website (discover.bio-rad.com)
This site includes links to technical notes, manuals, product information, and technical support.
Table 1 lists other Bio-Rad resources and how to locate what you need:
Table 1. Bio-Rad resources
Resource How to Contact
Local Bio-Rad Laboratories
representatives
Technical notes and literature Go to the Bio-Rad website (www.bio-rad.com) or Gene
Technical specialists Bio-Rad’s Technical Support department is staffed with
Find local information and contacts on the Bio-Rad website
by selecting your country on the home page
(www.bio-rad.com). Find the nearest international office
listed on the back of this manual
Expression Gateway (www.bio-rad.com/genomics/). Type a
search term in the Search box and select Literature to find
links to technical notes, manuals, and other literature
experienced scientists to provide customers with practical
and expert solutions
To find local technical support on the phone, contact your
nearest Bio-Rad office. For technical support in the United
States and Canada, call 1-800-424-6723 (toll-free phone),
and select the technical support option
Writing Conventions Used in this Manual
This manual explains how to safely set up and operate the CFX96 system or the CFX384 system. The
manual uses the writing conventions listed in Table 2.
Table 2. Conventions used in this manual
Convention Meaning
TIP: Provides helpful information and instructions, including information
explained in further detail elsewhere in this manual
NOTE: Provides important information, including information explained in
further detail elsewhere in this manual
WARNING! Explains very important information about something that might
damage the researcher, damage an instrument, or cause data loss
X > Y Select X and then select Y from a toolbar, menu or software window
For information about safety labels used in this manual and on the CFX96 system or the CFX384 system,
see, “Safety and Regulatory Compliance” on page iii.
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CFX96 and CFX384 Systems Manual
Safety and Regulatory Compliance
For safe operation of the CFX96 system or the CFX384 system, we strongly recommend that you follow
the safety specifications listed in this section and throughout this manual.
Safety Warning Labels
Warning labels posted on the instrument and in this manual warn you about sources of injury or harm.
Refer to Table 3 to review the meaning of each safety warning label.
Table 3. Meaning of safety warning labels
CAUTION: Risk of danger! This symbol identifies components that pose a risk of personal
injury or damage to the instrument if improperly handled. Wherever this symbol appears,
consult the manual for further information before proceeding
CAUTION: Hot surface! This symbol identifies components that pose a risk of personal
injury due to excessive heat if improperly handled
Instrument Safety Warnings
The warning labels shown in Table 4 also display on the instrument, and refer directly to the safe use of
the CFX96 system or the CFX384 system.
Table 4. Instrument Safety Warning Labels
Icon Meaning
Warning about risk of harm to body or equipment.
Operating the CFX96 or CFX384 real-time PCR detection system before reading this
manual can constitute a personal injury hazard. For safe use, do not operate this instrument
in any manner unspecified in this manual. Only qualified laboratory personnel trained in the
safe use of electrical equipment should operate this instrument. Always handle all
components of the system with care, and with clean, dry hands
Warning about risk of burning.
A thermal cycler generates enough heat to cause serious burns. Wear safety goggles or
other eye protection at all times during operation. Always allow the sample block to return
to idle temperature before opening the lid and removing samples. Always allow maximum
clearance to avoid accidental skin burns
Warning about risk of explosion.
The sample blocks can become hot enough during the course of normal operation to cause
liquids to boil and explode
NOTE: For information about the C1000™ thermal cycler, refer to the C1000 thermal cycler
instruction manual.
iii
Safe Use Specifications and Compliance
Table 5 lists the safe use specifications for the CFX96 system or the CFX384 System. Shielded cables
(supplied) must be used with this unit to ensure compliance with the Class A FCC limits.
