CFX96 and CFX384 Real-Time PCR Detection Systems: Instruction Manual
CFX96 and CFX384 Real-Time PCR Detection Systems: Instruction Manual
CFX96 and CFX384 Real-Time PCR Detection Systems: Instruction Manual
Catalog # 184-5384
# 185-5384
# 184-5096
# 185-5096
Copyright ©2010 Bio-Rad Laboratories, Inc. Reproduction in any form, either print or electronic, is
prohibited without written permission of Bio-Rad Laboratories, Inc.
Adobe and Reader are trademarks of Adobe Systems Incorporated. Cy is a trademark of GE Healthcare
Group companies. CAL Fluor and Quasar are trademarks of Biosearch Technologies, Inc. SYBR® and
Texas Red are trademarks of Invitrogen Corporation. Excel, Microsoft, PowerPoint, Windows, and
Windows Vista are trademarks of Microsoft Corporation. EvaGreen is a trademark of Biotium, Inc. Bio-
Rad Laboratories, Inc., is licensed by Biotium, Inc., to sell reagents containing EvaGreen dye for use in
real-time PCR, for research purposes only. FAM and ROX are trademarks of Applera 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.
This CFX96 or CFX384 detection module, when combined with a C1000™ thermal cycler for which the applicable
real-time 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, human in vitro diagnostics, and all applied fields except 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 real-time 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: U.S. Patent Nos. 6,767, 512 and 7,074,367.
Bio-Rad Resources
Table 1 lists Bio-Rad resources and how to locate what you need.
Table 1. Bio-Rad resources
Resource How to Contact
Local Bio-Rad Laboratories Find local information and contacts on the Bio-Rad website
representatives by selecting your country on the home page
(www.bio-rad.com). Find the nearest international office
listed on the back of this manual
Technical notes and literature Go to the Bio-Rad website (www.bio-rad.com). Type a
search term in the Search box and select Literature to find
links to technical notes, manuals, and other literature.
Technical specialists Bio-Rad’s Technical Support department is staffed with
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 800-424-6723
(toll-free phone) and select the technical support option.
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 Instruction Manual
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
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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 handling biohazardous materials.
When handling biohazardous samples, adhere to the recommended precautions and
guidelines, and comply with any local guidelines specific to your laboratory and location.
NOTE: For information about the C1000™ thermal cycler, refer to the C1000 thermal cycler
instruction manual.
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
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CFX96 and CFX384 Systems Instruction Manual
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
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.
Hazards
The CFX96 and CFX384 real-time PCR detection systems are designed to operate safely when used in
the manner prescribed by the manufacturer. If the CFX96 or CFX384 system or any of its associated
components are used in a manner not specified by the manufacturer, the inherent protection provided by
the instrument may be impaired. Bio-Rad Laboratories, Inc. is not liable for any injury or damage caused
by the use of this equipment in any unspecified manner, or by modifications to the instrument not
performed by Bio-Rad or an authorized agent. Service of the CFX96 or CFX384 system should be
performed only by Bio-Rad personnel.
Biohazards
The CFX96 and CFX384 systems are laboratory products. However, if biohazardous samples are present,
adhere to the following guidelines and comply with any local guidelines specific to your laboratory and
location.
GENERAL PRECAUTIONS
• Always wear laboratory gloves, coats, and safety glasses with side shields or goggles
• Keep your hands away from your mouth, nose, and eyes
• Completely protect any cut or abrasion before working with potentially infectious materials
• Wash your hands thoroughly with soap and water after working with any potentially infectious
material before leaving the laboratory
• Remove wristwatches and jewelry before working at the bench
• Store all infectious or potentially infectious material in unbreakable leak-proof containers
• Before leaving the laboratory, remove protective clothing
• Do not use a gloved hand to write, answer the telephone, turn on a light switch, or touch
anything that other people may touch without gloves
• Change gloves frequently. Remove gloves immediately when they are visibly contaminated
• Do not expose materials that cannot be properly decontaminated to potentially infectious
material
• Upon completion of the operation involving biohazardous material, decontaminate the work
area with an appropriate disinfectant (for example, a 1:10 dilution of household bleach)
• No biohazardous substances are exhausted during normal operations of this instrument
SURFACE DECONTAMINATION
WARNING! To prevent electrical shock, always turn off and unplug the instrument prior to performing
decontamination procedures.
v
The following areas can be cleaned with any hospital-grade bactericide, virucide, or fungicide
disinfectant:
• Outer lid and chassis
• Inner reaction block surface and reaction block wells
• Control panel and display
To prepare and apply the disinfectant, refer to the instructions provided by the product manufacturer.
Always rinse the reaction block and reaction block wells several times with water after applying a
disinfectant. Thoroughly dry the reaction block and reaction block wells after rinsing with water.
WARNING! Do not use abrasive or corrosive detergents or strong alkaline solutions. These agents can
scratch surfaces and damage the reaction block, resulting in loss of precise thermal control.
Chemical Hazards
The CFX96 or CFX384 system contains no potentially hazardous chemical materials.
Electrical Hazards
The CFX96 or CFX384 system poses no uncommon electrical hazard to operators if installed and
operated properly without physical modification and connected to a power source of proper specification.
Transport
Before moving or shipping the C1000™ thermal cycler, or CFX96 or CFX384 optical reaction module,
decontamination procedures must be performed. Always move or ship the C1000 thermal cycler chassis
and CFX96 or CFX384 optical reaction module in separate containers with the supplied packaging
materials which will protect the instrument from damage. If appropriate containers cannot be found,
contact your local Bio-Rad office.
Storage
The CFX96 or CFX384 system can be stored under the following conditions:
• Temperature range: –20 to 60oC
• Relative humidity: maximum 80%
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CFX96 and CFX384 Systems Instruction Manual
Disposal
The CFX96 or CFX384 real-time PCR detection system contains electrical or electrical materials; it should
be disposed of as unsorted waste and must be collected separately, according to European Union
Directive 2002/96/CE on waste and electronic equipment — WEEE Directive. Before disposal, contact
your local Bio-Rad representative for country-specific instructions.
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CFX96 and CFX384 Systems Instruction Manual
Table of Contents
Bio-Rad Resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii
Writing Conventions Used in This Manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii
Safety and Regulatory Compliance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii
Hazards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Table of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
Chapter 4. Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Protocol Editor Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Protocol Editor Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Temperature Control Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Protocol AutoWriter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
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Table of Contents
Chapter 5. Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Plate Editor Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Select Fluorophores Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Well Loading Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Experiment Settings Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Well Selector Right-Click Menu Items. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Well Groups Manager Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Plate Spreadsheet View Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
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CFX96 and CFX384 Systems Instruction Manual
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
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Table of Contents
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CFX96 and CFX384 Systems Instruction Manual
1 System Installation
Read this chapter for information about setting up the CFX96™ or CFX384™ system:
• Unpacking the optical reaction module (page 1)
• System requirements (page 1)
• System overview (page 2)
• Setting up the system (page 4)
• Installing CFX ManagerTM software (page 6)
• Software files (page 8)
• Running experiments (page 8)
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 - 31oC. Relative humidity maximum of 80%,
noncondensing.
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System Installation
• USB cable. If the 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.
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 optical reaction module is
located on the back.
• C1000TM thermal cycler base. The C1000 base includes a user interface to control the
system when running in stand-alone mode, the power button, and ports (both on the
back panel) to connect to a computer
Indicator LED
Open button
Front panel
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CFX96 and CFX384 Systems Instruction Manual
When open, the CFX96 or CFX384 system includes the features shown in Figure 2.
Block
Close button
WARNING! Avoid touching the inner lid or block: These surfaces can be hot.
• 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 144).
The back panel of the C1000 chassis includes these features (Figure 3):
• Power switch. Press the power switch to turn on 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 S1000TM thermal cycler
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System Installation
WARNING! Avoid contact with the back panel of the C1000 cycler during operation.
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CFX96 and CFX384 Systems Instruction Manual
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).
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System Installation
5. Check that the module is completely and evenly seated in the C1000 base. 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.
6. Plug the power cord into the back of the C1000 base (Figure 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 on the C1000 front panel to remove the red shipping screw from
the inner heater lid.
• Open the optical module lid by pressing the button below the Bio-Rad logo.
• Turn the screw counterclockwise to remove it from the hole in the inner heated lid that
corresponds to well A1 for the CFX96 or to the well adjacent to the left side of B1 for
the CFX384.
10.Close the optical module lid by pressing the button positioned in front of the block.
11.Press the Screw Removed button to confirm the shipping screw has been removed.
NOTE: If the shipping screw is not removed at this step, it will be detected by CFX
Manager software. Follow instructions to remove the screw (page 18).
TIP: The shipping screw must be in place when the module is shipped. Save this
screw in a safe place for future shipping.
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CFX96 and CFX384 Systems Instruction Manual
3. The software launch page should appear automatically. Double-click Install Software on
the software launch page (Figure 6).
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 (CD drive):\Bio-Rad
CFX, then open and follow instructions in the Readme.txt file. See “Installing the
Software Manually” on page 146.
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.
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. Click Next.
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System Installation
4. Instruct the wizard to Install the software automatically. Click Next to continue
installing the drivers.
5. Click Finish at the software installation completion screen when the drivers are installed.
Software Files
CFX Manager software stores information about runs in specific files (Table 7):
Table 7. Open these file types with CFX Manager software.
File Type Extension How to View and Edit File
Protocol .prcl Select in Run Setup and edit in Protocol Editor
Plate .pltd Select in Run 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 .zpcr Contains fluorescence readings from stand-alone
file operation that are converted into a data file
LIMS .plrn Contains plate setup and protocol information required to
conduct a LIMS compatible run
Running Experiments
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CFX96 and CFX384 Systems Instruction Manual
• Click the Open Lid button located on software’s Start Run tab (see “Start Run Tab”
on page 27), or press the lid button on the front of the system (Figure 1) to start
opening the motorized lid.
• 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 27). If needed, edit the well contents before,
during, or after the run.
WARNING! When running the CFX96 system, always balance the tube strips or cut
microplates in the wells (Figure 7). 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.
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System Installation
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CFX96 and CFX384 Systems Instruction Manual
11
CFX Manager™ Software
Menu Bar
The menu bar of the main software window provides the items listed in Table 8.
Table 8. Menu bar items in the main software window.
Menu Item Command Function
File New Create a new protocol, plate, run, 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 a Run Open the Run Setup window with the protocol and
plate from a completed run to quickly repeat the
run.
Exit Exit the software program.
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CFX96 and CFX384 Systems Instruction Manual
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CFX Manager™ Software
Toolbar Buttons
Click a button in the toolbar of the main software window (Table 9) for quick access to
common software commands.
Table 9. 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.
Open a Gene Study Open a browser window to locate a gene study file
(.mgxd extension) and open it in the Gene Study
window.
Create a New Gene Open the Gene Study window to add files and create
Study a new study.
Master Mix Calculator Open the Master Mix Calculator window to set up
reaction mixes.
Startup Wizard Open the Startup Wizard which links you to common
software functions.
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CFX96 and CFX384 Systems Instruction Manual
Startup Wizard
The Startup Wizard automatically appears when CFX Manager software is first opened. If it is
not shown, click the Startup Wizard button on the main software window toolbar.
