BIO RAD CFX96, CFX384 Instruction Manual

CFX96™ and CFX384™ Real-Time PCR Detection Systems

Instruction Manual

Catalog # 184-5384 # 185-5384 # 184-5096 # 185-5096

Excel, Microsoft, PowerPoint, Windows, and Windows Vista are trademarks of Microsoft Corporation. Adobe and Reader are trademarks of Adobe Systems Incorporated. Cy is a trademark of GE Healthcare group companies. Fluor and Quasar are trademarks of Biosearch Technologies, Inc. SYBR and Texas Red are trademarks of Invitrogen Corporation. Bio-Rad Laboratories, Inc. is licensed by Invitrogen Corporation to sell reagents containing SYBR Green I for use in real-time PCR, for research purposes only. Other brands or product names are trademarks of their respective holders.

LICENSE NOTICE TO PURCHASER

Bio-Rad Real-time thermal cycler CFX96 or CFX384 is licensed real-time thermal cycler(s) under Applera's United States Patent No. 6,814,934 B1 for use in research and for all other fields except the fields of human diagnostics and veterinary diagnostics.

This CFX96 or CFX384 detection module, when combined with a C1000 thermal cycler for which the applicable realtime thermal cycler royalty fee has been paid, constitutes a real-time thermal cycler licensed under U.S. Patent No. 6,814,934 and corresponding claims in any Canadian counterpart patent thereof owned by Applera Corporation, for use solely in research and all applied fields except human and veterinary in vitro diagnostics. These license rights are effective only if this detection module is combined with a Bio-Rad thermal cycler for which the applicable realtime thermal cycler royalty fee has been paid and not with any other thermal cycler. No rights are conveyed expressly, by implication or estoppel to any patents on real-time methods, including but not limited to 5' nuclease assays, or to any patent claiming a reagent or kit. For further information on purchasing license rights, contact the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California, 94404, USA.

This product is covered by one or more of the following U.S. patents, their foreign counterparts, or their foreign patents pending, owned by Eppendorf AG: US Patent Nos. 6,767, 512 and 7,074,367. No rights are conveyed expressly, by implication or estoppel to any patents on PCR methods, or to any patent claiming a reagent or kit

Bio-Rad’s Hard-Shell® plates are covered by one or more of the following U.S. patents, patents pending, or their foreign counterparts, owned by Eppendorf AG: US Patent Nos. 7,347,977, 6,340,589, 6,528,302 and US application 2007246858, 20080084004, 20030180192 and 20060120927

Bio-Rad Resources

Bio-Rad provides many resources for scientists. The following websites contain useful information about running PCR and real-time PCR experiments:

• Gene Expression Gateway (www.bio-rad.com/genomics/)

This site provides rich technical resources on a wide variety of methods and applications related to PCR, real-time PCR, and gene expression

• Life Science Research website (discover.bio-rad.com)

This site includes links to technical notes, manuals, product information, and technical support.

Table 1 lists other Bio-Rad resources and how to locate what you need:

Table 1. Bio-Rad resources

Resource How to Contact

Local Bio-Rad Laboratories representatives

Technical notes and literature Go to the Bio-Rad website (www.bio-rad.com) or Gene

Technical specialists Bio-Rad’s Technical Support department is staffed with

Find local information and contacts on the Bio-Rad website by selecting your country on the home page (www.bio-rad.com). Find the nearest international office listed on the back of this manual

Expression Gateway (www.bio-rad.com/genomics/). Type a search term in the Search box and select Literature to find links to technical notes, manuals, and other literature

experienced scientists to provide customers with practical and expert solutions

To find local technical support on the phone, contact your nearest Bio-Rad office. For technical support in the United States and Canada, call 1-800-424-6723 (toll-free phone), and select the technical support option

Writing Conventions Used in this Manual

This manual explains how to safely set up and operate the CFX96 system or the CFX384 system. The manual uses the writing conventions listed in Table 2.

Table 2. Conventions used in this manual

Convention Meaning

TIP: Provides helpful information and instructions, including information

explained in further detail elsewhere in this manual

NOTE: Provides important information, including information explained in

further detail elsewhere in this manual

WARNING! Explains very important information about something that might

damage the researcher, damage an instrument, or cause data loss

X > Y Select X and then select Y from a toolbar, menu or software window

For information about safety labels used in this manual and on the CFX96 system or the CFX384 system, see, “Safety and Regulatory Compliance” on page iii.

CFX96 and CFX384 Systems Manual

Safety and Regulatory Compliance

For safe operation of the CFX96 system or the CFX384 system, we strongly recommend that you follow the safety specifications listed in this section and throughout this manual.

Safety Warning Labels

Warning labels posted on the instrument and in this manual warn you about sources of injury or harm. Refer to Table 3 to review the meaning of each safety warning label.

Table 3. Meaning of safety warning labels

CAUTION: Risk of danger! This symbol identifies components that pose a risk of personal

injury or damage to the instrument if improperly handled. Wherever this symbol appears, consult the manual for further information before proceeding

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

Instrument Safety Warnings

The warning labels shown in Table 4 also display on the instrument, and refer directly to the safe use of the CFX96 system or the CFX384 system.

Table 4. Instrument Safety Warning Labels

Icon Meaning

Warning about risk of harm to body or equipment.

Operating the CFX96 or CFX384 real-time PCR detection system before reading this manual can constitute a personal injury hazard. For safe use, do not operate this instrument in any manner unspecified in this manual. Only qualified laboratory personnel trained in the safe use of electrical equipment should operate this instrument. Always handle all components of the system with care, and with clean, dry hands

Warning about risk of burning. A thermal cycler generates enough heat to cause serious burns. Wear safety goggles or other eye protection at all times during operation. Always allow the sample block to return to idle temperature before opening the lid and removing samples. Always allow maximum clearance to avoid accidental skin burns

Warning about risk of explosion. The sample blocks can become hot enough during the course of normal operation to cause liquids to boil and explode

NOTE: For information about the C1000™ thermal cycler, refer to the C1000 thermal cycler instruction manual.

iii

Safe Use Specifications and Compliance

Table 5 lists the safe use specifications for the CFX96 system or the CFX384 System. Shielded cables (supplied) must be used with this unit to ensure compliance with the Class A FCC limits.

Table 5. Safe Use Specifications

Safe Use Requirements Specifications

Temperature Indoor use

Altitude Up to 2,000 meters above sea level

Ambient temperature of 15—31 maximum of 80% noncondensing

REGULATORY COMPLIANCE

This instrument has been tested, and found to be in compliance with all applicable requirements of the following safety and electromagnetic standards:

• IEC 61010-1:2001 (2nd Ed.), EN61010-1:2001 (2nd Ed). Electrical Equipment For Measurement, Control, and Laboratory Use - Part 1: General Requirements

• IEC 61010-2-010:2005, EN61010-2-010:2003. Safety requirements for electrical equipment for measurement, control and laboratory use. Part 2-010: Particular requirements for laboratory equipment for the heating of materials

• IEC 61010-2-081:2001+A1, EN61010-2-081:2002+A1. Safety requirements for electrical equipment for measurement, control and laboratory use. Part 2-081: Particular requirements for automatic and semi-automatic laboratory equipment for analysis and other purposes (includes Amendment 1)

• EN 61326-1:2006 (Class A) Electrical equipment for measurement, control and laboratory use. EMC requirements, Part 1: General requirements

C. Relative humidity

This equipment generates, uses, and can radiate radio frequency energy and, if not installed and used in accordance with the instruction manual, may cause harmful interference to radio communications. Operation of this equipment in a residential area is likely to cause harmful interference in which case the user will be required to correct the interference at his own expense.

CFX96 and CFX384 Systems Manual

Bio-Rad Resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii

Writing Conventions Used in this Manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii

Safety and Regulatory Compliance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii

Chapter 1. System Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Unpacking the Optical Reaction Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

System Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

System Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Setting up the system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Installing CFX Manager Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Running Experiments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Chapter 2. Introduction to CFX Manager Software . . . . . . . . . . . . . . . . . . 11

Main Software Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Startup Wizard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Detected Instruments Pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Instrument Properties Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Software Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Tips and Tricks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Chapter 3. Running Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Experiment Setup Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Protocol Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Plate Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Start Run Tab. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Run Details Window. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Instrument Summary Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Chapter 4. Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Protocol Editor Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Protocol Editor Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Temperature Control Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Protocol AutoWriter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Table of Contents

Chapter 5. Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Plate Editor Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Select Fluorophores Window. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Experiment Settings Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Well Groups Manager Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

Plate Spreadsheet View Window. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

Chapter 6. Stand-Alone Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Control Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Main Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

Experiment Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Exporting Data for Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

Creating a Data File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

Setting Up Email . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Chapter 7. Data Analysis Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Data Analysis Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Data Analysis Menu Bar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

Quantitation Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

Data Analysis Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

Well Selectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Charts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

Spreadsheets. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

Chapter 8. Data Analysis Windows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Quantitation Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Quantitation Data Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Melt Curve Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

Melt Curve Data Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

End Point Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

Allelic Discrimination Tab. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

QC Tab. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

Reports for Data Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

Chapter 9. Gene Expression Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

Plate Setup for Gene Expression Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

Gene Expression Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

Experiment Settings Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

Gene Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

Gene Study Data Spreadsheet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

Gene Study Report Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

Frequently Asked Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

Gene Expression Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

CFX96 and CFX384 Systems Manual

Chapter 10. Users and Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

User Preferences Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

Email Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

Files Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

Protocol Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

Plate Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

Data Analysis Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

Gene Expression Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

QC Tab. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

User Administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

Chapter 11. Resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

Calibration Wizard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

Instrument Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

Application Log . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

Software Help Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

Instruments, Parts, and Accessories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

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Table of Contents

viii

CFX96 and CFX384 Systems Manual

1 System Installation

Read this chapter for information about setting up the CFX96 or CFX384 system:

• Unpacking the optical reaction module (below)

• System requirements (page 1)

• System overview (page 2)

• Setting up the system (page 3)

• Installing CFX Manager software (page 6)

• Running experiments (page 9)

Unpacking the Optical Reaction Module

Your CFX96 or CFX384 optical reaction module shipment includes these components:

• Optical reaction module

•USB cable

• CFX Manager

• Instruction manual

• CFX Manager software uick guides for system installation, protocol, plate, data analysis, and gene expression analysis

Remove all packing materials and store them for future use. If any items are missing or damaged, contact your local Bio-Rad office.

software installation CD

System Requirements

To operate the CFX96 or CFX384 system, use the following power sources and cables:

• Input power. 100—240 VAC, 50—60 Hz

• Indoor use. Ambient temperature of 15—31

(non-condensing)

• USB cable. If the CFX96 system is going to be controlled by a computer via a USB cable, the provided cable from Bio-Rad is sufficiently shielded for use.