Table 5. Safe Use Specifications
Safe Use Requirements Specifications
Temperature Indoor use
Altitude Up to 2,000 meters above sea level
Ambient temperature of 15—31
maximum of 80% noncondensing
REGULATORY COMPLIANCE
This instrument has been tested, and found to be in compliance with all applicable requirements of the
following safety and electromagnetic standards:
• IEC 61010-1:2001 (2nd Ed.), EN61010-1:2001 (2nd Ed). Electrical Equipment For
Measurement, Control, and Laboratory Use - Part 1: General Requirements
• IEC 61010-2-010:2005, EN61010-2-010:2003. Safety requirements for electrical equipment
for measurement, control and laboratory use. Part 2-010: Particular requirements for
laboratory equipment for the heating of materials
• IEC 61010-2-081:2001+A1, EN61010-2-081:2002+A1. Safety requirements for electrical
equipment for measurement, control and laboratory use. Part 2-081: Particular requirements
for automatic and semi-automatic laboratory equipment for analysis and other purposes
(includes Amendment 1)
• EN 61326-1:2006 (Class A) Electrical equipment for measurement, control and laboratory
use. EMC requirements, Part 1: General requirements
o
C. Relative humidity
This equipment generates, uses, and can radiate radio frequency 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.
iv
CFX96 and CFX384 Systems Manual
Table of Contents
Bio-Rad Resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii
Writing Conventions Used in this Manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii
Safety and Regulatory Compliance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii
Chapter 1. System Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Unpacking the Optical Reaction Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
System Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
System Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Setting up the system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Installing CFX Manager Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Running Experiments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Chapter 2. Introduction to CFX Manager Software . . . . . . . . . . . . . . . . . . 11
Main Software Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Startup Wizard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Detected Instruments Pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Instrument Properties Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Software Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Tips and Tricks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Chapter 3. Running Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Experiment Setup Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Protocol Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Plate Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Start Run Tab. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Run Details Window. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Instrument Summary Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Chapter 4. Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Protocol Editor Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Protocol Editor Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Temperature Control Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Protocol AutoWriter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
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Table of Contents
Chapter 5. Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Plate Editor Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Select Fluorophores Window. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Experiment Settings Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Well Groups Manager Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Plate Spreadsheet View Window. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Chapter 6. Stand-Alone Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Control Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Main Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Experiment Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Exporting Data for Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Creating a Data File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Setting Up Email . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Chapter 7. Data Analysis Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Data Analysis Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Data Analysis Menu Bar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Quantitation Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Data Analysis Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Well Selectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Charts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Spreadsheets. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Chapter 8. Data Analysis Windows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Quantitation Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Quantitation Data Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Melt Curve Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Melt Curve Data Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
End Point Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Allelic Discrimination Tab. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
QC Tab. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Reports for Data Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Chapter 9. Gene Expression Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Plate Setup for Gene Expression Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Gene Expression Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Experiment Settings Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Gene Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Gene Study Data Spreadsheet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
Gene Study Report Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Frequently Asked Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Gene Expression Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
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CFX96 and CFX384 Systems Manual
Chapter 10. Users and Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Log in or Select User . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
User Preferences Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
Email Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Files Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
Protocol Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
Plate Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Data Analysis Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
Gene Expression Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
QC Tab. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
User Administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Chapter 11. Resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Calibration Wizard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Instrument Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Application Log . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Software Help Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
Instruments, Parts, and Accessories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
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Table of Contents
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CFX96 and CFX384 Systems Manual
1 System Installation
Read this chapter for information about setting up the CFX96 or CFX384 system:
• Unpacking the optical reaction module (below)
• System requirements (page 1)
• System overview (page 2)
• Setting up the system (page 3)
• Installing CFX Manager software (page 6)
• Running experiments (page 9)
Unpacking the Optical Reaction Module
Your CFX96 or CFX384 optical reaction module shipment includes these components:
• Optical reaction module
•USB cable
• CFX Manager
• Instruction manual
• CFX Manager software uick guides for system installation, protocol, plate, data
analysis, and gene expression analysis
Remove all packing materials and store them for future use. If any items are missing or
damaged, contact your local Bio-Rad office.
Tm
software installation CD
System Requirements
To operate the CFX96 or CFX384 system, use the following power sources and cables:
• Input power. 100—240 VAC, 50—60 Hz
• Indoor use. Ambient temperature of 15—31
(non-condensing)
• USB cable. If the CFX96 system is going to be controlled by a computer via a USB
cable, the provided cable from Bio-Rad is sufficiently shielded for use.
