CFX96™ and CFX96 Deep Well

Real-Time PCR Detection Systems

Instruction Manual
185-5095-IVD
184-5097-IVD
185-4095-IVD
184-4095-IVD

Manual revision: October 2014

Software revision: 1.6
Copyright ©2014 Bio-Rad Laboratories, Inc. Reproduction in any form, either print or electronic, is
prohibited without written permission of Bio-Rad Laboratories, Inc.
Excel, Microsoft, PowerPoint, Windows, Windows 7 and Windows 8 are trademarks of Microsoft
Corporation. 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 Life Technologies Corporation. Bio-Rad Laboratories, Inc. is
licensed by Life Technologies Corporation to sell reagents containing SYBR® Green I for use in real-time
PCR, for research purposes only. EvaGreen is a trademark of Biotium, Inc. FAM, ROX, and VIC are
trademarks of Applera Corporation. Other brands or product names are trademarks of their respective
holders.

NOTICE REGARDING BIO-RAD THERMAL CYCLERS AND REAL-TIME SYSTEMS

This product is covered by one or more of the following U.S. patents or their foreign counterparts owned by
Eppendorf AG: U.S. Patent Numbers 6,767,512 and 7,074,367.
Bio-Rad’s Hard-Shell® plates are covered by one or more of the following U.S. patents, patents pending, or their
foreign counterparts, owned by Eppendorf AG: US Patent Nos. 7,347,977, 6,340,589, 6,528,302 and US application
2007246858, 20080084004, 20030180192 and 20060120927.
Instruction Manual Information
This manual contains information on how to safely set up and operate the CFX96™ real-time PCR
detection system that carries the CE-IVD mark (catalog number 1855095–IVD) as well as information on
how to safely set up and operate the CFX96™ Deep Well real-time PCR detection system (catalog
number 1854095–IVD). These two systems will be referred to as the CFX system in this instruction
manual. Additional information on CFX Manager™ software can be found in the CFX96 and CFX96 Deep
Well Instruction Manual for life science research (catalog numbers 1855095, 1844095).

Writing Conventions Used in this Manual

The manual uses the writing conventions listed in Table 1.
Table 1. Conventions used in this manual.
Convention Meaning
TIP: Provides helpful information and instructions, including information
explained in further detail elsewhere in this manual.
NOTE: Provides important information, including information explained in
further detail elsewhere in this manual.
WARNING! Explains very important information about something that might
damage the researcher, damage an instrument, or cause data loss.
X>Y Select X and then select Y from a toolbar, menu or software window.

For information about safety labels used in this manual and on the CFX system, see, “Safety and
Regulatory Compliance” on page iii.

Bio-Rad Resources
Table 2 lists Bio-Rad resources and how to locate what you need.
Table 2. 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 1-800-424-
6723 (toll-free phone), and select the technical support
option.

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 CFX96 and CFX96 Deep Well Systems Instruction Manual

Safety and Regulatory Compliance

For safe operation of the CFX system, we strongly recommend that you follow the safety specifications
listed in this section and throughout this manual.
The CFX96 system is approved for use as in vitro diagnostic (IVD) medical equipment.

Safety Warning Labels

Warning labels posted on the instrument and in this manual warn you about sources of injury or harm.
Refer to Table 3 to review the meaning of each safety warning label.
Table 3. Meaning of safety warning labels.
CAUTION: Biohazard! This symbol identifies components that may become contaminated
with biohazardous material.

CAUTION: Risk of danger! This symbol identifies components that pose a risk of personal
injury or damage to the instrument if improperly handled. Wherever this symbol appears,
consult the manual for further information before proceeding.

CAUTION: Hot surface! This symbol identifies components that pose a risk of personal
injury due to excessive heat if improperly handled.

Instrument Safety Warnings

The warning labels shown in Table 4 also display on the instrument and refer directly to the safe use of the
system.
Table 4. Instrument safety warning labels.
Icon Meaning
Warning about risk of harm to body or equipment.
Operating the CFX 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.

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Table 4. Instrument safety warning labels. (continued)
Icon Meaning
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.

Warning about risk of burning.

A thermal cycler generates enough heat to cause serious burns. Wear safety goggles or
other eye protection at all times during operation. Always allow the sample block to return
to idle temperature before opening the lid and removing samples. Always allow maximum
clearance to avoid accidental skin burns.
Warning about risk of explosion.
The sample blocks can become hot enough during the course of normal operation to cause
liquids to boil and explode.

Safe Use Specifications and Compliance

Table 5 lists the safe use specifications for the CFX system. Shielded cables (supplied) must be used with
this unit to ensure compliance with the Class A FCC limits.
Table 5. Safe use specifications.
Safe Use Requirements Specifications
Temperature Indoor use Ambient temperature 15–31oC. Relative humidity maximum
80%, noncondensing
Altitude Up to 2,000 meters above sea level

REGULATORY COMPLIANCE
This instrument has been tested, and found to be in compliance with all applicable requirements of the
following safety and electromagnetic standards:
• IEC 61010-1:2001 (2nd Ed.), EN61010-1:2001 (2nd Ed). Electrical Equipment For
Measurement, Control, and Laboratory Use - Part 1: General Requirements
• IEC 61010-2-010:2005, EN61010-2-010:2003. Safety requirements for electrical equipment
for measurement, control and laboratory use. Part 2-010: Particular requirements for
laboratory equipment for the heating of materials
• IEC 61010-2-081:2001+A1, EN61010-2-081:2002+A1. Safety requirements for electrical
equipment for measurement, control and laboratory use. Part 2-081: Particular requirements
for automatic and semi-automatic laboratory equipment for analysis and other purposes
(includes Amendment 1)
• EN 61326-1:2006 (Class A) Electrical equipment for measurement, control and laboratory
use. EMC requirements, Part 1: General requirements
• EN 61010-2-101. Safety requirements for electrical equipment for measurement, control and
laboratory use. Particular requirements for in vitro diagnostic (IVD) medical equipment
• EN 61326-2-6 (Class A) Electrical equipment for measurement, control and laboratory use.
EMC requirements. Part 2-6. Particular requirements. In vitro diagnostic (IVD) medical
equipment

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 CFX96 and CFX96 Deep Well Systems Instruction Manual

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 CFX system is designed to operate safely when used in the manner prescribed by the manufacturer.
If the 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 authorized agent. Service of the
system should be performed only by Bio-Rad personnel.

Biohazards
The CFX system is a laboratory product. 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)

SPECIFIC PRECAUTIONS
• All patient samples are a potential biohazard and should be handled accordingly using universal
precautions
• 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.
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

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 • 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 time 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.

DISPOSAL OF BIOHAZARDOUS MATERIAL

The CFX system contains no potentially hazardous chemical materials. Dispose of the following
potentially contaminated materials in accordance with laboratory local, regional and national regulations:
• Clinical samples
• Reagents
• Used reaction vessels or other consumables that may be contaminated

Chemical Hazards
The CFX system contains no potentially hazardous chemical materials.

Explosive or Flammability Hazards

The CFX system poses no uncommon hazard related to flammability or explosion when used in a proper
manner as specified by Bio-Rad Laboratories.

Electrical Hazards
The CFX 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 optical reaction module, decontamination
procedures must be performed. Always move or ship the C1000 thermal cycler chassis and optical
reaction module in separate containers with the supplied packaging materials that will protect the
instrument from damage. If appropriate containers cannot be found, contact your local Bio-Rad office.

Storage
The CFX system can be stored under the following conditions:
• Temperature range –20 to 60oC
• Relative humidity maximum 80%

Disposal
The CFX system contains electrical or electrical materials; it should be disposed of as unsorted waste and
must be collected separately according to the 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 CFX96 Deep Well Systems Instruction Manual

Table of Contents
Chapter 1. System Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Unpacking the Optical Reaction Module. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
System Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
System Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Setting Up the System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Installing CFX Manager Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Software Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Running Experiments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Chapter 2. CFX Manager™ Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Main Software Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Startup Wizard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Detected Instruments Pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Instrument Properties Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Chapter 3. Running Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Experiment Setup Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Protocol Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Plate Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Start Run Tab. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Run Details Window. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Chapter 4. Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Protocol Editor Window. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Protocol Editor Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Temperature Control Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Protocol AutoWriter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Chapter 5. Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Plate Editor Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Select Fluorophores Window. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Well Loading Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Experiment Settings Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Well Groups Manager Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Plate Spreadsheet View Window. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Chapter 6. Stand-Alone Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

C1000 Thermal Cycler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Creating a New Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Exporting Data for Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Creating a Data File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

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Chapter 7. Data Analysis Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Data Analysis Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Data Analysis Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Quantitation Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Data Analysis Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Well Selectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Charts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Spreadsheets. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Chapter 8. Data Analysis Windows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Quantitation Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Quantitation Data Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Melt Curve Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Melt Curve Data Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
End Point Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Allelic Discrimination Tab. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
QC Tab. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Run Information Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Data File Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Chapter 9. Gene Expression Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Plate Setup for Gene Expression Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Gene Expression Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Experiment Settings Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Gene Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Gene Study Data Spreadsheet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Gene Study Report Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

Chapter 10. Users and Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

Log In or Select User . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
User Preferences Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Configure Email Notification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
User Administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

Chapter 11. Resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

LIMS Integration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Calibration Wizard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Instrument Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Application Log . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

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 CFX96 and CFX96 Deep Well Systems Instruction Manual

1 System Installation
Read this chapter for information about setting up the CFX system:
• Unpacking the optical reaction module (page 1)
• System requirements (page 1)
• System overview (page 2)
• Setting up the system (page 3)
• Installing CFX Manager™ software (page 5)
• Software files (page 7)
• Running experiments (page 7)

Unpacking the Optical Reaction Module

The optical reaction module shipment includes these components:
• Optical reaction module
• USB cable
• CFX Manager software installation CD
• Instruction manual
Remove all packing materials and store them for future use. If any items are missing or
damaged, contact your local Bio-Rad office.

System Requirements
To operate the CFX system, use the following power sources and cables:
• Input power. 100–240 VAC, 50–60 Hz
• Indoor use. Ambient temperature 15–31oC. Relative humidity maximum 80%, non-
condensing
• USB cable. If the CFX system is going to be controlled by a computer via a USB cable,
the cable provided by 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.

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System Installation

System Overview
The CFX 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 CFX module is located on the back.
• C1000™ thermal cycler base. The C1000 base includes a user interface to control the
system when running in stand-alone mode and a power button and ports (both on back
panel) to connect to a computer

Indicator LED

Open button

Front panel

Figure 1. Front view of the CFX system.

When open, the CFX system includes the features shown in Figure 2.

Inner lid with heater plate

Block
Close button

Figure 2. Inside view of the CFX system.

WARNING! Avoid touching the inner lid or block: These surfaces can be hot.

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 CFX96 and CFX96 Deep Well Systems Instruction Manual

• 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 101).
The back panel of the C1000 chassis includes these features (Figure 3):
• Power switch. Press the power switch to turn on the power to the system
• Power input. Plug in the power cord here
• Ethernet port. Connect an ethernet cable to email run logs and stand-alone data files
• USB connections. Use these ports to connect the CFX system to a computer or to
connect an S1000™ thermal cycler

Power Power Ethernet

port USB connections
switch input

Figure 3. Back panel of C1000 thermal cycler.

WARNING! Do not touch the back of the CFX system when the instrument is on.

Setting Up the System

The CFX system should be installed on a clean, dry, level surface with sufficient cool airflow to
run properly. The CFX system can be run in two modes: stand-alone or software controlled. If
you are running the system under software-controlled mode, make sure there is sufficient
space for a computer during setup.
Position the C1000 thermal cycler base so that the power switch, which is located on the back
panel, is easily accessible.
To insert the CFX optical reaction module into the reaction module bay of the C1000
chassis, follow these instructions:
1. Place the C1000 chassis in a suitable location with the locking bar down. Remove any
previously installed reaction modules.

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System Installation

2. Lift the optical reaction module using the handle indents above the side air vents
(Figure 4).

Figure 4. Lifting the optical reaction module into the C1000 chassis.

3. Position the module in the reaction module bay of the C1000 chassis, leaving about 2 cm
of space in the front. When in the chassis bay, the optical module should be covering the
Bio-Rad logo in front of the bay of the C1000 chassis.

4. Reach around and pull up the locking bar of the C1000 thermal cycler until it is flush with
the sides of the module bay. This action moves the module forward, locking it into place
(Figure 5)

Figure 5. Locking the optical module into place.

5. Check that the module is completely and evenly seated in the C1000 base. There should
be no extra space between the module and the base; the space should be even.

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 CFX96 and CFX96 Deep Well Systems Instruction Manual

6. Plug the power cord into the back of the C1000 base (Figure 3 on page 3) and into an
appropriate three-pronged electrical outlet. Press the power switch on the back panel of
the C1000 thermal cycler to start the system.

7. Follow the instructions in the front panel of the C1000 thermal cycler to remove the red
shipping screw from the inner heater lid (Figure 6). Turn the screw counterclockwise to lift
it out of the hole.

Figure 6. Instructions to remove the shipping screw.

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 15).
TIP: The shipping screw must be in place when the module is shipped. Save this
screw in a safe place for future shipping.

8. Remove the shipping plate from the thermal cycler block to operate.

Installing CFX Manager Software

CFX Manager software is run on a personal computer (PC) and is required to analyze data from
the CFX system and to control the CFX system in software-controlled mode. Table 6 lists
computer system requirements for CFX Manager software (version 1.6 or higher).
Table 6. Computer requirements for CFX Manager software version 1.6 or higher
System Minimum Recommended
Operating system Windows 7 and Windows 8 Windows 7 or Windows 8
Drive CD-ROM drive CD-RW drive
Hard drive 10 GB 20 GB
Processor speed 2.0 GHz 2.0 GHz
RAM 2 GB RAM 2 GB RAM
Screen resolution 1,024 x 768 with true-color mode 1280 x 1024 with true-color mode
USB USB 2.0 Hi-Speed port USB 2.0 Hi-Speed port

To install CFX Manager software:

1. Make sure you are logged in with administrative privileges. The software must be
installed on the computer by a user with administrative privileges.

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System Installation

2. Place the CFX Manager software CD in the computer’s CD drive.

3. The software launch page should appear automatically. Double-click Install Software on
the software launch page (Figure 7).