NOTE: For a full list of the safety and compliance requirements for this instrument, see “Safety and Regulatory Compliance” on page iii.

C. Relative humidity maximum of 80%

System Installation

Open button

Indicator LED

Front panel

Close button

Block

Inner lid with heater plate

System Overview

The CFX96 system or CFX384 system includes two components:

• Optical reaction module. This module includes an optical system to collect fluorescent data and a thermal cycler block

NOTE: The serial number of the CFX96 or CFX384 module is located on the back.

• C1000

thermal cycler base. The C1000 base includes a user interface to control the

system when running in stand-alone mode and the power button and ports (both on back panel) to connect to a computer

Figure 1. Front view of the CFX96 system.

When open, the CFX96 or CFX384 system includes the features shown in Figure 2.

Figure 2. Inside view of the CFX96 system.

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

CFX96 and CFX384 Systems Manual

Power

Power input

switch

USB connections

Ethernet port

• Inner lid with heater plate. The heater lid maintains temperature on the top of the consumable to prevent sample evaporation. Avoid touching or otherwise contaminating the heater plate. Never poke anything through the holes; the optics shuttle system could be damaged

•Block. Load samples in this block before the run

• Close button. Press this button on the inside of the lid to close the motorized lid

WARNING! Prevent contamination of the instrument by spills, and never run a

reaction with an open or leaking sample lid. For information about general cleaning and maintenance of the instrument, see “Instrument Maintenance” (page 133).

The back panel of the C1000 chassis includes these features (Figure 3):

• Power switch. Press the power switch to turn on the power to the system

• Power input. Plug in the power cord here

• Ethernet port. Connect an ethernet cable to email run logs and stand-alone data files

• USB connections. Use these ports to connect the CFX96 system or CFX384 system to

a computer or to connect an S1000

thermal cycler

Figure 3. Back panel of C1000 thermal cycler.

WARNING! Avoid contact with the back panel of the C1000 during operation.

Setting up the system

The CFX96 or CFX384 real-time PCR detection system should be installed on a clean, dry, level surface with sufficient cool airflow to run properly. The CFX96 system or CFX384 system can be run in two modes: stand-alone or software-controlled. If you are running the system under software-controlled mode, make sure there is sufficient space for a computer during setup.

To insert the CFX96 or CFX384 optical reaction module into the reaction module bay of the C1000 chassis, follow these instructions:

1. Place the C1000 chassis in a suitable location with the locking bar down. Remove any previously installed reaction modules.

System Installation

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

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

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

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

Figure 5. Locking the optical module into place.

CFX96 and CFX384 Systems Manual

Check space

5. Check that the module is completely and evenly seated in the C1000 base. As shown in Figure 6, check the space around the bottom of the module. There should be no extra space between the module and the base; the space should be even.

Figure 6. Check for even space between module and base.

NOTE: If the space between the module and C1000 base is uneven, reinstall the module starting from Step 2 (page 4).

6. Plug the power cord into the back of the C1000 base (Figure 3 on page 3), and into an appropriate three-pronged electrical outlet.

7. Press the power switch on the back panel of the C1000 thermal cycler to start the system.

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

Figure 7. Instructions to remove the shipping screw.

System Installation

NOTE: If the shipping screw is not removed at this step, it will be detected by CFX Manager software. Follow the instructions in the software to remove the screw (page 19).

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

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

Installing CFX Manager Software

CFX Manager software is run on a PC computer with either the Windows XP or Windows Vista operating system and is required to analyze real-time PCR data from the CFX96 system or CFX384 system. This software can also be used to control the CFX96 system or CFX384 system in software-controlled mode. Table 6 lists the computer system requirements for the software on Windows XP and Windows Vista.

Table 6. Computer requirements for CFX Manager software

System Minimum Recommended

Operating system Windows XP Professional SP2 and

Above or Windows Vista Home Premium and above

Drive CD-ROM drive CD-RW drive

Hard drive 10 GB 20 GB

Processor speed 2.0 GHz 2.0 GHz

RAM 1 GB RAM (2 GB for Windows

Vista)

Screen resolution 1024 x 768 with true-color mode 1280 x 1024 with true-color mode

USB USB 2.0 Hi-Speed port USB 2.0 Hi-Speed port

Internet browser Internet Explorer Internet Explorer

Software Microsoft Office Suite

Windows XP Professional SP2 and above or Windows Vista Home Premium and above

2 GB RAM

NOTE: Running a CFX96 or CFX384 system with CFX Manager Software on a PC computer with a Windows 64-bit Operating Systems is not supported due to incompatible USB Drivers. A PC computer with a 64-bit processor (like Intel) on a 32-bit Windows Operating System is supported.

To install CFX Manager software:

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

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

CFX96 and CFX384 Systems Manual

3. The software launch page should appear automatically. Double-click Install Software on the software launch page (Figure 8).

Figure 8. Software installation screen.

TIP: Click the Documentation button to find searchable PDF copies of the instrument manuals and other documentation.

4. Follow the instructions on screen to complete installation. When completed, the Bio-Rad CFX manager software icon will appear on the desktop of the computer.

5. If the launch page does not appear automatically, double-click on (CD drive):\Bio-Rad CFX, then open and follow instructions in the Readme.txt file. See “Installing the Software Manually” on page 136.

Installing the Drivers

If the CFX96 system or CFX384 system is going to be run in Software-controlled mode, drivers must be installed on the computer. Use only the supplied USB cable, which is sufficiently shielded to prevent data loss.

To install the system drivers:

1. Connect the C1000 chassis to the computer by plugging a USB cable into the USB 2.0 A port located on the back of the chassis (Figure 3 on page 3), and then connecting the cable into the USB 2.0 B port located on the computer.

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

System Installation

3. On the first screen, select Yes, this time only to instruct the Windows operating system to connect to Windows Update to search for software (Figure 9). Click Next.

Figure 9. Found New Hardware Wizard.

4. Instruct the wizard to “ installing the drivers (Figure 10).

Install the software automatically

.” Click Next to continue

Figure 10. Software (Driver) installation screen.

5. Click Finish at the software installation completion screen when the drivers are installed.

Running Experiments

Recommended Plastic Consumables

Run only 384-well plates in the CFX384 system. For optimal results, Bio-Rad recommends the following consumables for the CFX384 system (catalog numbers are provided in bold):

• HSP-3805. Low-profile 384-well Hard-Shell® plates with clear shell and white wells

• HSP-3866. Low-profile 384-well Hard-Shell plates with black shell and white wells

• MSB-1001. Microseal ‘B’ adhesive seals, optically clear

The CFX96 system accepts both low profile 0.2 ml plates and tubes. For optimal results, BioRad recommends the following consumables:

• MLL-9601. Low-profile 96-well unskirted plates with clear wells

• MLL-9651. Low-profile 96-well unskirted plates with white wells

• HSP-9601. Hard-Shell 96-well skirted plates with white shell and clear wells

• HSP-9655. Hard-Shell 96-well skirted plates with white shell and white wells

• TLS-0801. Low-profile 0.2 ml 8-tube strips without caps, clear wells

• TLS-0851. Low-profile 0.2 ml 8-tube strips without caps, white wells

• TCS-0803. Optical flat 8-cap strips, for 0.2 ml tubes and plates

• MSB-1001. Microseal ‘B’ adhesive seals, optically clear

CFX96 and CFX384 Systems Manual

Loading the Block

To load your reactions in the block, follow these suggestions:

• Click the Open Lid button located on software’s Start Run tab (see “Start Run Tab” on page 24), or press the lid button on the front of the system (Figure 1) to start opening the motorized lid.

WARNING! The lid moves slowly at first, and then increases speed when it opens or closes.

• Place the 0.2 ml microplate or tube strips with sealed lids in the block. Check that the tubes are completely sealed to prevent leakage. For optimal results load sample volumes of 10–25 μl for the CFX96 system and load sample volumes of 5–20 μl for the CFX384 system.

NOTE: For accurate data analysis, check that the orientation of reactions in the block is exactly the same as the orientation of the well contents in the software Plate tab (see “Plate Tab” on page 23). If needed, edit the well contents before, during, or after the run. As shown in Figure 11 on page 10, rotating the microplate by 180 well.

causes the software to expect the reading for well H3 to be an empty A10

System Installation

Well H3

Well A10 (180

rotation)

(correct)

Filled tube strip

Tube strip for balance

Figure 11. Plate Editor window showing plate orientation.

WARNING! When running the CFX96 system, always balance the tube strips or cut

microplates in the wells (Figure 12). For example, if you run one tube strip on the left side of the block, run an empty tube strip (with caps) on the right side of the block to balance the pressure applied by the heated lid.

Figure 12. Balance the tube strips or cut microplates in the block.

WARNING! Be sure that nothing is blocking the lid when it closes. Although there

is a safety mechanism to prevent the lid from closing if it senses an obstruction, do not place anything in the way of the closing lid.