NOTE: For a full list of the safety and compliance requirements for this instrument,
see “Safety and Regulatory Compliance” on page iii.
o
C. Relative humidity maximum of 80%
1
System Installation
Open button
Indicator LED
Front panel
Close button
Block
Inner lid with heater plate
System Overview
The CFX96 system or CFX384 system includes two components:
• Optical reaction module. This module includes an optical system to collect fluorescent
data and a thermal cycler block
NOTE: The serial number of the CFX96 or CFX384 module is located on the back.
• C1000
Tm
thermal cycler base. The C1000 base includes a user interface to control the
system when running in stand-alone mode and the power button and ports (both on
back panel) to connect to a computer
Figure 1. Front view of the CFX96 system.
When open, the CFX96 or CFX384 system includes the features shown in Figure 2.
Figure 2. Inside view of the CFX96 system.
WARNING! Avoid touching the inner lid or block: These surfaces can be hot.
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CFX96 and CFX384 Systems Manual
Power
Power
input
switch
USB connections
Ethernet
port
• Inner lid with heater plate. The heater lid maintains temperature on the top of the
consumable to prevent sample evaporation. Avoid touching or otherwise contaminating
the heater plate. Never poke anything through the holes; the optics shuttle system could
be damaged
•Block. Load samples in this block before the run
• Close button. Press this button on the inside of the lid to close the motorized lid
WARNING! Prevent contamination of the instrument by spills, and never run a
reaction with an open or leaking sample lid. For information about general cleaning
and maintenance of the instrument, see “Instrument Maintenance” (page 133).
The back panel of the C1000 chassis includes these features (Figure 3):
• Power switch. Press the power switch to turn on the power to the system
• Power input. Plug in the power cord here
• Ethernet port. Connect an ethernet cable to email run logs and stand-alone data files
• USB connections. Use these ports to connect the CFX96 system or CFX384 system to
a computer or to connect an S1000
Tm
thermal cycler
Figure 3. Back panel of C1000 thermal cycler.
WARNING! Avoid contact with the back panel of the C1000 during operation.
Setting up the system
The CFX96 or CFX384 real-time PCR detection system should be installed on a clean, dry,
level surface with sufficient cool airflow to run properly. The CFX96 system or CFX384 system
can be run in two modes: stand-alone or software-controlled. If you are running the system
under software-controlled mode, make sure there is sufficient space for a computer during
setup.
To insert the CFX96 or CFX384 optical reaction module into the reaction module bay of
the C1000 chassis, follow these instructions:
1. Place the C1000 chassis in a suitable location with the locking bar down. Remove any
previously installed reaction modules.
3
System Installation
2. Lift the optical reaction module using the handle indents above the side air vents
(Figure 4).
Figure 4. Lifting the optical reaction module into the C1000 chassis.
3. Position the module in the reaction module bay of the C1000 chassis, leaving about 2 cm
of space in the front. When in the chassis bay, the optical module should be covering the
Bio-Rad logo in front of the bay of the C1000 chassis.
4. Reach around and pull up the locking bar of the C1000 until it is flush with the sides of
the module bay. This action moves the module forward, locking it into place (Figure 5)
Figure 5. Locking the optical module into place.
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CFX96 and CFX384 Systems Manual
Check
space
Check
space
Check
space
5. Check that the module is completely and evenly seated in the C1000 base. As shown in
Figure 6, check the space around the bottom of the module. There should be no extra
space between the module and the base; the space should be even.
Figure 6. Check for even space between module and base.
NOTE: If the space between the module and C1000 base is uneven, reinstall the
module starting from Step 2 (page 4).
6. Plug the power cord into the back of the C1000 base (Figure 3 on page 3), and into an
appropriate three-pronged electrical outlet.
7. Press the power switch on the back panel of the C1000 thermal cycler to start the
system.
8. Follow the instructions in the C1000 front panel to remove the red shipping screw from
the inner heater lid (Figure 7). Turn the screw counterclockwise to lift it out of the hole.
Figure 7. Instructions to remove the shipping screw.
5
System Installation
NOTE: If the shipping screw is not removed at this step, it will be detected by CFX
Manager software. Follow the instructions in the software to remove the screw
(page 19).
TIP: The shipping screw must be in place when the module is shipped. Save this
screw in a safe place for future shipping.
