Bio-Rad 10000070438 Console Operation & Maintenance Manual

CFX96™ Dx and

CFX96 Deep Well Dx Systems

Operation Manual

1845097-IVD
1844095-IVD
1841000-IVD
12007917

Manual revision: January 2019
Software revision: 3.1

ETL LISTED

CONFORMS TO

UL Std. 61010-1
UL Std. 61010-2-010
UL Std. 61010-2-101

3120330

UL Std. 61010-2-081

CERTIFIED TO

CAN/CSA Std. C22.2 NO. 61010-1-12
CAN/CSA Std. C22.2 NO. 61010-2-010
CAN/CSA Std. C22.2 NO. 61010-2-101
CAN/CSA Std. C22.2 NO. 61010-2-081:2015

CFX96™ Dx and CFX96 Deep Well Dx

Systems

Operation Manual

Version 3.1

Bio-Rad Technical Support

For technical assistance, please contact your local Bio-Rad representative.

Notice

No part of this publication may be reproduced or transmitted in any form or by any means,
electronic or mechanical, including photocopy, recording, or any information storage or retrieval
system, without permission in writing from Bio-Rad.

Bio-Rad reserves the right to modify its products and services at any time. This guide is subject to
change without notice. Although prepared to ensure accuracy, Bio-Rad assumes no liability for
errors or omissions, or for any damage resulting from the application or use of this information.

The following are trademarks of Bio-Rad Laboratories, Inc.: Bio-Rad, CFX Manager, CFX96,
C1000.

Bio-Rad thermal cyclers and real-time systems are covered by one or more of the following U.S.
patents or their foreign counterparts owned by Eppendorf AG: U.S. Patent Numbers 6,767,512 and
7,074,367.

Copyright © 2018 by Bio-Rad Laboratories, Inc. All rights reserved.