CFX96 and CFX384 Systems Manual

2 Introduction to CFX Manager

Software

Read this chapter for information about getting started with CFX Manager software.

• Main software window (below)

• Startup Wizard (page 15)

• Detected Instruments pane (page 16)

• Instrument Properties window (page 18)

• Software files (page 20)

• Tips and tricks (page 20)

Main Software Window

For instructions about running the system, refer to one of the CFX Manager software quick guides that ships with the system:

• Installation quick guide

• Protocol quick guide

• Plate quick guide

• Data Analysis quick guide

• Gene Expression Analysis quick guide

TIP: See the software Help for more guides about running experiments.

Get started in the main software window by using these features (Figure 13):

• Status bar. View the current software and instrument status (page 12)

•Menu bar. Select software commands (page 12), such as creating or opening files

• Toolbar buttons. Click these buttons (page 14) to open software files, the Startup

Wizard (page 18), the Experiment Setup window (page 21)

• Detected Instruments pane. View a list of attached instruments (page 15)

Introduction to CFX Manager Software

• Startup Wizard window. Access common software commands (page 16)

Figure 13. The main software window.

Status Bar

The status bar at the bottom of the main software window shows the status of the software. View the left side of the status bar (Figure 14) to see the current status of instruments.

Figure 14. Left side of status bar in main software window.

View the right side of the status bar (Figure 15) to see the current user name, date, and time.

Figure 15. Right side of status bar in the main software window.

Click and drag the right corner of the status bar to resize the main window.

Menu Bar

The menu bar of the main software window provides the items listed in Figure 16:

Figure 16. Menu bar in the main software window.

CFX96 and CFX384 Systems Manual

Select the commands shown in the menu bar (Table 7).

Table 7. Menu bar items in the main software window

Menu Item Command Function

File New Create a new protocol, plate, experiment, or Gene

Study

Open Open existing files, including protocol (.prcl), plate

(.pltd), data (.pcrd), and Gene Study (.mgxd) files, stand-alone run files (.zpcr)

Recent Data Files View a list of the ten most recently viewed data

files, and select one to open in Data Analysis

Repeat an Experiment Open the Experiment Setup window with the

protocol and plate from a completed run to quickly repeat the run

Exit Exit the software program

View Application Log Display the application log for the software

Run Reports Select a run report to review from a list

Startup Wizard Open the Startup Wizard

Experiment Setup Open the Experiment Setup window

Instrument Summary Open the Instrument Summary window

Detected Instruments Show or hide the Detected Instruments pane

Toolbar Show or hide the main software window toolbar

Status Bar Show or hide the main software window status bar

User Select User Open the Select User window to change software

users

Change Password Change your user password

User Preferences Open the User Preferences window

User Administration Manage users in the User Administration window

Tools Dye Calibration Wizard Open the Dye Calibration window to calibrate an

instrument for a new fluorophore

Protocol AutoWriter Open the Protocol AutoWriter window to create a

new protocol

Ta Calculator Open the Ta Calculator window to calculate the

annealing temperature of primers

View Block Status Log View a log of the thermal cycler block

Application Data Folder Open the Application Data folder to view software

files

User Data Folder Open the Data folder to view protocol, plate, and

data files

Properties All Instruments

Zip Data and Log Files Choose and condense selected files in a zipped

Options Configure software email settings

View properties of all detected instruments, including serial numbers

file for storage or to email

Introduction to CFX Manager Software

Table 7. Menu bar items in the main software window (continued)

Menu Item Command Function

Windows Cascade Arrange software windows on top of each other

Tile Vertical Arrange software windows from top to bottom

Tile Horizontal Arrange software windows from right to left

Close All Close all open software windows

Help Contents Open the software Help for more information

Index View the index in the software Help

Search Search the software Help

Gene Expression Gateway Web site

PCR Reagents Website View a website that lists Bio-Rad consumables for

PCR Plastic Consumables Website

Software Updates Check for software updates from Bio-Rad

About Open a window to see the software version

about running PCR and real-time PCR

Open a web site to find information about running PCR and real-time PCR experiments

PCR and real-time PCR reagents

View a website that lists Bio-Rad consumables for PCR and real-time PCR experiments

Toolbar Buttons

Click a button in the toolbar of the main software window (Table 8) for quick access to common software commands.

NOTE: To show or hide the toolbar, select View > Toolbar in the menu bar.

Table 8. Toolbar buttons in the main software window

Button Button Name Function

Open a Data File Open a browser window to locate a data file (*.pcrd

extension) and open it in the Data Analysis window (page 69)

Open a Gene Study Open a browser window to locate a Gene Study file

(.mgxd extension) and open it in the Gene Study window (page 101)

Create a New Gene Study

Print Print the current software window

Open the Gene Study window (page 101) to add files and create a new study

Startup Wizard Open the Startup Wizard that links you to common

software functions (page 16)

CFX96 and CFX384 Systems Manual

Table 8. Toolbar buttons in the main software window (continued)

Button Button Name Function

Experiment Setup Open the Experiment Setup window to run an

experiment (page 21)

Protocol AutoWriter Open the Protocol AutoWriter window to create a new

protocol (page 37)

Select User Open the Select User window to change software

users (see “Log in or Select User” on page 121)

User Preferences Open the User Preferences window (page 121)

Help Open the software Help window for more information

about running PCR and real-time PCR

Startup Wizard

The Startup Wizard automatically appears when CFX Manager software is first opened (Figure 17). If it is not shown, click the Startup Wizard button on the main software window toolbar.

Figure 17. Startup Wizard window.

Options in the Startup Wizard include the following:

Introduction to CFX Manager Software

• Create a new Experiment (page 21). Set up the protocol and plate to begin a new experiment.

NOTE: Select the appropriate instrument in the pull down list to make sure the default plate settings match the instrument to be used for the experiment.

• Repeat an Experiment. Set up an experiment with the protocol and plate from a completed run. If needed, you can edit the experiment before the run

• Open a Data File (page 69). Open a data file to analyze results

• Open a Gene Study (page 101). Open a multi-file gene expression study to analyze

results from multiple gene expression experiments

• Open User Preferences (page 121). Open the User Preferences window to customize software settings

Detected Instruments Pane

CFX Manager software runs these 1000-series instruments:

• CFX96 and CFX384 real-time PCR systems. Systems include a C1000 thermal cycler and an optical reaction module

• C1000 thermal cycler. The most versatile of the instruments run by CFX Manager software. Add and remove these PCR reaction modules to the C1000 chassis in seconds:

• 96-well fast reaction module, gradient enabled

• 384-well reaction module

• Dual 48/48 fast reaction module, gradient enabled

• S1000 thermal cycler connected to a C1000 thermal cycler. For more information, see S1000 and C1000 thermal cycler instruction manuals

TIP: Locate PDF copies of the instruction manuals by opening the Documents folder on the CFX Manager software installation CD.

You can simultaneously run up to four instruments with CFX Manager software, including a combination of CFX96 system and CFX384 systems, or a mix of real-time PCR systems and thermal cyclers.

Viewing Detected Instruments

Connected instruments appear in the Detected Instruments pane (page 17). This list shows each instrument as an icon named with the serial number (default). The list of instruments also shows individual blocks (Block A and Block B) for each dual block reaction module.

TIP: Right-click any instrument in the Detected Instruments pane to open the Instrument Properties window and rename the instrument.

Figure 18 shows four detected instruments:

• One C1000 thermal cycler (C48FSIM00) with a dual 48/48 reaction module

• One S1000 thermal cycler (S96FSIM01) with a 96-well block, which is connected to the C1000 thermal cycler called C48FSIM00

• One CFX384 system (CFX384SIM03)

CFX96 and CFX384 Systems Manual

• One CFX96 system (CFX96SIM02)

Figure 18. Instruments listed at the top of the Detected Instruments pane.

Right-click on the instrument icon or block to select one of these options:

•View Status. Open the Run Details window to check the status of the selected instrument block

• Flash Block Indicator. Flash the indicator LED on the instrument

• Open Lid. Open a motorized lid on the selected instrument block

•Close Lid. Close a motorized lid on the selected instrument block

• Rename. Change the name of the instrument

•Properties. Open the Instrument Properties window

• Collapse All. Collapse the list of instruments in the Detected Instruments pane

• Expand All. Expand the list of instruments in the Detected Instruments pane

You can also control a block by clicking an instrument block icon in the Detected Instrument pane and then clicking a button in the Selected Instrument pane (Figure 19).

Figure 19. Buttons at the bottom of the Detected Instruments pane.

• Click View Status to open the Run Details window to check the status of the selected instrument block

• Click Open Lid to open the motorized lids on the selected instrument

• Click Close Lid to close the motorized lids on the selected instrument

• Click View Summary to open the Instrument Summary window

Introduction to CFX Manager Software

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

Instrument Properties Window

To open the Instrument Properties window to view information about an instrument, right-click on the instrument icon in the Detected Instruments pane (Figure 18 on page 17). The window includes three tabs (Figure 20):

•Properties. View serial numbers and the C1000 thermal cycler name

• Shipping Screw. Remove the shipping screw to run the instrument, or install the

shipping screw when you want to transport the instrument

• Calibrated Dyes. View the list of calibrated fluorophores

Figure 20. Instrument Properties window.

Properties Tab

The default name for an instrument is the C1000 thermal cycler serial number, which appears in many locations, including the Detected Instruments pane (Figure 18).

To rename an instrument for ease of identification, follow these instructions:

• In the Instrument Properties tab, type a name in the Rename box at the top of the Properties tab and hit the Rename button to save the new name

NOTE: In the Detected Instrument pane, right-click the instrument icon and select Rename to open the Instrument Properties window.