9. Remove the shipping plate from the thermal cycler block to operate.
Installing CFX Manager Software
CFX Manager software is run on a PC computer with either the Windows XP or Windows Vista
operating system and is required to analyze real-time PCR data from the CFX96 system or
CFX384 system. This software can also be used to control the CFX96 system or CFX384
system in software-controlled mode. Table 6 lists the computer system requirements for the
software on Windows XP and Windows Vista.
Table 6. Computer requirements for CFX Manager software
System Minimum Recommended
Operating system Windows XP Professional SP2 and
Above or Windows Vista Home
Premium and above
Drive CD-ROM drive CD-RW drive
Hard drive 10 GB 20 GB
Processor speed 2.0 GHz 2.0 GHz
RAM 1 GB RAM (2 GB for Windows
Vista)
Screen resolution 1024 x 768 with true-color mode 1280 x 1024 with true-color mode
USB USB 2.0 Hi-Speed port USB 2.0 Hi-Speed port
Internet browser Internet Explorer Internet Explorer
Software Microsoft Office Suite
Windows XP Professional SP2 and
above or Windows Vista Home
Premium and above
2 GB RAM
NOTE: Running a CFX96 or CFX384 system with CFX Manager Software on a PC
computer with a Windows 64-bit Operating Systems is not supported due to
incompatible USB Drivers. A PC computer with a 64-bit processor (like Intel) on a
32-bit Windows Operating System is supported.
To install CFX Manager software:
1. The software must be installed on the computer by a user with administrative privileges.
Make sure you are logged in with administrative privileges.
2. Place the CFX Manager software CD in the computer’s CD drive.
6
CFX96 and CFX384 Systems Manual
3. The software launch page should appear automatically. Double-click Install Software on
the software launch page (Figure 8).
Figure 8. Software installation screen.
TIP: Click the Documentation button to find searchable PDF copies of the
instrument manuals and other documentation.
4. Follow the instructions on screen to complete installation. When completed, the Bio-Rad
CFX manager software icon will appear on the desktop of the computer.
5. If the launch page does not appear automatically, double-click on (CD drive):\Bio-Rad
CFX, then open and follow instructions in the Readme.txt file. See “Installing the
Software Manually” on page 136.
Installing the Drivers
If the CFX96 system or CFX384 system is going to be run in Software-controlled mode,
drivers must be installed on the computer. Use only the supplied USB cable, which is
sufficiently shielded to prevent data loss.
To install the system drivers:
1. Connect the C1000 chassis to the computer by plugging a USB cable into the USB 2.0 A
port located on the back of the chassis (Figure 3 on page 3), and then connecting the
cable into the USB 2.0 B port located on the computer.
2. If it is not already turned on, turn on the system using the power switch on the back of
the C1000 chassis. Follow the instructions in the Found New Hardware Wizard that
launches after the instrument is first detected by the computer.
7
System Installation
3. On the first screen, select Yes, this time only to instruct the Windows operating system
to connect to Windows Update to search for software (Figure 9). Click Next.
Figure 9. Found New Hardware Wizard.
4. Instruct the wizard to “
installing the drivers (Figure 10).
Install the software automatically
.” Click Next to continue
Figure 10. Software (Driver) installation screen.
5. Click Finish at the software installation completion screen when the drivers are installed.
8
Running Experiments
Recommended Plastic Consumables
Run only 384-well plates in the CFX384 system. For optimal results, Bio-Rad recommends the
following consumables for the CFX384 system (catalog numbers are provided in bold):
• HSP-3805. Low-profile 384-well Hard-Shell® plates with clear shell and white wells
• HSP-3866. Low-profile 384-well Hard-Shell plates with black shell and white wells
• MSB-1001. Microseal ‘B’ adhesive seals, optically clear
The CFX96 system accepts both low profile 0.2 ml plates and tubes. For optimal results, BioRad recommends the following consumables:
• MLL-9601. Low-profile 96-well unskirted plates with clear wells
• MLL-9651. Low-profile 96-well unskirted plates with white wells
• HSP-9601. Hard-Shell 96-well skirted plates with white shell and clear wells
• HSP-9655. Hard-Shell 96-well skirted plates with white shell and white wells
• TLS-0801. Low-profile 0.2 ml 8-tube strips without caps, clear wells
• TLS-0851. Low-profile 0.2 ml 8-tube strips without caps, white wells
• TCS-0803. Optical flat 8-cap strips, for 0.2 ml tubes and plates
• MSB-1001. Microseal ‘B’ adhesive seals, optically clear
CFX96 and CFX384 Systems Manual
Loading the Block
To load your reactions in the block, follow these suggestions:
• Click the Open Lid button located on software’s Start Run tab (see “Start Run Tab”
on page 24), or press the lid button on the front of the system (Figure 1) to start
opening the motorized lid.