Table of Contents

Safety and Regulatory Compliance 11

Safety Warning Labels 11

Safe Use Specifications and Compliance 12

Regulatory Compliance 12

Hazards 13

Biohazards 13

Chemical Hazards 15

Explosive or Flammability Hazards 15

Electrical Hazards 15

Transport 15

Battery 15

Disposal 16

Warranty 16

Chapter 1 Introduction 17

CFX Dx PCR Detection Systems 17

Intended Use 18

Finding Out More 18

Chapter 2 Setting Up the C1000 Dx Thermal Cycler 19

Site Requirements 19

Bench Space Requirements 19

Environment Requirements 20

Power Requirements 20

System Overview 21

Front View 21

Back View 22

Optical Reaction Modules 23

Recommended Sample Volumes 23

Installing the C1000 Dx Thermal Cycler 24

Operation Manual | iii

Table of Contents

Unpacking and Setting Up the C1000 Dx Thermal Cycler 24

Attaching the Optical Reaction Module 25

Removing the Shipping Screw 26

Loading Sample Plates 27

Detecting Connected Instruments 29

Detaching the Reaction Module 30

Shutting Down the C1000 Dx Thermal Cycler 30

Chapter 3 Installing CFX Manager Dx Software 31

System Requirements 32

Installing CFX Manager Dx Software 33

Detecting Connected Instruments 33

Software Files 34

Recommended Cybersecurity Measures 34

Chapter 4 The Workspace 37

The Home Window 38

The Startup Wizard 39

The Protocol Editor Window 40

The Plate Editor Window 41

The Data Analysis Window 42

Chapter 5 The Home Window 43

The Home Window 44

File Menu Commands 45

View Menu Commands 45

User Menu Commands 46

Run Menu Commands 46

Tools Menu Commands 47

Help Menu Commands 47

Toolbar Commands 48

The Startup Wizard 49

Status Bar 49

Detected Instruments Pane 50

Viewing the Properties of an Instrument 53

Before You Begin 55

iv | CFX96 Dx and CFX96 Deep Well Dx Systems

Table of Contents

Setting User Preferences 55

Creating a Reaction Master Mix 69

Calibrating New Dyes 72

Chapter 6 Creating Protocols 75

Protocol Editor Window 76

File Menu Commands 76

Settings Menu Command 77

Tools Menu Commands 77

Toolbar Commands 77

Protocol Editing Controls 78

Creating a Protocol in the Protocol Editor 80

Opening a New Protocol File in the Protocol Editor 80

Opening an Existing Protocol in the Protocol Editor 81

Setting Up a New Protocol 82

Adding Steps to a Protocol 84

Inserting a Gradient Step 84

Inserting a GOTO Step 86

Inserting a Melt Curve Step 86

Adding or Removing a Plate Read Step 88

Changing Step Options 88

Deleting a Step 89

Copying, Exporting, or Printing a Protocol 89

Creating a Protocol with the Protocol AutoWriter 90

Using the Ta Calculator 92

About the Ta Calculator 92

Chapter 7 Preparing Plates 97

Plate Editor Window 98

File Menu Commands 99

Settings Menu Commands 99

Editing Tools Menu Commands 99

Toolbar Commands 100

Creating a Plate File Using the Plate Editor 101

Opening a New Plate File in the Plate Editor 101

Opening an Existing Plate File in the Plate Editor 103

Operation Manual | v

Table of Contents

Setting Up a New Plate File 104

Assigning Optional Parameters to the Plate File 110

Assigning a Target to Wells 110

Assigning a Sample Name to Wells 112

Assigning Biological Sets to Wells 113

Assigning Replicate Numbers to Wells 115

Assigning a Dilution Series to Standard Sample Types 116

Copying Well Contents into Another Well 117

Adding a Note to a Well 117

Clearing Wells of All Content 118

Changing Experiment Settings 119

Creating Well Groups 121

Changing Trace Styles 124

Viewing the Plate in Spreadsheet Format 125

Creating a Plate Layout Using the Plate Setup Wizard 127

Using the Plate Setup Wizard 127

Chapter 8 Running Experiments 131

Accessing the Run Setup Window 131

The Run Setup Window 132

Protocol Tab 133

Plate Tab 135

Start Run Tab 137

Running an Experiment 138

Run Details Dialog Box 140

Run Status Tab 140

Real-time Status Tab 142

Time Status Tab 144

Performing PrimePCR Experiments 145

Chapter 9 Data Analysis Overview 147

Data Analysis Window 147

Data Analysis Toolbar 148

Data Analysis Menu Bar 149

Tab Details 152

Step Number Selector 152

vi | CFX96 Dx and CFX96 Deep Well Dx Systems

Table of Contents

Viewing Well Groups in Data Analysis 153

Changing Well Contents after a Run 153

Data Analysis Settings 154

Adjusting the Threshold 154

Baseline Settings 154

Analysis Mode 155

Cycles to Analyze 156

Well Selector 157

Well Selector Right-Click Menu Items 158

Temporarily Excluding Wells from Analysis 159

Charts 160

Common Right-Click Menu Items for Charts 160

Copying Chart Data to the Clipboard 160

Modifying the Baseline Threshold Settings 161

Sorting Target and Sample Data 162

Magnifying an Area in the Chart 163

Copying Charts into a Microsoft File 163

Spreadsheets 164

Common Right-Click Menu Items for Spreadsheets 164

Export 166

Exporting All Data Sheets 166

Creating a Custom Export File 167

Exporting to a LIMS Folder 168

Exporting Seegene-Formatted Data 168

Chapter 10 Data Analysis Details 169

Quantification Tab 170

Fluorophore Options 170

Trace Styles Dialog Box 171

Log Scale Option 172

Standard Curve Chart 173

Amplification Chart Menu Options 173

Quantification Tab Spreadsheet 174

Quantification Data Tab 175

Results Spreadsheet 175

Operation Manual | vii

Table of Contents

Standard Curve Results Spreadsheet 177

Plate Spreadsheet 178

RFU Spreadsheet 178

Melt Curve Tab 179

Adjusting Melt Curve Data 181

Melt Curve Data Tab 182

Melt Peaks Spreadsheet 182

Plate Spreadsheet 183

RFU Spreadsheet 184

-d(RFU)/dT Spreadsheet 185

End Point Tab 186

Results Data 187

Adjusting the End Point Data Analysis 188

RFU Spreadsheet for End Point Analysis 188

Allelic Discrimination Tab 189

Adjusting Data for Allelic Discrimination 190

Chart Menu Options 191

Allelic Discrimination Spreadsheet 191

Custom Data View Tab 192

Creating a Custom Data View 193

QC Tab 194

Changing QC Criteria 194

Excluding Wells That Fail QC 194

Run Information Tab 195

Data Analysis Reports 196

Data Analysis Report Categories 197

Creating a Data Analysis Report 200

Creating Well Group Reports 201

Chapter 11 Gene Expression Analysis 203