The Properties tab displays important serial numbers for the connected instrument, including the thermal cycler and reaction module. The firmware versions are also displayed.

CFX96 and CFX384 Systems Manual

Shipping Screw Tab

The Shipping Screw tab includes instructions for installing or removing the red shipping screw. To prevent damage to the optical reaction modules, install the shipping screw any time you ship the CFX96 system or CFX384 system.

NOTE: If the shipping screw is detected by the software, the Instrument Properties window automatically opens with the Shipping Screw tab in front. Follow the instructions to remove the screw.

The information in this tab changes depending on whether the shipping screw is installed or removed. For example, to install the shipping screw, click the Install Shipping Screw button and follow the instructions in the tab (Figure 21).

Figure 21. Instructions for installing the shipping screw.

Calibrated Dyes Tab

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

Figure 22. Calibrated Dyes tab in the Instrument Properties window.

Introduction to CFX Manager Software

Software Files

CFX Manager software stores information about experiments in specific files (Table 9):

Table 9. Open these file types with CFX Manager software

File Type Extension How to View and Edit File

Protocol .prcl Select in Experiment Setup and edit in Protocol Editor

Plate .pltd Select in Experiment Setup and edit in Plate Editor

Data .pcrd View and analyze in Data Analysis window

Gene Study .mgxd View and analyze in Gene Study window

Stand-alone pre-data file

.zpcr Contains fluorescence readings from stand-alone

Tips and Tricks

Tips and tricks for using CFX Manager software are listed below.

• Open any Protocol, Plate, Data, or Gene Study file by dragging it from a folder to an open software window

• Print or export the information shown in many windows by right-clicking a chart, spreadsheet, or well selector

• Change the size of any window by clicking and dragging the edges

• Open the User Preferences window to choose default settings that activate every time you log in to the software

• Add data files to a Gene Study by dragging from a folder to an open Gene Study window

• Open multiple Data Analysis and Gene Study files at the same time

• Click the Settings or Tools menus to find advanced functions

• To add or delete files from the Express Load menu, add or delete the files (.prcl and.pltd extensions) in the ExpressLoad folder, select Tools > User Data Folder in the menu bar of the main software window

• To view all the information loaded into one well in a plate, double-click the well to open the Well Info window

• Right-click any graph or chart to change viewing and data analysis options

• Edit well contents before, during, or after the run

• Select a well group to view and analyze a subset of the wells in the plate. Select each well group by name in the Well Group pull-down menu in the toolbar

operation that is converted into a data file

CFX96 and CFX384 Systems Manual

3 Running Experiments

Read this chapter for information about running experiments using CFX Manager software:

• Experiment Setup window (below)

•Protocol tab (page22)

• End point only runs (page 22)

• Plate tab (page 23)

• Start Run tab (page 24)

• Run Details window (page 26)

• Instrument Summary Window (page 28)

Experiment Setup Window

The Experiment Setup window provides quick access to the files and settings needed to set up and run an experiment. To open the Experiment Setup window, follow one of these options:

• Click Create a New Experiment option in the Startup Wizard (page 16)

• Click the Experiment Setup button in the main software toolbar (page 14)

•Select File > New > Experiment in the main software menu bar (page 12)

The Experiment Setup window includes three tabs:

•Protocol. Click the Protocol tab to select an existing protocol to run or edit, or to create a new protocol in the Protocol Editor window (page 31)

•Plate. Click the Plate tab to select an existing plate to run or edit, or to create a new plate in the Plate Editor window (page 41)

• Start Run. Click the Start Run tab (page 24) to check the run settings, select one or more instrument blocks, and begin the run

NOTE: If the protocol currently selected in the Protocol tab does not include a step with a plate read for real-time PCR analysis, then the Plate tab is hidden. To view the Plate tab, add a “Plate Read” (page 34) in at least one step in the protocol.

NOTE: Start a new experiment from a previous run by selecting File > Repeat an Experiment in the main software menu bar. Then select the data file (.pcrd) for the experiment you want to repeat.

Running Experiments

The Experiment Setup window opens with the Protocol tab in front (Figure 23). To open another tab, click that tab or click Prev and Next buttons at the bottom of the window.

Figure 23. 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 23). A protocol file contains the instructions for the instrument temperature steps, as well as instrument options that control the ramp rate and lid temperature.

Select one of the following options to select an existing protocol, create a new protocol, or edit the currently selected protocol:

• Create New button. Open the Protocol Editor to create a new protocol

• Select Existing button. Open a browser window to select and load an existing protocol

file (.prcl extension) into the Protocol tab

• Express Load pull-down menu. Quickly select a protocol to load it into the Protocol tab

TIP: To add or delete protocols in the Express Load menu, add or delete files (.prcl extension) in the ExpressLoad folder. To locate this folder, select Too ls > U se r

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.

Figure 24 shows the default end point protocol.

Figure 24. End Point Only protocol.

CFX96 and CFX384 Systems Manual

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. Figure 25 shows the step temperature is changed to 55.0oC.

Plate Tab

The Plate tab shows a preview of the selected plate file loaded in the Experiment Setup (Figure 26). 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

• Express Load pull-down menu. Quickly select a plate to load it into the Plate tab

Figure 25. Change the sample volume on the Start Run tab.

(.pltd extension) into the Plate tab

TIP: To add or delete plates in the Express Load menu, add or delete files (.pltd extension) in the ExpressLoad folder. To locate this folder, select Too ls > U se r Data Folder in the menu bar of the main software window.

Running Experiments

• Edit Selected button. Open the currently selected plate in the Plate Editor

Figure 26. Plate tab window.

Start Run Tab

The Start Run tab (Figure 27) 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

• 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 27. The Start Run tab.

CFX96 and CFX384 Systems Manual

By default, the Start Run on Selected Block(s) pane shows the block name, block type and status of each instrument detected by the software. These descriptions can not be edited.

NOTE: You can override the Sample Volume loaded in the Protocol file by selecting the volume in the spreadsheet cell and typing a new volume.

NOTE: A run ID can be entered for each block by selecting the cell and typing an ID or by selecting the cell and scanning with a bar code reader.

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 lid temperature by selecting the text and typing a new temperature

Table 10 shows additional options on the right-click menu in the Start Run on Selected Block(s) pane:

Table 10. Start Run on Selected Block(s) right-click menu options

Right-Click Option Function

Copy Copy selected text

Copy as Image Copy an image of the Start Run on Selected Block(s) pane

Print... Print the current view of the list of selected blocks

Print Selection... Print the column that is currently selected

Export to Excel... Export the list of blocks to an Excel spreadsheet file

Export to Text... Export the list of blocks to a text file

Find... Find text in the list of blocks

Sort... Sort up to three columns in the list of blocks

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

Running Experiments

Figure 28 shows the features of the Run Details window.

Figure 28. Run Details window.

Run Status Tab

The Run Status tab (Figure 28) shows the current status of a run in progress in the Run Details window and provides buttons (page 26) 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 11 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 11. 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

CFX96 and CFX384 Systems Manual

Table 11. 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 protocols ends, which may alter your data

Real-Time Status Tab

The Real-Time Status tab (Figure 29) 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.

TIP: Click the View/Edit Plate button to open the Plate Editor window. During the run, you can enter more information about the contents of each well in the plate.

Figure 29. The Real-time Status tab displays the data during a run.

Running Experiments

Replacing a Plate File

During a run, replace the plate file by clicking the Replace Plate button (Figure 29) in the Realtime 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 (Figure 30).

Figure 30. The Time Status tab displays a count-down timer for the current run.

Instrument Summary Window

The Instrument Summary window shows a list of the detected instruments and their status. Open the Instrument Summary by clicking the View Summary button (Figure 19 on page 17) in the Detected Instrument pane.

Figure 31 shows the Instrument Summary window, including the Block Name list and the current status of all detected instruments. Select one or more blocks and click the buttons in the tool bar to change the status of each instrument.

Figure 31. Instrument Summary window.

CFX96 and CFX384 Systems Manual

Instrument Summary Toolbar

The Instrument Summary toolbar includes buttons and functions listed in Table 12.

Table 12. Toolbar buttons in the Instrument Summary window

Button Button Name Function

Set Up Experiment Set up an experiment on the selected

block by opening the Experiment Setup window

Stop Stop the current run on selected blocks

Pause Pause the current run on selected blocks

Resume Resume the run on selected blocks

Flash Block Indicator Flash the indicator LED on the lid of the

selected blocks

Open Lid Open a selected block motorized lid

Close Lid Close a selected block motorized lid

Hide Selected Blocks Hide the selected blocks in the

Instrument Summary list

Show All Blocks Show the selected blocks in the

Instrument Summary list

Show Select which blocks to show in the list.

Select one of the options to show all detected blocks, all idle blocks, all the blocks that are running with the current user, or all running blocks

Right-Click Menu

Right-click in the Instrument Summary window to change the list of options that appear:

• Block Name. View the name of the block

•Type. View the size and type of the block

• Status. View the current status of the block

•User. View the current user who is logged in to the software

• Remaining. View the time remaining in the current run

Running Experiments

• Copy. Copy the entire list

• Copy as Image. Copy the list as an image file

•Print. Print the list

• Print Selection. Print only the selected cells in the list

• Export to Excel. Export the list as an Excel formatted file

• Export to Text. Export the list as a text file

•Find. Find text in the list

•Sort. Sort the list by selecting up to three columns of data in the Sort window

4 Protocols

Read the following chapter for information about creating and editing protocol files:

• Protocol Editor window (below)

• Protocol Editor controls (page 33)

• Temperature control mode (page 37)

• Protocol AutoWriter (page 37)

CFX96 and CFX384 Systems Manual

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.