Figure 7. Software installation screen.

4. Follow the instructions on screen to complete installation. When completed, the Bio-Rad
CFX manager software icon will appear on the desktop of the computer.
5. If the launch page does not appear automatically, double-click on (CD drive):\Bio-Rad
CFX, then open and follow instructions in the Readme.txt file. See “Installing the
Software Manually” on page 103.

Installing the Drivers

If the CFX system is going to be run in Software-controlled mode, drivers must be installed
on the computer. Use only the supplied USB cable, which is sufficiently shielded to prevent
data loss.
To install the system drivers:
1. Connect the C1000 chassis to the computer by plugging a USB cable into the USB 2.0 A
port located on the back of the chassis (Figure 3 on page 3), and then connecting the
cable into the USB 2.0 B port located on the computer.

2. If it is not already turned on, turn on the system using the power switch on the back of
the C1000 chassis. Follow the instructions in the Found New Hardware Wizard that
launches after the instrument is first detected by the computer.
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.

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.

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 CFX96 and CFX96 Deep Well Systems Instruction Manual

Software Files
CFX Manager software stores information about experiments 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 Experiment Setup and edit in Protocol Editor
Plate .pltd Select in Experiment Setup and edit in Plate Editor
Data .pcrd View and analyze in Data Analysis window
LIMS .plrn Contains plate setup and protocol information required to
conduct a LIMS compatible run
Gene Study .mgxd View and analyze in Gene Study window
Stand-alone pre-data .zpcr Contains fluorescence readings from stand-alone
file operation that is converted into a data file

Running Experiments

Recommended Plastic Consumables

The CFX system accepts low profile 0.2 ml plates and tubes. Bio-Rad recommends the
following consumables for optimal results:
• MLL-9601. Low-profile 96-well unskirted plates with clear wells
• MLL-9651. Low-profile 96-well unskirted plates with white wells
• HSP-9601. Hard-Shell 96-well skirted plates with white shell and clear wells
• HSP-9655. Hard-Shell 96-well skirted plates with white shell and white wells
• TLS-0801. Low-profile 0.2 ml 8-tube strips without caps, clear wells
• TLS-0851. Low-profile 0.2 ml 8-tube strips without caps, white wells
• TCS-0803. Optical flat 8-cap strips, for 0.2 ml tubes and plates
NOTE: High profile plates can also be used with the CFX96 Deep Well system.

Plate Seals:
• MSB-1001. Microseal® ‘B’ adhesive seals, optically clear (strong adhesive-based)
• MSC-1001. Microseal ‘C’ optical seals, optically clear (pressure-activated adhesivebased)

Loading the Block

To load your reactions in the block, follow these suggestions:
• Click the Open Lid button located on software’s Start Run tab (see “Start Run Tab”
on page 19), 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 10–125 µl for the CFX96 Deep Well
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. If needed, edit the well contents before, during, or after the run.

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System Installation

WARNING! When running the CFX system, always balance the tube strips or cut
microplates in the wells (Figure 8). 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. Failure to balance the pressure
can result in sample evaporation and failed runs.

Filled tube Tube strip for

strip balance

Figure 8. Balance the tube strips or cut microplates in the block.

WARNING! Be sure that nothing is blocking the lid when it closes. Although there
is a safety mechanism to prevent the lid from closing if it senses an obstruction, do
not place anything in the way of the closing lid.

Shutting Down the System

To shut down the CFX system, follow these suggestions:
• After a run, click the open lid button on the front of the CFX system to access the
samples loaded in the thermal cycler block.
• Remove the samples from the block and click the close lid button to close the lid of
the CFX system.
• Press the power switch on the back panel of the C1000 thermal cycler to power
down the system.

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 CFX96 and CFX96 Deep Well Systems Instruction Manual

2 CFX Manager™ Software

Read this chapter for information about getting started with CFX Manager software.
• Main software window (page 9)
• Startup Wizard (page 12)
• Detected Instruments pane (page 13)
• Instrument Properties window (page 14)

Main Software Window

Features available in the main software window are provided in Figure 9.

Figure 9. The main software window.

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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, experiment, or Gene Study
Open Open existing files, including protocol (.prcl), plate (.pltd),
data (.pcrd), and Gene Study (.mgxd) files, LIMS
(.plrn), stand-alone run files (.zpcr)
Recent Data Files View a list of the ten most recently viewed data files, and
select one to open in Data Analysis
Repeat an Open the Experiment Setup window with the protocol
Experiment and plate from a completed run to quickly repeat the run
Exit Exit the software program
View Application Log Display the application log for the software
Run Reports Select a run report to review from a list
Startup Wizard Open the Startup Wizard
Experiment Setup Open the Experiment Setup window
Instrument Open the Instrument Summary window
Summary
Detected Show or hide the Detected Instruments pane
Instruments
Toolbar Show or hide the main software window toolbar
Status Bar Show or hide the main software window status bar
User Select User Open the Select User window to change software users
Change Password Change your user password
User Preferences Open the User Preferences window
User Manage users in the User Administration window
Administration
Tools Dye Calibration Open the Dye Calibration window to calibrate an
Wizard instrument for a new fluorophore
Protocol Open the Protocol AutoWriter window to create a new
AutoWriter protocol
Ta Calculator Open the Ta Calculator window to calculate the
annealing temperature of primers
View Block Status View a log of the thermal cycler block
Log
Application Data Open the Application Data folder to view software files
Folder
User Data Folder Open the Data folder to view protocol, plate, and data
files
Properties All View properties of all detected instruments, including
Instruments serial numbers
Zip Data and Log Choose and condense selected files in a zipped file for
Files storage or to email
Options Configure software email

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 CFX96 and CFX96 Deep Well Systems Instruction Manual

Table 8. Menu bar items in the main software window.

Menu Item Command Function
Windows Cascade Arrange software windows on top of each other
Tile Vertical Arrange software windows from top to bottom
Tile Horizontal Arrange software windows from right to left
Close All Close all open software windows
Help Contents Open the software Help for more information about
running PCR and real-time PCR
Index View the index in the software Help
Search Search the software Help
Gene Expression Open a website to find information about running PCR
Gateway Website and real-time PCR experiments
PCR Reagents View a website that lists Bio-Rad consumables for PCR
Website and real-time PCR reagents
PCR Plastic View a website that lists Bio-Rad consumables for PCR
Consumables and real-time PCR experiments
Website
Software Updates Check for software updates from Bio-Rad
About Open a window to see the software version

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
(page 53)

Open a Gene Study Open a browser window to locate a Gene Study file
(.mgxd extension) and open it in the Gene Study
window (page 87)

Create a New Gene Open the Gene Study window (page 87) to add files
Study and create a new study

Print Print the current software window

Startup Wizard Open the Startup Wizard that links you to common
software functions (page 12)

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CFX Manager™ Software

Table 9. Toolbar buttons in the main software window.

Button Button Name Function
Experiment Setup Open the Experiment Setup window to run an
experiment (page 17)

Protocol AutoWriter Open the Protocol AutoWriter window to create a new

protocol (page 30)

Select User Open the Select User window to change software

users (see “Log In or Select User” on page 91)

User Preferences Open the User Preferences window (page 92)

Help Open the software Help window for more information

about running PCR and real-time PCR

Startup Wizard
The Startup Wizard automatically appears when CFX Manager software is first opened
(Figure 9). 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 Experiment (page 17). Set up the protocol and plate to begin a new
experiment.
• Repeat an Experiment. Set up an experiment with the protocol and plate from a
completed run. If needed, you can edit the experiment before the run
• Open a Data File (page 53). Open a data file to analyze results
• Open a Gene Study (page 86). Open a multifile gene expression study to analyze
results from multiple gene expression experiments
• Open User Preferences (page 92). Open the User Preferences window to customize
software settings

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 CFX96 and CFX96 Deep Well Systems Instruction Manual

Detected Instruments Pane

Connected instruments appear in the Detected Instruments pane (Figure 10). This list shows
each instrument as an icon named with the serial number (default).

Figure 10. Instruments listed in the Detected Instruments pane.

Right-click on the instrument icon or block to select one of these options:
• View Status. Open the Run Details window to check the status of the selected
instrument block
• Flash Block Indicator. Flash the indicator LED on the instrument
• Open Lid. Open a motorized lid on the selected instrument block
• Close Lid. Close a motorized lid on the selected instrument block
• Rename. Change the name of the instrument
• Properties. Open the Instrument Properties window
• Collapse All. Collapse the list of instruments in the Detected Instruments pane
• Expand All. Expand the list of instruments in the Detected Instruments pane
You can also control a block by clicking an instrument block icon in the Detected Instrument
pane and then clicking a button in the Selected Instrument pane (Figure 11).

Figure 11. Buttons at the bottom of the Detected Instruments pane.

• Click View Status to open the Run Details window to check the status of the
selected instrument block
• Click Open Lid to open the motorized lids on the selected instrument
• Click Close Lid to close the motorized lids on the selected instrument
• Click View Summary to open the Instrument Summary window

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CFX Manager™ Software

If only one instrument is detected, then the View Summary button does not appear. To view
the Instrument Summary window for a single instrument, select View > Instrument Summary.

Status Bar
The left side of the status bar at the bottom of the main software window shows the current
status of instruments. View the right side of the status bar to see the current user name, date,
and time.
Click and drag the right corner of the status bar to resize the main window.

Instrument Properties Window

To open the Instrument Properties window to view information about an instrument, right-click
on the instrument icon in the Detected Instruments pane (Figure 10 on page 13).

Properties Tab
The Properties tab displays important serial numbers for the connected instrument, including
the thermal cycler and reaction module. The firmware versions are also displayed. The default
name for an instrument is the C1000 thermal cycler serial number, which appears in many
locations, including the Detected Instruments pane (Figure 12).
To rename an instrument for ease of identification, follow these instructions:
• In the Instrument Properties tab, type a name in the Rename box at the top of the
Properties tab and hit the Rename button to save the new name

Figure 12. Instrument Properties window.

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 CFX96 and CFX96 Deep Well Systems Instruction Manual

Shipping Screw Tab

The Shipping Screw tab includes instructions for installing or removing the red shipping screw.
To prevent damage to the optical reaction modules, install the shipping screw any time you
ship the CFX system.
NOTE: If the shipping screw is detected by the software, the Instrument Properties
window automatically opens with the Shipping Screw tab in front. Follow the
instructions to remove the screw. You can not perform a run with the shipping
screw installed.
The information in this tab changes depending on whether the shipping screw is installed or
removed. For example, to install the shipping screw, click the Install Shipping Screw button
and follow the instructions in the tab (Figure 13).

Figure 13. Instructions for installing the shipping screw.

Calibrated Dyes Tab

Open the Calibrated Dyes tab to view the list of calibrated fluorophores and plates for the
selected instrument (Figure 14). Click an Info button to see detailed information about a
calibration.

Figure 14. Calibrated Dyes tab in the Instrument Properties window.

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CFX Manager™ Software

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 CFX96 and CFX96 Deep Well Systems Instruction Manual

3 Running Experiments
Read this chapter for information about running experiments using CFX Manager™ software:
• Experiment Setup window (page 17)
• Protocol tab (page 18)
• Plate tab (page 19)
• Start Run tab (page 19)
• Run Details window (page 20)

Experiment Setup Window

The Experiment Setup window provides quick access to the files and settings needed to set
up and run an experiment. To open the Experiment Setup window, follow one of these options:
• Click Create a New Experiment option in the Startup Wizard (page 12)
• Click the Experiment Setup button in the main software toolbar (page 17)
• Select File > New > Experiment in the main software menu bar (page 10)
The Experiment Setup window includes three tabs:
• Protocol. Click the Protocol tab to select an existing protocol to run or edit, or to create
a new protocol in the Protocol Editor window (page 25)
• Plate. Click the Plate tab to select an existing plate to run or edit, or to create a new
plate in the Plate Editor window (page 33)
• Start Run. Click the Start Run tab (page 19) to check the run settings, select one or
more instrument blocks, and begin the run
NOTE: If the protocol currently selected in the Protocol tab does not include a step
with a plate read for real-time PCR analysis, then the Plate tab is hidden. To view
the Plate tab, add a “Plate Read” (page 27) in at least one step in the protocol.
NOTE: Start a new experiment from a previous run by selecting File > Repeat an
Experiment in the main software menu bar. Then select the data file (.pcrd) for the
experiment you want to repeat.

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Running Experiments

The Experiment Setup window opens with the Protocol tab in front (Figure 15). To open
another tab, click that tab or click Prev and Next buttons at the bottom of the window.

Figure 15. Experiment 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 the Experiment Setup
(Figure 15). A protocol file contains the instructions for the instrument temperature steps and
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 Express Load 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

End Point Only Runs

To run a protocol that contains only an end point data acquisition step, select Options > End
Point Only Run from Options in the menu bar of the Experiment Setup window. The default
end point protocol, which includes two cycles of 60.0°C for 30 seconds, is loaded into the
Protocol tab. To change the step temperature or sample volume for the end point only run,
click the Start Run tab and edit the Step Temperature or Sample Volume.

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 CFX96 and CFX96 Deep Well Systems Instruction Manual

Plate Tab
The Plate tab shows a preview of the selected plate file loaded in the Experiment Setup
(Figure 16). In a real-time PCR experiment, 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 Express Load 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

Figure 16. Plate tab window.

Start Run Tab

The Start Run tab (Figure 17) includes a section for checking information about the run that is
going to be started, including the selected protocol and plate files, and a section for selecting
the instrument block.
• Run Information pane. View the selected Protocol file, Plate file, and data acquisition
Scan Mode setting. Enter optional notes about the experiment in the Notes box

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Running Experiments

• 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 experiment

Figure 17. The Start Run tab.

To add or remove run parameters from the spreadsheet in the Start Run on Selected Block(s)
pane, right-click on the list and select an option in the menu to display. Choose the value to
change by clicking the text inside the cell to select it and then typing in the cell or by selecting
a new parameter from the pull-down menu. Editable parameters include:
• Lid Temperature. View the temperature of the lid. Override the default lid temperature
by selecting the text and typing a new temperature
WARNING! Changing the lid temperature can impact experiment results and may
result in failed runs.