WARNING! The lid moves slowly at first, and then increases speed when it opens
or closes.
• Place the 0.2 ml microplate or tube strips with sealed lids in the block. Check that
the tubes are completely sealed to prevent leakage. For optimal results load sample
volumes of 10–25 μl for the CFX96 system and load sample volumes of 5–20 μl for
the CFX384 system.
NOTE: For accurate data analysis, check that the orientation of reactions in the
block is exactly the same as the orientation of the well contents in the software
Plate tab (see “Plate Tab” on page 23). If needed, edit the well contents before,
during, or after the run. As shown in Figure 11 on page 10, rotating the microplate
by 180
well.
o
causes the software to expect the reading for well H3 to be an empty A10
9
System Installation
Well H3
Well A10
(180
o
rotation)
(correct)
Filled tube
strip
Tube strip for
balance
Figure 11. Plate Editor window showing plate orientation.
WARNING! When running the CFX96 system, always balance the tube strips or cut
microplates in the wells (Figure 12). For example, if you run one tube strip on the
left side of the block, run an empty tube strip (with caps) on the right side of the
block to balance the pressure applied by the heated lid.
Figure 12. Balance the tube strips or cut microplates in the block.
WARNING! Be sure that nothing is blocking the lid when it closes. Although there
is a safety mechanism to prevent the lid from closing if it senses an obstruction, do
not place anything in the way of the closing lid.
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CFX96 and CFX384 Systems Manual
2 Introduction to CFX Manager
Software
Read this chapter for information about getting started with CFX Manager software.
• Main software window (below)
• Startup Wizard (page 15)
• Detected Instruments pane (page 16)
• Instrument Properties window (page 18)
• Software files (page 20)
• Tips and tricks (page 20)
Main Software Window
For instructions about running the system, refer to one of the CFX Manager software quick
guides that ships with the system:
• Installation quick guide
• Protocol quick guide
• Plate quick guide
• Data Analysis quick guide
• Gene Expression Analysis quick guide
TIP: See the software Help for more guides about running experiments.
Get started in the main software window by using these features (Figure 13):
• Status bar. View the current software and instrument status (page 12)
•Menu bar. Select software commands (page 12), such as creating or opening files
• Toolbar buttons. Click these buttons (page 14) to open software files, the Startup
Wizard (page 18), the Experiment Setup window (page 21)
• Detected Instruments pane. View a list of attached instruments (page 15)
11
Introduction to CFX Manager Software
• Startup Wizard window. Access common software commands (page 16)
Figure 13. The main software window.
Status Bar
The status bar at the bottom of the main software window shows the status of the software.
View the left side of the status bar (Figure 14) to see the current status of instruments.
Figure 14. Left side of status bar in main software window.
View the right side of the status bar (Figure 15) to see the current user name, date, and time.
Figure 15. Right side of status bar in the main software window.
Click and drag the right corner of the status bar to resize the main window.
Menu Bar
The menu bar of the main software window provides the items listed in Figure 16:
12
Figure 16. Menu bar in the main software window.
CFX96 and CFX384 Systems Manual
Select the commands shown in the menu bar (Table 7).