Options in the Startup Wizard include the following:
• Create a new Run (page 25). Set up the protocol and plate to begin a new run.
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 run.
• Repeat a Run. Set up an run with the protocol and plate from a completed run. If
needed, you can edit the run before the starting
• Open a Data File (page 69). Open a data file to analyze results
• Open a Gene Study (page 113). Open a multi-file gene expression study to analyze
results from multiple gene expression runs
• Open User Preferences (page 126). Open the User Preferences window to customize
software settings
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CFX Manager™ Software
• One S1000 thermal cycler (S96FSIM01) with a 96-well block, which is connected to
the C1000 thermal cycler called C48FSIM00
• One CFX384 system (CFX384SIM03)
• One CFX96 system (CFX96SIM02)
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CFX96 and CFX384 Systems Instruction Manual
• 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
If only one instrument is detected, the View Summary button does not appear. To view the
Instrument Summary window for a single instrument, select View > Instrument Summary.
Status Bar
The left side of the status bar at the bottom of the main software window shows the current
status of the instruments. View the right side of the status bar to see the current user name,
date, and time. Click and drag the lower right corner of the status bar to resize the main
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 9).
To rename an instrument for ease of identification, follow these instructions:
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CFX Manager™ Software
• 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
The Properties tab displays important serial numbers for the connected instrument, including
the thermal cycler and reaction module. The firmware versions are also displayed.
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CFX96 and CFX384 Systems Instruction Manual
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CFX Manager™ Software
2. Edit the default target name by highlighting the target name in the drop-down target list,
entering a new target name in the Target box, and pressing Enter on the keyboard.
3. Enter the starting and final concentrations for your forward and reverse primers and any
probes.
4. Additional targets can be added by clicking the New button. To delete targets, select the
target using the drop-down target list and click Remove.
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CFX96 and CFX384 Systems Instruction Manual
WARNING! Removing a target from the target list also removes it from any master
mixes calculations it is used in.
5. Adjust the Supermix Concentration, Reaction Volume Per Well, Excess Reaction Volume,
template volume that will be added to each well, and the Number of Reactions that will
be run.
6. Check the checkbox next to the target (only one can be chosen per SYBR® Green/
EvaGreen master mix) or targets (for probe multiplex reactions). The calculated volumes
of the components required for the master mix are listed.
8. Click the Set as Default button to set the quantities input in the Target and Master Mix
Setup sections as new defaults.
9. To save the contents of the Master Mix Calculator window click OK.
Scheduler
Use the Scheduler to reserve access to an instrument(s). To access the Scheduler click the
Scheduler button in the toolbar (Table 9) or select Tools > Scheduler from the main window.
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CFX Manager™ Software
2. To add a new instrument, select View > Instrument Details or click the Manage
Instruments button below the Instruments list (Figure 15) in the scheduler main window.
In the Instrument Details window, enter the instrument name in the Name column.
Choose a model from the drop-down menu or leave it blank to schedule instrument
types not listed. Entering base and optical head serial numbers is optional.
3. To add a new user, select View > User Details or click the Manage Users button below
the Users list. In the User Details window (Figure 16), enter the new user name in the
Name column. An email address can be entered so that optional electronic notifications
can be sent.
NOTE: The SMTP server needs to be set up in order for electronic notifications to be
enabled.
4. To remove an instrument or user, open the appropriate details window and check the
corresponding box in the Delete column.
WARNING! All events associated with this instrument or user will be removed from
the calendar.
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CFX96 and CFX384 Systems Instruction Manual
2. Select the instrument and user from the drop-down list (Figure 17).
3. Adjust the start and end times. Once an event appears in the calendar view, it can be
moved to another time period by clicking and dragging the entry to a new position in the
calendar.
5. To include an email or a pop-up reminder that will appear at a specified time prior to the
start of an event, check the Reminder checkbox and choose an advance notification
time period for the drop-down list.
WARNING! Scheduler must be running for reminders to be activated. Minimizing
the Scheduler window will enable pop-up and email reminders to occur at the
scheduled time. Selecting Close will quit the Scheduler.
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CFX Manager™ Software
Cleanup events
Select Tools > Cleanup Events to delete events from the calendar older than the period of
time specified in the Scheduler Options window (below).
WARNING! All events older than the specified date will be deleted.
Scheduler Options
Select Tools > Options to define Scheduler display, cleanup, and launch settings. Click
Restore Defaults to restore the Scheduler default settings.
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CFX96 and CFX384 Systems Instruction Manual
3 Performing Runs
Read this chapter for information about performing runs using CFX Manager™ software:
• Run Setup window (page 25)
• Protocol tab (page 26)
• End point only runs (page 26)
• Plate tab (page 27)
• Start Run tab (page 27)
• Run Details window (page 28)
• Instrument Summary window (page 31)
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Performing Runs
The Run Setup window opens with the Protocol tab in front (Figure 19). To open another tab,
click that tab or click the Prev or Next button at the bottom of the window.
Figure 19. Run Setup window, including the Protocol, Plate, and Start Run tabs.
Protocol Tab
The Protocol tab shows a preview of the selected protocol file loaded in Run Setup (Figure 19).
A protocol file contains the instructions for the instrument temperature steps as well as
instrument options that control the ramp rate and lid temperature.
Select one of the following options to select an existing protocol, create a new protocol, or edit
the currently selected protocol:
• Create New button. Open the Protocol Editor to create a new protocol
• Select Existing button. Open a browser window to select and load an existing protocol
file (.prcl extension) into the Protocol tab
• Express Load pull-down menu. Quickly select a protocol to load it into the Protocol tab
TIP: To add or delete protocols in the Express Load menu, add or delete files (.prcl
extension) in the ExpressLoad folder. To locate this folder, select Tools > User
Data Folder in the menu bar of the main software window
• Edit Selected button. Open the currently selected protocol in the Protocol Editor
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CFX96 and CFX384 Systems Instruction Manual
Plate Tab
The Plate tab shows a preview of the selected plate file loaded in Run Setup (Figure 20). In a
real-time PCR run, the plate file contains a description of the contents of each well, the scan
mode, and the plate type. CFX Manager software uses these descriptions for data collection
and analysis.
Select one of the following options to select an existing plate, create a new plate, or edit the
currently selected plate:
• Create New button. Open the Plate Editor to create a new plate
• Select Existing button. Open a browser window to select and load an existing plate file
(.pltd extension) into the Plate tab
• Express Load pull-down menu. Quickly select a plate to load it into the Plate tab
TIP: To add or delete plates in the Express Load menu, add or delete files (.pltd
extension) in the ExpressLoad folder. To locate this folder, select Tools > User
Data Folder in the menu bar of the main software window.
• Edit Selected button. Open the currently selected plate in the Plate Editor
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Performing Runs
• Start Run on Selected Block(s) pane. Select one or more blocks, edit run parameters
(if necessary), and then click the Start Run button to begin the run
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CFX96 and CFX384 Systems Instruction Manual
• Real-time Status tab. View the real-time PCR fluorescence data as they are collected
• Time Status tab. View a full-screen countdown timer for the protocol
Figure 22 shows the features of the Run Details window.
Figure 22. Run Details window showing the Run Status tab.
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Performing Runs
Skip the current step in the protocol. If you skip a GOTO step,
the software verifies that you want to skip the entire GOTO
loop and proceed to the next step in the protocol.
Flash the LED on the selected instrument to identify the
selected blocks.
Stop the run before the protocols ends, which may alter your
data.
Figure 23. The Real-time Status tab displays the data during a run.
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CFX96 and CFX384 Systems Instruction Manual
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Performing Runs
Flash Block Indicator Flash the indicator LED on the lid of the
selected blocks.
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CFX96 and CFX384 Systems Instruction Manual
4 Protocols
Read the following chapter for information about creating and editing protocol files:
• Protocol Editor window (page 33)
• Protocol Editor controls (page 35)
• Temperature control mode (page 38)
• Protocol AutoWriter (page 39)
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Protocols
• Protocol Editor buttons. Edit the protocol by clicking one of the buttons to the left of
the text view
Figure 25. Protocol Editor window with buttons for editing protocols.
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CFX96 and CFX384 Systems Instruction Manual
2. Edit the temperature or hold time by clicking the default value in the graphic or text view
and entering a new value.
3. (Optional) Click the Step Options button to enter an increment or extend option to the
step (page 38).
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Protocols
2. Click the Add Plate Read to Step button to add a plate read to the selected step. If the
step already contains a plate read, the text on the button changes so that the same
button reads Remove Plate Read. Click to remove a plate read from the selected step.
2. Make sure the plate size for the gradient matches the block type of the instrument, 96
well or 384 well. Select the plate size for the gradient by selecting Tools > Gradient
Calculator in the Protocol Editor menu bar.
3. Edit the gradient temperature range by clicking the default temperature in the graphic or
text view and entering a new temperature. Alternatively, click the Step Options button to
enter the gradient range in the Step Options window (page 38).
4. Edit the hold time by clicking the default time in the graphic or text view and entering a
new time.
Figure 26 shows the inserted gradient step. The temperatures of each row in the gradient are
charted on the right side of the window.
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CFX96 and CFX384 Systems Instruction Manual
2. Edit the GOTO step number or number of GOTO repeats by clicking the default number
in the graphic or text view and entering a new value.
Figure 26 shows an inserted GOTO step at the end of the protocol. Notice that the GOTO loop
includes steps 2 through 4.
2. Edit the melt temperature range or increment time by clicking the default number in the
graphic or text view and entering a new value. Alternatively, click the Step Options
button to enter the gradient range in the Step Options window (page 38).
NOTE: You cannot insert a melt curve step inside a GOTO loop.
NOTE: The melt curve step includes a 30 sec hold at the beginning of the step that
is not shown in the protocol.
Figure 27 shows a melt curve step added after step 6.
Step Options
To change a step option for the selected step:
1. Select a step by clicking on the step in the graphic or text view.
2. Click the Step Options button to open the Step Options window.
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Protocols
Figure 28 shows the selected step with a gradient of 10oC. Notice that some options are not
available in a gradient step. A gradient step cannot include an increment or ramp rate change.
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CFX96 and CFX384 Systems Instruction Manual
• Calculated mode. When you enter a sample volume between 1 and 50 μl (96-well block)
or between 1 and 30 μl (384-well block) the thermal cycler calculates the sample
temperature based on the sample volume. This is the standard mode
• Block mode. When you enter a sample volume of zero (0) μl, the thermal cycler records
the sample temperature as the same as the measured block temperature
Protocol AutoWriter
Open the Protocol AutoWriter to quickly write protocols for PCR and real-time PCR runs. To
open the Protocol AutoWriter, select one of these options:
• Click the Protocol AutoWriter button in the main software window toolbar
• Select Tools > Protocol AutoWriter from the menu bar in the main software window
Figure 29 shows a protocol (bottom of window) written by the Protocol AutoWriter.
2. Enter the Annealing Temperature (Ta) and Amplicon Length in the boxes within the
Enter Target Values/Enzymes pane. If you do not know the annealing temperature for
primers, click the Ta Calculator button to enter the primer sequences and calculate the
annealing temperature. For information about the calculations used in the Ta Calculator
see Breslauer et al. 1986.