Buttons for Controlling the Instrument

Click the following buttons in the Start Run tab to remotely operate the selected instruments:
• Start Run. Start the experiment on the selected instrument blocks
• Flash Block Indicator. Flash the indicator LED on the selected instrument blocks
• Open Lid. Open motorized lid on selected instrument blocks
• Close Lid. Close motorized lid on selected instrument blocks

Run Details Window

When you click the Start Run button, CFX Manager software prompts you to save the name of
the data file and then opens the Run Details window. Review the information in this window to
monitor the progress of a run.
• Run Status tab. Check the current status of the protocol, open the lid, pause a run, add
repeats, skip steps, or stop the run
• 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

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 CFX96 and CFX96 Deep Well Systems Instruction Manual

Figure 18 shows the features of the Run Details window.

Figure 18. Run Details window.

Run Status Tab

The Run Status tab (Figure 18) shows the current status of a run in progress in the Run Details
window and provides buttons (page 21) to control the lid and change the run in progress.
• Run Status pane. Displays the current progress of the protocol, including the current
step, current GOTO repeat, block temperature, remaining hold time for the current step,
sample temperature, lid and shuttle temperature
• Run Status buttons. Click one of the buttons to remotely operate the instrument or to
interrupt the current protocol
• Run Information pane. Displays experiment details

RUN STATUS TAB BUTTONS

Click one of the buttons listed in Table 10 to operate the instrument from the software, or to
change the run that is in progress.
NOTE: Changing the protocol during the run, such as adding repeats, does not change the
protocol file associated with the run. These actions are recorded in the Run Log.
Table 10. Run Status buttons and their functions
Button Function
Open the motorized lid on selected instruments
WARNING! Opening the lid during a run pauses the
run during the current step and might alter the data.
Close the motorized lid on selected instruments

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Running Experiments

Table 10. Run Status buttons and their functions (continued)

Button Function
Add more repeats to the current GOTO step in the protocol.
This button is only available when a GOTO step is running.

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

Pause the protocol

NOTE: This action is recorded in the Run Log.

Resume a protocol that was paused

Stop the run before the protocol ends

WARNING! Stopping a run prematurely may alter
your data and may result in a failed experiment.

Real-Time Status Tab

The Real-time Status tab (Figure 19) shows real-time PCR data collected at each cycle during
the protocol after the first two plate reads. This tab also shows the well selector and text
describing the protocol status at the bottom of the window.

Figure 19. The Real-time Status tab displays the data during a run.

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 CFX96 and CFX96 Deep Well Systems Instruction Manual

REPLACING A PLATE FILE

During a run, replace the plate file by clicking the Replace Plate button (Figure 19) in the Real-
time Status tab. Select the new plate file (.pltd) from the list in the windows browser.
NOTE: CFX Manager software checks the scan mode and plate size for the plate
file; these must match the run settings that were started during the experiment.
TIP: Replacing a plate file is especially useful if you start a run with a Quick Plate
file in the Express Load folder.

Time Status Tab

The Time Status tab shows a countdown timer for the current run.

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Running Experiments

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 CFX96 and CFX96 Deep Well Systems Instruction Manual

4 Protocols
Read the following chapter for information about creating and editing protocol files:
• Protocol Editor window (page 25)
• Protocol Editor controls (page 27)
• Temperature control mode (page 30)
• Protocol AutoWriter (page 30)

Protocol Editor Window

A protocol instructs the instrument to control the temperature steps, lid temperature, and other
instrument options. Open the Protocol Editor window to create a new protocol or to edit the
protocol currently selected in the Protocol tab. Once a Protocol is created or edited in the
Protocol Editor, click OK to load the protocol file into the Experiment Setup window and run it.
WARNING! Always confirm the loaded protocol is correct in the Start Run tab
before beginning the run. Performing a run with the wrong protocol could alter the
data or result in a failed run.

Opening the Protocol Editor

To open the Protocol Editor, follow one of these options:
• To create a new protocol, select File > New > Protocol or click the Create New
button in the Protocol tab (page 18)
• To open an existing protocol, select File > Open > Protocol, or click the Open
Existing button in the Protocol tab (page 18)
• To edit the current protocol in the Protocol tab, click the Edit Selected button in the
Protocol tab (page 18)
TIP: To change the default settings in the Protocol Editor window, enter the
changes in the Protocol tab in the User Preferences window (page 92)

Protocol Editor Window

The Protocol Editor window (Figure 20) includes the following features:
• Menu bar. Select settings for the protocol
• Toolbar. Select options for editing the protocol

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Protocols

• Protocol. View the selected protocol in a graphic (top) and text (bottom) view. Click the
temperature or dwell time in the graphic or text view of any step to enter a new value
• Protocol Editor buttons. Edit the protocol by clicking one of the buttons to the left of
the text view

Figure 20. Protocol Editor window with buttons for editing protocols.

Protocol Editor Menu Bar

The menu bar in the Protocol Editor window provides the menu items listed in Table 11
Table 11. Protocol Editor menu bar
Menu Item Command Function
File Save Save the current protocol
Save As Save the current protocol with a new name or in a new
location
Close Close the Protocol Editor
Settings Lid Settings Open the Lid Settings window to change or to set the Lid
Temperature
Tools Gradient Select the block type for a gradient step.
Calculator
Run-time Select the instrument and scan mode to be used for
Calculator calculating the estimated run time in the Experiment Setup
window

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 CFX96 and CFX96 Deep Well Systems Instruction Manual

The toolbar in the Protocol Editor window provides quick access for important functions.
Table 12 lists the function of the Protocol Editor toolbar buttons.
Table 12. Protocol Editor toolbar buttons
Toolbar Button and Menus Name Function
Save Save the current protocol file

Print Print the selected window

Insert Step Select After or Before to insert a step

relative to the currently highlighted step

Sample Volume Enter a sample volume in µl between 0 and

50. Sample volume determines the
Temperature Control mode (page 30). Enter
zero (0) to select Block mode
Run Time View an estimated run time based on the
protocol steps and ramp rate
Help Open the software Help for more
information about protocols

Protocol Editor Controls

The Protocol Editor window includes buttons for editing the protocol on the bottom left of the
screen. First, select and highlight a step in the protocol by left clicking it with the mouse
pointer. Then click one of the Protocol Editor buttons at the bottom left side of the Protocol
Editor window. The location for inserting a new step Before or After the currently selected
step is determined by the status of the Insert Step box located in the toolbar.

Insert Step Button

To insert a temperature step before or after the currently selected step:
1. Click the Insert Step button.

2. Edit the temperature or hold time by clicking the default value in the graphic or text view,
and entering a new value.

Add or Remove a Plate Read

To add a plate read to a step or to remove a plate read from a step:
1. Select the step by clicking the step in either the graphical or text view.

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 now the same
button reads Remove Plate Read. Click to remove a plate read from the selected step.

27
Protocols

Insert Gradient Button

To insert a gradient step before or after the currently selected step:
1. Insert a temperature gradient step by clicking the Insert Gradient button.

2. Edit the gradient temperature range by clicking the default temperature in the graphic or
text view, and entering a new temperature.

3. Edit the hold time by clicking the default time in the graphic or text view, and entering a
new time.
Figure 21 shows the inserted gradient step. The temperatures of each row in the gradient are
charted on the right side of the window.

Figure 21. Protocol with inserted gradient step.

Insert GOTO Button

To insert a GOTO step before or after the selected step:
1. Click the Insert GOTO button.

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 21 shows an inserted GOTO step at the end of the protocol. Notice that the GOTO loop
includes steps 2 through 4.

Insert Melt Curve Button

To insert a melt curve step before or after the selected step:
1. Click the Insert Melt Curve button.

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 CFX96 and CFX96 Deep Well Systems Instruction Manual

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 29).
NOTE: You cannot insert a melt curve step inside a GOTO loop.
NOTE: The melt curve step includes a 30 second hold at the beginning of the step
that is not shown in the protocol.
Figure 22 shows a melt curve step added after step 6.

Figure 22. Protocol with inserted melt curve step.

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.

3. Add or remove options by entering a number, editing a number, or clicking a check box.
TIP: To hold a step forever (an infinite hold), enter zero (0.00) for the time.
Figure 23 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.

Figure 23. Step option for a gradient.

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Protocols

The Step Options window lists the following options you can add or remove from steps:
• Plate Read. Check the box to include a plate read
• Temperature. Enter a target temperature for the selected step
• Gradient. Enter a gradient range for the step
• Increment. Enter a temperature to increment the selected step; the increment amount is
added to the target temperature with each cycle
• Ramp Rate. Enter a rate for the selected step; the range depends on the block size
• Time. Enter a hold time for the selected step
• Extend. Enter a time to extend the selected step. The extend amount is added to the
hold time with each cycle
• Beep. Check the box to include a beep at the end of the step
TIP: When you enter a number that is outside the option range, the software
changes the number to the closest entry within the range.

Delete Step Button

To delete a step in the protocol:
1. Select a step in the graphic or text view.

2. Click the Delete Step button to delete the selected step.

WARNING! You cannot undo this function.

Temperature Control Mode

The instrument uses one of two temperature control modes to determine when the sample
reaches the target temperature in a protocol.
Enter a sample volume in the protocol editor to select a temperature control mode:
• Calculated mode. When you enter a sample volume between 1 and 50 µl, the thermal
cycler calculates the sample temperature based on 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
experiments. 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

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 CFX96 and CFX96 Deep Well Systems Instruction Manual

Figure 24 shows a protocol (bottom of the window) written by the Protocol AutoWriter.

Figure 24. Protocol AutoWriter window with a new protocol.

Creating a Protocol With the Protocol AutoWriter

The Protocol AutoWriter window uses information about your reaction to automatically
generate a protocol file. Follow these steps to use the Protocol AutoWriter to create a new
protocol:
1. Click the Protocol AutoWriter button on the toolbar to open the Protocol AutoWriter
window.

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.

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.

7. Click OK to save the new protocol, or click Cancel to close the window without saving
the protocol.
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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Protocols

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5 Plates
Read this chapter for information about creating and editing plate files:
• Plate Editor window (page 33)
• Select Fluorophores window (page 35)
• Well loading controls (page 36)
• Experiment settings window (page 38)
• Well Groups Manager window (page 40)
• Plate Spreadsheet View window (page 42)

Plate Editor Window

A plate file contains run parameters, such as scan mode and fluorophores, and well contents
and instructs the instrument about how to analyze the data. Open the Plate Editor window to
create a new plate or to edit the plate currently selected in the Plate tab. Once a plate file is
created or edited in the Plate Editor, click OK to load the plate file into the Experiment Setup
window and run it.
To run an experiment, you must load at least one well with sample type and fluorophore.
TIP: Click the Plate Loading Guide button to open the Plate Loading Guide
window from the toolbar for a quick overview of instructions to load a plate.

Opening the Plate Editor

To open the Plate Editor window (Figure 25), follow one of these options:
• To create a new plate, select File > New > Plate, or click the Create New button in
the Plate tab (page 19)
• To open an existing plate, select File > Open > Plate, or click the Open Existing
button in the Plate tab (page 19)
• To edit the current plate in the Plate tab, click the Edit Selected button in the Plate
tab (page 19)
• To open the plate associated with a data file in the Data Analysis window (page 53),
click View/Edit Plate on the toolbar

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Plates

Figure 25. Plate Editor window.

Plate Size and Type

The software applies these plate settings to all the wells during the experiment:
• Plate Size. Select a Plate Size under Settings menu that represents the size of the
reaction module block of your instrument. Choosing the instrument type, from the pull
down menu option on the Startup Wizard will change the default plate size loaded in the
Plate tab of the Experiment Settings window. In the Plate Editor, select the plate size
from the Settings menu (see Table 13 on page 35). Plate size cannot be changed during
or after the experiment
• Plate Type. Select Clear Wells or White Wells from the Plate Type under the Settings
menu. Make sure the fluorophore being used in the experiment is calibrated for the
selected plate type
NOTE: CFX instruments are factory calibrated for many fluorescent dye and plate
combinations. Calibration is specific to the instrument, dye, and plate type. To
calibrate a new combination of dye and plate type on an instrument, select Tools >
Calibration Wizard (see “Calibration Wizard” on page 99)

Scan Mode
The CFX system excites and detects fluorophores in six channels and uses 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:

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 CFX96 and CFX96 Deep Well Systems Instruction Manual

• All Channels. Includes channels 1 through 5 on the CFX system

• SYBR/FAM only. Includes only channel 1, and provides a fast scan
• FRET. Includes only the FRET channel and provides a fast scan

Plate Editor Toolbar

The toolbar in the Plate Editor provides quick access to important plate loading functions:
Table 13 lists the functions available in the Plate Editor toolbar.
Table 13. Toolbar items in the Plate Editor.
Toolbar Item Name Function
Save Save the current plate file

Print Print the selected window

Zoom Increase or decrease magnification in plate view

Scan Mode Select a scan mode to instruct the instrument what

channels to collect fluorescence data from during a run.
Select All Channels (default), SYBR/FAM only, or FRET

Well Groups Open the Well Groups Manager window and set up well
groups for the current plate

Help Open the software Help for information about plates

Plate Loading Show a quick guide about how to set up a plate and load
Guide the wells

Select Fluorophores Window

The Select Fluorophores window lists fluorophores that can be selected to load into the Plate
Editor well loading controls. To open the Select Fluorophores window, click the Select
Fluorophores button on the right side of the Plate Editor.
NOTE: The fluorophores listed depend on the scan mode; when SYBR/FAM only is
chosen, only channel 1 fluorophores are shown in the Select Fluorophores window.
NOTE: You cannot add or remove fluorophores in this list; you must calibrate the
new fluorophores on an instrument in the Calibration Wizard (page 99). After
calibration, the new fluorophore is added to the Select Fluorophore window.

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Plates

Click the Selected check box next to the fluorophore name to add or remove the fluorophores
to the list on the right side of the Plate Editor window.
In this example, SYBR is selected from the list of available fluorophores (Figure 26).

Figure 26. Select Fluorophores window.

• Click the Color box next to the fluorophore name and select a new color to represent
each fluorophore in the Plate Editor window and Data Analysis charts
NOTE: Before beginning the run, the software verifies that the fluorophores you
specified in the plate are calibrated on that instrument. You cannot run a plate if it
includes fluorophores that have not been calibrated on that instrument.