Plate Setup for Gene Expression Analysis 203

Guided Plate Setup 204

Gene Expression Charts 205

Bar Chart 206

Sorting Target and Sample Data 208

viii | CFX96 Dx and CFX96 Deep Well Dx Systems

Table of Contents

Adjusting Gene Expression Data 208

Experiment Settings 210

Target Stability Value 212

Right-Click Menu Options 213

Data Spreadsheet 214

Show Details Option 214

Clustergram 217

Settings 217

Right-Click Menu Options 217

Data Spreadsheet 217

Scatter Plot 218

Settings 218

Right-Click Menu Options 218

Data Spreadsheet 218

Results 219

Gene Study 220

Inter-Run Calibration 220

Gene Study Dialog Box 220

Study Setup Tab 220

Preparing a Gene Study 221

Study Analysis Tab 222

Creating a Gene Study Report 223

Gene Study Report Categories 223

Appendix A Data Analysis Calculations 225

Reaction Efficiency 225

Relative Quantity 225

Relative Quantity When a Control Is Selected 226

Standard Deviation of Relative Quantity 226

Efficiency Corrected Cq (CqE) 227

Mean Efficiency Corrected Cq (MCqE) 227

Normalization Factor 227

Normalized Expression 228

Normalized Expression When a Control Is Selected 228

Standard Deviation for the Normalized Expression 229

Operation Manual | ix

Table of Contents

Normalized Expression Scaled to Highest Expression Level 230

Normalized Expression Scaled to Lowest Expression Level 230

Normalized Expression Scaled to Average Expression Level 230

Standard Deviation for the Scaled Normalized Expression 231

Regulation 231

Corrected Values Formulas 232

Appendix B Managing CFX Manager Dx Users and Roles 233

Managing Users 233

Adding and Removing Users 233

Managing the Rights of Roles 235

Logging In to CFX Manager Dx Software 236

Changing Users 236

Changing User Passwords 237

Viewing Your Role and Rights 237

Appendix C LIMS Integration 239

Creating LIMS-Compatible Data Files 239

Setting Up LIMS Folder and Data Export Options 239

Creating a LIMS Protocol 240

Creating a LIMS File 241

Starting a LIMS Run 244

Exporting Data to a LIMS 244

Appendix D Troubleshooting CFX Manager Dx Software Connection Issues 245

Application Log 245

Troubleshooting 246

Power Failure 246

Retrieving Files to the CFX Manager Dx Computer 248

Installing CFX Manager Dx Software Manually 248

Reinstalling the Drivers 248

Appendix E References 249

x | CFX96 Dx and CFX96 Deep Well Dx Systems

Safety and Regulatory Compliance

For safe operation of the CFX96™ Dx System or CFX96 Deep Well Dx System with CFX Manager™

Dx Software, referred to as the CFX Dx system in this document, Bio-Rad strongly recommends that

you follow the safety specifications listed in this section and throughout this manual.

Important: The CFX96 Dx and CFX96 Deep Well Dx systems are approved for use as invitro

diagnostic (IVD) medical devices.

Safety Warning Labels

Warning labels posted on the instrument and in this manual warn you about sources of injury or

harm. Table 1 defines each safety warning label.

Table 1. Meaning of safety warning labels

Icon Meaning

Warning about risk of harm to body or equipment

Operating the CFX Dx system before reading this manual can constitute a personal

injury hazard. For safe use, do not operate this instrument in any manner unspecified in

this manual. Only qualified laboratory personnel trained in the safe use of electrical

equipment should operate this instrument. Always handle all components of the system

with care and with clean, dry hands.

Warning about handling biohazardous materials

When handling biohazardous samples, adhere to the recommended precautions and

guidelines and comply with any local guidelines specific to your laboratory and location.

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.

Operation Manual | 11

Safety and Regulatory Compliance

Safe Use Specifications and Compliance

Table 2 lists the safe use specifications for Bio-Rad’s CFX Dx real-time PCR detection systems.

The supplied shielded cables must be used with these instruments to ensure compliance with the

Class A FCC limits.

Table 2. Conditions for safe use

Usage Aspect Conditions for Safe Use

Rated input power 100–240 VAC, 50–60 Hz, 850 W max

Overvoltage category II

Fuses 10 A, 250 V, 5 x 20 mm, fast blow (qty. 2)

Environment Indoor use only

Usage temperature 15–31°C

Storage temperature –20 to 60°C

Relative humidity Up to 80% (non-condensing)

Altitude Up to 2,000 meters above sea level

Pollution degree 2

Regulatory Compliance

The CFX Dx real-time PCRdetection system has been tested and found to be in compliance with

all applicable requirements of the following safety and electromagnetic standards:

n IEC 61010-1:2010 (3rd ed.), EN61010-1:2010 (3rd ed.). Electrical equipment for

measurement, control, and laboratory use - Part 1: General requirements

n IEC 61010-2-010:2014, EN 61010-2-010:2014. Safety requirements for electrical equipment

for measurement, control, and laboratory use. Part 2-010: Particular requirements for

laboratory equipment for the heating of materials

n IEC 61010-2-081:2015, EN 61010-2-081:2015. 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)

12 | CFX96 Dx and CFX96 Deep Well Dx Systems

n IEC 61010-2-101:2015 (2nd ed.). Safety requirements for electrical equipment for

measurement, control, and laboratory use. Particular requirements for in vitro diagnostic (IVD)

medical equipment

n IEC 61326-1:2012 (Class A), EN 61326-1:2013 (Class A). Electrical equipment for

measurement, control, and laboratory use. EMC requirements, Part 1: General requirements

n IEC 61326-2-6:2012, EN 61326-2-6:2013 (Class A). Electrical equipment for measurement,

control, and laboratory use. EMC requirements. Particular requirements for in vitro diagnostic

(IVD) medical equipment

Important: This equipment generates, uses, and can radiate radio frequency energy and, if

not installed and used in accordance with the provided instructional documentation, may

cause harmful interference to radio communications. Operation of the systems in a residential

area is likely to cause harmful interference, in which case users will be required to correct the

interference at their own expense.