Table 7. Menu bar items in the main software window
Menu Item Command Function
File New Create a new protocol, plate, experiment, or Gene
Study
Open Open existing files, including protocol (.prcl), plate
(.pltd), data (.pcrd), and Gene Study (.mgxd) files,
stand-alone run files (.zpcr)
Recent Data Files View a list of the ten most recently viewed data
files, and select one to open in Data Analysis
Repeat an Experiment Open the Experiment Setup window with the
protocol and plate from a completed run to quickly
repeat the run
Exit Exit the software program
View Application Log Display the application log for the software
Run Reports Select a run report to review from a list
Startup Wizard Open the Startup Wizard
Experiment Setup Open the Experiment Setup window
Instrument Summary Open the Instrument Summary window
Detected Instruments Show or hide the Detected Instruments pane
Toolbar Show or hide the main software window toolbar
Status Bar Show or hide the main software window status bar
User Select User Open the Select User window to change software
users
Change Password Change your user password
User Preferences Open the User Preferences window
User Administration Manage users in the User Administration window
Tools Dye Calibration Wizard Open the Dye Calibration window to calibrate an
instrument for a new fluorophore
Protocol AutoWriter Open the Protocol AutoWriter window to create a
new protocol
Ta Calculator Open the Ta Calculator window to calculate the
annealing temperature of primers
View Block Status Log View a log of the thermal cycler block
Application Data Folder Open the Application Data folder to view software
files
User Data Folder Open the Data folder to view protocol, plate, and
data files
Properties All
Instruments
Zip Data and Log Files Choose and condense selected files in a zipped
Options Configure software email settings
View properties of all detected instruments,
including serial numbers
file for storage or to email
13
Introduction to CFX Manager Software
Table 7. Menu bar items in the main software window (continued)
Menu Item Command Function
Windows Cascade Arrange software windows on top of each other
Tile Vertical Arrange software windows from top to bottom
Tile Horizontal Arrange software windows from right to left
Close All Close all open software windows
Help Contents Open the software Help for more information
Index View the index in the software Help
Search Search the software Help
Gene Expression
Gateway Web site
PCR Reagents Website View a website that lists Bio-Rad consumables for
PCR Plastic
Consumables Website
Software Updates Check for software updates from Bio-Rad
About Open a window to see the software version
about running PCR and real-time PCR
Open a web site to find information about running
PCR and real-time PCR experiments
PCR and real-time PCR reagents
View a website that lists Bio-Rad consumables for
PCR and real-time PCR experiments
Toolbar Buttons
Click a button in the toolbar of the main software window (Table 8) for quick access to
common software commands.
NOTE: To show or hide the toolbar, select View > Toolbar in the menu bar.
Table 8. Toolbar buttons in the main software window
Button Button Name Function
Open a Data File Open a browser window to locate a data file (*.pcrd
extension) and open it in the Data Analysis window
(page 69)
Open a Gene Study Open a browser window to locate a Gene Study file
(.mgxd extension) and open it in the Gene Study
window (page 101)
Create a New Gene
Study
Print Print the current software window
Open the Gene Study window (page 101) to add files
and create a new study
14
Startup Wizard Open the Startup Wizard that links you to common
software functions (page 16)
CFX96 and CFX384 Systems Manual
Table 8. Toolbar buttons in the main software window (continued)
Button Button Name Function
Experiment Setup Open the Experiment Setup window to run an
experiment (page 21)
Protocol AutoWriter Open the Protocol AutoWriter window to create a new
protocol (page 37)
Select User Open the Select User window to change software
users (see “Log in or Select User” on page 121)
User Preferences Open the User Preferences window (page 121)
Help Open the software Help window for more information
about running PCR and real-time PCR
Startup Wizard
The Startup Wizard automatically appears when CFX Manager software is first opened
(Figure 17). If it is not shown, click the Startup Wizard button on the main software window
toolbar.
Figure 17. Startup Wizard window.
Options in the Startup Wizard include the following:
15
Introduction to CFX Manager Software
• Create a new Experiment (page 21). Set up the protocol and plate to begin a new
experiment.
NOTE: Select the appropriate instrument in the pull down list to make sure the
default plate settings match the instrument to be used for the experiment.