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Protocols
3. Select an enzyme type from the list of options (iTaq™, iProof™, or Other).
4. Add parameters in the Additional Parameters (Optional) pane if you want to add a
Gradient Range, Hot Start Activation temperature, or Final Extension time in the
protocol.
5. Select a protocol speed (Standard, Fast, or Ultrafast) by moving the sliding bar in the
Type pane. When you move the sliding bar, the software adjusts the total run time. Select
Real-time PCR to tell the software to collect fluorescence data.
6. Review the protocol in the Preview pane and total run time. Make changes as needed.
TIP: Enter the lid temperature and sample volume before each run by editing the
parameters in the Start Run tab (see “Start Run Tab” on page 27).
7. Click OK to save the new protocol or click Cancel to close the window without saving
the protocol.
TIP: To edit a protocol written with the Protocol AutoWriter, open the protocol file
(.prcl extension) in the Protocol Editor window (page 33).
NOTE: Bio-Rad Laboratories does not guarantee that running a protocol written in
the Protocol AutoWriter window will always result in a PCR product.
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CFX96 and CFX384 Systems Instruction Manual
5 Plates
Read this chapter for information about creating and editing plate files:
• Plate Editor window (page 41)
• Plate size and type (page 44)
• Scan mode (page 44)
• Select Fluorophores window (page 45)
• Well loading controls (page 45)
• Well Groups Manager window (page 50)
• Plate Spreadsheet View window (page 52)
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Plates
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CFX96 and CFX384 Systems Instruction Manual
Well Groups Open the Well Groups Manager window and set up well
groups for the current plate.
Trace Styles Select the colors and symbol used for the amplification
traces.
43
Plates
Plate Loading Show a quick guide about how to set up a plate and load
Guide the wells.
Scan Mode
The CFX96 system excites and detects fluorophores in six channels. The CFX384 system
excites and detects fluorophores in five channels. Both systems use multiple data acquisition
scan modes to collect fluorescence data from during a run.
Select one of these scan modes in the Plate Editor window toolbar:
• All Channels. Includes channels 1 through 5 on the CFX96 system or channels 1
through 4 on the CFX384 system
• SYBR/FAM only. Includes only channel 1 and provides a fast scan
• FRET. Includes only the FRET channel and provides a fast scan
Trace Styles
During plate setup and while a run is in progress, the color of the amplification traces can be
modified. These colors will be displayed as the data are being collected and the traces can be
viewed in the Real-time Status window. For more information on Trace Styles, see page 84.
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CFX96 and CFX384 Systems Instruction Manual
In this example, SYBR® is selected from the list of available fluorophores (Figure 31).
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Plates
• Sample Name. One identifier or condition that corresponds to the sample in each
loaded well, such as “0 hr,” “1 hr,” or “2 hr”
TIP: Target names and sample names must match between wells to compare data
in the Gene Expression tab of the Data Analysis window. Each name must contain
the same punctuation and spacing. For example, “Actin” is not the same as
“actin,” and “2hr” is not the same as “2 hr.” To facilitate consistency in names,
enter them in the Target and Sample Names Libraries in the Plate tab of the User
Preferences window (page 129).
• Biological Set Name. Select Tools > Show Biological Set Name to show this pane
in the well loading controls and then enter Biological Set names for one or more
wells
Select a well to load contents into by left-clicking in the plate view. Hold down the mouse
button and drag to select multiple wells. The buttons and lists on the right side of the plate
view include all the options needed to load the wells (Table 17).
Table 17. Options for loading the plate and wells in the Plate Editor.
Option Function
After selecting wells, the Sample Type must be loaded
first. Select a Sample Type from the pull-down menu to
load it in the selected wells, including Unknown,
Standard, NTC (no template control), Positive Control,
Negative Control, and NRT (no reverse transcriptase).
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CFX96 and CFX384 Systems Instruction Manual
Table 17. Options for loading the plate and wells in the Plate Editor. (continued)
Option Function
To load replicate numbers, selected wells must contain
identical well contents. If they do not, the software
disables this loading control.
Select Tools > Show Well Notes to show this pane. Enter
notes about one or more wells by selecting the wells and
typing the notes in the pull-down menu. Any notes you
add appear in the spreadsheet on the Quantification Data
tab.
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Plates
Table 17. Options for loading the plate and wells in the Plate Editor. (continued)
Option Function
Select Tools > Show Biological Set Name to show this
pane. Enter biological set information about one or more
wells by selecting the wells and typing a biological set
name in the pull-down menu. Entering Biological Set
Name information enables sample analysis in one of four
configurations defined by the Biological Set Analysis
Options.
Click the Experiment Settings button to open the
Experiment Settings window to manage the lists of
Targets and Samples and to set up a gene expression run.
Click the Clear Replicate # button to clear the replicate
numbers in the selected wells.
NOTE: Well contents can also be copied and pasted into other wells. To do this,
highlight the well that is to be copied (only one well can be copied at a time), right-
click, and select Copy Well. Highlight the wells into which content is be pasted and
select Paste Well. Depress and hold the control key to select non-contiguous wells
to paste content into.
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CFX96 and CFX384 Systems Instruction Manual
Figure 32 shows the Targets tab with the analysis settings shown.
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Plates
• Click a cell in the Color column to change the color of the targets graphed in the
Gene Expression chart
• Enter a number for the efficiency of a target. The software will calculate the relative
efficiency for a target using Auto Efficiency if the data for a target include a standard
curve. Alternatively, type a previously determined efficiency
To adjust the settings for a sample in the Samples tab:
• Click a color in the Color column to change the color of the samples graphed in the
Gene Expression chart
• Click a box in the Show Graph column to show the sample in the Gene Expression
chart using a color selected in the Color column
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CFX96 and CFX384 Systems Instruction Manual
2. Click Add to create a new group. The pull-down menu shows the group name as Group
1 for the first group.
3. Select the wells that will compose the well group in the plate view by clicking and
dragging across the group of wells. Selected wells turn blue in color (Figure 34).
4. (Optional) Change the name of the group by selecting the group name in the pull-down
menu and typing a new name.
6. (Optional) Delete well groups by selecting the group name in the pull-down list and
clicking the Delete button.
7. Click OK to finish and close the window, or click Cancel to close the window without
making changes.
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Plates
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CFX96 and CFX384 Systems Instruction Manual
6 Stand-Alone Operation
Read this chapter for information about running the CFX96™ system or CFX384™ system in
stand-alone mode:
• Control panel (page 53)
• Main menu (page 54)
• Creating a New Run (page 55)
• Exporting data for analysis (page 61)
• Creating a data file (page 63)
• Setting up email (page 64)
Control Panel
The CFX96 system or the CFX384 system can perform real-time PCR runs without a computer.
You can export the fluorescence data acquired during a run using the USB thumb key or
choose to have the data emailed directly to you if the C1000™ base is connected to the
Internet (see Exporting Data Using Email on page 62). The data require CFX Manager™
software for analysis. The control panel on the C1000 thermal cycler provides access to all the
functions needed to run the instrument. Figure 36 shows the components of the control panel.
Protocol
Alphanumeric
LCD AutoWriter key
keys
Navigation
Command keys
keys
USB port
(below)
Function keys
Figure 36. Thermal cycler control panel.
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Stand-Alone Operation
The control panel contains five sets of keys with the functions listed in table Table 19.
Table 19. Functions of keys on the control panel.
Key Function
COMMAND KEYS
RUN Select and run a protocol
EDIT Select and change a protocol
STATUS View the status of one or more running protocols
VIEW Switch between graphic and text view of a protocol
FUNCTION KEYS
F1, F2, F3, or F4 Function key buttons’ names and functions change on
each screen
ALPHANUMERIC KEYS
1 through 9 Enter numbers or letters of the alphabet. Press a key
multiple times to switch to each associated letter
0, INCUBATE Insert a zero, (infinity), or start instant incubation
decimal point (.) Enter a decimal point
minus sign (-) Enter a minus sign
PROTOCOL AUTOWRITER
Launch the Protocol AutoWriter
NAVIGATION KEYS
RIGHT arrow Move cursor to the right
LEFT arrow Move cursor to the left
UP arrow Move cursor up
DOWN arrow Move cursor down
ENTER Confirm a setting
BACK Cancel a function. Delete a letter, number, or word
Main Menu
When it starts, the CFX96 system or CFX384 system runs a self-test to verify proper functions
and then displays the main menu. Use the main menu to begin operating the instrument. The
main menu provides access to all system operations, displays the date and time, the name of
the logged-in user, the system status, the type of reaction module and thermal cycler name,
and any attached S1000™ thermal cyclers (Figure 37).
NOTE: To rename the thermal cycler, open the files library (Files [F2] button) and
then select Rename Cycler.
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CFX96 and CFX384 Systems Instruction Manual
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Stand-Alone Operation
2. To change the target temperature and the hold time in a temperature step, press the
arrow keys to navigate among steps and to select a parameter (temperature or time).
Press the alphanumeric keys to enter a new number for each parameter you highlight.
TIP: Connect a computer mouse via a USB port on the C1000 chassis to navigate.
NOTE: Press the VIEW key to switch between graphic and text view of the
protocol.
3. (Optional) To insert a new step, select the Insert (F1) button. To delete a step, select the
Delete (F3) button (Figure 38).
4. (Optional) To change step options, select the Options (F4) button (Figure 38). In the Step
Options window, select a parameter to change, including the temperature and time of
the step, or add/remove a plate read to the step (Figure 39).
NOTE: Press the alphanumeric keys to enter a Gradient Range ranging from 1 to
24oC.
TIP: Once a step has a gradient, you can edit the upper and lower temperatures in
the graphic or text view without opening the Options screen.
5. The GOTO step instructs the thermal cycler to repeat a set of steps in a loop to create
the cycles in the PCR run. Select a GOTO step, press the arrow keys to select and then
edit the step number in a GOTO step or to change the number of repeats.
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CFX96 and CFX384 Systems Instruction Manual
The sample volume you enter determines the temperature control mode that is used
during a run
TIP: Entering a sample volume from 1 to 50 selects Temperature Control mode,
which is the standard mode. Entering zero (0) selects Block mode. Temperature
mode is the recommended mode because it most accurately represents the actual
sample temperature.
• To change the default lid temperature, select the lid temperature box (LID) by
pressing the arrow keys (Figure 38). Use the alphanumeric keys to enter a new
temperature. For the CFX96 system, use a lid temperature of 105oC; for the CFX384
system use a lid temperature of 95oC
NOTE: Heating the lid prevents condensation in the sealed reaction vessels.
NOTE: The C1000 thermal cycler can store up to 20 real-time PCR runs.
2. Enter a protocol name if you have already not done so or edit the name previously
created in the Protocol window. Use arrow keys to select a destination folder (Figure 40).
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Stand-Alone Operation
3. Click Edit Filename (F1) and type a new name in the box (Figure 41).
5. Click Run (F2) to continue and run the protocol (Figure 42).
6. Edit the Sample Volume or Lid Temperature that will be used for the run (Figure 43).
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CFX96 and CFX384 Systems Instruction Manual
9. Select a Scan Mode to instruct the instrument in which channels to collect fluorescence
data during a run (Figure 44).