Well Loading Controls

A plate file contains information about the contents of each well loaded with sample for an
experiment. After the run, the software links the well contents to the fluorescence data
collected during the protocol and applies the appropriate analysis in the Data Analysis window.
For example, wells loaded with standard sample type are used to generate a standard curve.
Consider the following guidelines for well contents:
• Target Name. One or more targets of interest (genes or sequences) in each loaded well.
Each target is assigned to one fluorophore
• Sample Name. One identifier or condition that corresponds to the sample in each
loaded well, such as 0 hr, 1 hr, or 2 hr

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 CFX96 and CFX96 Deep Well Systems Instruction Manual

Select a well to load contents into by left clicking with the mouse pointer 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 14).
Table 14. 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).

Click a Load box to add a fluorophore to the selected

wells; each fluorophore corresponds to a target name. To
add fluorophores to the Load list, select them in the
Select Fluorophores window.

To distinguish between multiple targets, select a name in

the Target Name pull- down menu and press the Enter
key to load the target name in the well. To add a new
target name to the pull-down menu in the current plate
only, type a name in the pull-down box and press the
Enter key. To delete a target name, select it, press the
Delete key, and press the Enter key.
For gene expression analysis or to distinguish between
multiple samples, select a Sample Name from the pull-
down menu to load that sample name in the selected
wells. To add a new sample name to the pull-down menu
in the current plate, type a new name in the pull-down box
and press the Enter key. To delete a sample name, select
it in the menu, press the Delete key on your keyboard,
and then press Enter.
To load replicate numbers, selected wells must contain
identical well contents. If they do not, the software
disables this loading control. Click the Load box to add a
Replicate # to the selected wells.

In the Replicate Series pane you can apply a replicate

series to a set of selected wells. Enter the Replicate
Group Size by selecting a number that represents the
number of samples (wells) in each group of replicates.
Select a Starting Replicate # to add replicates.

You can load replicate groups with replicate numbers

progressing from left to right (Horizontal), or progressing
from top to bottom (Vertical).

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Plates

Table 14. Options for loading the plate and wells in the Plate Editor.
Option Function
Enter a concentration to the selected wells with standard
sample type by editing or typing a number in the
Concentration box. To apply the concentration to one
fluorophore in the well, select a single fluorophore from
the pull-down menu (<All>) under the concentration box.
To delete a concentration, select it, press the Back Space
key on your keyboard, and then press Enter.

Select multiple wells with a Standard sample type to

activate the Dilution Series button.
Click the Dilution Series button to enter a dilution series
for the concentration of Standard samples and load a
standard curve.

Enter the Starting Concentration for the dilution series,

the Replicates from (starting replicate number) and to
(ending replicate number), and the Dilution Factor
(amount to change the concentration with each replicate
group). Select Increasing for a dilution series that
increases, or select Decreasing for a dilution series that
decreases. Finally, select the fluorophore used for the
dilution series from the pull-down menu, and click Apply.

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 Quantitation Data
tab (page 68).
Select Tools > Show Collection Name to show this
pane. Enter sample collection information about one or
more wells by selecting the wells and typing a collection
name in the pull-down menu. Any collection name you
add to wells appear in the Gene Expression Analysis
window and enables sample grouping 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
experiment.
Click the Clear Replicate # button to clear the replicates
numbers in the selected wells.

Click the Clear Wells button to permanently clear all

content in the selected wells

Experiment Settings Window

To open the Experiment Settings window, follow one of these options:

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 CFX96 and CFX96 Deep Well Systems Instruction Manual

• In the Plate Editor, click the Experiment Settings button

• While analyzing data in the Data Analysis window, click the Experiment Settings
button in the Gene Expression tab
Open the Experiment Settings window to view or change the list of Targets and Samples
(Figure 27) or to set the gene expression analysis sample group to be analyzed if Collection
Names have been added to the wells.
• Targets. A list of target names for each PCR reaction, such as a genes or sequences of
interest. Click the Reference column to assign reference genes in an experiment
• Samples. A list of sample names that indicate the source of the target, such as a sample
taken at 1 hour (1 hr), or taken from a specific individual (mouse1). Click the Control
column to assign the control condition for an experiment
Figure 27 shows the Targets tab with the analysis settings shown.

Figure 27. Targets tab in Experiment Settings window.

Figure 28 shows the Sample Tab with the analysis settings shown.

Figure 28. Samples tab in Experiment Settings window.

To adjust the lists in these tabs, use the following functions:

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Plates

• Add a target or sample name by typing a name in the New box and clicking Add
• Remove a target or sample name from the list by clicking the Select to Remove box
for that row, and then clicking the Remove checked item(s) button
• Select the target as a reference for gene expression data analysis by clicking the box
in the Reference column next to the name for that target
• Select the sample as a control sample for gene expression data analysis by clicking
the box in the Control column next to the name for that sample
Click the Show Analysis Settings box in the Experiment Settings window to view or change
analysis parameters applied in the Gene Expression tab.
To adjust target parameters:
• 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 includes 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 that is selected in the Color column

Sample Name Grouping Option

Loading Collection Names in the wells enables samples to be analyzed in one of four
configurations defined by the Sample Name Grouping Option. These options are available
from the pull down menu in the Experiment Settings tab.
• Target vs. Sample
• Target vs. Collection
• Target vs. Sample_Collection
• Target vs. Collection_Sample

Well Groups Manager Window

Well groups divide a single plate into subsets of wells that can be analyzed independently in
the Data Analysis window. Once well groups are set up, select one in the Data Analysis
window to analyze the data in an independent group. For example, set up well groups to
analyze multiple experiments run in one plate or to analyze each well group with a different
standard curve.
NOTE: The default well group is All Wells.

Create Well Groups

To create well groups in the Well Groups Manager window, follow these instructions:
1. Click the Well Groups button in the toolbar of the Plate Editor.

2. Click Add to create a new group. The pull-down menu shows the group name as Group
1 for the first group.

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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 29).

4. (Optional) Change the name of the group by selecting the group name in the pull-down
menu and typing a new name.

5. (Optional) Create more well groups by repeating steps 1 and 2.

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.

Figure 29. Color of wells in the Well Group Manager window.

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Plates

Plate Spreadsheet View Window

The Plate Spreadsheet View window shows the contents of a plate in the Plate Editor. Open
the Plate Spreadsheet View window (Figure 30) by selecting Tools > Show Spreadsheet View
in the Plate Editor menu bar.

Figure 30. Plate Spreadsheet View window.

Open the spreadsheet view to import or export the well contents to Excel or to another tab-
delimited format:
• Click Import Template to import well contents from a comma delimited file
• Click Export Template to export well contents in Excel file (.csv format)
Sort or edit a column by selecting it and using these methods:
• Sort the spreadsheet according to the data in one column by clicking the diamond
next to a column name
• Edit the contents of a column that has an asterisk (*) at top by clicking and typing in
each well
NOTE: Select the units for the standard curve data in the Quantity column by
opening the Plate Editor and selecting Settings > Units in the menu bar. After the
plate runs, the data from these standards appear in the Standard Curve chart of the
Quantitation tab (Data Analysis window) with the units you select. Open the
spreadsheet view to import or export the plate contents to Excel or another tab-
delimited format.
Right-click on the spreadsheet to select one of these options from the right-click menu:
• Copy. Copy the entire spreadsheet
• Copy as Image. Copy the spreadsheet as an image file
• Print. Print the spreadsheet
• Print Selection. Print only the selected cells
• Export to Excel. Export the file as an Excel formatted file
• Export to Text. Export the file as a text file
• Find. Find text in the spreadsheet
• Sort. Sort the spreadsheet by selecting up to three columns of data in the Sort window

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 CFX96 and CFX96 Deep Well Systems Instruction Manual

6 Stand-Alone Operation
Read this chapter for information about running the CFX system in stand-alone mode:
• C1000™ thermal cycler (page 43)
• Creating a new experiment (page 45)
• Exporting data for analysis (page 49)
• Creating a data file (page 51)

C1000 Thermal Cycler

The CFX system can run real-time PCR experiments without a computer. You can export the
fluorescence data acquired during a run using the USB thumb key. You can also choose to
have the data emailed directly to you if the C1000 base is attached to the internet and the
email functionality has been configured (see the C1000 thermal cycler Instruction Manual for
information on how to configure the email settings). The data requires 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 31 shows the components of the control panel:
Protocol
Alphanumeric
LCD AutoWriter key
keys

Navigation
Command keys
keys

USB port
(below)

Function keys
Figure 31. The C1000 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 15:
Table 15. Functions of keys on control panel.

Key Function
COMMAND KEYS
RUN Select and run a protocol
EDIT Select and change 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
Protocol AutoWriter key 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 the CFX system starts, it runs a self-test to verify proper functions and then displays the
main menu. 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.
NOTE: The C1000 thermal cycler stores up to 20 real-time PCR experiment runs
using a date/time stamp on the runs. When 20 runs are stored on the C1000, older
run data is deleted when a new run is stored.

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 CFX96 and CFX96 Deep Well Systems Instruction Manual

Figure 32. Start-up screen on the front panel.

To initiate the functions in the main menu, press the associated function keys (F1 through F4):
• Log In (F1). Log in to the C1000 thermal cycler. Once you log in the button name
changes to Log Off
• Files (F2). View the files and folders in the file library
• Utilities (F3). Open the Utilities menu
• New Protocol (F4). Create a new protocol

Creating a New Experiment

1. Select New Protocol (F4) in the start up screen to begin (Figure 33).

Figure 33. Default real-time PCR protocol.

2. To change the target temperature and the hold time in a temperature step, press the
arrow keys to navigate between 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.

3. (Optional) To insert a new step, select the Insert (F1) button. To delete a step, select the
Delete (F3) button (Figure 33).

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Stand-Alone Operation

4. (Optional) To change step options, select the Options (F4) button (Figure 33). 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 34).

Figure 34. Step Options window.

NOTE: Press the alphanumeric keys to enter a Gradient Range spanning from 1 to
24oC.

5. The GOTO step instructs the thermal cycler to repeat a set of steps in a loop to create
the cycles in the PCR experiment. 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.

6. (Optional) To change the default sample volume, select the sample volume box (Vol)
(Figure 33 on page 45). Use the alphanumeric keys to enter a new sample volume in
microliters. The sample volume you enter determines the temperature control mode that
is used during a run.

7. (Optional) To change the default lid temperature, select the lid temperature box (Lid) by
pressing the arrow keys (Figure 33 on page 45). Use the alphanumeric keys to enter a
new temperature. The default lid temperatures for the CFX96 modules is105oC.
NOTE: Heating the lid prevents condensation in the sealed reaction vessels.

8. When creating a new protocol, you have the option to save it with a name. Use the arrow
keys to navigate to the Protocol Name box and then press the alphanumeric keys
multiple times to enter a letter or number to type a new protocol name.

9. Press ENTER to accept the name.

Running the Protocol

1. To begin the run, click Done (F2) in the Protocol window (Figure 33 on page 45).
TIP: Alternatively, click the RUN command key to start the run without saving or
editing the name of the protocol.

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 CFX96 and CFX96 Deep Well Systems Instruction Manual

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 35).

Figure 35. Saving a protocol.

3. Click Edit Filename (F1). Type a new name in the box and click Save (F2) (Figure 36).

Figure 36. Entering a protocol name.

4. Click Run (F2) to continue and run the protocol (Figure 37).

Figure 37. Protocol successfully saved.

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Stand-Alone Operation

5. Edit the Sample Volume and Lid Temperature. A Sample ID or User can also be
recorded for the run (Figure 38). Click OK (F1) to proceed.

Figure 38. Editing sample volume and lid temperature.

6. Select a Scan Mode to collect fluorescence data during a run (Figure 39).

Figure 39. Scan mode and data file name.

Scan modes detect calibrated fluorophores in these channels:
• All Channels. Collects data from channels 1 through 5 on the CFX96 and CFX96 Deep
Well systems
• SYBR/FAM. Collects data only from channel 1 and provides a fast scan
• FRET. Collects data only from the FRET channel and provides a fast scan

7. 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 enter a letter or number to type a new data file (.zpcr) name.

8. Click the OK (F1) button to start the run.

Running a Previously Saved Protocol

• To change an existing protocol, press the EDIT key to open the file library and select
a protocol to edit

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• To run an existing protocol, press the RUN command key and select a previously
saved protocol from the file library

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 a run, skip a step, or access the main menu (Figure 40)
• 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

Figure 40. Monitoring run status.

Exporting Data for Analysis

When the run is finished, the fluorescence data need to be transferred to a computer running
CFX Manager software for analysis. The stand-alone data file is automatically saved to the
RT_DATA folder located in the SYSTEM folder (Figure 41).

Figure 41. RT_DATA folder stores real-time PCR runs.

If a USB key has been placed in a USB key port on the C1000 thermal cycler, the data (.zpcr)
will automatically be saved to the root directory of the USB key.

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Stand-Alone Operation

If a USB key is not in the thermal cycler at the end of the run, follow these instructions:
1. Press the Files (F2) button on the main screen to access the file folders.

2. Navigate to the RT_DATA folder and then press the right arrow key to open the folder.

3. Select the file using the up and down arrow keys.

4. Press Export File (F1) to export the run data (.zpcr) to the USB key (Figure 42).

Figure 42. Exporting stand-alone run data to a USB key.

5. Click Yes (F1) to confirm the export.

You can choose to email your data to you directly from the C1000 thermal cycler after the run
completes by configuring the email settings (see the C1000 thermal cycler instruction manual
for information on configuring the email settings).
To send an email with attached data (.zpcr) at the end of a run, follow these instructions:
1. Select Options (F4) in the Run information screen (Figure 38 on page 48).

2. Using the arrow keys select the Send email notification option.

3. Click OK (F1) to return to the Run information screen.

4. Use the arrow keys to navigate to the Email Address box and then use the alphanumeric
keys to enter the email address.

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5. Click OK (F1) to continue to run the assay.

Figure 43. Confirming export to USB key.