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 22)

• To open an existing protocol, select File > Open > Protocol, or click the Open Existing button in the Protocol tab (page 22)

• To edit the current protocol in the Protocol tab, click the Edit Selected button in the Protocol tab (page 22)

TIP: To change the default settings in the Protocol Editor window, enter the changes in the Protocol tab in the user Preferences window (page 124)

Protocol Editor Window

The Protocol Editor window (Figure 32) includes the following features:

•Menu bar. Select settings for the protocol

• Toolbar. Select options for editing the protocol

•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

Protocols

• Protocol Editor buttons. Edit the protocol by clicking one of the buttons to the left of the text view

Figure 32. 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 13

Table 13. 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 set the Lid

Temperature

Tools Gradient

Calculator

Run-time Calculator

The toolbar (Figure 33) in the Protocol Editor window provide quick access for important functions:

Select the block type for a gradient step. Choose 96 Wells or 384 Wells

Select the instrument and scan mode to be used for calculating the estimated run time in the Experiment Setup window

Figure 33. Protocol Editor toolbar.

CFX96 and CFX384 Systems Manual

Table 14 lists the function of the Protocol Editor toolbar buttons:

Table 14. 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 steps in a

position relative to the currently highlighted step

Sample Volume Enter a sample volume in μl between 0 and

50 (for 96-well block), or between 0 and 30 (for 384-well block) Sample volume determines the Temperature Control mode (page 37). 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. 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 to change the protocol. 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.

Protocols

3. (Optional) Click the Step Options button to enter an increment or extend option to the step (page 36). Figure 34 shows the new step that was inserted after step 2.

Figure 34. Protocol with inserted step.

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.

In Figure 34, notice that the camera icon in the graphic view (top) shows that step 4 includes a plate read.

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. Make sure the plate size for the gradient matches the block type of the instrument, 96 well or 384 well. Select the plate size for the gradient by selecting Tools > Gradient Calculator in the Protocol Editor menu bar.

3. Edit the gradient temperature range by clicking the default temperature in the graphic or text view, and entering a new temperature. Alternatively, click the Step Options button to enter the gradient range in the Step Options window (page 36)

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

CFX96 and CFX384 Systems Manual

Figure 35 shows the inserted gradient step. The temperatures of each row in the gradient are charted on the right side of the window.

Figure 35. 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 35 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.

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 36).

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.

Protocols

Figure 36 shows a melt curve step added after step 6:

Figure 36. 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 37 shows the selected step with a gradient of 10 available in a gradient step. A gradient step cannot include an increment or ramp rate change.

Figure 37. Step option for a gradient.

C. Notice that some options are not

NOTE: A gradient runs with the lowest temperature in the front of the block (row H) and the highest temperature in the back of the block (row A).

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

CFX96 and CFX384 Systems Manual

• 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.

TIP: The sample volume can be changed before a run by editing the Sample Volume parameter in the Start Run tab (see “Start Run Tab” on page 24).

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 (96-well block) or between 1 and 30 μl (384-well block) the thermal cycler calculates the sample temperature based on the sample volume. This is the standard mode

• Block mode. When you enter a sample volume of zero (0) μl, the thermal cycler records the sample temperature as the same as the measured block temperature

Protocol AutoWriter

Open the Protocol AutoWriter to quickly write protocols for PCR and real-time PCR 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

Protocols

Figure 38 shows a protocol (bottom of window) written by the Protocol AutoWriter.

Figure 38. Protocol AutoWriter window with a new protocol.

Protocol AutoWriter Overview

The Protocol AutoWriter window uses information about your reaction to automatically generate a protocol file. Enter the following information about your PCR experiment:

• Annealing Temperature (Ta) or primer sequence. Enter the annealing temperature for the primers. If the annealing temperature is unknown, click the Ta Calculator button to enter the primer sequence to calculate the annealing temperature in the Ta Calculator window

• Amplicon Length (bp). Enter the expected length of the PCR product

• Enzyme. Select the DNA polymerase enzyme (iTaq, iProof, or Other) (iTag

Polymerase; iProof including hot start activation time, and final extension time

• Run time and type. Enter a speed (Standard, Fast, Ultrafast) to adjust the total run time, and select the type of PCR (Real-time PCR or PCR)

The run time for any protocol is influenced by the number of steps and cycles, the incubation time at each step, and the time it takes to reach uniformity at the target temperature. To reduce the overall run time, the Protocol AutoWriter makes one or more of the following changes:

• Reduces the total number of protocol steps

• Reduces the number of GOTO repeats

• Minimizes the hold time in each temperature step

• Minimizes the ramp time between steps by reducing the temperature change from one step to the next

high fidelity DNA Polymerase); enter additional information

DNA

CFX96 and CFX384 Systems Manual

For example, a typical PCR protocol includes the following three sets of steps with a total run time of 1.5 to 2.0 hours:

1. Initial template denaturation and enzyme activation (95°C for 3-10 minutes).

2. Cycles of three temperature steps (30 to 40 cycles): Denaturation of template (94-95°C for 15-30 seconds), annealing of primers (anneal for 15-30 seconds), and extension of product (72°C for 15-60 seconds).

3. Final extension (72°C for 10 minutes).

The Protocol AutoWriter might make these modifications to shorten a typical protocol:

• Change the initial template denaturation and enzyme activation step from 95°C for 3 minutes to 98°C for 30 seconds

• Change the denaturation step in each cycle from 95°C for 30 seconds to 92°C for 1 second

• Combine the annealing and extension steps into a single step at 70°C for 20 seconds

NOTE: Combining the annealing and extension steps imposes limits on the melting temperature of the primers. If the melting temperatures of the primers do not fall within the specified range, adjust the primers. For example, shorten the primers by 2 to 3 basepairs (bp), or redesign them to adjust the melting temperature.

Create a Protocol With the Protocol AutoWriter

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 C a l c u l at o r 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.

TIP: Enter the lid temperature and sample volume before each run by editing the parameters in the Start Run tab (see “Start Run Tab” on page 24).

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

TIP: To edit a protocol written with the Protocol AutoWriter, open the protocol file (.prcl extension) in the Protocol Editor window (page 31).

NOTE: Bio-Rad Laboratories does not guarantee that running a protocol written in the Protocol AutoWriter window will always result in a PCR product.

Protocols

5 Plates

Read this chapter for information about creating and editing plate files:

• Plate Editor window (next section)

• Plate size and type (page 44)

• Scan mode (page 45)

• Select Fluorophores window (page 45)

• Well loading controls (page 46)

• Well Groups Manager window (page 50)

CFX96 and CFX384 Systems Manual

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 a real-time PCR experiment, you must load the minimal required information in the Plate Editor: at least one well must contain a loaded sample type and fluorophore.

TIP: Change the well contents before, during, and after running the experiment. However, the scan mode and plate size cannot be changed during or after the run.

Open the Plate Editor

To open the Plate Editor window (Figure 39), 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 23)

• To open an existing plate, select File > Open > Plate, or click the Open Existing button in the Plate tab (page 23)

• To edit the current plate in the Plate tab, click the Edit Selected button in the Plate tab (page 23)

• To open the plate associated with a data file, in the Data Analysis window (page 69), click View/Edit Plate on the toolbar

Plates

Figure 39. Plate Editor window.

Plate Editor Window

The Plate Editor window includes the following features:

•Menu bar. Select settings for the plate size, plate type, number conventions, and units (page 43)

NOTE: A 96-well plate collects data for up to 5 channels (All Channels scan). A 384-well plate collects data for up to four channels (All Channels scan).

• Toolbar. Select settings for the Scan Mode (page 45) or Well Groups (page 50)

• Plate Loading Guide. Open the Plate Loading Guide window from the toolbar for a

quick overview of instructions to load a plate (page 42)

• Plate view. View the current well contents. Load wells by using the plate loading options on the right side of the plate view (Figure 39)

• Well loading controls. Choose the contents to load in the wells (page 46) from the controls on the right side of the plate view

The Plate Editor example shows some loaded wells with unknowns (Unk) and standards (Std) (Figure 39). The bottom of the plate lists the plate type (BR Clear) and a selector to View Ta rg e t Name or Concentration when wells are loaded with Standard sample type.

CFX96 and CFX384 Systems Manual

Plate Editor Menu Bar

The menu bar in the Plate Editor window provides the menu items shown in Table 15.

Table 15. Menu bar items in the Plate Editor

Menu Item Command Function

File Save Save the plate files

Save As Save the plate file with a new file name

Exit Exit the Plate Editor

Settings Plate Size Select a plate size that reflects the number of wells in

the instrument block. Choose 384-well for the CFX384, 96-well for the CFX96 NOTE: Plate Size must be the same as the block size in the instrument on which the experiment will be run

Plate Type Choose the type of wells in the plate that holds your

samples, including BR White and BR Clear. For accurate data analysis, the plate type must be the same as the plate well type used in the experiment NOTE: You must calibrate new plate types (page 131).

Number Convention Select or cancel the selection for Scientific Notation

Units Select the units to show in the spreadsheets when

performing quantitation of unknowns versus a standard curve. Select copy number, fold dilution, micromoles, nanomoles, picomoles, femtomoles, attomoles, milligrams, micrograms, nanograms, picograms, femtomoles, attograms, or percent

Tools Show Spreadsheet

View

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

Show Well Notes Select to show this pane in the well loading controls.

Show Collection Name

Help Help Contents Open the Help for more information about plates

Show the plate information in a spreadsheet view for export or printing

load the wells

Enter notes about one or more wells

Select to show this pane in the well loading controls. Select to enter collection names for one or more wells

Plate Editor Toolbar

The toolbar in the Plate Editor provides quick access to important plate loading functions:

TIP: To change the default settings in the Plate Editor window, open the User Preferences window and enter the changes in the Plate tab.

Plates

Table 16 lists the functions available in the Plate Editor toolbar.