10.A default stand-alone data file name is created prior to the run. If you wish to change the
name, use the arrow keys to navigate to the Data File Name box, then press the
alphanumeric keys to type a new data file (.zpcr) name.
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Stand-Alone Operation
Monitoring a Run
When a run begins, the run status window appears. Review the information in this window to
monitor the progress of a run.
• Status. Press the STATUS command key to check the current status of the protocol,
pause the run, cancel the run, skip a step, or access the main menu (Figure 45)
• Time Status. Press the VIEW command key to see a full-screen count-down timer for
the protocol. Press the VIEW key again to switch back to the Status screen
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CFX96 and CFX384 Systems Instruction Manual
2. Use the up and down arrow keys to navigate to the RT_DATA folder and then press the
right arrow key to open the folder.
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Stand-Alone Operation
4. Press the Export File (F1) button to export a copy of the run data (.zpcr) to the USB key,
as shown in Figure 47.
5. Use the arrow keys to navigate to the folder on the USB key in which to save the file.
2. Using the arrow keys to select the Send email notification option.
4. Use the arrow keys to navigate to the Email Address box and then use the alphanumeric
keys to enter an email address.
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CFX96 and CFX384 Systems Instruction Manual
2. In the Run File Processor window, click the Select Plate button to import the name of
the plate file the software will use to create the data file (Figure 50).
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Stand-Alone Operation
NOTE: CFX Manager software checks the scan mode and plate size for the plate
file; these must match the current run settings that were started during the run.
TIP: Load a Quick Plate file to quickly access data from all the wells.
Setting up Email
After a run, a .zpcr file can be emailed directly to a computer running CFX Manager software.
To configure the outgoing email from the C1000 thermal cycler, follows these instructions:
1. Connect an ethernet cable to the port in the back of the C1000 chassis.
2. On the main menu, select Log In (F1) to log in to the thermal cycler as the administrator
(Figure 37 on page 55).
NOTE: The logged in user name appears under the date and time when you return
to the main window.
3. Select Utilities (F3) on the main screen (Figure 37 on page 55) to launch the Utilities
Menu.
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CFX96 and CFX384 Systems Instruction Manual
5. In the Administrator Settings Menu, select 9: SMTP Server Settings (Figure 51).
8. Type the name of the server in the text box using the virtual keypad.
NOTE: The SMTP server name will use the following nomenclature:
SMTP.YourInstitution.com. Do not use Bio-Rad.com in the name.
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Stand-Alone Operation
9. Click Save (F1) to save the name of the SMTP server (Figure 54).
10.The added server name will appear in the SMTP Server Names pull-down menu, as
shown in Figure 55.
11.Select Set Current Server (F3) to set the current server to be used for email (Figure 55).
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CFX96 and CFX384 Systems Instruction Manual
12.Use the arrow keys to select the Test Current Server button and click the Enter
navigation button (Figure 56).
13.Type an email address in the text box and select Test Server (F1) (Figure 57).
14.The C1000 thermal cycler will send an email to the entered address as a test of the
SMTP server configuration.
NOTE: Some SMTP servers do not allow attachments and others allow
attachments only up to certain sizes. If you will use CFX Manager software or the
C1000 chassis to email data files and/or reports, you may want to test your
server's ability to email attachments by checking the Test Attachment box and
setting the attachment size in MB with up to 5 megabytes (MB) or more.
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Stand-Alone Operation
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CFX96 and CFX384 Systems Instruction Manual
69
Data Analysis Overview
The Data Analysis window displays multiple tabs (Figure 58), each tab showing the analyzed
data for a specific analysis method or run-specific information. Tabs display only if the data
collected in the run are available for that type of analysis.
Well Group Select an existing well group name from the pull-down
menu. The default selection is All Wells.
View/Edit Plate Open the Plate Editor to view and edit the contents of
the wells.
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CFX96 and CFX384 Systems Instruction Manual
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Data Analysis Overview
Table 21. Menu bar items in the Data Analysis window. (continued)
Menu Item Command Function
Export RDML File Open a Save As window to specify an
RDML file name and location.
Custom Export... Open the Custom Export window in which
the fields to be exported and the file
format can be specified.
Export to LIMS Folder... Open a window to save data in a pre-
determined format to the LIMS folder.
Tools Reports... Open the Report for this data file.
Well Group Reports... Open the Well Group Report window to
generate reports for specified well groups.
Import Fluorophore Select a calibration file to apply to the
Calibration... current data file.
Replace Plate... Replace the current plate file in the data
analysis.
Quantification Tab
Each tab in the Data Analysis window displays data in charts and spreadsheets for a specific
analysis method and includes a well selector to select the data you want to show. The Data
Analysis window opens with the Quantification tab (Figure 60) in front. The Amplification chart
data in this tab should be used to determine the appropriate analysis settings for the run.
Figure 60. Quantification tab in the Data Analysis window with Group 1 selected.
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CFX96 and CFX384 Systems Instruction Manual
NOTE: The software links the data in the panes of each Data Analysis tab. For
example, highlighting a well by placing the mouse pointer over the well in the well
selector view highlights the data in all the other panes.
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Data Analysis Overview
2. Select Settings > Baseline Thresholds in the menu bar to open the Baseline Thresholds
window.
3. Adjust the crossing threshold (Figure 62) for the fluorophore by clicking User Defined
and entering a threshold number.
Baseline Settings
The software automatically sets the baseline individually for each well. Select the Baseline
Setting to determine the method of baseline subtraction for all fluorescence traces. Select
Settings > Baseline Setting to choose one of these three options:
• No Baseline Subtraction. The software displays the data as relative fluorescence
traces. Some analysis is not possible in this analysis mode and therefore the software
does not display the Gene Expression, End Point, and Allelic Discrimination tabs
• Baseline Subtracted. The software displays the data as baseline subtracted traces for
each fluorophore in a well. The software must baseline subtract the data to determine
quantification cycles, construct standard curves, and determine the concentration of
unknown samples. To generate a baseline subtracted trace, the software fits the best
straight line through the recorded fluorescence of each well during the baseline cycles
and then subtracts the best fit data from the background subtracted data at each cycle
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CFX96 and CFX384 Systems Instruction Manual
• Baseline Subtracted Curve Fit. The software displays the data as baseline subtracted
traces and the software smoothes the baseline subtracted curve using a centered mean
filter. This process is performed so that each Cq is left invariant
Along with the latter two options above, the following can also be selected:
• Apply Fluorescent Drift Correction. For wells that have abnormally drifting RFU values
during the initial few cycles of a run, the software derives an estimated baseline from
adjacent wells for which a horizontal baseline was successfully generated
2. Select Settings > Baseline Threshold to open the Baseline Threshold window.
To adjust the begin and end baseline cycle for each well:
1. In the Baseline Cycles pane, select one or more wells by clicking the row number,
clicking the top left corner to select all wells, holding down the Control key to select
multiple individual wells, or holding down the shift key to select multiple wells in a row.
2. Adjust the Baseline Begin cycle and Baseline End cycle for all selected wells, or
change the Begin and End cycle number at the bottom of the spreadsheet (Figure 62).
3. To revert the settings back to the last saved values, click Reset All User Defined
Values.
Analysis Mode
Data can be analyzed and displayed grouped by either fluorophore or target name. To choose
the data analysis mode, select Settings > Analysis Mode or make a selection from the
Analysis Mode drop-down menu in the toolbar.
When Fluorophore is chosen, data traces are displayed by fluorophore as indicated in the
plate setup for that run. Individual fluorophore data are displayed in the amplification and
standard curve chart (if available) by checking the appropriate fluorophore selector
checkboxes located below the amplification chart.
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Data Analysis Overview
Cycles to Analyze
To restrict data analysis to a specified range of cycles, select Settings > Cycles to Analyze.
Select the starting cycle and the ending cycle using the arrow buttons or by typing in the
desired values and pressing Enter. Click the Restore Defaults button to return to the cycles
originally used for analysis.
NOTE: Removing cycles from the beginning of a run can have a significant impact
on baselining.
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CFX96 and CFX384 Systems Instruction Manual
Well Selectors
Click the wells in the well selector to show or hide the data in the charts or spreadsheets
throughout the Data Analysis window:
• To hide one well, highlight and click the individual well. To show that well, highlight
and click the well again
• To hide multiple wells, click and drag across the wells you want to select. To show
those wells, click and drag across the wells again
• Click the top left corner of the plate to hide all the wells. Click the top left corner
again to show all wells
• Click the start of a column or row to hide those wells. Click the column or row again
to show the wells
Only wells loaded with content (entered in the Plate Editor) can be selected in the well selector,
and their color shows if they are selected. As shown in Figure 65, the well selector shows
these three types of wells:
• Selected, loaded wells (blue). These wells contain a loaded Unk (unknown) sample
type. The data from these wells appear in the Data Analysis window
• Unselected, loaded wells (light gray). These wells contain loaded Std and Pos sample
types. The data from unselected wells do not appear in the Data Analysis window
• Empty wells (dark gray). These wells were not loaded in the Plate Editor window
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Data Analysis Overview
3. Click Exclude Wells in Analysis (Figure 67) to exclude the selected wells. This checkbox
is at the bottom of the Plate Editor controls on the right side of the window.
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CFX96 and CFX384 Systems Instruction Manual
4. The excluded well(s) are marked with an asterisk (*) in the Plate Editor window.
Alternatively, to permanently remove wells from analysis, clear the contents from wells in the
Plate Editor by clicking the Clear Wells button.
WARNING! You will have to reenter any well content that are cleared.
Charts
Each chart in the Data Analysis window displays the data in a different graph and includes
options for adjusting the data. To magnify an area of the chart, select an area by clicking and
dragging the mouse. The software resizes the chart and centers it on the selected area.
TIP: Return the chart to a full view by right-clicking on the chart and selecting Set
Scale to Default from the right-click menu.
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Data Analysis Overview
NOTE: Menu items that apply to specific charts are described in the next chapter,
Data Analysis Windows (page 83).
Spreadsheets
The spreadsheets shown in Data Analysis include options for sorting and transferring the data.
Sort the columns by one of these methods:
• Click and drag a column to a new location in the selected table
• Click the column header to sort the data in Ascending or Descending order
To sort up to three columns of data in the Sort window, follow these steps:
1. Right-click on the spreadsheet to open the menu and select Sort.
2. In the Sort window, select the first column title to sort. Sort the data in Ascending or
Descending order.
3. Select more than one column title by selecting the title in the pull-down menu. Select
Ascending or Descending to sort the column in that order.
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CFX96 and CFX384 Systems Instruction Manual
Export
Four export options are accessible from the Export drop-down menu.
Custom Export
Select Export > Custom Export to open a window in which the fields to be exported and the
file format can be customized (Figure 68).
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Data Analysis Overview
3. Click the Export button to open a Save As window to specify the file name and location
for the exported file.
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CFX96 and CFX384 Systems Instruction Manual
Quantification Tab
Use the data in the Quantification tab (Figure 69) to set the data analysis conditions, including
the baseline settings for individual wells and the threshold settings. The Quantification tab
shows data in these four views:
• Amplification chart. Shows the relative fluorescence units (RFUs) for each well at every
cycle. Each trace in the chart represents data from a single fluorophore in one well
• Standard curve. This graph is shown only if the run includes wells designated as
Sample Type Standard. It shows a standard curve with the threshold cycle plotted
against the log of the starting quantity. The legend shows the Reaction Efficiency (E) for
each fluorophore in the wells with a standard sample type
• Well selector. Selects the wells with the fluorescence data you want to show
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Data Analysis Windows
Figure 69. Layout for the Quantification tab in the Data Analysis window.