• Repeat an Experiment. Set up an experiment with the protocol and plate from a
completed run. If needed, you can edit the experiment before the run
• Open a Data File (page 69). Open a data file to analyze results
• Open a Gene Study (page 101). Open a multi-file gene expression study to analyze
results from multiple gene expression experiments
• Open User Preferences (page 121). Open the User Preferences window to customize
software settings
Detected Instruments Pane
CFX Manager software runs these 1000-series instruments:
• CFX96 and CFX384 real-time PCR systems. Systems include a C1000 thermal cycler
and an optical reaction module
• C1000 thermal cycler. The most versatile of the instruments run by CFX Manager
software. Add and remove these PCR reaction modules to the C1000 chassis in
seconds:
• 96-well fast reaction module, gradient enabled
• 384-well reaction module
• Dual 48/48 fast reaction module, gradient enabled
• S1000 thermal cycler connected to a C1000 thermal cycler. For more information,
see S1000 and C1000 thermal cycler instruction manuals
TIP: Locate PDF copies of the instruction manuals by opening the Documents
folder on the CFX Manager software installation CD.
You can simultaneously run up to four instruments with CFX Manager software, including a
combination of CFX96 system and CFX384 systems, or a mix of real-time PCR systems and
thermal cyclers.
Viewing Detected Instruments
Connected instruments appear in the Detected Instruments pane (page 17). This list shows
each instrument as an icon named with the serial number (default). The list of instruments also
shows individual blocks (Block A and Block B) for each dual block reaction module.
TIP: Right-click any instrument in the Detected Instruments pane to open the
Instrument Properties window and rename the instrument.
Figure 18 shows four detected instruments:
• One C1000 thermal cycler (C48FSIM00) with a dual 48/48 reaction module
• One S1000 thermal cycler (S96FSIM01) with a 96-well block, which is connected to
the C1000 thermal cycler called C48FSIM00
• One CFX384 system (CFX384SIM03)
16
CFX96 and CFX384 Systems Manual
• One CFX96 system (CFX96SIM02)
Figure 18. Instruments listed at the top of the Detected Instruments pane.
Right-click on the instrument icon or block to select one of these options:
•View Status. Open the Run Details window to check the status of the selected
instrument block
• Flash Block Indicator. Flash the indicator LED on the instrument
• Open Lid. Open a motorized lid on the selected instrument block
•Close Lid. Close a motorized lid on the selected instrument block
• Rename. Change the name of the instrument
•Properties. Open the Instrument Properties window
• Collapse All. Collapse the list of instruments in the Detected Instruments pane
• Expand All. Expand the list of instruments in the Detected Instruments pane
You can also control a block by clicking an instrument block icon in the Detected Instrument
pane and then clicking a button in the Selected Instrument pane (Figure 19).
Figure 19. Buttons at the bottom of the Detected Instruments pane.
• Click View Status to open the Run Details window to check the status of the
selected instrument block
• Click Open Lid to open the motorized lids on the selected instrument
• Click Close Lid to close the motorized lids on the selected instrument
• Click View Summary to open the Instrument Summary window
17
Introduction to CFX Manager Software
If only one instrument is detected, then the View Summary button does not appear. To view
the Instrument Summary window for a single instrument, select View > Instrument Summary.
Instrument Properties Window
To open the Instrument Properties window to view information about an instrument, right-click
on the instrument icon in the Detected Instruments pane (Figure 18 on page 17). The window
includes three tabs (Figure 20):
•Properties. View serial numbers and the C1000 thermal cycler name
• Shipping Screw. Remove the shipping screw to run the instrument, or install the
shipping screw when you want to transport the instrument
• Calibrated Dyes. View the list of calibrated fluorophores
18
Figure 20. Instrument Properties window.
Properties Tab
The default name for an instrument is the C1000 thermal cycler serial number, which appears
in many locations, including the Detected Instruments pane (Figure 18).
To rename an instrument for ease of identification, follow these instructions:
• In the Instrument Properties tab, type a name in the Rename box at the top of the
Properties tab and hit the Rename button to save the new name
NOTE: In the Detected Instrument pane, right-click the instrument icon and select
Rename to open the Instrument Properties window.
The Properties tab displays important serial numbers for the connected instrument, including
the thermal cycler and reaction module. The firmware versions are also displayed.