Table 16. 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 Guide

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

Plate Size and Type

The software applies these plate settings to all the wells during the experiment:

• Plate Size. Select a plate size that represents the size of the reaction module block of your instrument. Choosing the instrument type, CFX96 or CFX384, 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 15). Plate size can not be changed during or after the experiment

•Plate Type. Select clear or white wells from the Settings menu. Make sure the fluorophore being used in the experiment is calibrated for the selected plate type

NOTE: CFX96 and CFX384 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 131)

Scan Mode

The CFX96 system excites and detects fluorophores in six channels. The CFX384 system excites and detects fluorophores in five channels. Both systems use multiple data acquistion scan modes to collect fluorescence data from during a run.

Select one of these scan modes int he Plate Editor window toolbar:

• All Channels. Includes channels 1 through 5 on the CFX96 system or channels 1 through 4 on the CFX384 system

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

•FRET. Includes only the FRET channel and provides a fast scan

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 131). After calibration, the new fluorophore is added to the Select Fluorophore window.

CFX96 and CFX384 Systems Manual

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 40).

Figure 40. 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.

Plates

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.

When setting up a gene expression experiment, 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”

TIP: Target names and sample names must match between wells to compare data in the Gene Expression tab in the Data Analysis window. Each name must contain the same punctuation and spacing. For example, “Actin” is not the same as “actin”, and “2hr” is not the same as “2 hr”. To facilitate consistency in names, enter them in the Target and Sample Names Libraries in the Plate tab in the User Preferences window (page 125).

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 17).

Table 17. Options for loading the plate and wells in the Plate Editor

Option Function

After selecting wells, the Sample Type must be loaded first. Select a Sample Type from the pull-down menu to load it in the selected wells, including Unknown, Standard, NTC (no template control), Positive Control, Negative Control, and NRT (no reverse transcriptase)

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

For gene expression analysis or to distinguish between multiple targets, select a name in the Tar g e t Na m e pulldown menu and press the Enter key to load the target name in the well. To delete a target name, select it, press the Delete key, and press the Enter key

TIP: 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

CFX96 and CFX384 Systems Manual

Table 17. Options for loading the plate and wells in the Plate Editor (continued)

Option Function

For gene expression analysis or to distinguish between multiple samples, select a Sample Name from the pulldown menu to load that sample name in the selected wells. To delete a sample name, select it in the menu, press the Delete key on your keyboard, and then press Enter.

TIP: 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 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. Click the Clear Replicate # button to clear the replicate number from selected cells.

TIP: To load multiple replicate numbers across a series of wells, click the Replicate Series button.

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.

NOTE: You can load replicate groups with replicate numbers progressing from left to right (Horizontal), or progressing from top to bottom (Vertical).

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

Plates

Table 17. Options for loading the plate and wells in the Plate Editor (continued)

Option Function

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 78)

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 clear all content in the selected wells

NOTE: Clicking the Clear Wells button permanently removes the content of the wells.

Experiment Settings Window

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

• 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 41) or to set the gene expression analysis sample group to be analyzed if Collection Names have been added to the wells.

CFX96 and CFX384 Systems Manual

• 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 41 shows the Targets tab with the analysis settings shown.

Figure 41. Targets tab in Experiment Settings window.

Figure 42 shows the Sample Tab with the Analysis Settings shown.

Figure 42. Samples tab in Experiment Settings window.

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 box for that row, and then clicking the Remove checked items(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

Plates

• 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.

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 43).

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

CFX96 and CFX384 Systems Manual

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 43. Color of wells in the Well Group Manager window.

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 44) by selecting Tools > Show Spreadsheet View in the Plate Editor menu bar.

Figure 44. Plate Spreadsheet View window.

Plates

Open the spreadsheet view to import or export the well contents to Excel or to another tabdelimited 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 tabdelimited 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

CFX96 and CFX384 Systems Manual

Function keys

Protocol

Command

Alphanumeric

LCD

USB port

(below)

keys

Navigation keys

AutoWriter key

keys

6 Stand-Alone Operation

Read this chapter for information about running the CFX96 system or CFX384 system in stand-alone mode:

• Control panel (below)

• Main menu (page 54)

• Experiment setup (page 55)

• Exporting data for analysis (page 61)

• Creating a data file (page 64)

• Setting up email (page 65)

Control Panel

The control panel on the C1000 thermal cycler provides access to all the functions needed to run the instrument utilizing the following three components:

• Liquid crystal display (LCD): Displays the main menu and other screens

•Keypad: Navigate screens and enter commands with these keys

• USB port: Connect a USB drive, mouse, or keyboard.

Figure 45 shows the components of the control panel:

Figure 45. Thermal cycler control panel.

Stand-Alone Operation

The control panel contains five sets of keys with the functions listed in table Table 18:

Table 18. 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

ALPHANUMERIC KEYS

1 through 9 Enter numbers or letters of the alphabet. Press a key

0, INCUBATE

decimal point (.) Enter a decimal point

minus sign (-) Enter a minus sign

Protocol AutoWriter

each screen

multiple times to switch to each associated letter

Insert a zero, (infinity), or start instant incubation

NAVIGATION KEYS

RIGHT arrow Move cursor to the right

LEFT arrow Move cursor to the left

UP arrow Move cursor up

DOWN arrow Move cursor down

ENTER Confirm a setting

BACK Cancel a function. Delete a letter, number, or word

Main Menu

When it starts, the CFX96 system or CFX384 system runs a self-test to verify proper functions, and then displays the main menu. Use the main menu to begin operating the instrument. The main menu provides access to all system operations, displays the date and time, the name of the logged-in user, the system status, the type of reaction module and thermal cycler name, and any attached S1000 thermal cyclers(Figure 46).

NOTE: To rename the thermal cycler, open the files library (Files (F2) button) and then select Rename Cycler.

Launch the Protocol AutoWriter

CFX96 and CFX384 Systems Manual

Figure 46. 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

Experiment Setup

The CFX96 system or the CFX384 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 “Exporting Data Using Email” on page 62). The data requires CFX Manager software for analysis.

NOTE: The C1000 thermal cycler stores up to 20 real-time PCR experiment runs.

Stand-Alone Operation

To create a new experiment:

1. Select New Protocol (F4) in the start up screen to open a new protocol template (Figure 47).

NOTE: By default, the protocol template contains a plate read step when a CFX96 or a CFX384 optical reaction module is inserted in the C1000 chassis.

TIP: To change an existing protocol, press the EDIT command key to open the file library and select a protocol to edit.

Figure 47. 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.

NOTE: Press the VIEW key to switch between graphic and text view of the protocol.

3. (Optional) To insert a new step, select the Insert (F1) button. To delete a step, select the Delete (F3) button (Figure 47).

4. (Optional) To change step options, select the Options (F4) button (Figure 47). 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 48)

Figure 48. Step Options window.

CFX96 and CFX384 Systems Manual

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

TIP: Once a step has a gradient, you can edit the upper and lower temperatures in the graphic or text view without opening the Options screen.

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

Entering a Protocol Name:

• 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 (Figure 47 on page 56).

• Press ENTER to accept the name.

Changing run parameters:

• To change the default sample volume, select the sample volume box (Vol) (Figure 47 on page 56). 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

TIP: Entering a sample volume from 1 to 50 selects Temperature Control mode, which is the standard mode. Entering zero (0) selects Block mode. Temperature mode is the recommended mode because it most accurately represents the actual sample temperature.

• To change the default lid temperature, select the lid temperature box (LID) by pressing the arrow keys (Figure 47 on page 56). Use the alphanumeric keys to enter a new temperature. For the CFX96 system, use a lid temperature of 105 CFX384 system use a lid temperature of 95

NOTE: Heating the lid prevents condensation in the sealed reaction vessels.

C; for the

Running the Protocol:

1. To run the protocol, click the Done (F2) button in the Protocol window (Figure 47 on page

56).

TIP: Alternatively, click the RUN command key to start the run without saving or editing the name of the protocol.

Stand-Alone Operation

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 49).

3. Click Edit Filename (F1) and type a new name in the box (Figure 50).

Figure 49. Saving a protocol.

Figure 50. Entering a protocol name.

4. Click Save (F2) (Figure 50).

CFX96 and CFX384 Systems Manual

5. Click Run (F2) to continue and run the protocol (Figure 51).

Figure 51. Protocol successfully saved.

6. Edit the Sample Volume or Lid Temperature that will be used for the run (Figure 52).

7. Enter the Sample ID or User to be recorded in the Run information screen.

Figure 52. Editing sample volume and lid temperature.

8. Click OK (F1) to proceed.

Stand-Alone Operation

9. Select a Scan Mode to instruct the instrument in which channels to collect fluorescence data during a run (Figure 53).

Scan modes detect calibrated fluorophores in these channels:

• All Channels. Collects data from channels 1 through 5 on the CFX96 system, or channels 1 through 4 on the CFX384 system

•SYBR/FAM only. Collects data only from channel 1 on either system, and provides a fast scan

•FRET. Collects data only from the FRET channel on either system and provides a fast scan

Figure 53. Scan mode and data file name.

10.A default stand-alone data file name is created prior to the run. If you wish to change the name, use the arrow keys to navigate to the Data File Name box, then press the alphanumeric keys to enter a letter or number to type a new data file (.zpcr) name.

11.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

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

NOTE: Press the RUN key from any screen to open the file library and select a folder to locate a protocol file to run

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 54)

•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 54. Monitoring run status.

Exporting Data for Analysis

CFX96 and CFX384 Systems Manual

When the run is finished, the fluorescence data needs 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 55).

Figure 55. RT_DATA folder stores real-time PCR runs.

Data can be transferred to the analysis computer using a USB key, or it can be emailed directly to the computer using the SMTP server once an email connection is configured.

NOTE: The C1000 stores up to 20 real-time PCR experiment runs.