Fluorophore Selector
To select the fluorophore data to display in the Quantification tab charts and spreadsheets,
click the fluorophore selector below the Amplification chart. Click the box next to the
fluorophore name to show or hide the fluorophore data throughout the data analysis window.
2. Click the Trace Styles button in the Data Analysis toolbar, select Settings > Trace
Styles in the Data Analysis menu bar, or right-click on a trace and select Trace Styles.
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Data Analysis Windows
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CFX96 and CFX384 Systems Instruction Manual
Baseline Thresholds... Open the Baseline Thresholds window to change the baseline
or thresholds of each fluorophore (changes appear in the
Amplification chart in the Quantification tab)
Results Spreadsheet
Select a Results spreadsheet (Figure 73) to see data for each well in the plate.
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Data Analysis Windows
The Results spreadsheet includes the types of information listed in Table 27.
Table 27. Results spreadsheet content.
Information Description
Well Well position in the plate
Fluor Fluorophore detected
Target Amplification target name (gene)
Content Sample type and Replicate #
Sample Sample description
Biological Set Name Name of the biological set
Cq Quantification cycle
Cq Mean Mean of the quantification cycle for the replicate group
Cq Std. Dev Standard deviation of the quantification cycle for the replicate
group
Starting Quantity (SQ) Estimate of the starting quantity of the target
Log Starting Quantity Log of the starting quantity
SQ Mean Mean of the starting quantity
SQ Std. Dev Standard deviation of the starting quantity
Set Point Temperature of sample in the well for a gradient step
Sample Note One round of denaturation, annealing, and extension or one
round of annealing and extension steps in a protocol
Figure 74. Standard Curve Results spreadsheet in the Quantification Data tab.
These values can be copied and pasted into a document by right-clicking and selecting Copy
or a file can be created by choosing one of the Export options.
Information Description
Fluor (or Target) Fluorophore (or Target) detected
Efficiency % Reaction efficiency
Slope Slope of the standard curve
y-intercept Point at which the curve intercepts the y-axis
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Information Description
R^2 Coefficient of determination
Plate Spreadsheet
Select the Plate spreadsheet to see a plate map of the data for one fluorophore at a time.
Select each fluorophore by clicking a tab at the bottom of the spreadsheet. Figure 75 shows
the Plate spreadsheet as a plate map.
RFU Spreadsheet
Select the RFU spreadsheet to see the relative fluorescence units (RFU) readings for each well
acquired at each cycle of the run. Select individual fluorophores by clicking a tab at the bottom
of the spreadsheet. The well number appears at the top of each column and the cycle number
appears to the left of each row (Figure 76).
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The software plots the RFU data collected during a melt curve as a function of temperature. To
analyze melt peak data, the software assigns a beginning and ending temperature to each
peak by moving the threshold bar. The floor of the peak area is specified by the position of the
melt threshold bar. A valid peak must have a minimum height relative to the distance between
the threshold bar and the height of the highest peak.
Open the Melt Curve tab (Figure 77) to determine the Tm of amplified PCR products. This tab
shows the melt curve data in these four views:
• Melt Curve. View the real-time data for each fluorophore as RFUs per temperature for
each well
• Melt Peak. View the negative regression of the RFU data per temperature for each well
• Well selector. Select wells to show or hide the data
• Peak spreadsheet. View a spreadsheet of the data collected in the selected well
NOTE: This spreadsheet only shows as many as two peaks for each trace. To see
more peaks, click the Melt Curve Data tab (page 91).
Figure 77. Layout of the Melt Curve tab in the Data Analysis window.
Adjust the Melt Curve data by any of these methods:
• Click and drag the threshold bars in the Melt Peak chart to include or exclude peaks
in data analysis
• Select Positive in the Peaks pull-down menu to show the spreadsheet data for the
peaks above the Melt Threshold line or select Negative to view the spreadsheet data
for the peaks below the Melt Threshold line
• Open the Trace Styles window to change the color of the traces in the Melt Curve
and Melt Peak charts
• Select a number in the Step Number selector (page 73) to view the Melt Curve data
at another step in the protocol. The list shows more than one step if the protocol
includes plate read (camera icon) in two or more melt curve steps
• Select wells in the well selector to focus on subsets of the data
• Select a well group (page 73) 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
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Figure 78. Melt Peaks spreadsheet in the Melt Curve Data tab.
The Melt Peaks spreadsheet (Figure 78) includes the types of information shown in Table 29.
Table 29. Melt Peaks spreadsheet content.
Information Description
Well Well position in the plate
Fluor Fluorophore detected
Content Sample Type listed in the Plate Editor window
Target Amplification target (gene)
Sample Sample Name listed in the Plate Editor window
Melt Temperature The melting temperature of each product, listed as one peak (highest) per
row in the spreadsheet
Peak Height Height of the peak
Begin Temperature at the beginning of the peak
Temperature
End Temperature Temperature at the end of the peak
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Plate Spreadsheet
Select the Plate spreadsheet (Figure 79) to view melt curve data in a plate format.
RFU Spreadsheet
Select the RFU spreadsheet to view the fluorescence for each well at each cycle acquired
during the melt curve (Figure 80).
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-d(RFU)/dT Spreadsheet
Select the -d(RFU)/dT spreadsheet to view the types of data shown in Figure 81.
Figure 81. The -d(RFU)/dT spreadsheet in the Melt Curve Data tab.
Table 32 lists the types of information shown in the -d(RFU)/dT spreadsheet.
Table 32. -d(RFU)/dT spreadsheet content.
Information Description
Well number (A1, A2, A3, A4, Well position in the plate for the loaded wells
A5...)
-d(RFU)/dT Negative rate of change in RFU as temperature (T) changes
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The End Point tab shows the average RFU values to determine whether or not the target was
amplified by the last (end) cycle. Use these data to determine if a specific target sequence is
present (positive) in a sample. Positive targets have higher RFU values than the cutoff level you
define.
TIP: To create an end point protocol, open the Protocol tab (Run Setup window)
and select Options > End Point Only Run.
The software displays these data in the End Point tab:
• Settings. Adjust data analysis settings
• Results. Shows the results immediately after you adjust the Settings
• Well Selector. Select the wells with the end point data you want to show
• Well spreadsheet. Shows a spreadsheet of the end RFU collected in the selected wells
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• Well spreadsheet. Shows a spreadsheet listing the allelic discrimination data collected
in the selected wells
Figure 83. Layout of the Allelic Discrimination tab in the Data Analysis window.
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• Change a call manually by highlighting a row in the spreadsheet and then selecting
an option in the Call Selected Alleles list (including Allele 1, Allele 2, Heterozygote,
None, Unknown, Control 1, or Control 2)
• Click the Restore Default Thresholds button to restore the vertical and horizontal
bars to their original position, which are indicated by the numbers next to the bars
• Select the Cq Display Mode to view the data as threshold levels. Select RFU
Display Mode to view the data in relative fluorescence units at the selected cycle
• Select Normalize Data to normalize the RFU data shown in the chart and
spreadsheet
Normalization changes the data on the chart to a range from 0 to 1 on both axes. To normalize
the data, the plate must contain wells with “no template control” (NTC) sample types for both
Allele 1 and Allele 2. For this plot, the RFU data are normalized to the NTC values as a linear
combination of Allele 1- and Allele 2-specific RFUs. This plot is an effective way to present
RFU data.
The calculation for normalized RFU follows the formulas presented in Livak et al. (1995).
A1
Normalized A 1 = --------------------------------------------------------------
A 1 + A 2 + x ( NTC A1 + A2 )
Where:
• A1 represents RFU for Allele 1
• A2 represents RFU for Allele 2
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The Load a Preset View drop-down list offers a selection of display format templates. The
default view displayed is dependent on the file being analyzed. For example, if Melt Curve data
are present, the Amp+Melt default view is displayed.
The data view can be further customized by:
• Selecting an alternate preset view from the drop-down list
• Using the drop-down menu located at the top of an individual pane
• Using the Rows and Columns drop-down selection options
• Changing individual pane dimensions by clicking and dragging the bars at the
periphery of each pane
Customized views can be saved as new preset templates by clicking Save as Preset. Existing
presets can be deleted, renamed, or the default preset views restored using Manage Presets.
QC Tab
Open the QC tab to quickly assess the quality of the run data based on the rules defined in the
QC tab in the User Preferences window (see QC Tab on page 132 for more information).
The QC tab is divided into four areas (Figure 85):
• Amplification chart. Shows the RFU for each well at every cycle. Each trace in the chart
represents data from a single fluorophore in one well
• QC rules table. Shows the available QC rules and the settings that define each rule.
Applied QC rules are indicated by a checkmark. A QC rule can be removed by
unchecking the Use box
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• Well selector. Selects the wells with the fluorescence data you want to show
• QC Rule Summary. Shows the selected QC rule and highlights wells that fail the rule
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2. Click the Report button in the Data Analysis toolbar to open the Report window.
3. Change the options you want to include in the report. The report opens with default
options selected. Click the checkboxes in the report options list to change whole
categories or individual options within a category.
NOTE: The data that appear in the report are dependent on the current selections
within the tabs of the Data Analysis window. For example, a quantification run
might not contain a standard curve, and therefore those data do not appear in the
Data Analysis window or in the data report.
4. The ordering of categories and items within a report can be changed by clicking and
dragging these to the desired relative position. Items can be reordered only within the
categories to which they belong.
5. Click the Update Report button to update the Report Preview with any changes.
6. Print or save the report. Click the Print Report button in the toolbar to print the current
report. Select File > Save to save the report as a PDF- (Adobe Acrobat Reader file),
MHT- (Microsoft document), or MHTML- (Microsoft document) formatted file and select a
location to store the file. Select File > Save As to save the report with a new name or in a
new location.
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7. (Optional) Create a report template with the information you want. To save the current
report settings in a template, select Template > Save or Save As. Then load the report
template the next time you want to make a new report.
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Table 35. Data analysis report categories in the options list. (continued)
Category Option Description
Allelic Discrimination
Analysis Settings Includes display mode, fluorophores,
cycle, thresholds, and normalized data
Allelic Discrimination Copy of the allelic discrimination chart
Chart
Data Spreadsheet listing the data in each well
End Point
Analysis Settings Includes fluorophore, end cycles to
average, mode, lowest RFU value,
highest RFU value, and cut off value
Data Spreadsheet listing the data in each well
QC Parameters
Data Spreadsheet listing the parameters for
each QC rule
2. From the Well Groups Reports window (Figure 88) the Well Groups, Amplification Steps,
and Melt Steps to be included in the reports can be specified by checking the
appropriate box.
3. The destination folder can be changed to another location by clicking the ... button.
4. Select Choose a Report Template to choose a template other the default. Click the ...
button to browse for the template file.
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5. Once the reports have been generated, the destination folder can be opened and the
reports viewed by checking the appropriate box.