Hazards

Hazards

The CFX Dx real-time PCR detection system is designed to operate safely when used in the

manner prescribed by the manufacturer. If the CFX Dx real-time PCR detection system or any of its

associated components is used in a manner not specified by the manufacturer, the inherent

protection provided by the instrument may be impaired. Bio-Rad Laboratories, Inc. is not liable for

any injury or damage caused by the use of this equipment in any unspecified manner, or by

modifications to the instrument not performed by Bio-Rad or an authorized agent. Service of the

CFX Dx real-time PCR detection system should be performed only by trained Bio-Rad personnel.

Biohazards

The CFX Dx real-time PCR detection system is a laboratory product. However, if biohazardous

samples are present, adhere to the following guidelines and comply with any local guidelines

specific to your laboratory and location.

Note: No biohazardous substances are exhausted during normal operations of this

instrument.

General Precautions

n Always wear laboratory coat, laboratory gloves, and safety glasses with side shields or

goggles.

n Keep your hands away from your mouth, nose, and eyes.

n Completely protect any cut or abrasion before working with potentially infectious materials.

Operation Manual | 13

Safety and Regulatory Compliance

n Wash your hands thoroughly with soap and water after working with any potentially infectious

material before leaving the laboratory.

n Remove wristwatches and jewelry before working at the bench.

n Store all infectious or potentially infectious material in unbreakable leak-proof containers.

n Before leaving the laboratory, remove protective clothing.

n Do not use a gloved hand to write, answer the telephone, turn on a light switch, or touch

anything that other people may touch without gloves.

n Change gloves frequently. Remove gloves immediately when they are visibly contaminated.

n Do not expose materials that cannot be properly decontaminated to potentially infectious

material.

n Upon completion of an operation involving biohazardous material, decontaminate the work

area with an appropriate disinfectant (for example, a 1:10 dilution of household bleach).

Specific IVD Precautions

n All patient samples are a potential biohazard and should be handled accordingly using

universal precautions.

n No biohazardous substances are exhausted during normal operation of this instrument.

Surface Decontamination

WARNING! To prevent electrical shock, always turn off and unplug the instrument

prior to performing decontamination procedures.

The following areas can be cleaned with any hospital-grade bactericide, virucide, or fungicide

disinfectant:

n Outer lid and chassis

n Inner reaction block surface and reaction block wells

n Control panel and display

To prepare and apply the disinfectant, refer to the instructions provided by the product

manufacturer. Always rinse the reaction block and reaction block wells several times with water

after applying a disinfectant. Thoroughly dry the reaction block and reaction block wells after

rinsing with water.

14 | CFX96 Dx and CFX96 Deep Well Dx Systems

Important: Do not use abrasive or corrosive detergents or strong alkaline solutions. These

agents can scratch surfaces and damage the reaction block, resulting in loss of precise

thermal control.

Disposal of Biohazardous Material

Dispose of the following potentially contaminated materials in accordance with laboratory local,

regional, and national regulations:

n Clinical samples

n Reagents

n Used reaction vessels or other consumables that may be contaminated

Chemical Hazards

The CFX Dx real-time PCRdetection system contains no potentially hazardous chemical materials.

Hazards

Explosive or Flammability Hazards

The CFX Dx real-time PCRdetection system poses no uncommon hazard related to flammability or

explosion when used in a proper manner as specified by Bio-Rad Laboratories.

Electrical Hazards

The CFX Dxreal-time PCR detection system poses no uncommon electrical hazard to operators if

installed and operated properly without physical modification and connected to a power source of

proper specification.

Transport

Before moving or shipping the CFX Dx real-time PCR detection system or its optical reaction

module or its thermal cycler base, decontamination procedures must be performed. Always move

or ship the CFX Dx real-time PCR detection system and optical reaction modules in separate

containers with the supplied packaging materials, which will protect the instrument from damage. If

appropriate containers cannot be found, contact your local Bio-Rad office.

Battery

The CFX Dx system thermal cycler uses a 3 V lithium-metal coin cell battery and a 4.8 V nickel-

metal hydride rechargeable battery pack to maintain time settings and run data in the event of AC

power loss. If the time and/or run data do not remain set after the unit is turned off, it may be an

Operation Manual | 15

Safety and Regulatory Compliance

indication that the batteries are getting weak. If this occurs, contact Bio-Rad Technical Support for

assistance.

Do not attempt to change the batteries. Contact Bio-Rad Technical Support.