CFX96 and CFX384 Systems Manual
Shipping Screw Tab
The Shipping Screw tab includes instructions for installing or removing the red shipping screw.
To prevent damage to the optical reaction modules, install the shipping screw any time you
ship the CFX96 system or CFX384 system.
NOTE: If the shipping screw is detected by the software, the Instrument Properties
window automatically opens with the Shipping Screw tab in front. Follow the
instructions to remove the screw.
The information in this tab changes depending on whether the shipping screw is installed or
removed. For example, to install the shipping screw, click the Install Shipping Screw button
and follow the instructions in the tab (Figure 21).
Figure 21. Instructions for installing the shipping screw.
Calibrated Dyes Tab
Open the Calibrated Dyes tab to view the list of calibrated fluorophores and plates for the
selected instrument. Click an Info button to see detailed information about a calibration.
Figure 22. Calibrated Dyes tab in the Instrument Properties window.
19
Introduction to CFX Manager Software
Software Files
CFX Manager software stores information about experiments in specific files (Table 9):
Table 9. Open these file types with CFX Manager software
File Type Extension How to View and Edit File
Protocol .prcl Select in Experiment Setup and edit in Protocol Editor
Plate .pltd Select in Experiment Setup and edit in Plate Editor
Data .pcrd View and analyze in Data Analysis window
Gene Study .mgxd View and analyze in Gene Study window
Stand-alone pre-data
file
.zpcr Contains fluorescence readings from stand-alone
Tips and Tricks
Tips and tricks for using CFX Manager software are listed below.
• Open any Protocol, Plate, Data, or Gene Study file by dragging it from a folder to an
open software window
• Print or export the information shown in many windows by right-clicking a chart,
spreadsheet, or well selector
• Change the size of any window by clicking and dragging the edges
• Open the User Preferences window to choose default settings that activate every
time you log in to the software
• Add data files to a Gene Study by dragging from a folder to an open Gene Study
window
• Open multiple Data Analysis and Gene Study files at the same time
• Click the Settings or Tools menus to find advanced functions
• To add or delete files from the Express Load menu, add or delete the files (.prcl
and.pltd extensions) in the ExpressLoad folder, select Tools > User Data Folder in
the menu bar of the main software window
• To view all the information loaded into one well in a plate, double-click the well to
open the Well Info window
• Right-click any graph or chart to change viewing and data analysis options
• Edit well contents before, during, or after the run
• Select a well group to view and analyze a subset of the wells in the plate. Select each
well group by name in the Well Group pull-down menu in the toolbar
operation that is converted into a data file
20
CFX96 and CFX384 Systems Manual
3 Running Experiments
Read this chapter for information about running experiments using CFX Manager software:
• Experiment Setup window (below)
•Protocol tab (page22)
• End point only runs (page 22)
• Plate tab (page 23)
• Start Run tab (page 24)
• Run Details window (page 26)
• Instrument Summary Window (page 28)
Experiment Setup Window
The Experiment Setup window provides quick access to the files and settings needed to set
up and run an experiment. To open the Experiment Setup window, follow one of these options:
• Click Create a New Experiment option in the Startup Wizard (page 16)
• Click the Experiment Setup button in the main software toolbar (page 14)
•Select File > New > Experiment in the main software menu bar (page 12)
The Experiment Setup window includes three tabs:
•Protocol. Click the Protocol tab to select an existing protocol to run or edit, or to create
a new protocol in the Protocol Editor window (page 31)
•Plate. Click the Plate tab to select an existing plate to run or edit, or to create a new
plate in the Plate Editor window (page 41)
• Start Run. Click the Start Run tab (page 24) to check the run settings, select one or
more instrument blocks, and begin the run
NOTE: If the protocol currently selected in the Protocol tab does not include a step
with a plate read for real-time PCR analysis, then the Plate tab is hidden. To view
the Plate tab, add a “Plate Read” (page 34) in at least one step in the protocol.
NOTE: Start a new experiment from a previous run by selecting File > Repeat an
Experiment in the main software menu bar. Then select the data file (.pcrd) for the
experiment you want to repeat.
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