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Gene Expression
With the use of stringently qualified controls in your reactions, you can perform a gene
expression run to normalize the relative differences in a target concentration among samples.
Typically, message levels for one or more reference genes are used to normalize the
expression levels of a gene of interest. Reference genes take into account loading differences
or other variations represented in each sample and they should not be regulated in the
biological system being studied.
Open the Gene Expression tab to evaluate relative differences between PCR reactions in two
or more wells. For example, you can evaluate relative numbers of viral genomes or relative
number of transfected sequences in a PCR reaction. The most common application for gene
expression study is the comparison of cDNA concentration in more than one reaction to
estimate the levels of steady state messenger RNA.
The software calculates the relative expression level of a target with one of these scenarios:
• Relative expression level of a target sequence (Target 1) relative to another target
(Target 2). For example, the amount of one gene relative to another gene under the
same sample treatment
• Relative expression level of one target sequence in one sample compared to the
same target under different sample treatments. For example, the relative amount of
one gene relative to itself under different temporal, geographical, or developmental
conditions
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Figure 91. Layout of the Gene Expression tab in the Data Analysis window.
TIP: Right-click on the chart to select right-click menu options. Select Sort from
this menu to rearrange the order of the Target and Sample names in the chart.
2. Review the data in the Quantification tab of the Data Analysis window. Make adjustments
to the data, such as changing the threshold and the Analysis Mode.
4. Choose a control in the Samples tab of the Experiment Settings window. If a control is
assigned, the software normalizes the relative quantities for all genes to the control
quantity, which is set to 1.
5. Select reference genes for this run in the Target tab of the Experiment Settings window.
Gene expression analysis requires one reference among the targets in your samples.
6. Select Normalized Expression (ΔΔCq) if it is not already selected and then view the
expression levels in the Gene Expression tab.
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Relative Quantity
By definition, relative quantity (ΔCq) data are not normalized. This method is used to quantitate
samples that do not include any reference genes (targets). Typically, researchers are confident
in one of the following considerations when they set up their run:
• Each sample represents the same amount of template in each biological sample,
possibly the same mass of RNA or cDNA in each well
• Any variance in the amount of biological sample loaded will be normalized after the
run by some method in the data analysis outside of the software. For example, a
researcher might choose to simply divide the relative quantity value by the
normalizing factor, possibly the mass of nucleic acid loaded for each sample, or the
number of cells from which the nucleic acid was isolated
Select Relative Quantity (ΔCq) from the drop-down menu in the chart controls of the Gene
Expression tab to run a Relative Quantity (ΔCq) analysis.
TIP: To compare results to data from other gene expression runs, open a new Gene
Study (page 115) or add a data file to an existing Gene Study.
GRAPH DATA
Graph data options allow you to present the data in the graph with one of these two options:
• Relative to control. Graph the data with the axis scaled from 0 to 1. If you assign a
control in your run, select this option to quickly visualize upregulation and
downregulation of the target
• Relative to zero. Graph the data with the origin at zero
X-AXIS OPTIONS
The X-axis option allows you to select the x-axis data of the Gene Expression graph:
• Target. Select this option to graph the target names on the x-axis
• Sample. Select this option to graph the sample names on the x-axis
Y-AXIS OPTIONS
The Y-axis option allows you to show the Gene Expression graph in one of these three scales:
• Linear. Select this option to show a linear scale
• Log 2. Select this option to evaluate samples across a large dynamic range
• Log 10. Select this option to evaluate samples across a very large dynamic range
SCALING OPTIONS
Select Normalized Gene Expression (ΔΔCq) to activate the scaling options in the Gene
Expression graph. Select one of these scaling options to calculate and present your data in a
manner that best suits your run design:
• Unscaled expression. This option presents the unscaled normalized gene expression
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• Highest expression. Scale the normalized gene expression to the highest for each
target by dividing the expression level of each sample by the highest level of expression
in all the samples. This scaling option uses the scaled to highest formula
• Lowest expression. Recalculate the normalized gene expression for each target by
dividing the expression level of each sample by the lowest level of expression in all the
samples. This scaling option uses the scaled to lowest formula
ERROR TYPE
Select an option for the type of error calculations (error bars) in the Gene Expression graph:
• Standard Error of the Mean (default, SEMs)
• Standard Deviation (Std Devs)
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Table 39. Information in the Gene Expression spreadsheet with Show Details selected.
Information Description
Unscaled Expression Calculated unscaled expression
Unscaled Expression SD Calculated standard deviation unscaled expression
Corrected Unscaled Calculated standard deviation of the unscaled expression
Expression SD
Unscaled Expression SEM Calculated standard error of the mean unscaled expression
Corrected Unscaled Calculated standard error of the mean of the unscaled
Expression SEM expression
Expression Relative expression level
Wells Well number in the plate
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Figure 93. Targets tab in the Experiment Settings window with Analysis Settings
selected.
To adjust the settings for a sample in the Samples tab:
• Click a color in the Color column to change the color of the samples graphed in the
Gene Expression chart
• Click a box in the Show Chart column to show the sample in the Gene Expression
chart using a color that is selected in the Color column
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Figure 94 shows the samples with the Show Chart option selected.
Figure 94. Samples tab in the Experiment Settings window with Analysis Settings
selected.
Gene Study
Create a Gene Study to compare gene expression data from one or more real-time PCR
experiments using an inter-run calibrator to normalize between the experiments. Create a
Gene Study by adding data from one or more data files (.pcrd extension) to the Gene Study;
the software groups them into a single file (.mgxd extension).
NOTE: The maximum number of samples you can analyze in a Gene Study is
limited by the size of the computer's RAM and virtual memory.
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To find the dominant inter-run calibrator, the software calculates the average of the ΔCq values
for all inter-run calibrators of a given target and then uses a multitiered algorithm to determine
the dominant inter-run calibrator within all the data. The algorithm for finding the dominant
inter-run calibrator includes the following hierarchy:
1. Set the dominant calibrator to the target with the highest number of common replicate
groups in a given pair-wise comparison.
2. If any target has the same number of common replicate groups, then set the dominant
calibrator to the target with the smallest range of ΔCq values in pair-wise comparisons.
The range is examined by comparing the absolute value of the difference between the
maximum and minimum ΔCq for the inter-run calibrators of a given target.
3. If any target has an identical range as the ΔCq values, then set the dominant calibrator to
the target with the smallest absolute value of average ΔCq for eligible inter-run calibrator
samples.
4. If any target has identical average ΔCq absolute values, then set the dominant calibrator
to the replicate group with the smallest ΔCq.
NOTE: The first data file imported into the Gene Study will always serve as the
“hub” file for pair-wise data comparison during inter-run calibration.
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The Study Setup tab lists the data files in the Gene Study, as described in Table 40.
Table 40. Study Setup tab in the Gene Study window.
Column Title Description
File Name Name of the run data file (.pcrd extension)
File Folder Directory that stores the data file for each run in the Gene
Study
Date Created Date the run data were collected
Well Group Name Name of the well group that was selected when the file was
added to the Gene Study
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• Y-axis options. Change the labels on the y-axis of the graph, including Linear, Log 2, or
Log 10
• Scaling Options. Choose Highest value, Lowest value, or leave the data Unscaled.
This option is available only when your samples do not contain controls
• Graph Error. Select the multiplier for standard deviation bars in the graph, including ± 1,
2, or 3
• Experiment Settings button. Choose the show options for targets and samples in the
Experiment Settings window
• Show Details checkbox. Click Show Details to add more columns of data to the chart
Highlighting a sample in the Gene Expression chart highlights the corresponding cell in the
spreadsheet below the chart (Figure 96).
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Table 41. Information in the spreadsheet on the Study Analysis tab. (continued)
Information Description
Expression Normalized Gene Expression (ΔΔCq) or Relative Quantity
(ΔCq), depending on the selected mode
Expression SEM (or SD) Standard Error of the Mean or Standard Deviation, depending
on the selected option
Corrected Expression SEM Corrected value calculation for Standard Error of the Mean
(or SD) (SEM) or Standard Deviation (SD) of the relative expression,
depending on the selected option
Mean Cq Mean of the quantification cycle
Cq SEM (or SD) Standard Error of the Mean or Standard Deviation of the
quantification cycle, depending on the selected option
2. Select Tools > Reports to open the Gene Study report window.
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3. Click the checkboxes in the report options list to select and remove options to choose
the data to display. Select the options shown in Table 43.
Table 43. Categories for a Gene Study report.
Category Option Description
Header Title, subtitle, and logo for the report
Report Information Date, user name, data file name, data file
path, and the selected well group
Gene Study File List List of all the data files in the Gene Study
Notes Notes about the data report
Analysis Parameters A list of the selected analysis parameters
Chart Gene Expression chart showing the data
Target Names List of targets in the Gene Study
Sample Names List of samples in the Gene Study
Data Spreadsheet that shows the data
Inter-Run Calibration Inter-run calibration data
4. Fill in the text for the report by entering text and images in option panes (Figure 98).
Figure 98. Example of Header and Logo options in a Gene Study report.
5. Click the Update Report button to update the report preview pane. The report preview
pane shows a view of the Report.
6. Print or save the report. Click the Print button in the toolbar to print the current report.
Select File > Save to save the report as a PDF- (Adobe Acrobat Reader file), MHT-
(Microsoft document), or MHTML- (Microsoft document) formatted file and select a
location to store the file. Select File > Save As to save the report with a new name or in a
new location.
7. Create a report template once you create a report with the content you want to include in
all reports. To create a template, select Template > Save or Save As and save the
current report as a template.
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Reaction Efficiency
Evidence suggests that using accurate measure of efficiencies for each primer and probe sets
will give you more accurate results when analyzing gene expression data. The default value of
efficiency used in the gene expression calculations is 100%. To evaluate the reaction
efficiency, generate a standard curve using serial dilutions of a representative sample across a
relevant dynamic range and then record the efficiency for subsequent gene expression
analysis. If your run includes a standard curve, then the software automatically calculates the
efficiency and displays it under the Standard Curve on the Quantification tab when Auto
Efficiency is checked in the Targets tab in the Experiment Settings window.