Creating a Data File

The stand-alone run data (.zpcr) data need to be converted into a data file (.pcrd) by CFX
Manager software to be analyzed. To create a data file from a stand-alone run:
1. Click and drag the .zpcr file from the USB key directory over the main software window,
or Select File > Open > Stand-alone Run from the main software window menu options
to select the file name.

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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 44).

Figure 44. Assigning a plate file.

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
experiment. Load a Quick Plate file to quickly access data from all the wells.

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7 Data Analysis Overview

Read this chapter for information about data analysis:
• Data analysis precautions (page 53)
• Data Analysis window (page 54)
• Quantitation tab (page 57)
• Data analysis settings (page 58)
• Well selectors (page 60)
• Charts (page 62)
• Spreadsheets (page 62)

Data Analysis Precautions

There are many ways in which the data generated from the system can be interpreted.
Occasionally, the data can be interpreted incorrectly. In order to prevent data
misinterpretation, please follow these guidelines when inferring a result from the data:
1. Confirm that the sample data is correct. If a barcode was entered, check that the
barcode of the assay or plate matches that of the sample.

2. Confirm that the appropriate assay was run using the appropriate protocol.

3. Check that the controls were included in the assay and generated the expected result.

4. Confirm that the assay was run to completion.

5. Check that the appropriately diluted samples were loaded into the correct wells and
assigned the correct dye, Be sure that the samples were run in duplicate at a minimum.
6. Check to optimize an examination procedure in order to improve its performance
characteristics.

7. Care should be taken to note if any variable in the protocol was modified as this could
alter the call for the assay. These variables can include changes due to:
• Manually adjusting the baseline
• Manually adjusting the threshold
• Cq determination mode
• Removal of outliers
• Removal or changes to QC check rules

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Data Analysis Overview

Data Analysis Window

During data analysis, changing the way the data are displayed by changing the contents of
wells in the Plate Editor never changes the fluorescence data that were collected from each
well during the run. Once the module collects fluorescence data you cannot delete those data,
but you can choose to remove data from view and analysis.
To change the content of wells after a run, open the Plate Editor by clicking the Edit/View
Plate button at the top of the Data Analysis window.
TIP: You can add or edit information about the contents of the well before, during,
or after you run the real-time PCR experiment. You must assign the scan mode and
plate size before the run, and these parameters cannot change after the run.
CFX Manager™ software processes real-time PCR data automatically at the end of each run,
and opens the Data Analysis window to display these data. Choose one of these methods to
open existing data files in the Data Analysis window:
• Drag a data file (.pcrd extension) over the main software window and release it
• Select File > Open > Data File in the main software window to select a file in the
Windows browser
• Click the Data Analysis button in the main software window toolbar to select a file in
the Windows browser
• Select File > Recent Data Files to select from a list of the ten most recently opened
data files
The Data Analysis window displays up to nine tabs (Figure 45). Each tab shows the analyzed
data for a specific analysis method:

Figure 45. All the tabs that can display in the Data Analysis window.
The software only displays a tab in the Data Analysis window if the data are collected in the
run and are available for that type of analysis.

Data Analysis Toolbar

The toolbar in the Data Analysis window provides quick access to important data analysis
functions.Table 16 lists the functions of buttons in the toolbar.
Table 16. Toolbar in the Data Analysis window.
Toolbar button Name Function
Save Save the current data file

Print Print the selected window

Trace Style Open Trace Style window

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Table 16. Toolbar in the Data Analysis window. (continued)

Toolbar button Name Function
Report Open a Report for the current data file

View/Edit Plate Open the Plate Editor to view and edit the contents of
the wells
Well Groups... Select a well group name from the pull-down menu.
The default selection is All Wells
Help Open the software Help site for more information
about data analysis

Data Analysis Menu Bar

Table 17 lists the functions of items in the menu bar.
Table 17. Menu bar items in Data Analysis window.
Menu Item Command Function
File Save Save the file
Save As Save the file with a new name
Repeat Experiment Extract the protocol and plate file from the
current experiment to rerun it
Exit Exit the Data Analysis window
View Run Log Open a Run Log window to view the run
log of a data file
Settings Analysis Mode Select Baseline Subtraction method for
the selected well groups in the data
C(t) Determination Mode Select Regression or Single-Threshold
mode to determine how C(t) values are
calculated for each trace
Baseline Thresholds Open the Baseline Thresholds window to
adjust the baseline or the threshold
Trace Styles Open the Trace Styles window
View/Edit Plate Open the Plate Editor to view and edit the
plate
Mouse Highlighting Turn on or off the simultaneous
highlighting of data with the mouse
pointer

TIP: If the Mouse Highlighting is turned off,

then hold down the Control key to
temporarily turn on the highlighting
Display Threshold Values Display the value of the threshold line in
the chart
Tools Reports Open the Report for this data file

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Data Analysis Overview

Table 17. Menu bar items in Data Analysis window. (continued)

Menu Item Command Function
Import Fluorophore Calibration Select a calibration file to apply to the
current data file
Replace Plate Replace the current plate file in the data
analysis
Export All Data Sheets to Excel Export all the spreadsheet views from
every tab to a separate Excel formatted
file
Help Open software Help for more information
about data analysis

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Quantitation Tab
Each tab in the Data Analysis window displays data in charts and spreadsheets for a specific
analysis method, with a well selector to select the data you want to show. The Data Analysis
window opens with the Quantitation tab (Figure 46) in front. The Amplification chart data in
this tab should be used to determine the appropriate analysis settings for the experiment.
NOTE: The Amplification chart shows the relative fluorescence (RFU) for each well
at every cycle. Each trace in the chart represents data from a single fluorophore in
one well.

Figure 46. Layout for the Quantitation tab in the Data Analysis window.
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.

Step Number Selector

The CFX system can acquire fluorescence data at multiple protocol steps; the software
maintains the data acquired at each step independently. The software displays the Step
Number selector below the Standard Curve chart on the Quantitation tab whenever a protocol
contains more than one data collection step. When you select a step, the software applies that
selection to all the data that are shown in the Data Analysis window. Figure 47 shows the data
collection step number is 3 for all the data.

Figure 47. Step Number selection in the Data Analysis window.

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Data Analysis Overview

Viewing Well Groups in Data Analysis

Wells in the plate can be grouped into subsets for independent analysis using well groups.
When you create well groups in the Well Groups Manager window in the Plate Editor
(page 40), group names appear in the Data Analysis window within the Well Groups list on the
toolbar.
By default, the well group All Wells is selected when the Data Analysis Window is first opened,
with the data in all wells with content shown in the charts and spreadsheets.
Figure 48 shows Group 2 selected in the Well Groups menu. Only the wells in that well group
appear loaded in the well selector and data for these wells only are included in the data
analysis calculations.

Figure 48. Data Analysis window with Group 2 selected.

Data Analysis Settings

The Amplification chart data in the Quantitation tab shows the relative fluorescence (RFU) for
each well at every cycle. Each trace in the chart represents data from a single fluorophore in
one well. These data are used to determine C(t) values for each well on a per fluorophore
basis. The software uses one of two modes to determine C(t) values:
• Regression. This mode applies a multivariable, nonlinear regression model to individual
well traces and then uses this model to compute an optimal C(t) value
• Single Threshold. This mode uses a single threshold value to calculate the C(t) value
based on the threshold crossing point of individual fluorescence traces

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Adjusting the Threshold

In Single-Threshold mode, adjust the threshold for a fluorophore by clicking on the threshold
line in the Amplification chart and moving the mouse pointer vertically. Alternatively, specify an
exact crossing threshold for the selected fluorophore by following these instructions:
1. Select Settings > Baseline Thresholds in the menu bar to open the Baseline Thresholds
window.

2. Adjust the crossing threshold (Figure 49) for the fluorophore by clicking User Defined
and entering a threshold number.

Figure 49. Baseline Thresholds window.

3. Click OK to confirm the change and close the window.

Baseline Settings
The software automatically sets the baseline individually for each well. Once you select the
wells for analysis, check the baseline settings in these wells. Open the Baseline Thresholds
window (Figure 49) to change the default baseline for selected wells. To open this window:
1. Select Settings > Baseline Thresholds to open the Baseline Thresholds 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 49).

3. Click OK to confirm the change and close the window.

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Data Analysis Overview

Select the Analysis Mode

Select the Analysis Mode to determine the method of baseline subtraction for all fluorescence
traces. Select Settings > Analysis Mode 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
• 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
threshold 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
• 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 C(t) is left invariant

Well Selectors
Click the wells in the well selector to show or to 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 50, 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

Figure 50. Three well colors appear in a well selector.

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Temporarily Exclude Wells from Analysis

RIGHT-CLICK OPTION
1. Right-click on the well in the well selector, on a fluorescence trace, or on a point plotted
on the standard curve.

2. Choose Exclude Well XX from Analysis from the menu options.

Figure 51. Right-click to exclude a well from analysis.

PLATE EDITOR OPTIONS

1. Click the View/Edit Plate button on the toolbar in the Data Analysis window.

2. Select one or more wells in the well selector view.

3. Click Exclude Wells in Analysis (Figure 52) to exclude the selected wells. This checkbox
is at the bottom of the Plate Editor controls on the right side of the window.

Figure 52. Exclude Wells in Analysis checkbox at bottom of the pane.

4. Excluded wells 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 is cleared.

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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.

Common Right-Click Menu Items for Charts

Right-click menu items are available on all charts. Some of the available items are present for
all charts, and these items can be used to change how the data are displayed or to easily
export the data from a chart (Table 18.)
Table 18. Right-click menu items for charts.
Item Function
Copy Copy the chart into the clipboard
Save Image As... Save the chart image in the selected image file type. Select
from these formats: PNG (default), GIF, JPG, TIF, or BMP
Page Setup... Preview and select page setup for printing
Print... Print the chart
Show Point Values Show the point values when the mouse moves over a point
on the chart.
Set Scale to Default Return to default chart view after magnifying the chart
Chart Options... Open the Chart Options window to change the chart,
including changing the title, selecting limits for the x and y
axes, showing grid lines, and showing minor ticks in the
axes
NOTE: Menu items that apply to specific charts are described in the next chapter
“Data Analysis Windows” (page 65).

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.

4. Click OK to sort the data, or click Cancel to stop sorting.

Highlight the data on the associated charts and well selector by holding the mouse pointer
over a cell. If you click in the cell, you can copy the contents to paste into another software
program.

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Common Right-Click Menu Items for Spreadsheets

Right-click any spreadsheet view to select the items shown in Table 19.
Table 19. Right-click menu items for spreadsheets
Item Function
Copy Copy the contents of the selected wells to a clipboard, then
paste the contents into a spreadsheet such as Excel
Copy as Image Copy the spreadsheet view as an image file, and paste it
into a file that accepts an image file such as text, image, or
spreadsheet files
Print... Print the current view
Print Selection... Print the current selection
Export to Excel... Export the data to an Excel spreadsheet
Export to Text... Export the data to a text editor
Export to XML Export the data to an XML file
Export to HTML Export the data to an HRML file
Find... Search for text
Sort... Sort the data in up to three columns

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 CFX96 and CFX96 Deep Well Systems Instruction Manual

8 Data Analysis Windows

Read this chapter for more information about the tabs in the Data Analysis window:
• Quantitation tab (page 65)
• Quantitation Data tab (page 68)
• Melt Curve tab (page 69)
• Melt Curve Data tab (page 70)
• End Point tab (page 70)
• Allelic Discrimination tab (page 72)
• QC tab (page 74)
• Run Information tab (page 74)
• Data file reports (page 75)

Quantitation Tab
Use the data in the Quantitation tab (Figure 46 on page 57) to set the data analysis conditions,
including the baseline settings for individual wells and the threshold settings. The Quantitation
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 only shown if the experiment includes wells designated
as Sample Type Standard. 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
• Spreadsheet. Shows a spreadsheet of the data collected in the selected wells

Fluorophore Selector
To select the fluorophore data to display in the Quantitation tab charts and spreadsheets, click
the fluorophore selector below the Amplification chart (Figure 53). Click the box next to the
fluorophore name to show or hide the fluorophore data throughout the data analysis window.

Figure 53. Fluorophore selector with FAM selected.

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Trace Styles Window

Open the Trace Styles window (Figure 54) to adjust the appearance of traces in the
amplification and melt curve charts in the Quantitation and Melt Curve tabs.
To open this window, follow these steps:
1. Select only one fluorophore in the fluorophore selection boxes.

2. Click the Trace Styles button in the Data Analysis toolbar, or select Settings > Trace
Styles in the Data Analysis menu bar.

Figure 54. Trace Styles window.

Use the tools in the Trace Styles window to adjust appearance of traces and preview the
changes in the well selector at the bottom of the window.
• Select a specific set of wells by using the well selector at the bottom of the window.
Alternatively, select wells that contain one sample type in the pull-down menu in the
Wells column
• Click the box in the Color column to select a color for the wells
• Select a symbol from the pull-down menu in the Symbol column
• Click Show Contents to show the sample types in each well, or click Show
Symbols to show the selected Symbols in each well.

Log Scale Option

Click the Log Scale box at the bottom of the Amplification chart to view the fluorescence
traces in a semi-log scale, as shown in Figure 55.

Figure 55. Log Scale option selected in Amplification chart.

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Standard Curve Chart

The software creates a Standard Curve chart (Figure 56) in the Quantitation tab if the data
include sample types defined as standard (Std) for one fluorophore in the experiment.

Figure 56. Standard Curve chart.

The Standard Curve chart displays the following information:
• Name for each curve (the fluorophore name)
• Color of each fluorophore
• Reaction efficiency (E). Use this statistic to optimize a multiplex reaction, and
equalize the data for a standard curve
NOTE: The reaction efficiency describes how much of your target is being
produced with each cycle in the protocol. An efficiency of 100% means that you
are doubling your target with each cycle.
• Coefficient of determination, R2 (written as R^2). Use this statistic to determine how
correctly the line describes the data (goodness of fit)

Chart Right-Click Menu Options

In addition to the common right-click menu options to copy, print, and export charts, Table 20
lists the menu options available only on the Amplification chart.
Table 20. Amplification chart specific right-click menu options.
Menu Option Function
Show Threshold Values Display the threshold value for each amplification curve on the
chart
Trace Styles... Open the Trace Styles window to change trace styles that
appear on the Quantitation and Melt Curve tabs
Baseline Thresholds... Open the Baseline Thresholds window to change baseline or
thresholds of each fluorophore (changes appear in
Amplification chart in Quantitation tab)

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Quantitation Data Tab

The Quantitation Data tab shows spreadsheets that describe the quantitation data collected in
each well. Select one of the three options to show the data in different formats:
• Results. Displays a spreadsheet view of the data
• Plate. Displays a view of the data in each well as a plate map
• RFU. Choose this spreadsheet to show the RFU quantities in each well for each cycle
TIP: Right-click any spreadsheet for options, including the sort option.