Disposal

The CFX Dx real-time PCR detection system contains electrical materials; they should be disposed

of as unsorted waste and must be collected separately, according to European Union Directive

200296CE on waste and electronic equipment — WEEE Directive. Before disposal, contact your

local Bio-Rad representative for country-specific instructions.

Warranty

The CFX Dx real-time PCR detection system and its associated accessories are covered by a

standard Bio-Rad warranty. Contact your local Bio-Rad office for the details of the warranty.

16 | CFX96 Dx and CFX96 Deep Well Dx Systems

Chapter 1 Introduction

Bio-Rad’s CFX Dx real-time PCR amplification systems for in vitro diagnostics (IVD) feature the

latest technological advances, allowing for PCRquantification with standard curve, gene

expression analysis, allelic discrimination, and end-point analysis.

The CFX Dx systems are comprised of two hardware modules and software:

n CFX96™ Dx or CFX96 Deep Well Dx Optical Reaction Module (ORM)

n C1000™ Dx Thermal Cycler

n CFX Manager™ Dx Software

When used with CFX Manager Dx software you can

n Generate immediate results with the Startup Wizard

n Enter or edit well information before, during, or after a run

n Interpret complex data and make sense of your gene expression study with tools such as

PrimePCR™ controls analysis, and the reference gene selector tool

n Prepare comprehensive reports of your real-time PCR data

CFX Dx PCR Detection Systems

Table 3 lists Bio-Rad’s IVD PCR products that ship with CFX Dx systems.

Note: A CFX Dx system ships with the CFX Manager Dx Software, the C1000 Dx Thermal

Cycler, and either the CFX96 Dx or CFX96 Deep Well Dx Optical Reaction Module.

Table 3. CFX IVD PCR Detection Systems

Catalog # Description

1845097-IVD CFX96 Dx ORM *

1844095-IVD CFX96 Deep Well Dx ORM

1841000-IVD C1000 Dx Thermal Cycler

12007917 CFX Manager Dx Software v3.1

* Optical Reaction Module

Operation Manual | 17

Chapter 1 Introduction

Intended Use

CFX96 Dx System and CFX96 Deep Well Dx System with CFX Manager Dx Software are intended

to perform fluorescence-based PCR to detect and quantitate nucleic acid sequences. The systems

and software are intended for in vitro diagnostic use by trained laboratory technicians. The systems

are intended to be used with third party diagnostic nucleic acid tests which have been

manufactured and labeled for diagnostic purposes.

Finding Out More

This document explains how to safely set up and operate the CFX96 Dx and CFX96 Deep Well Dx

real-time PCR detection systems, which carry the CE-IVD mark. These systems are referred to as

the CFX Dx system in this document. In addition, this document explains how to use CFX Manager

Dx software with the CFX Dx system.

Tip: Click the Bio-Rad logo in the upper right corner of any CFX Manager Dx software window

to launch Bio-Rad’s website. This site includes links to technical notes, manuals, product

information, and technical support. This site also provides many technical resources on a wide

variety of methods and applications related to PCR, real-time PCR, and gene expression.

18 | CFX96 Dx and CFX96 Deep Well Dx Systems

Chapter 2 Setting Up the C1000 Dx Thermal

Cycler

This chapter explains how to set up the CFX Dx system's C1000 Dx thermal cycler at your site.

Tip: Before setting up the thermal cycler, familiarize yourself with the thermal cycler and its

optical reaction module, ports, and accessories.

Site Requirements

The tables in this section list the room, environment, and power requirements necessary to

successfully install and use the CFX Dx system thermal cycler.

Note: Install your CFX Dx system thermal cycler on a flat, dry surface with sufficient cool

airflow for it to run properly.

Bench Space Requirements

Table 4. CFX Dx system thermal cycler bench space requirements

Item Specification

Input power Up to 850 W, maximum

Frequency 50–60 Hz, single phase

USB ports 5 A, 1 B

Dimensions W: 13 in; 33 cm

D: 18 in; 46 cm

H: 14 in; 36 cm

Weight 47 lb; 21 kg

Operation Manual | 19

Chapter 2 Setting Up the C1000 Dx Ther mal Cycler

Environment Requirements

Table 5. CFX Dx system thermal cycler environment requirements

Parameter Range Humidity Range

Operating conditions 15–31°C

59–87.8° F

Storage conditions 15–31°C

59–87.8° F

0–80% RH, non-condensing

0–80% RH, non-condensing

Power Requirements

Power to the CFX Dx system thermal cycler must be stable and within specifications to ensure

proper operation. The power cable connected to the power inlet port must be rated for 7 amps or

more.