Exporting Data Using the USB Key

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.

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.

Stand-Alone Operation

2. Use the up and down arrow keys to navigate to the RT_DATA folder and then press the right arrow key to open the folder.

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

4. Press the Export File (F1) button to export a copy of the run data (.zpcr) to the USB key, as shown in Figure 56.

Figure 56. Exporting stand-alone run data to a USB key.

5. Use the arrow keys to navigate to the folder on the USB in which to save the file.

6. Click Ye s (F1) to confirm the export (Figure 57).

Figure 57. Confirming export to USB key.

Exporting Data Using Email

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 “Setting Up Email” on page 65).

CFX96 and CFX384 Systems Manual

To send an email with attached data (.zpcr) at the end of a run, follow these instructions:

1. After saving the name of the protocol or selecting a protocol out of the File Library using the Run command key, select Options (F4) in the Run information screen (Figure 58).

Figure 58. Run information screen.

2. Using the arrow keys to select the Send email notification option (Figure 59).

Figure 59. Selecting email notification.

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 name of an email address.

Stand-Alone Operation

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

Creating a Data File

Figure 60. Confirming export to USB key.

The stand-alone run data (.zpcr) data needs to be converted into a data file (.pcrd) by CFX Manager software in order to be analyzed. Follow these instructions 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.

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 61).

Figure 61. 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.

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

Setting Up Email

After a run, a .zpcr file can be emailed directly to a computer running CFX Manager software. To configure the outgoing email from the C1000 thermal cycler, follows these instructions:

1. Connect an ethernet cable to the port in the back of the C1000 chassis.

2. On the main menu, select Log In (F1) to log in to the thermal cycler as the administrator (Figure 46 on page 55).

NOTE: The logged in user name appears under the date and time when you return to the main window.

3. Select Utilities (F3) on the main screen (Figure 46 on page 55) to launch the utilities menu.

4. In the Utilities Menu, select 5: Administrator Settings (Figure 62).

CFX96 and CFX384 Systems Manual

Figure 62. Utilities Menu.

Stand-Alone Operation

5. In the Administrator Settings Menu, select 9: SMTP Server Settings (Figure 62).

6. Contact your network administrator for your the SMTP server name.

NOTE: The SMTP server name is provided by your ISP.

7. Select Add Server Name (F1) (Figure 64).

Figure 63. Administrator Settings Menu window.

Figure 64. SMTP server setup window.

8. Type the name of the server in the text box using the virtual keypad.

NOTE: The SMTP server name will use the following nomenclature: SMTP.YourInstitution.com.

Do not use Bio-Rad.com in the name

CFX96 and CFX384 Systems Manual

9. Click Save (F1) to save the name of the SMTP server (Figure 65).

Figure 65. Saving server name.

10.The added server name will appear in the SMTP Server Names pull-down menu, as shown in Figure 66.

Figure 66. Added server name.

11.Select Set Current Server (F3) to set the current server to be used for email (Figure 66).

Stand-Alone Operation

12.Use the arrow keys to select the Test Current Server button and click the Enter navigation button (Figure 67).

13.Type an email address in the text box and select Test Server (F1) (Figure 68).

Figure 67. Test the current server.

Figure 68. Enter email to test the current server.

14.The C1000 thermal cycler will send an email to the entered address as a test of the SMTP server configuration.

NOTE: Some SMTP servers do not allow attachments, and others allow attachments only up to certain sizes. If you will use CFX Manager software or the C1000 chassis to email data files and/or reports, you may want to test your server's ability to email attachments by checking the Test Attachment box, and setting the attachment size in MB with up to 5 megabytes (MB) or more.

CFX96 and CFX384 Systems Manual

7 Data Analysis Overview

Read this chapter for information about data analysis:

• Data Analysis window (below)

• Quantitation tab (page 72)

• Well groups (page 73)

• Data analysis settings (page 73)

• Well selectors (page 75)

• Charts (page 78)

• Spreadsheets (page 79)

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

Data Analysis Overview

The Data Analysis window displays up to nine tabs (Figure 69). Each tab shows the analyzed data for a specific analysis method:

The software only displays a tab in the Data Analysis window if the data are collected in the run and data are available for that type of analysis. For example, the Melt Curve and Melt Curve Data tabs do not appear if the experiment does not include a melt curve step.

Data Analysis Toolbar

The toolbar in the Data Analysis window (Figure 70) provides quick access to important data analysis functions.

Table 19 lists the functions of buttons in the toolbar.

Table 19. Toolbar in the Data Analysis window

Toolbar button Name Function

Figure 69. All the tabs that can display in the Data Analysis window.

Figure 70. Toolbar in the Data Analysis window.

Save Save the current data file

Print Print the selected window

Trace Style Open Trace Style window

Report Open a Report for the current data file

View/Edit Plate Open the Plate Editor to view and edit the contents of

Well Groups... Select a well group name from the pull-down menu.

Help Open the software Help site for more information

the wells

The default selection is All Wells

about data analysis

Data Analysis Menu Bar

The menu bar in the Data Analysis window (Figure 71) provides these menu items:

Figure 71. Menu bar in the Data Analysis window.

Table 20 lists the functions of items in the menu bar.

Table 20. 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

Exit Exit the Data Analysis window

View Run Log Open a Run Log window to view the run

Settings Analysis Mode Select Baseline Subtraction method for

C(t) Determination Mode Select Regression or Single-Threshold

Baseline Thresholds Open the Baseline Thresholds window to

Trace Styles Open the Trace Styles window

View/Edit Plate Open the Plate Editor to view and edit the

Mouse Highlighting Turn on or off the simultaneous

CFX96 and CFX384 Systems Manual

current experiment to rerun it

log of those data file

the selected well groups in the data

mode to determine how C(t) values are calculated for each trace

adjust the baseline or the threshold

plate

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

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

Data Analysis Overview

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 72) 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 72. 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 CFX96 system or CFX384 system can acquire fluorescence data at multiple protocol steps; the software maintains the data acquired at each step independent. 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 73 shows the data collection step number is 3 for all the data.

Figure 73. Step Number selection in the Data Analysis window.

CFX96 and CFX384 Systems Manual

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 50), group names appear in the Data Analysis window within the Well Groups list on the toolbar.

TIP: To open the Plate Editor, click the View/Edit Plate button in the Data Analysis window 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 74 shows Group 2 selected in the Well Groups menu. Only the wells in that well group appear loaded with content in the well selector and data only for these wells are included in the data analysis calculations.

Figure 74. 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

Data Analysis Overview

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 one fluorophore in the fluorophore selector in the Quantitation tab (Figure 72) by clicking the boxes next to the fluorophore name located under the Amplification chart.

2. Select Settings > Baseline Thresholds in the menu bar to open the Baseline Thresholds window.

3. Adjust the crossing threshold (Figure 75) for the fluorophore by clicking User Defined and entering a threshold number.

Figure 75. Baseline Thresholds window.

4. 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 75) to change the default baseline for selected wells. To open this window:

1. Select one fluorophore in the fluorophore selector in the Quantitation tab (Figure 72) by clicking the boxes next to the fluorophore name located under the Amplification chart.

2. 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 75).

CFX96 and CFX384 Systems Manual

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

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, and therefore the software does not display the Gene Expression, End Point, and Allelic Discrimination tabs

• Baseline Subtracted. The software displays the data as baseline subtracted traces for each fluorophore in a well. The software must baseline subtract the data to determine 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 76, 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

Data Analysis Overview

• Empty wells (dark gray). These wells were not loaded in the Plate Editor window

Well Selector Right-Click Menu Items

Right-click any well selector view to select the items listed in Table 21.

Table 21. Right-click menu items in the well selectors

Item Function

Copy Copy the content of the well to a clipboard, including Sample

Copy as Image Copy the well selector view as an image

Print... Print the well selector view

Print Selection... Print the current selection

Export to Excel... Export the data to an Excel spreadsheet

Figure 76. Three well colors appear in a well selector.

Type and optional Replicate #

Exclude Wells From Analysis

To exclude any wells from data analysis temporarily, follow these instructions:

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.

CFX96 and CFX384 Systems Manual

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

Figure 77. Right-click to exclude a well from analysis.

NOTE: Unselect the Exclude Well from Analysis from the right-click menu to reinclude the well.

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 78) to exclude the selected wells. This checkbox is at the bottom of the Plate Editor controls on the right side of the window.

Figure 78. Exclude Wells in Analysis checkbox at bottom of the pane.

Data Analysis Overview

In Figure 79, one well (under the pointer) was excluded from data analysis in the Plate Editor. Notice that the excluded well is marked with an asterisk (*).

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.

Figure 79. Excluded well (marked with *) in the Plate Editor.

Charts

Each chart in the Data Analysis window displays the data in a different graph and includes options for adjusting the data. To magnify an area of the chart, select an area by clicking and dragging the mouse. The software resizes the chart and centers it on the selected area.

TIP: Return the chart to a full view by right-clicking on the chart and selecting Set Scale to Default from the right-click menu.

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 22)

Table 22. 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 81).

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.

CFX96 and CFX384 Systems Manual

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.

Common Right-Click Menu Items for Spreadsheets

Right-click any spreadsheet view to select the items shown in Table 23.

Table 23. 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 Html file

Find... Search for text

Sort... Sort the data in up to three columns

Data Analysis Overview

CFX96 and CFX384 Systems Manual

8 Data Analysis Windows

Read this chapter for more information about the tabs in the Data Analysis window:

• Quantitation tab (below)

• Quantitation Data tab (page 84)

• Melt Curve tab (page 87)

• Melt Curve Data tab (page 89)

• End Point tab (page 91)

• Allelic Discrimination tab (page 93)

•QC tab (page93)

• Run Information tab (page 96)

• Data file reports (page 97)

Quantitation Tab

Use the data in the Quantitation tab (Figure 80) 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

Data Analysis Windows

• Spreadsheet. Shows a spreadsheet of the data collected in the selected wells

Figure 80. Layout for the Quantitation tab in Data Analysis window.