Results Spreadsheet
Select a Results spreadsheet (Figure 57) to see data for each well in the plate.

Figure 57. Quantitation Data tab with Results spreadsheet selected.

NOTE: All Std. Dev (standard deviation) calculations apply to the replicate groups
assigned in the wells in the Plate Editor window. The calculations average the C(t)
value for each well in the replicate group.
The Results spreadsheet includes the type of information listed in Table 21.
Table 21. Results spreadsheet content.
Information Description
Well Well in the plate
Fluor Fluorophore detected
Content Sample type and replicate number
Target Amplification target name (gene)
Sample Sample description
Threshold Cycle (C(t)) Threshold cycle
C(t) Mean Mean of the threshold cycle for the replicate group
C(t) Std. Dev Standard deviation of the threshold 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

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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 58 shows
the Plate spreadsheet as plate map.

Figure 58. Plate spreadsheet in Quantitation Data tab.

RFU Spreadsheet
Select the RFU spreadsheet to see the RFU readings for each well acquired at each cycle of
the experiment. 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 59).

Figure 59. RFU spreadsheet in the Quantitation Data tab.

Melt Curve Tab

Open the Melt Curve tab (Figure 60) to determine the melting temperature (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 70).

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Data Analysis Windows

Figure 60. 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 Peak Type 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

Melt Curve Data Tab

The Melt Curve Data tab shows the data from the Melt Curve tab in multiple spreadsheets that
include all the melt peaks for each trace. Select one of four options to show the melt curve
data in different spreadsheets.
• Melt Peaks. List all the data, including all the melt peaks, for each trace
• Plate. List a view of the data and contents of each well in the plate, including the
content, sample name, peak 1 and peak 2
• RFU. List the RFU quantities at each temperature for each well
• –d(RFU)/dT. List the negative rate of change in RFU as the temperature (T) changes for
each well. This is a first regression plot for each well in the plate

End Point Tab

Open the End Point tab to analyze final relative fluorescence units (RFUs) for the sample wells.
The software compares the RFU levels for wells with unknown samples to the RFU levels for
wells with negative controls, and scores the unknown as a Positive or Negative. Positive
samples have an RFU value that is greater than the average RFU value of the negative controls
plus the Cut Off Value.
To analyze the end point data, the plate must contain negative controls, or the software cannot
make the call. Run one of these two types of protocols:
• Run a Quantitation protocol. Set up a standard protocol. After running the experiment,
open the Data Analysis window, adjust the data analysis settings in the Quantitation tab,
and then click the End Point tab to pick an end point cycle

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• Run an End Point Only protocol. Load the End Point Only protocol in the Plate tab of
the Experiment Setup window, select or create a plate, and run the experiment
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.
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

Figure 61. Layout of the End Point analysis tab.

The Results list includes this information:
• Lowest RFU value. Lowest RFU value in the data
• Highest RFU value. Highest RFU value in the data
• Negative Control Average. Average RFU for the wells that contain negative controls
• Cut Off Value. Calculated by adding the tolerance (RFU or Percentage of Range listed in
the Settings) and the average of the negative controls. Samples with RFUs that are
greater than the cutoff value will be called “Positive”. To adjust the cutoff value, change
the RFU or Percentage of Range
The Cut Off Value is calculated using this formula:
Cut Off Value = Negative Control Average + Tolerance

Select a tolerance by one of these methods:

• RFUs (default). Select this method to use an absolute RFU value for the tolerance. The
minimum RFU tolerance value is 2. The maximum is the absolute value of the highest
RFU value minus the absolute value of the lowest RFU value. The default RFU tolerance
value is 10% of the total RFU range
• Percent of Range. Select this method to use a percentage of the RFU range for the
tolerance. The minimum percent of range is 1 percent. The maximum percent of range is
99 percent. The default percent of range is 10 percent

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Adjusting the End Point Data Analysis

Adjust the information shown in the End Point tab by following these methods:
• Choose a Fluorophore from the pull-down list to view the data
• Choose an End Cycle to Average value to set the number of cycles that the
software uses to calculate the average end point RFU
• Select RFUs to view the data in relative fluorescence units
• Select Percentage of Range to view the data as a percentage of the RFU range

Allelic Discrimination Tab

The Allelic Discrimination tab assigns the genotypes to wells with unknown samples using the
RFU or C(t) of positive control samples (Figure 62). Use this data to identify sample genotypes,
including Allele 1, Allele 2, Heterozygote, Unknown, Control 1, or Control 2.
NOTE: The data for allelic discrimination must come from multiplex experiments.
Allelic discrimination analysis requires the following minimal well contents:
• Two fluorophores in each well, except the wells that contain positive controls can
contain only one fluorophore
• One fluorophore that is common to all wells in the well group
• NTC (no template control) samples if you want to normalize the data.

Figure 62. Layout of the Allelic Discrimination tab in the Data Analysis window.

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Adjusting Data for Allelic Discrimination

The software automatically assigns a genotype to wells with unknown samples based on the
positions of the vertical and horizontal threshold bars and then lists genotype calls in the
spreadsheet view. To automatically call genotypes, the software uses positive controls (when
available), or estimates the thresholds. The software takes an average C(t) or RFU for the
positive controls to automatically set the threshold lines for discrimination of the alleles.
Adjust the positions of the threshold bars by clicking and dragging them, and the software
automatically adjusts the calculations to make new genotype assignments:
• If the experiment contains three controls in the plate, then the positions of the
threshold bars are based on the mean and the standard deviation of the RFU or C(t)
of the controls
• If the number of controls is less than three, then the positions of the threshold bars is
determined by the range of RFU or threshold cycle values in the selected fluorophore
Adjust allelic discrimination data by following any of these methods:
• Click and drag the threshold bars in the Allelic Discrimination chart to adjust the calls
in the spreadsheet
• Select a fluorophore for each axis in the chart (X: and Y:) in the settings options on
the bottom right of the window
• 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 positions, which are indicated by the numbers next to the bars
• Select the C(t) 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 calculations for normalized RFUs follow 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

• represents the mean RFU

NTCA1 + A2 represents the sum of RFUs for the NTC sample of Allele 1 and Allele 2

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QC Tab
Open the QC tab to quickly assess the quality of the experimental data based on the rules
defined in the QC tab in the User Preferences window (see “QC Tab” on page 96).
The software displays the currently applied QC rules and the settings that define each rule
(Figure 63). The rule description also displays wells that fail a selected rule.
NOTE: You can turn on or turn off rules by clicking the check box next to the rule in
the Use Rule column.

Figure 63. QC tab layout.

Run Information Tab

The Run Information tab (Figure 64) shows the protocol and other information about the run for
each experiment. You can also enter and edit the run Notes by typing in the Notes box or enter
and edit the data ID for the run by typing in the ID box.

Figure 64. Layout of the Run Information tab in the Data Analysis window.

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Data File Reports

The Report window (Figure 65) shows information about the current data file in the Data
Analysis window. To open a report, select Tools > Reports, or click the Reports button on the
toolbar in the Data Analysis window.
The Report window shows these three sections:
• Menu and toolbar. Select options to format, save, and print the report or template
• Options list (top, left side of window). Select options to show in the report
• Options pane (bottom, left side of window). Enter information about a selected option
• Preview pane (right side of window). View the current report in a preview

Figure 65. Example of a Report window for a data file.

TIP: The layout of the report can define the type of information that appears in any
report if you save the report as a template. Select Template > Save or Save As to
save the layout of the current report as a template.

Create a Data Analysis Report

To create a report in the Data Analysis window, follow these steps:
1. Make final adjustments to the well contents, selected wells, charts, and spreadsheets in
the Data Analysis window before creating the report.

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 check boxes 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 quantitation
experiment might not contain a standard curve, and therefore those data do not
appear in the Data Analysis window or in the data report.

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Data Analysis Windows

4. Click the Update Report button to update the Report Preview with any changes.

5. 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.

6. (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.

Data Analysis Report Categories

A report can include any of the options in each category described in Table 22, depending on
the type of data in the Data Analysis window.
Table 22. Data analysis report categories in the options list.
Category Option Description
Header Title, subtitle and logo for the report
Report Information Experiment date, user name, data file
name, data file path, and selected well
group
Notes Notes about the data report
Experiment Setup
Run Information Includes the experiment date, user, data
file name, data file path, and the selected
well group
Protocol Text view of the protocol steps and
options
Plate Display Show a plate view of the information in
each well of the plate
Quantitation
Analysis Settings Includes the step number when data
were collected, the analysis mode, and
the baseline subtraction method
Amplification Chart Copy of the amplification chart for
experiments that include quantitation
data
Standard Curve Chart Copy of the standard curve chart
Data Spreadsheet listing the data in each well
Gene Expression
Analysis Settings Includes the analysis mode, chart data,
scaling option, and chart error
Chart Copy of the gene expression chart
Target Names Chart of the names
Sample Names Chart of the names
Data Spreadsheet listing the data in each well
Melt Curve

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Table 22. Data analysis report categories in the options list. (continued)
Category Option Description
Analysis Settings Includes the melt step number and
threshold bar setting
Melt Curve Chart Copy of the melt curve chart
Melt Peak Chart Copy of the melt peak chart
Data Spreadsheet listing the data in each well
Allelic Discrimination
Analysis Settings Includes display mode, fluorophores,
cycle, thresholds, and normalized data
Chart Copy of the allelic discrimination 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 cutoff value
Data Spreadsheet listing the data in each well

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9 Gene Expression Analysis

Read this chapter for information about performing Gene Expression Analysis:
• Gene Expression (page 79)
• Plate setup for gene expression analysis (page 80)
• Gene Expression tab (page 80)
• Experiment Settings window (page 85)
• Gene Study (page 86)
• Gene Study Data spreadsheet (page 89)
• Gene Study Report window (page 90)

Gene Expression
With the use of stringently qualified controls in your reactions, you can run a gene expression
experiment to normalize the relative differences in a target concentration between 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
numbers 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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Plate Setup for Gene Expression Analysis

To perform gene expression analysis, the contents of the wells must include the following:
• Two or more targets. The two targets that represent different amplified genes or
sequences in your samples
• One or more reference targets. At least one target must be a reference target for
normalized expression. Assign all reference targets in the Experiment Settings window
(page 38) to analyze the data in Normalized Expression mode (C(t)). Experiments that
do not contain a reference must be analyzed using Relative Expression mode (C(t))
• Common samples. Your reactions must include common samples (minimum of two
required) to view your data plotted in the Gene Expression tab. These samples represent
different treatments or conditions for each of your target sequences. Assign a control
sample (optional) in the Experiment Settings window (page 38)
The requirements for Gene Expression setup in the Plate Editor depend on whether reaction
contents are singleplex PCR with one fluorophore in the reactions or multiplex PCR with
more than one fluorophore in the reactions.
Figure 66 shows an example of the minimum contents of the wells for a singleplex gene
expression experiment.

Figure 66. Example of well contents in a singleplex gene expression experiment.

Figure 67 shows an example of the minimum contents of the wells for a multiplex gene
expression experiment.

Figure 67. Example of well contents in a multiplex gene expression experiment.

Gene Expression Tab

The Gene Expression tab in the Data Analysis window shows the relative expression of targets
in these two views:
• Gene Expression chart. Shows the real-time PCR data as normalized expression
(C(t)) or relative quantity (C(t))
• Spreadsheet. Shows a spreadsheet of the gene expression data
TIP: Right-click any chart or spreadsheet for options. Click the View/Edit Plate
button to open the Plate Editor, and change well contents in the plate.

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Figure 68. 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.

Normalized Gene Expression

To normalize data, use the measured expression level of one or more reference genes (targets)
as a normalization factor. Reference genes are targets that are not regulated in the biological
system being studied, such as actin, GAPDH, or Histone H3.
To set up normalized gene expression (C(t)) analysis, follow these steps:
1. Open a data file (.pcrd extension).

2. Review the data in the Quantitation tab of the Data Analysis window. Make adjustments
to the data, such as changing the threshold and the Analysis Mode.

3. Click the Gene Expression tab.

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 experiment 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 (C(t)) if it is not already selected, and then view the
expression levels in the Gene Expression tab.

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Relative Quantity
Select Relative Quantity (C(t)) from the pull-down menu in the chart controls of the Gene
Expression tab to run a Relative Quantity (C(t)) analysis. By definition, relative quantity (C(t))
data are not normalized. This method is used to quantitate samples that do not include any
reference genes (targets). Typically, the researcher is confident in one of the following
considerations when setting up the experiment:
• 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 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.

Adjusting Gene Expression Data

After selecting your analysis method, adjust the data you view in the Gene Expression tab by
changing the settings options to the right of the chart.