Table 6. CFX Dx system power requirements

Item Specification

Mains input voltage 100–240 VAC; 50–60 Hz, single phase

Maximum power usage <850 watts

Number of power sockets Aminimum of 2 power sockets:

n 1 socket for the thermal cycler

n 1 socket for the computer running CFX Manager Dx

software

20 | CFX96 Dx and CFX96 Deep Well Dx Systems

System Overview

The illustrations in this section display the main components of the C1000 Dx thermal cycler base.

Front View

System Overview

LEGEND

1.

2.

3.

4.

5.

6.

7.

Optical reaction module

thermal cycler block. The CFX Dx real-time PCR detection systems support either a CFX96™

Dx or CFX96 Deep Well Dx module.

Status LED

Lid button

— indicates when the block is in use.

— opens or closes the optical reaction module lid and seals the reaction chamber.

C1000™ Dx thermal cycler base

accommodates CFX96 Dx and CFX96 Deep Well optical reaction modules.

Front panel display and buttons

— includes an optical system to collect fluorescent data and a

— provides system power and communi cation, and

— allows control of the system in stand-alone mode.

Important: To ensure IVD gene study data integrity, the CFX Manager Dx software does

not support data generated by the thermal cycler in stand-alone mode.

Heated inner lid

Sample/reaction block

— maintains the lid temperature to prevent condensation and evaporation.

— holds reaction vessel, including tubes and microplates.

Operation Manual | 21

Chapter 2 Setting Up the C1000 Dx Ther mal Cycler

Back View

LEGEND

1.

2.

3.

4.

5.

6.

7.

8.

Ethernet port

USB Type B port

Manager Dx software.

USB Type A ports

— connects the C1000Dxthermal cycler to your network.

— connect the C1000Dxthermal cycler to a computer running CFX

— transfer data to and from a USB flash drive.

Important: To ensure IVD gene study data integrity, the CFX Manager Dx software does

not support data generated by the thermal cycler in stand-alone mode.

Serial test port

Cooling vents

— for service testing only.

— cools thermal cycler.

Important: Do not block the cooling vents. For optimal operation, ensure that air can

circulate behind the thermal cycler base.

Power input

Power switch

Fuses

specifications.

— AC mains power; use provided power cord.

— rocker switch to turn thermal cycler on and off.

— see

Safe Use Specifications and Compliance on page 12

for fuse

22 | CFX96 Dx and CFX96 Deep Well Dx Systems

Optical Reaction Modules

The C1000 Dx thermal cycler is compatible with the following Bio-Rad optical reaction modules for

real-time PCR.

n CFX96 Dx optical reaction module

n CFX96 Deep Well Dx optical reaction module

The chosen CFX Dx optical reaction module and the thermal cycler are shipped in separate boxes.

CFX Manager Dx software is shipped with the optical reaction module.

Important: The optical reaction module is calibrated with the thermal cycler base with which it

ships. Therefore, do not use the optical reaction module with any other thermal cycler base, or

the thermal cycler base with any other optical reaction module.

Both optical reaction modules include a fully adjustable heated lid that is capable of running

reliably with a broad range of reaction vessels. Each optical reaction module contains cooling fans

for fast heating and cooling.

OpticalReaction Modules

Each CFX Dx optical reaction module comprises the following components:

n Heated inner lid — maintains the lid temperature to prevent condensation and evaporation.

n Sample/reaction block — holds reaction vessels, including tubes and microplates.

n Lid button — opens and closes the lid and seals the reaction.

n Status LED — when on, indicates that the block is in use.

Recommended Sample Volumes

When using the C1000 Dx thermal cycler, the maximum sample volume is determined by the type

of reaction module used. Table 7 lists the recommended volumes to use with each reaction

module.

Table 7. Size and volume limit for reaction modules

Recommended Sample Volume, µl

Number of Wells Number of Blocks

96-well 1 10–50

96–deep well 1 10–125

(Upper Limit)

Operation Manual | 23

Chapter 2 Setting Up the C1000 Dx Ther mal Cycler

Installing the C1000 Dx Thermal Cycler

The C1000 Dx thermal cycler base ships in a separate box from the optical reaction module. The

package includes:

n C1000 Dx thermal cycler base

n Power cord

n 1 USB cable

To install the C1000 Dx thermal cycler:

1. Unpack and set up the C1000 Dx thermal cycler base.

2. Attach the reaction module to the base.

3. Remove the shipping screw.

This section explains these tasks in detail.

Unpacking and Setting Up the C1000 Dx Thermal Cycler

Important: Before operating the thermal cycler, read the information in Safety and Regulatory

Compliance on page 11 and Safety Warning Labels on page 11.

Tip: During setup, ensure that you have sufficient space near the thermal cycler for a computer

on which to run the CFX Manager Dx software.

To unpack and set up the thermal cycler base

1. Locate the package containing the thermal cycler base.

2. Remove the base from the packing material.

Tip: Store the packing material for future use. If any item is missing or damaged, contact
your local Bio-Rad office.