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 81). Click the box next to the fluorophore name to show or hide the fluorophore data throughout the data analysis window.

Figure 81. Fluorophore selector with FAM selected.

Trace Styles Window

Open the Trace Styles window (Figure 82) 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 (Figure 81) under the Amplification chart.

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

CFX96 and CFX384 Systems Manual

Figure 82. 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, including Unknown, Standard, NTC (no template control), Positive Control, Negative Control, or NRT (no reverse transcriptase control) sample types

• 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

• Click Remove Symbols to remove all the added symbols from all wells

• Click Restore Default Colors to return to the default trace colors

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 83.

Figure 83. Log Scale option selected in Amplification chart.

TIP: To magnify any area of the chart, click and drag the mouse across an area. To return to a full view, right-click and select Set Scale to Default from the menu.

Data Analysis Windows

Standard Curve Chart

The software creates a Standard Curve chart (Figure 84) in the Quantitation tab if the data include sample types defined as standard (Std) for one fluorophore in the experiment.

Figure 84. 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, R correctly the line describes the data (goodness of fit)

(written as R^2). Use this statistic to determine how

Chart Right-Click Menu Options

In addition to the common right-click menu options to copy, print and export charts, Table 24 lists the menu options available only on the Amplification chart.

Table 24. 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)

Quantitation tab Spreadsheet

Table 25 shows the type of data shown in the spreadsheet at the bottom right side of the Quantitation tab:

Table 25. Quantitation tab spreadsheet content

Information Description

Well Well position in the plate

Fluor Fluorophore detected

Content A combination of the Sample Type (required) and Replicate #

Sample Sample Name loaded in the Plate Editor wells

C(t) Threshold cycle for each trace

TIP: To make changes to the Content and Sample, open the Plate Editor by clicking the View/Edit Plate button.

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.

CFX96 and CFX384 Systems Manual

(optional) loaded in the Plate Editor

Results Spreadsheet

Select a Results spreadsheet (Figure 85) to see data for each well in the plate.

Figure 85. 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 26.

Table 26. Results spreadsheet content

Information Description

Well Well position in the plate

Fluor Fluorophore detected

Content Sample type and replicate number

Data Analysis Windows

Table 26. Results spreadsheet content (continued)

Information Description

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

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

Plate Spreadsheet

group

round of annealing and extension steps in a protocol

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 86 shows the Plate spreadsheet as plate map.

Figure 86. Plate spreadsheet in Quantitation Data tab.

The Plate spreadsheet includes the type of information shown in Table 27, including the selected fluorophore in the plate map.

Table 27. Plate spreadsheet contents

Information Description

Content Sample type and replicate number

Sample Sample description

Copy Number Starting number of targets in the sample

RFU Relative fluorescence units

CFX96 and CFX384 Systems Manual

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 87).

Figure 87. RFU spreadsheet in the Quantitation Data tab.

The RFU spreadsheet includes the type of information shown in Table 28.

Table 28. RFU spreadsheet contents

Information Description

Well number (A2, A3, A4, A5, A6...)

Cycle One round of denaturation, annealing, and extension, or one

Melt Curve Tab

For DNA-binding dyes and noncleavable hybridization probes, the fluorescence is brightest when the two strands of DNA anneal. Therefore, as the temperature rises towards the melting temperature (Tm), fluorescence decreases at a constant rate (constant slope). At the Tm, there is a dramatic reduction in the fluorescence with a noticeable change in slope. The rate of this change is determined by plotting the negative first Regression of fluorescence versus temperature (-d(RFU)/dT). The greatest rate of change in fluorescence results in visible peaks and represents the Tm of the double-stranded DNA complexes.

The software plots the RFU data collected during a melt curve as a function of temperature. To analyze melt peak data, the software assigns a beginning and ending temperature to each peak by moving the threshold bar. The floor of the peak area is specified by the position of the melt threshold bar. A valid peak must have a minimum height relative to the distance between the threshold bar and the height of the highest peak.

Open the Melt Curve tab (Figure 88) 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 89).

Well data, listed by position in the plate for all the loaded wells

round of annealing and extension steps in a protocol

Data Analysis Windows

Figure 88. Layout of the Melt Curve tab in the Data Analysis window.

Adjusting Melt Curve Data

Adjust the Melt Curve data by any of these methods:

• Click and drag the threshold bars in the Melt Peak chart to include or exclude peaks in data analysis

•Select Positive in the Peaks pull-down menu to show the spreadsheet data for the peaks above the Melt Threshold line, or select Negative to view the spreadsheet data for the peaks below the Melt Threshold line

• Open the Trace Styles window to change the color of the traces in Melt Curve and Melt Peak charts.

• Select a number in the Step Number selector (page 82) to view the Melt Curve data at another step in the protocol. The list shows more than one step if the protocol includes plate read (camera icon) in two or more melt curve steps

• Select wells in the well selector to focus on subsets of the data

• Select a well group (page 73) to view and analyze a subset of the wells in the plate. Select each well group by name in the Well Group pull-down menu in the toolbar

Melt Curve tab Spreadsheet

Table 29 shows the type of information in the spreadsheet at the bottom right side of the Melt Curve tab.

Table 29. Melt Curve tab spreadsheet contents

Information Description

Well Well position in the plate

Fluor Fluorophore detected

Content A combination of Sample Type and Replicate #

Sample Sample Name loaded in the Plate Editor

Melt Temp The temperature of the melt peak for each well. Only the two

highest peaks are displayed in this spreadsheet.

Peak Height The highest point of the melt peak (-d(RFU)/dT)

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 these 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

•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. This

is a first regression plot for each well in the plate

Melt Peaks Spreadsheet

Select the Melt Peaks spreadsheet (Figure 89) to view melt curve data.

CFX96 and CFX384 Systems Manual

Figure 89. Melt Peaks spreadsheet in Melt Curve Data tab.

The Melt Peaks spreadsheet (Figure 89) includes the type of information shown in Table 30.

Table 30. Melt Peaks spreadsheet content

Information Description

Well Well position in the plate

Fluor Fluorophore detected

Content Sample Type listed in the Plate Editor window

Target Amplification target (gene)

Sample Sample Name listed in the Plate Editor window

Melt Temperature The melting temperature of each product, listed as one peak (highest) per

row in the spreadsheet

Peak Height Height of the peak

Begin Temperature

End Temperature Temperature at the end of the peak

Temperature at the beginning of the peak

Data Analysis Windows

Plate Spreadsheet

Select the Plate spreadsheet (Figure 90) to view melt curve data in a plate format:

NOTE: To adjust the peak that the software calls, adjust the threshold line in the Melt Peak chart on the Melt Curve tab.

The Plate spreadsheet includes the types of information shown in Table 31.

Table 31. Plate spreadsheet content

Figure 90. Plate spreadsheet in Melt Curve Data tab.

Information Description

Content A combination of Sample Type (required) and Replicate #

(optional)

Sample Sample description

Peak 1 First melt peak (highest)

Peak 2 Second (lower) melt peak

RFU Spreadsheet

Select the RFU spreadsheet to view the fluorescence for each well at each cycle acquired during the melt curve (Figure 91).

Figure 91. RFU spreadsheet in Melt Curve Data tab.

CFX96 and CFX384 Systems Manual

Table 32 lists the type of information shown in the RFU spreadsheet.

Table 32. RFU spreadsheet content

Information Description

Well number (A1, A2, A3, A4, A5...)

Temperature Melting temperature of the amplified target. Plotted as one

Well position in the plate for the loaded wells

well per row, and multiple wells for multiple products in the same well

-d(RFU)/dT Spreadsheet

Select the -d(RFU)/dT spreadsheet to view the type of data shown in Figure 92.

Figure 92. The -d(RFU)/dT spreadsheet in the Melt Curve Data tab.

Table 33 lists the type of information shown in the -d(RFU)/dT spreadsheet.

Table 33. -d(RFU)/dT spreadsheet content

Information Description

Well number (A1, A2, A3, A4, A5...)

-d(RFU)/dT Negative rate of change in RFU as temperature (T) changes

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 “calls” 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

• 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

Well position in the plate for the loaded wells

BIO RAD CFX96, CFX384 Instruction Manual

Specifications and Main Features

Frequently Asked Questions

User Manual

Bio-Rad Resources

Writing Conventions Used in this Manual

Safety and Regulatory Compliance

Table of Contents

1 System Installation

Unpacking the Optical Reaction Module

System Requirements

System Overview

Setting up the system

Installing CFX Manager Software

Running Experiments

Main Software Window

Startup Wizard

Detected Instruments Pane

Instrument Properties Window

Software Files

Tips and Tricks

3 Running Experiments

Experiment Setup Window

Protocol Tab

Plate Tab

Start Run Tab

Run Details Window

Instrument Summary Window

4 Protocols

Protocol Editor Window

Protocol Editor Controls

Temperature Control Mode

Protocol AutoWriter

5 Plates

Plate Editor Window

Select Fluorophores Window

Experiment Settings Window

Well Groups Manager Window

Plate Spreadsheet View Window

6 Stand-Alone Operation

Control Panel

Main Menu

Experiment Setup

Exporting Data for Analysis

Creating a Data File

Setting Up Email

7 Data Analysis Overview

Data Analysis Window

Data Analysis Menu Bar

Quantitation Tab

Data Analysis Settings

Well Selectors

Charts

Spreadsheets

8 Data Analysis Windows

Quantitation Tab

Quantitation Data Tab

Melt Curve Tab

Melt Curve Data Tab

End Point Tab