GRAPH DATA
Graph data options allow you to present the data in the graph with one of two options:
• Relative to control. Graph the data with the axis scaled from 0 to 1. If you assign a
control in your experiment, 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 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 (C(t)) 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 experimental design:
• Unscaled expression. This option presents the unscaled normalized gene expression
• 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

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• 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 options 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)

CHART ERROR BAR MULTIPLIER

Select a multiplier for the error bars in the Gene Expression graph. Select one of these
integers: 1 (default), 2, or 3. The type of multiplier changes when you select the Error Type:
• SEMs for Standard Error of the Mean
• Std Devs for Standard Deviations

TARGET STABILITY VALUE

Open this window whenever more than 1 reference gene is used. The software calculates two
quality parameters for the reference genes:
• Coefficient of Variation (CV) of normalized reference gene relative quantities. Lower
CV values denotes higher stability
• M-value. A measure of the reference gene expression stability

Right-Click Menu Options for Gene Expression Graph

Right-click on the Gene Expression graph to select the items shown in Table 23.
Table 23. Right-click menu items.
Item Function
Copy Copy the chart to a clipboard
Save as Image Save the graph in the chart view as an image file. The default
image type is PNG. The other selections for image file types
include GIF, JPG, TIF, and BMP
Page Setup... Select a page setup for printing
Print... Print the chart view
Show Point Values Display the relative quantity of each point on the graph when
you place the cursor over that point
Set Scale to Default Set the chart view back to the default settings after magnifying
it
Chart Options... Open the Chart Options window to adjust the graph
Sort Sort the order that samples or targets appear on the chart x-
axis
User Corrected Std Devs Calculate the error bars using the corrected standard deviation
formula
Use Solid Bar Colors Display solid bars in the graph
x-axis labels Choose to display x-axis labels horizontal or angled

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Gene Expression Spreadsheet

Table 24 describes the information shown in the Gene Expression spreadsheet.
Table 24. Description of information in the spreadsheet on the Gene Expression tab.
Information Description
Target Target Name (amplified gene) selected in the Experiment
Settings window
Sample Sample Name selected in the Experiment Settings window
Ctrl Control sample, when the Sample Name is selected as a
control in the Experiment Settings window
Expression Normalized Gene Expression (C(t)) or Relative quantity
(C(t)) 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 (C(t)) Mean of the threshold cycle
C(t) SEM (or SD) Standard Error of the Mean or Standard Deviation of the
threshold cycle, depending on the selected option

Show Details Option

When you click the Show Details check box, Table 25 also shows this information.
Table 25. Information in Gene Expression spreadsheet with Show Details selected.
Information Description
Data Set Fluorescence data from one fluorophore in the data file
Relative Quantity Calculated relative quantity of samples
Relative Quantity SD Standard deviation of the relative quantity calculation
Corrected Relative Quantity Calculated standard deviation of the corrected relative
SD quantity
Unscaled Expression Calculated unscaled expression
Unscaled Expression SD Calculated standard deviation of the unscaled expression
Corrected Unscaled Corrected standard deviation of the unscaled expression
Expression SD
Expression Relative expression level
Wells Well number in the plate

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Experiment Settings Window

Open the Experiment Settings window by clicking the Experiment Settings button in the
Gene Expression tab. In this window, view or change the list of Targets and Samples, select
reference genes, select control samples, or set the Gene Expression Analysis sample group to
be analyzed if Collection Names have been added to the wells (Figure 69).

Figure 69. Experiment Settings window with Targets tab selected.

To adjust the lists in these tabs, use the following functions:
• Add a target or sample name by typing a name in the New box, and clicking Add
• Remove a target or sample name from the list by clicking the Select to Remove
Name box for that row, and then clicking the Remove checked item(s) button
• Select the target as a reference for gene expression data analysis by clicking the box
in the Reference column next to the Name for that target
• Select the sample as a control sample for gene expression data analysis by clicking
the box in the Control column next to the name for that sample

Sample Name Grouping Option

Loading Collection Names in the wells enables samples to be analyzed in one of four
configurations defined by the Sample Name Grouping Option. These options are available
from the pull-down menu in the Experiment Settings tab.
• Target vs. Sample. Only the well sample name is used in the gene expression
calculations
• Target vs. Collection. Only the well collection name is used in the calculations
• Target vs. Sample_Collection. The sample name and collection name are combined to
make a single name that is used in the calculations
• Target vs. Collection_Sample. The collection name and sample name are combined to
make a single name that is used in the calculations

Show Analysis Settings in Experiment Settings

Click the Show Analysis Settings box in the Experiment Settings window to view or change
analysis parameters applied in the Gene Expression tab:
• Click a cell in the Color column to change the color of the targets graphed in the
Gene Expression chart

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• 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 includes a
standard curve. Alternatively, type a previously determined efficiency
Figure 70 shows the efficiency of all the targets, which appears if Auto Efficiency is selected.

Figure 70. Targets tab in 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

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 data 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 gene expression data must include a common sample in every data file
to create a Gene Study. The software uses the common sample to normalize the
data between experiments. Select the sample names in the Experiment Settings
window (page 38).
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.

Gene Study Inter-Run Calibration

All data within the Gene Study are normalized by inter-run calibrator to calculate the smallest
average C(t) value. When the data files within the Gene Study include more than one inter-run
calibrator, then the calibrator with the smallest average C(t) value becomes the dominant
inter-run calibrator. The dominant calibrator is used to adjust all C(t) values in the Gene Study.

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To find the dominant inter-run calibrator, the software calculates the average of the C(t)
values for all inter-run calibrators of a given target (gene), 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 C(t) values in pair-wise comparisons.
The range is examined by comparing the absolute value of the difference between the
maximum and minimum C(t) for the inter-run calibrators of a given target.

3. If any target has an identical range as the C(t) values, then set the dominant calibrator
to the target with the smallest absolute value of average C(t) for eligible inter-run
calibrator samples.

4. If any target has identical average C(t) absolute values, then set the dominant calibrator
to the replicate group with the smallest C(t).
NOTE: The first data file imported into the Gene Study will always serve as the hub
file for pairwise data comparison during inter-run calibration.

Gene Study Window

The Gene Study window includes two tabs:
• Study Setup tab. Click this tab to manage the experiments in the Gene Study. Adding or
removing data files in a Gene Study does not change the original data in that file
• Study Analysis tab. Click this tab to view the gene expression data for the combined
experiments
Figure 71 shows the Gene Study window.

Figure 71. Gene Study window.

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Study Setup Tab

Before importing data into a Gene Study, do the following in the Data Analysis window:
• Check that samples that contain the same content are named with the same name.
In a Gene Study, the software assumes that wells with the same Target or Sample
name contain the same samples
• Adjust the baseline and threshold (C(t)) in the Quantitation tab to optimize the data in
each experiment before you add them to a Gene Study
• Select the well group you want to include in the Gene Study
The Study Setup tab (Figure 71) shows a list of all the experiments in the Gene Study.
• Add experiments. Click the Add Data Files button to select a file from a browser
window. To quickly add experiments to a Gene Study, drag the data files (.pcrd
extension) to the Gene Study window
• Remove experiments from this Gene Study. Select one or more files in the list and
click Remove
• Add notes about the Gene Study. Type in the Notes box to add comments about the
files and analysis in this Gene Study
The Study Setup tab lists the data files in the Gene Study, as described in Table 26.
Table 26. Study Setup tab in Gene Study window.
Column Title Description
File Name Name of the experiment data file (.pcrd extension)
File Folder Directory that stores the data file for each experiment 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

TIP: To analyze one well group in the Gene Study, that well
group must be selected in the Data Analysis window before
importing the data file into the Gene Study
Step Protocol step that included the plate read to collect real-
time PCR data
Grid View Open a plate map of the plate with the data in each of the
experiments included in the Gene Study

Study Analysis Tab

The Study Analysis tab shows the data from all experiments that are added to the Gene Study.
Open this tab to analyze the data, and select these options for the Gene Expression chart:
• Mode. Select Normalized Expression (C(t)) or Relative Quantity (C(t))
• Graph Data. Select Relative to normal or Relative to control in the graph
• x-axis options. Select the labels on the x-axis of the graph, including Sample or Target
• 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 only available when your samples do not contain controls

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• 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 check box. 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 72).

Figure 72. Study Analysis tab in Gene Study window.

Gene Study Data Spreadsheet

The data spreadsheet in the Gene Study window lists information about each target and
sample in the Gene Study (Figure 72).
Table 27 describes the information shown in the Gene Study spreadsheet.
Table 27. Information in the spreadsheet on the Study Analysis tab.
Information Description
Target Target Name (amplified gene) selected in the Experiment
Settings window
Sample Sample Name selected in the Experiment Settings window
Ctrl Control sample, when the sample name is selected as a
control in the Experiment Settings window
Expression Normalized Gene Expression (C(t)) or Relative Quantity
(C(t)), depending on the selected mode
Expression SEM (or SD) Standard Error of the Mean or Standard Deviation, depending
on the selected option

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Table 27. Information in the spreadsheet on the Study Analysis tab. (continued)
Information Description
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 (C(t)) Mean of the threshold cycle
C(t) SEM (or SD) Standard Error of the Mean or Standard Deviation of the
threshold cycle, depending on the selected option

Show Details Data

Click the Show Details check box to show additional information. The spreadsheet adds the
information in the columns listed in Table 28.
Table 28. Information added to the spreadsheet when Show Details selected.
Information Description
Data Set Fluorescence data from one fluorophore in one data file
Relative Quantity Calculated relative quantity of samples
Relative Quantity SD Standard deviation of the relative quantity calculation
Corrected Relative Quantity Calculated standard deviation of the corrected relative quantity
SD
Unscaled Expression Calculated unscaled expression
Unscaled Expression SD Calculated standard deviation of the unscaled expression
Corrected Unscaled Corrected standard deviation of the unscaled expression
Expression SD
Expression Relative expression
Wells Well number in the plate

Gene Study Report Window

Open the Gene Study Report window to arrange the Gene Study data into a report. To create a
gene study report, follow these steps:
1. Adjust the Gene Study report data and charts as needed before creating a report.

2. Select Tools > Reports to open the Gene Study report window.

3. Click the check boxes in the report options list to select and remove options to choose
the data to display.

4. Click the Update Report button to update the report preview pane.The report preview
pane shows a view of the report.

5. 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.

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10 Users and Preferences

Read this chapter to learn more about managing software users and their preferences:
• Log In or Select User (page 91)
• User Preferences window (page 92)
• Configure email notification (page 93)
• User Administration (page 97)

Log In or Select User

CFX Manager™ software manages multiple users and their preferences. The current, logged in
software user is displayed at the top of the main software window (Figure 73).

Figure 73. User name displayed.

CFX Manager software manages who logs in to the software through the Login dialog box
(Figure 74). When you start the software, the Login dialog box opens automatically if there are
two or more users listed in the User Administration window.

Figure 74. Login dialog box.

Log in to the software, or switch users by following these steps:
1. Open the Login dialog box, if it is not already open, by clicking the Select User button in
the toolbar or selecting User > Select User in the menu bar.

2. Select a name from the User Name pull-down list. The default is “Admin” (administrator).

3. Type a password in the Password box.

4. Click OK to close the Login dialog box and open the software.

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5. To add a new user name and password, contact your software administrator.

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.

2. Enter the old password in the Old Password box.

3. Enter the new password in the New Password and the Confirm New Password boxes.

4. Click OK to confirm the change.

User Preferences Window

CFX Manager software tracks the preferences of each user that logs in to the software. To
change user preferences, open the User Preferences window using one of these methods:
• Click the User Preferences button in the main software window toolbar
• Select User > User Preferences in the main software window menu bar
• Click one of the tabs (Figure 75) to view or change preferences

Figure 75. User Preferences window with tabs.

Email Tab
Select the Email tab (Figure 75) to enter the email addresses where you want to receive
confirmation of the completion of the run. The software can send an attached data file or
report file with the email when the check boxes next to these options are checked.

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Configure Email Notification

Click the Configure Outgoing Email button to open the Options window (Figure 76) to
configure the SMTP server and send a test email from the computer. Input the following:
• SMTP Server Name. The name of the SMTP server as provided by your ISP
• Port. The port number of your SMTP server, as provided by your ISP; this is usually 25
• Use SSL. Whether to use Secure Sockets Layer. Some SMTP servers require this to be
used, others require that it not be used
• Use Default “From” Address. This can usually be left in the default checked state.
However, some SMTP servers require all sent email to have a “from” address that is from
a certain domain, i.e.<name>@YourCompany.com. If that is the case, this checkbox
must be unchecked, and a valid “from” email address must be supplied in the box
labeled “From” Address:
• Authentication Required. Many SMTP servers require authentication. If so, this
checkbox must be checked, and a User Name and Password must be supplied
• Test email. To test the email settings, enter one or more email addresses in Test Email
Address text box. Multiple email addresses can be separated by a comma. Then click
the Test Email button

Figure 76. Options to configure email.

NOTE: Some SMTP servers do not allow attachments, and others allow
attachments only up to certain sizes. If you will use CFX Manager software 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.

Files Tab
Select the Files tab to list the default locations for opening and saving files. Click the “...”
button to the right of each box to open a browser window and locate a folder
• 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)

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• File Selection for Experiment 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.

Protocol Tab
Select the Protocol tab 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 and select a
lid shutoff temperature
• Protocol AutoWriter. Selects default settings that appear in the Protocol AutoWriter

Plate Tab
Select the Plate tab in the User Preferences window (Figure 77) to specify the following default
settings for a new Plate file in the Plate Editor window:
• Plate Type. Select the default plate type
• Plate Size. Select the default plate size
• Units. Select the units used to describe the concentration of the starting template for
wells that contain standards.
• Scientific Notation. Select scientific notation to view concentration units in that notation
• Scan Mode. Select a default scan mode
• Fluorophores. Click check boxes to select the default fluorophores that appear in the
Plate Editor well loading controls
• Libraries. Enter the target and sample names used in your experiments. These names
appear in the lists in the Targets tab and Samples tab in the Experiment Settings window

Figure 77. Plate tab in the User Preferences window.

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Data Analysis Tab

Select the Data Analysis Tab in the User Preferences window to change the default settings
for data that appear in the Data Analysis window.

Figure 78. Data Analysis tab in the User Preferences window.

For the quantitation data, select the following settings:
• Analysis Mode. Select the default base lining method for the analysis mode. Choose
Baseline Subtracted Curve Fit, No Baseline Subtraction, or Baseline Subtracted
• C(t) Determination Mode. Select between Regression mode or Single Threshold mode
to determine how C(t) values are calculated for each fluorescence trace
• Log View. Select On to show a semi-logarithmic graph of the amplification data. Select
Off to show a linear graph
For the allelic discrimination data, select the following settings:
• Display Mode. Select RFU to show the data as a graph of the RFU, or select Threshold
Cycle to show a graph of threshold cycles
• Normalize Data. This selection is only available when RFU is selected. Select No to
show non normalized data. Select Yes to normalize the data to the control sample
For end point data, select the following settings. Select the number of end cycles to average
when calculating the end point calculations:
• PCR. Enter a number of cycles for PCR to average the end cycles for quantitation data
(default is 5)
• End Point Only Run. Enter a number of cycles for End Point Only Run to average the
end cycles for end point data (default is 2)

Gene Expression Tab

Select the Gene Expression tab in the User Preferences window to 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

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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 (Ct) or
relative expression (Ct)
• Error Bar. Select Std Dev. for standard deviation, or Std. Error Mean for the standard
error of the mean
• Std Devs. 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 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 (page 96).