3. Place the thermal cycler base on a flat, dry surface with sufficient cool airflow to run properly.

4. Locate the power cord in the shipping package and insert one end into the power inlet port on
back of the thermal cycler.

Important: Do not power on the instrument at this time.

5. Attach the IVD reaction module to the base. Proceed to Attaching the Optical Reaction Module

on page 25.

24 | CFX96 Dx and CFX96 Deep Well Dx Systems

Installing the C1000 Dx T hermal Cycler

Attaching the Optical Reaction Module

Bio-Rad ships the CFX96 Dx or CFX96 Deep Well optical reaction module with its C1000 Dx

thermal cycler base (but in a separate box). Carefully unpack the optical reaction module and

verify that the power and USB cables are included in the shipping container.

Important: Each optical reaction module is calibrated with the thermal cycler base with which

it ships. Therefore, do not use the optical reaction module with any other thermal cycler base.

Ensure that the C1000 Dx thermal cycler base rests on a flat, dry surface with sufficient cool airflow

to run properly.

To attach the reaction module to the thermal cycler base

1. Place the C1000 Dx thermal cycler in a suitable location with the locking bar down.

2. Lifting the optical reaction module using the handle indents above the side air vents, position

the module in the C1000 Dx reaction module bay, leaving about 2cm of space in the front.

When in the bay, the optical module should be covering the Bio-Rad logo in front of bay.

3. Pull up the locking bar until it is flush with the sides of the module bay. This action moves the

module forward, locking it into place.

Operation Manual | 25

Chapter 2 Setting Up the C1000 Dx Ther mal Cycler

4. Check that the module is completely and evenly seated in the C1000 Dx thermal cycler base.

There should be no extra space between the module and the base.

5. Plug the power cord into the back of the C1000 Dx thermal cycler base and into an

appropriate electrical outlet, and then press the power switch on the back panel of the C1000

Dx thermal cycler to start the system.

Removing the Shipping Screw

Important: Bio-Rad’s optical reaction modules ship with a red shipping screw inserted in the

inner lid to stabilize the optical reaction module during shipping. You must remove the

shipping screw before you can operate the optical reaction module.

To remove the shipping screw

1. The C1000 Dx thermal cycler recognizes that the shipping screw is inserted in the optical

reaction module and displays a message instructing you to remove the screw.

26 | CFX96 Dx and CFX96 Deep Well Dx Systems

Installing the C1000 Dx T hermal Cycler

2. Follow the instructions to remove the shipping screw. The following diagram shows the

location of the shipping screw.

Note: You must reinsert the shipping screw should you need to return the reaction

module for any reason. Save the screw in a safe and accessible place.

Loading Sample Plates

To ensure uniform heating and cooling of samples, plates must be in complete contact with the

reaction block. To ensure adequate contact, do the following:

n Confirm that the block is clean before loading samples.

n Firmly press the individual tubes, tube strips, or microplates into the block wells.

n When using one or a few tubes, use the tube frame (catalog #1849000 or #1849001) or load

at least one empty tube in each corner of the block to ensure the lid exerts even pressure on

individual tubes.

Operation Manual | 27

Chapter 2 Setting Up the C1000 Dx Ther mal Cycler

Loading Plates into the Optical Reaction Module

Important: When running the CFX Dx system, always balance the tube strips or add tube caps

to the corner wells to ensure the heated lid applies even pressure across the block.

To load plates into the reaction module

1. To open the motorized lid, do one of the following:

n In the Detected Instruments pane in CFX Manager Dx software, click Open Lid.

n On the Start Run tab in the software, click Open Lid.

n Press the lid button on the front of the instrument.

2. Place the microplate, individual tubes, or tube strips with sealed lids in the block.

Important: Verify that the tubes are completely sealed to prevent leakage.

Tip: For optimal results, load sample volumes of 10–25 µl for the CFX Dx system.

3. For accurate data analysis, verify that the orientation of reactions in the block is exactly the

same as the orientation of the well contents in the Plate tab in CFX Manager Dx software.

Tip: You can edit the well contents using CFX Manager Dx software before, during, or

after the run.

4. To close the motorized lid, do one of the following:

n Press the lid button on the instrument.

n In the Detected Instruments pane in the software, click Close Lid.

n On the Start Run tab in the software, click Close Lid.

Important: Ensure that nothing blocks 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 lid before closing.

28 | CFX96 Dx and CFX96 Deep Well Dx Systems

Installing the C1000 Dx T hermal Cycler

PCR Plastics and Reagents Consumables

To find and order recommended plastic consumables for the CFX Dx system go to the Bio-Rad

web site. You can access this site from Help>PCRPlasticConsumablesWebSite menu item in

the CFX Manager Dx software. Additionally, refer to the Plastics Selector and the Reagents

Selector to help you easily find and order plastic consumables and reagents for your specific

hardware and PCR needs.