The efficiency (E) in the efficiency formulas refers to the “efficiencies” as described by Pfaffl
(2001) and Vandesompele et al. (2002). In these publications, an efficiency of 2 (perfect
doubling with every cycle) is equivalent to 100% efficiency in this software. You have the
option to convert your efficiency calculations to those used in the software by using the
following mathematical relationships:
• E = (% Efficiency * 0.01) + 1
• % Efficiency = (E - 1) * 100
Relative Quantity
The relative quantity (ΔCq) for any sample (GOI) is calculated with this formula:
(C – C q (sample) )
Relative Quantity sample (GOI) = EGOIq (MIN)
Where:
• E = Efficiency of primer and probe set. This efficiency is calculated with the formula
(% Efficiency * 0.01) + 1, where 100% efficiency = 2
• Cq (MIN) = Average Cq for the Sample with the lowest average Cq for GOI
• Cq (sample) = Average Cq for the Sample
• GOI = Gene of interest (one target)
Where:
• E = Efficiency of primer and probe set. This efficiency is calculated with the formula
(% Efficiency * 0.01) + 1, where 100% efficiency = 2
• Cq (control) = Average Cq for the control sample
• Cq (sample) = Average Cq for any samples with a GOI
• GOI = Gene of interest (one target)
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Where:
• SD Relative Quantity = standard deviation of the relative quantity
• SD Cq sample = Standard deviation of the Cq for the sample (GOI)
• Relative Quantity = Relative quantity of the sample
• E = Efficiency of primer and probe set. This efficiency is calculated with the formula
(% Efficiency * 0.01) + 1, where 100% efficiency = 2
• GOI = Gene of interest (one target)
Normalization Factor
The denominator of the normalized expression equation is referred to as the normalization
factor. The normalization factor is the geometric mean of the relative quantities of all the
reference targets (genes) for a given sample, as described in this formula:
1
--
n
Normalization Factor sample (GOI) = (RQ sample (Ref 1) × RQ sample (Ref 2) × … × RQ sample (Ref n) )
Where:
• RQ = Relative quantity
• n = Number of reference targets
• GOI = Gene of interest (one target)
Normalized Expression
Normalized expression (ΔΔCq) is the relative quantity of your target (gene) normalized to the
quantities of the reference targets (genes or sequences) in your biological system. To select
reference targets, open the Experiment Settings window and click the reference column for
each target that serves as a reference gene.
The calculation for normalized expression is described in the following formula, which uses the
calculated Relative Quantity (RQ) calculation:
RQ sample (GOI)
Normalized Expression sample (GOI) = ----------------------------------------------------------------------------------------------------------------------------------------------------1-
--
n
( RQ sample (Ref 1) × RQ sample (Ref 2) × … × RQ sample (Ref n) )
Where:
• RQ = Relative Quantity of a sample
• Ref = Reference target in a run that includes one or more reference targets in each
sample
• GOI = Gene of interest (one target)
Provided that reference targets do not change their expression level in your biological system,
the calculation of normalized expression will account for loading differences or variations in
cell number that are represented in each of your samples.
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Where:
• RQ = Relative quantity of a sample
• SD = Standard deviation
• NF = Normalization factor
• Ref = Reference target
• n = Number of reference targets
When a control sample is assigned, you do not need to perform this re-scaling function on the
standard deviation, as shown in the following formula:
SD NF sample 2 SD RQ sample (GOI) 2
SD NE sample (GOI) = NE sample (GOI) × § ----------------------------· + § ---------------------------------------·
© NF sample ¹ © RQ sample (GOI) ¹
Where:
• NE = Normalized expression
• RQ = Relative quantity of a sample
• SD = Standard deviation
• GOI = Gene of interest (one target)
Where:
• GOI = Gene of interest (target)
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Where:
• GOI = Gene of interest (target)
Where:
• NE = Normalized expression
• SD = Standard deviation
• GOI = Gene of interest (target)
• MAX = Highest expression level
• MIN = Lowest expression level
Where
• n = Number of reference targets (genes)
• SD = Standard deviation
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The standard error for the normalization factor in the normalized expression formula is shown
here:
SE RQ sample (Ref 1) 2 SE RQsample (Ref 2) 2 SE RQ sample (Ref n) 2
SE NF n = NF n × § ---------------------------------------------------------· + § ---------------------------------------------------------· + … + § ---------------------------------------------------------·
© n × SE RQ sample (Ref 1)¹ © n × SE RQ sample (Ref 2)¹ © n × SE RQ sample (Ref n)¹
Where:
• n = Number of reference targets
• SE = Standard error
• NF = Normalized expression
• RQ = Relative quantity
The standard error for normalized gene of interest (GOI) formula is shown here:
SE NF n 2 SE GOI 2
SE GOI n = GOI n × § ----------------· + § -----------------·
© NF n ¹ © GOI ¹
Where:
• SE = Standard error
• GOI = Gene of interest (one target)
• NF = Normalization factor
• n = Number of reference targets
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2. Select a name from the User Name pull-down list. The default is “Admin” (administrator).
4. Click OK to close the Login dialog box and open the software.
5. To add a new user name and password, contact your software administrator.
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Users and Preferences
Change a Password
Change a password by following these steps:
1. Select User > Change Password from the main software window menu to open the
Change Password dialog box (Figure 100).
3. Enter the new password in the New Password and the Confirm New Password boxes,
respectively.
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TIP: Click the Restore Defaults button to restore all settings to the default settings
shown in this image. Then click OK to save the settings and close the window.
Email Tab
Select the Email tab (Figure 101) to enter the email addresses where you want to receive
confirmation of the completion of runs. The software can send an attached data file or report
file with the email when the checkboxes next to these options are checked.
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Data Files and/or Reports, you may want to test your server's ability to email
attachments by checking the Test Attachment box and setting the Attachment Size
in MB with up to 5 megabytes (MB) or more.
Files Tab
Select the Files tab (Figure 103) to list the default locations for opening and saving files.
• Default Folder for File Creation. Select a default folder where you want to save new
files. Select a location for each file type (Protocol, Plate, Data, or Gene Study file)
• File Selection for Run Setup. Select the default protocol and plate files that appear
when you open the Experiment Setup window
• Data File Prefix. Define the beginning text of the file name for data files. The default
setting instructs the software to create a file name that starts with the User (user name of
the user who is currently logged in to software), Date (file creation date), and Instrument
Name (instrument serial number or name)
Protocol Tab
Select the Protocol tab (Figure 104) in the User Preferences window to specify the default
settings for a new protocol file in the Protocol Editor window:
• Protocol Editor. Set the default settings that appear in the Protocol Editor. Select a
default Sample Volume to describe the volume of each sample in the wells (in μl) and
select a Lid Shutoff Temperature at which the lid heater turns off during a run
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• Protocol AutoWriter. Selects default settings that appear in the Protocol AutoWriter,
including default Annealing Temperature for experiments that use iProof™, iTaq™, or
other polymerases and the default amplicon length
Plate Tab
Select the Plate tab in the User Preferences window (Figure 105) to specify the following
default settings for a new Plate file in the Plate Editor window:
• Plate Type. Select the default plate well type from the list
• Plate Size. Select the default plate size from the list
• Units. Select the units used to describe the concentration of the starting template for
wells that contain standards. The software uses these units to create a standard curve in
the Data Analysis Quantification tab
• Scientific Notation. Select scientific notation to view concentration units in that notation
• Scan Mode. Select a default scan mode to set the number of channels to scan during a
run
• Fluorophores. Click checkboxes to select the default fluorophores that appear in the
Plate Editor well loading controls
• Libraries. Enter the target and sample names that you typically use in your experiments.
Enter target names to list genes and sequences, and enter sample names to list
conditions for experiment samples. These names appear in the lists of the Targets tab
and Samples tab in the Experiment Settings window
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Specify the default settings for a new Gene Expression data file:
• Relative to. Select a control or zero. To graph the gene expression data originating at 1
(relative to a control), select Control. When you assign a control sample in the
Experiment Setup window, the software automatically defaults to calculate the data
relative to that control. Select Relative to zero to instruct the software to ignore the
control, which is the default selection when no control sample is assigned in the
Experiment Settings window
• X-Axis. Graph the Target or the Sample on the x-axis
• Y-Axis. Graph Linear, Log 2, or Log 10 scale on the y-axis
• Scaling. Select a scaling option for the graph. Leave the graph unscaled. Alternatively,
choose a scaling option to scale to the Highest value or to the Lowest value
• Method. Set the default analysis mode, including normalized expression (ΔΔCq) or
relative expression (ΔCq)
• Error Bar. Select Std Dev. for standard deviation or Std. Error Mean for the standard
error of the mean
• Error Bar Multiplier. Select the standard deviation multiplier to graph the error bars. The
default is 1. Change the multiplier to either 2 or 3
QC Tab
Select the QC tab in the User Preferences window (Figure 108) to specify QC rules to apply to
data in Data Analysis Module. The software validates the data against the enabled tests and
the assigned values.
NOTE: Wells that fail a QC parameter can easily be excluded from analysis in the
QC module of the Data Analysis Window using the right-click menu option.
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Users and Preferences
The ordering of the items selected can be changed by highlighting the item and then using the
arrow buttons to the left of the Exported Columns list to move them up or down.
NOTE: Selecting Restore Defaults from any of the User Preferences tabs restores the default
factory settings for all user preferences options.
User Administration
Open the User Administration window in the main software window:
• Select Users > User Administration
• Click the User Administration button in the menu bar
If you log in as an Administrator, open the User Administration window to manage users and
user rights:
• Manage Users. Add or remove Users and assign each user a Role
• Manage Rights. Change rights for user roles (Principal, Operator, or Guest)
NOTE: Only users who are Administrators can edit this window. Other users can
only view it.
To assign a role to each user, select from the list of roles in the User Administration window
(Figure 110). In this example, the Guest user is given the added right to save files.
2. Select a user Role. These roles restrict the rights of each user. The default is Principal.
3. (Optional) Enter a Full Name and Password for the new software user.
4. Click OK to open a dialog box and confirm that you want to close the window.
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2. Click OK to open a dialog box and confirm that you want to close the window.
2. Click OK to open a dialog box and confirm that you want to close the window.
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11 Resources
Read this chapter to learn more about resources for the CFX96™ system or the CFX384™
system:
• LIMS integration (page 137)
• Calibration Wizard (page 142)
• Instrument maintenance (page 144)
• Application Log (page 146)
• Troubleshooting (page 146)
• References (page 149)
LIMS Integration
CFX Manager™ software can be configured for use with a Laboratory Information
Management System (LIMS). For LIMS integration, CFX Manager software requires plate setup
information generated by the LIMS platform (a LIMS file, *.plrn), a protocol file created using
CFX Manager software (*.prcl), a defined data export location, and a defined export format.
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2. At run completion, a LIMS data export file can be automatically generated in addition to
the CFX Manager software *.pcrd data file. Check the Automatically Export Data after
Run (Figure 111) box to have the data exported automatically once a run is completed.
3. Click the Data Export Settings button to specify the file format to be used for the
exported data and which information fields will be exported (Figure 112).
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2. Select either CFX96 Plate Import Template.csv or CFX384 Plate Import Template.csv
and import into your internal LIMS (Figure 113).
3. Using the LIMS, complete the template by filling in the required fields as listed in
Table 44.
4. Save the template with the file name extension .plrn directly to the designated LIMS file
folder location.
WARNING! Changing the file extension from .csv to .plrn is required for CFX
Manager software to recognize the file and start a LIMS run.
TIP: To quickly view the location of the designated LIMS file folder location, select
Tools > LIMS File Folder from the main software window menu bar.
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2. To start the run for a selected LIMS file, select an instrument and click Start Run
(Figure 114). The contents of the LIMS file and linked protocol file are used to complete
the protocol and plate tabs.
Figure 114. Run Setup window with a LIMS run ready to start.
Calibration Wizard
The CFX96 system is factory calibrated for commonly used fluorophores in white-well and
clear-well plates. The CFX384 system is factory calibrated for the same fluorophores in white-
well plates only (Table 45).
Table 45. Factory calibrated fluorophores, channels, and instruments.