Figure 79. QC tab in User Preferences.

Specify to add cut off values and to enable the following QC rules:
• Negative control with a C(t) less than XX. Input a C(t) cut-off value
• NTC (no template control) with a C(t) less than XX. Input a C(t) cut-off value
• NRT (no reverse transcriptase control) with a C(t) less than XX. Input a C(t) cut-off
value
• Positive control with a C(t) greater than XX. Input a C(t) cut-off value
• Unknown without a C(t)
• Standard without a C(t)
• Efficiency greater than XX. Input a reaction efficiency cutoff value that is calculated
for the standard curve

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• Efficiency less than XX. Input a reaction efficiency cutoff value that is calculated for
the standard curve
• Std Curve R^2 less than XX. Input a cutoff R^2 value for the standard curve
• Replicate group C(t) Std Dev greater than XX. Input a cutoff standard deviation that is
calculated for each replicate group

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 80). In this example, the Guest user is given the right to save files.

Figure 80. User Administration window with three users.

Adding and Removing Software Users

Only a software Administrator can add and remove users. To add software users in the
Manage Users pane, follow these steps:
1. Enter a User Name for the new software user.

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.

5. Click Yes to close the dialog box and window.

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To remove a software user, follow these steps:

1. In the Manage Users pane, click the box in the Delete list for each software user you
want to remove.

2. Click OK to open a dialog box and confirm that you want to close the window.

3. Click Yes to close the dialog box and window.

NOTE: The list of software users must always include one Administrator.

Assign Rights for User Roles

The User Administration window provides access to user roles and rights. The software
includes these four roles:
• Administrator (required). Each Administrator has all rights, and you cannot change
those rights. The Administrator can also add and remove software users and change the
rights for each role
• Principal. By default, each Principal has all rights
• Operator. By default, each Operator has all rights except skipping cycles and creating a
Gene Study
• Guest. By default, each Guest has no rights and can only read files
To specify the rights for each role, follow these steps. Only a software Administrator can
change the rights for any role:
1. In the Manage Rights pane, click a box under the name of the role to add or remove that
right. Click one or more rights in the list. To change all the rights for all the roles to the
default list, click Restore Default Rights.

2. Click OK to open a dialog box and confirm that you want to close the window.

3. Click Yes to close the dialog box and window.

To view your current user role and rights, select User > User Administration. Contact a
software administrator to modify the user settings, rights, and roles listed in the User
Administration window. A Principal, Operator, or Guest user can view only their own user
settings, rights, and roles.

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11 Resources
Read this chapter to learn more about resources for the CFX system:
• LIMS Integration (page 99)
• Calibration Wizard (page 102)
• Instrument maintenance (page 104)
• Application log (page 106)
• Troubleshooting (page 106)
• References (page 108)

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.

Creating a LIMS File

A LIMS file (*.plrn) contains the plate setup details and the protocol file name. CFX Manager
software will use the LIMS file to create a plate file that will be used in conjunction with the
named protocol file to start a run and generate data.

The following steps should be performed by a LIMS specialist.

1. Select the template file “CFX96 LIMS Plate Import Template.csv” located in the
“SupportFiles” folder: [C:\Program Files\Bio-Rad\CFXIVD\SupportFiles\CFX 96 LIMS Plate
Import Template.csv]. Use a text editor to edit template file.
2. Using the LIMS, complete the template by filling in the required fields as listed in Table 29.
3. Save the template with the file name extension .plrn directly to your LIMS file folder location.
Note: the .plrn files and the protocol files referenced in the .plrn files must exist in the
same folder location.

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The .plrn file is a format known as a comma separated value (CSV) file. It utilizes the comma ","
as the separator between fields. A comma should not be used within any field. Changing the
file extension from .csv to .plrn is required for CFX Manager software to recognize the file and
start a LIMS run.
Floating point numbers are parsed as US English, so the decimal mark used must be a period
and not a comma. Also, for floating point numbers, the thousands separator should not be
used.

Table 29. Definition of LIMS .csv file contents.

Column Row Description Content Purpose

A 1 Plate Header Do not edit Predefined

A,B,D 2 Field/Data/ Do not edit Predefined

Instruction

B 3 Version Do not edit Predefined

B 4 Plate Size Do not edit Predefined

B 5 Plate Type Enter “BR White”, “BR Clear”, or Required

other
calibrated plate type

B 6 Scan Mode Enter “SYBR/FAM Only”, “All Required

Channels” or “FRET”

B 7 Units Enter one of the following: “copy Required

number”, “fold dilution”,
“micromoles”,
“nanomoles”, “picomoles”,
“femtomoles”, “attomoles”,
“milligrams”, “micrograms”,
“nanograms”, “picograms”,
“femtograms”, “attograms”, or
“percent”

B 8 Run ID Enter short description or Optional

barcode
identifying this run. Note: Do not
use commas in run ID

B 9 Run Note Enter run description. Note: Do Optional

not use commas in run ID
B 10 Run Protocol Protocol file name (protocol file Required
and .plrn file must exist in same
folder)

A 11 & 12 Data File/TBD Do not edit Predefined

A 13 Plate Data Do not edit Predefined

A 14-110 Well Position Do not edit Predefined

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Column Row Description Content Purpose

B-G 14-110 Ch1 Dye, Ch2 Enter one calibrated dye name Required
Dye, Ch3 Dye, (for
Ch4 Dye, Ch5 example “FAM”) for each
Dye, FRET channel being
used

H Sample Type Enter one of the following sample Required

types:
“Unknown”, “Standard”,
“Positive
Control”, “Negative Control”,
“NTC”, or
“NRT”

I Sample Name Enter sample name Optional

J-O 14-110 CH1 Target, Enter target name for each Optional
CH2 Target, channel
CH3 Target, used
CH4 Target,
CH5 Target,
FRET Target
P Biological Set Enter biological set name Optional
Name

Q Replicate Enter a positive integer for each Optional

set of
replicates. The value cannot be
zero

R-W CH1 Quantity, Enter quantity values for any Required

CH2 Quantity, standards. for all
CH3 Quantity, Enter concentration in decimal standards
CH4 Quantity, form
CH5 Quantity,
FRET Quantity

X Well Note Enter well note Optional

Y-AD Ch1 Well Color, Enter any user-defined trace Not
Ch2 Well Color, style color enabled
Ch3 Well Color, in a 32-bit integer (ARGB)
Ch4 Well Color, decimal format
Ch5 Well Color,
FRET Well
Color

Initiating a LIMS Run

To initiate a LIMS run:
1. Open a LIMS file using one of the following methods:

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• Drag and drop the .plrn file onto the CFX Manager software window or desktop
icon
• Double-click on the desired .plrn file
• Select File > Open > LIMS file from the main software window menu bar
In the Experimental Setup Window, the Start Run tab is displayed. The Start Run tab includes
a section for checking information about the run that is going to be started, including the
selected protocol and plate file, and a section for selecting the instrument block.
Check the RUN Information. This information includes the protocol name, the plate name, and
optional added notes.

2. Click the Start Run button to begin running the experiment on the selected block.

Calibration Wizard
The CFX system is factory-calibrated for commonly used fluorophores in white-well and clear-
well plates (Table 30).
Table 30. Factory-calibrated fluorophores, channels, and instruments.

Fluorophore Channel Excitation Emission

(nm) (nm)

FAM, SYBR® Green I 1 450-490 515-530

VIC, HEX, CAL Fluor 2 515-535 560-580

Gold 540, Cal Fluor
Orange 560

ROX, Texas Red, CAL 3 560-590 610-650

Fluor Red 610, TEX 615

CY5, Quasar 670 4 620-650 675-690

Quasar 705, Cy5.5 5 672-684 705-730

The CFX system also includes a channel dedicated for FRET chemistry; this channel does not
require calibration for specific dyes.
To open the Calibration Wizard to calibrate the CFX 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 81).

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Figure 81 shows an example of the Dye Calibration window.

Figure 81. Dye Calibration window.

Calibrating the CFX System

To calibrate the CFX system in the Dye Calibration window:
1. In the Calibrate New or Existing Fluorophores pane, select the fluorophore you want to
calibrate from the pull-down list. If the fluorophore name is not included in the list, type
the name in the box to add it to the list.

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.

3. Select a Channel for the fluorophore.

4. Click Add to Plate to add the fluorophore. To clear the plate, click Clear Plate 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 Dye Plate Display.
Use this window as a guide for loading dyes into the plate.

7. Prepare a 96-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 4
wells with 50 µl (96-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.

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 the CFX Manager™ software completes the calibration run, a dialog box appears.
Click Yes to finish calibration and open the Dye Calibration Viewer.

11.Click OK to close the window.

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Instrument Maintenance
The CFX system includes a sensitive optical shuttle system 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. Only trained Bio-Rad service engineers can
clean the shuttle optical system.
Avoid contaminating the CFX system by following these suggestions:
• Always clean the outside of any container before placing it in the block
• Never run a reaction with a seal that is open, loose, punctured, or otherwise
damaged; doing so 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 (page 105)
• Never clean or otherwise touch the optical system behind the heater plate holes that
are in the inner lid (Figure 82 on page 105)
• Clean the outer lid and C1000™ thermal cycler base on a regular schedule (for
details see the C1000 thermal cycler instruction manual)

Cleaning the Optical Reaction Module

The block of the optical reaction and the C1000 thermal cycler base should be cleaned on a
regular schedule to remove any debris or dirt that might interfere with proper function. Clean
as soon as you discover debris and spilled liquids with a soft, lint-free cloth that is dampened
with water. Cleaning the instrument allows precise instrument function. For more detailed
information about cleaning the C1000 base, see the C1000 thermal cycler instruction manual.
WARNING! Never use cleaning solutions that are corrosive to aluminum. Avoid
scratching the surface of the C1000 reaction module bay. Scratches and damage
to this surface interfere with precise thermal control.
WARNING! Never pour water or other solutions in the C1000 reaction module bay.
Wet components can cause electrical shock when the thermal cycler is plugged in.
Clean the CFX optical reaction module as soon as you discover debris, dirt, or contamination
in the block or on the inner lid. Any dirt can interfere with the ability of the block to change
temperature quickly and collect accurate fluorescent data. To clean the reaction module,
follow these guidelines. Follow these suggestions for cleaning:
WARNING! To prevent electrical shock, always remove the reaction module from
the thermal cycler base, or unplug the base before cleaning the instrument.

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WARNING! Never touch or allow solutions to touch the optical system that is
located behind the heater plate holes in the inner lid (Figure 82).

Never touch anything

behind these holes

Block (96-well)

Figure 82. Heating plate holes in the inner lid.

• Clean the outer surface. Use a damp cloth or tissue to clean spills from the outside
case. If needed, use a mild soap solution and rinse the surface with a damp cloth. Clean
the cover to prevent corrosion
• Clean the cooling fins. Remove dust with a soft brush or damp cloth. Remove any
heavy dust that is deep in the vents with a vacuum cleaner. Use water and a soft, lint-free
cloth to remove debris that is stuck to the fins. Avoid scratching the surface. If needed,
use a mild soap solution and rinse well to remove residue completely. Cleaning the fins
improves precise sample heating and cooling
NOTE: Never use cleaning solutions that are corrosive to aluminum such as bleach
or abrasive cleansers.
• Use of oil in the wells is not recommended. If oil is used, the wells must be cleaned
thoroughly and often. Remove the oil when it is discolored or contains dirt. Use a
solution of 95% ethanol to clean oil. Do not allow oil to build up in the block.
• Clean the wells in the block. Clean spills immediately to prevent them from drying. Use
disposable plastic pipets with water (recommended), 95% ethanol, or a 1:100 dilution of
bleach in water. Also use a soft, lint-free cloth or paper towel and water to clean the
block. Always rinse the wells with water several times to remove all traces of cleaning
reagents
WARNING! Never clean the block with strong alkaline solutions (strong soap,
ammonia, or concentrated bleach). Never use corrosive or abrasive cleaning
solutions. These cleaning agents can damage the block and prevent precise
thermal control.
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.

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• 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 a new run, the instrument initiates a self-diagnostic test to verify that it is running within
the 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 83). 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.

Figure 83. Example of an Event Log file.

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.

Installing the Software Manually

If needed, install the software manually by following these instructions:
1. Insert the software CD.

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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Power Failure Options

In a power failure, the instrument and computer will shut down. After a short power failure, the
instrument will resume running a protocol, but the Application log will note the power failure.
Depending on the computer settings and the length of time that the power is off, the
instrument and software attempt to continue running depending on the protocol step:
• If the protocol is in a step with no plate read, then the protocol continues running as
soon as the instrument gets power again
• If the protocol is in a step with a plate read, then the instrument waits for the
software to restart and resume communication to collect the data. In this situation,
the protocol only continues if the software is not shut down by the computer. When
the computer and software start up again, the protocol continues
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 base.

2. Place 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 84.

Figure 84. 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 that are located at the front edge of the module. You should hear the locking
latch release from inside the module. Figure 85 shows the two large screws.

Figure 85. Remove these screws to unlock the optical module.

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4. Push the reaction module lid open. Notice that the latch (dark plastic) is no longer
attached. Remove your samples from the block.

5. Reassemble the reaction module with the lid open by replacing the locking latch and
securing it with the large screws. Figure 86 shows the locking latch in place.

Figure 86. Optical module locking latch.

References
Breslauer KJ et al. (1986). Predicting DNA duplex stability from the base sequence. Proc
Nat Acad Sci 83, 3746–50.
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.
Minpack Copyright Notice (1999) University of Chicago. All rights reserved
Redistribution and use in source and binary forms, with or without modification, are permitted
provided that the following conditions are met:
1. Redistributions of source code must retain the above copyright notice, this list of
conditions and the following disclaimer.
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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Bio-Rad
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