Fluorophores Channel Instrument
FAM, SYBR® Green I 1 CFX96 and CFX384
VIC, HEX, TET, CAL Fluor Gold 540 2 CFX96 and CFX384
ROX, Texas Red, CAL Fluor Red 610 3 CFX96 and CFX384
CY5, Quasar 670 4 CFX96 and CFX384
Quasar 705 5 CFX96 only
The CFX96 system or the CFX384 system also includes a channel dedicated to FRET
chemistry; this channel does not require calibration for specific dyes.
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To open the Calibration Wizard to calibrate the CFX96 or CFX384 real-time PCR system:
1. Select an instrument in the detected instruments pane.
2. Select Tool > Calibration Wizard to open the window and calibrate new dye and plate
combinations (Figure 115).
Figure 115 shows an example of the Dye Calibration window.
2. Select the Plate Type. If the plate type is not included in the list, type the name in the box
to add it to the list.
4. Click the Add to List button to add the fluorophore. To clear the plate, click Clear List to
remove all the fluorophores.
5. (Optional) Repeat steps 1- 6 to add each fluorophore you plan to calibrate for the plate.
6. When you finish adding fluorophores, click View Plate to open the Pure Dye Plate
Display. Use this window as a guide for loading dyes into the plate.
7. Begin preparing a 96- or 384-well plate for dye calibration by pipetting dye solution into
each well, following the pattern shown in the Pure Dye Plate Display. For each
fluorophore, fill four wells with 50 μl (96-well plate) or 30 μl (384-well plate) of 300 nM dye
solution. Notice that at least half the plate contains blank wells.
8. Seal the plate using the sealing method you will use in your experiment.
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9. Place the calibration plate in the block and close the lid. Then click Calibrate and click
OK to confirm that the plate is in the block.
10.When CFX Manager software completes the calibration run, a dialog box appears. Click
Yes to finish calibration and open the Dye Calibration Viewer.
Instrument Maintenance
Your CFX96 system or CFX384 system includes a sensitive optical shuttle system that moves
quickly during data collection and a sample block that must heat and cool very fast.
Contamination of these components can interfere with thermal cycling and data collection.
WARNING! Never allow a reaction to run with an open or leaking sample lid. The
reagents could escape and coat the block, inner lid, and optical head in the shuttle
system. Excessive dirt can dim the signal and fluorescence contamination can
create excessive background signal. The shuttle system cannot be cleaned, except
by trained Bio-Rad service engineers.
Avoid contaminating the CFX96 or CFX384 system by following these suggestions:
• Always clean the outside of any containers before placing them in the block
• Never run a reaction with a seal that is open, loose, punctured, or otherwise
damaged because you could contaminate the block, inner lid, and optical system
• Never run a PCR or real-time PCR reaction with volatile reagents that could explode
and contaminate the block, inner lid, and optical system
• Clean the block and inner lid periodically to prevent the buildup of dirt, biohazardous
material, or fluorescent solutions (see Cleaning the Optical Reaction Module on
page 144)
• Never clean or otherwise touch the optical system behind the heater plate holes are
in the inner lid (Figure 116)
• Clean the outer lid and C1000 base on a regular schedule (for details see C1000
thermal cycler instruction manual)
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WARNING! To prevent electrical shock, always remove the reaction module from
the thermal cycler base or unplug the base before cleaning the instrument.
WARNING! Never touch or allow solutions to touch the optical system that is
located behind the heated plate holes in the inner lid (Figure 116).
Block (96-well)
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WARNING! Bleach, ethanol, or soap that is left in the blocks could corrode the
block and destroy plastics during a run. After cleaning, always rinse the wells
thoroughly with water to remove all traces of cleaning reagents.
WARNING! Never heat the block after adding a cleaning solution. Heating the
block with cleaning solution will damage the block, reaction module, and thermal
cycler base.
• Clean the inner lid. Use a soft, lint-free cloth and water to remove debris and solutions
from the inner lid surface. Never use abrasive detergents or rough material that will
scratch the surface. Cleaning the inner lid improves precise sample heating and cooling.
Application Log
Before starting a new run, the CFX96 or CFX384 instrument initiates a self-diagnostic test to
verify that it is running within specifications. The software records results of this test in the Run
Log and Application Log file. If you notice a problem in one or more experiments, open the run
and application logs to find out when the problem started.
CFX Manager software tracks information about the state of an instrument during a run in the
Application Log (Figure 117). Use these logs to track events that occur on instruments and in
the software and for troubleshooting.
To open the Application log in the main software window, select View > Application Log.
Troubleshooting
Typically, software and instrument communication problems can be resolved by restarting your
computer and the system. Be sure to save any work in progress before restarting.
NOTE: Check that your computer has sufficient RAM and free hard drive space.
The minimum RAM is 2 GB and the minimum hard drive space is 20 GB.
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2. Right-click the software CD icon and select Explore to open the CD window.
3. Double-click the CFX_Manager folder to open the folder and then double-click
setup.exe to start the software installation wizard.
4. Follow the instructions on the wizard to install the software and then click Finish.
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If you want to open a locked motorized lid on a reaction module to remove your samples
during a power failure, follow these steps to remove the locking plate:
1. Remove the reaction module from the C1000 chassis by pushing down on the locking
bar of the C1000.
2. Position the module on the front of a desk so that the front of the module extends 2
inches over the edge of the desk as shown in Figure 120.
Figure 120. Setting up the Optical Module to remove the locking plate.
3. With an allen wrench, remove the two large screws from under the front edge of the
reaction module (below the button for opening the lid). Do not remove the two small
screws located at the front edge of the module. You should hear the locking latch release
from inside the module. Figure 121 shows the two large screws.
4. Push the reaction module lid open. Notice that the latch (dark plastic) is no longer
attached. Remove your samples from the block.
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5. Reassemble the reaction module with the lid open by replacing the locking latch and
securing it with the large screws. Figure 122 shows the locking latch in place.
References
Breslauer KJ, et al. (1986). Predicting DNA duplex stability from the base sequence. Proc
Nat Acad Sci 83, 3746–50.
Hellemans J, et al. (2007). qBase relative quantification framework and software for
management and automated analysis of real-time quantitative PCR data, Genome Biol,
8, R19.
Livak JL, et al. (1995). Towards fully automated genome-wide polymorphism screening.
Nature Genetics 9, 341–342 .
Pfaffl MW. (2001). A new mathematical model for relative quantification in real-time RT-
PCR. Nucleic Acids Research 29(9), 2002–2007.
Vandesompele J, et al. (2002). Accurate normalization of real-time quantitative RT-PCR
data by geometric averaging of multiple internal control genes. Genome Biology 3(7), 1–
12.
2. Redistributions in binary form must reproduce the above copyright notice, this list of
conditions, and the following disclaimer in the documentation and/or other materials
provided with the distribution.
3. The end-user documentation included with the redistribution, if any, must include the
following acknowledgment:
“This product includes software developed by the University of Chicago, as Operator of
Argonne National Laboratory.”
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Index
A Contact, ii
Block, 3
Add Repeats, 30 Mode, 39
Adding Button, 30
Beep, 38 Clear Replicate #, 48
Extend, 38 Clear Wells, 48
Increment, 38 Close lid, 3, 29
Ramp rate, 38 Delete step, 38
Repeats in run, 30 Experiment Settings, 48
Adjusting Flash Block Indicator, 30
Allelic Discrimination data, 96 Open lid, 29
Data analysis charts, 79 Pause, 30
End point data, 95 Protocol AutoWriter, 39
Gene expression data, 108 Protocol Editor, 35
Melt Curve Data, 90 Resume, 30
Threshold, 74 Show Analysis Settings, 49, 112
Trace color, 84 Skip Step, 30
Trace style, 84
Adjusting the Threshold, 77
All Channels scan mode, 44, 59
Allelic Discrimination, 95 C
Adjusting data, 96 C1000 Control Panel, 53
RFU, 95, 98 Calculated mode, 39
Spreadsheet, 97 Calibration
Amplicon length, 39
Inter-run in a Gene Study, 113
Analysis Mode, 75 Cancelling a run, 30
Annealing temperature CFX Manager
Protocol AutoWriter, 39 Files, 8
Apply Fluorescent Drift Correction, 75 CFX96 system, 2
Auto Efficiency, 50, 112
Overview, 2
Changing
Baseline, 75
B Sample volume, 26, 35
Thresholds, 75
Back panel, 3 Chart
Balance Data analysis, 79
Cut microplates, 9 Cleanup events, 24
How to, 9 Clear Replicate #, 48
Tube strips, 9 Clear Wells button, 48
Baseline Clearing wells in Plate Editor, 79
Window, 75 Close button for lid, 3
Baseline Settings, 74 Close Lid button, 29
Baseline Subtracted Closing the lid, 29
Curve Fit mode, 75 Coefficient of Variation, 109
Baseline Subtracted mode, 74 Color, 45, 50, 112
Baseline Threshold window, 75 Components in shipment, 1
Beep, 38 Concentration, 46, 47
Bio-Rad Laboratories Contact Bio-Rad Laboratories, ii
151
Index
Control Spreadsheet, 95
Relative quantity with, 119 Tab, 94
Creating End Point Only Run protocol, 26
Data report, 101 Enzyme type, 40
Protocol, 39 Excluding wells
Well groups, 51 Plate Editor, 78
Custom Data View, 97 Experiment Settings, 48
Custom Export, 81 Auto Efficiency, 50, 112
Cycles to Analyze, 76 Button, 48
Color, 50, 112
Show Analysis Settings button, 49, 112
Show Graph, 50, 112
D Targets tab, 48, 111
-d(RFU)/dT Export All Data Sheets to Excel, 71, 81
Spreadsheet, 93 Export RDML Files, 81
Data Export Template, 52
Files, 8 Export to LIMS Folder, 82
Data analysis Exporting Data to LIMS, 142
About, 69 Exporting Stand-Alone Data, 61
Adjusting, 69 Express Load
Charts, adjusting, 79 Protocol, 26, 27
Clearing wells, 79 Extend, 38
Data analysis, 95
End Point, 93
Excluding wells, 78
Formula, 119
F
Gene Expression, 105 Files
Melt Curve, 90 Data, 8
Menu bar, 71 Gene Study, 8
Plate content, 69 Plate, 8
Toolbar, 70 Protocol, 8
Well contents, 69 Software, 8
Well groups, 50 Flash Block Indicator button, 30
Well selector, 77 Fluorophore Analysis Mode, 75
Data file Fluorophores, 45
Report, 100 Formula, 120
Data report Data analysis, 119
Creating, 101 E (see reaction efficiency), 86
Delete Step button, 38 Normalized expression, 120, 121, 122
Deleting a step, 38 Normalized expression, scaled, 121
Detected Instruments, 12 Reaction Efficiency (E), 86
Dilution Series, 47 Relative quantity, 119
SD normalized expression, 122
Standard error, 122
Standard error normalized expression, 122
E FRET, 44, 59
Email function on the C1000, 62 Front view, 2
Email Setup in CFX Manager, 127
Email setup on the C1000, 64
Email Tab, 127
End Point
G
Adjusting data, 95 Gene expression
Changing sample volume in protocol, 26 Adjusting data, 108
RFU data analysis, 93 Data analysis, 105
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153
Index
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155
Index
U
Units, 43
Unpacking instrument, 1
USB
Connections, 3
156
Bio-Rad
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