Detecting Connected Instruments

During installation, the CFX Manager Dx software installer automatically installs the instrument

drivers onto the computer running the CFX Manager Dx software. CFX Manager Dx detects

connected instruments when you start the software.

Important: You must disconnect the C1000 Dx thermal cycler from the CFX Manager Dx

computer before you install the software. You do not need to turn off the thermal cycler during

the software installation.

To detect connected instruments

1. If you have not yet done so, insert the square (male) end of the supplied USB Type B cable

into the USBType B port located on the back of the base.

2. Insert the other (port) end into a USB port on the CFX Manager Dx computer.

3. If the thermal cycler is not already running, press the power switch on the back of the

instrument to turn it on.

4. Start CFX Manager Dx software.

The software automatically detects the connected instrument and displays its name in the Detected

Instruments pane in the Home window.

Note: If the instrument does not appear in the Detected Instruments pane, verify that the USB

cable is properly installed. To reinstall drivers, select Tools > Reinstall Instrument Drivers in

the Home window in CFX Manager Dx software.

Operation Manual | 29

Chapter 2 Setting Up the C1000 Dx Ther mal Cycler

Detaching the Reaction Module

Important: Power off the C1000 Dx thermal cycler before detaching a reaction module (see

Shutting Down the C1000 Dx Thermal Cycler on page 30). Cooling fins within the reaction module

might be hot immediately after running a protocol or incubation. Ensure that the fins are cool

before detaching the reaction module.

To detach the optical reaction module from the thermal cycler base

1. On the back of the thermal cycler base, push the locking bar down to unlock and release the

optical reaction module.

2. Carefully lift the optical reaction module out of the bay using the handle indents on each side.

3. Set the optical reaction module on a clean, flat surface where it cannot get bumped, scraped,

or dropped.

Shutting Down the C1000 Dx Thermal Cycler

To shut down the thermal cycler

1. After a run, press the open lid button on the front of the CFX optical reaction module to access

the samples loaded in the block.

2. Remove the samples from the block and press the close lid button to close the lid.

3. Press the power switch on the back panel of the C1000 Dx thermal cycler to power down the

system.

30 | CFX96 Dx and CFX96 Deep Well Dx Systems

Chapter 3 Installing CFX Manager Dx Software

This chapter explains how to install the CFX Manager™ Dx software.

CFX Manager Dx software is required to analyze real-time PCR data from the CFX96™ Dx and

CFX96 Deep Well Dx systems. You can also use this software to control these systems in software-

controlled mode.

For information about installing the CFX Dx system thermal cycler and optical reaction module, see

Setting Up the C1000 Dx Thermal Cycler on page 19.

Operation Manual | 31

Chapter 3 Installing CFX Manager Dx Software

System Requirements

Table 8 lists the minimum and recommended system requirements for the computer running the

CFX Manager Dx software (known as the CFX Manager Dx computer).

Table 8. Computer requirements for CFX Manager Dx software

System Minimum Recommended

Operating system Microsoft Windows 7 SP1

Pro

Any of the following:

n Microsoft Windows 7 SP2 Pro (32- and 64-bit)

n Microsoft Windows 10 Pro (64-bit only)

n Microsoft Windows 10 Enterprise (64-bit only)

Important: Secure Boot must be disabled on both Microsoft Windows 10 Pro and Enterprise.

Ports 2 USB 2.0 High-speed ports 2 USB 2.0 High-speed ports

Hard disk space 128 GB 128 GB

Processor speed 2.4 GHz, Dual Core 2.4 GHz, Quad Core

RAM 4 GB RAM 8 GB RAM

Screen resolution 1024 x 768 with true-color

mode

PDF reader Adobe PDF Reader or Windows PDF Reader

1280 x 1024 with true-color mode

from one of the supported Microsoft Office

Suites:

n 2007

n 2010

n 2013

32 | CFX96 Dx and CFX96 Deep Well Dx Systems

Installing CFX Manager Dx Software

Important: You must disconnect any connected instruments from the CFX Manager Dx

computer before you install or upgrade the software. You do not need to turn off the thermal

cycler during the software installation. Ensure that you have saved all runs and that no

experiments are running.

Note: If you are installing CFX Manager Dx software on Windows 10, verify that Secure Boot is

disabled before beginning the installation procedure.

To install CFX Manager Dx software

1. If necessary, disconnect any connected instruments from the computer.

Locate and disconnect the instrument’s USB cable on the CFX Manager Dx computer. The

end inserted in the instrument can remain in place.

2. Log in to the CFX Manager Dx computer with administrative privileges.

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

Installing CFX Manager Dx Softwar e

4. The software launch page should appear automatically. Double-click Install Software on the

software launch page.

Note: If the launch page does not appear automatically, navigate to the CD drive and

open the CFX_Manager folder and then double-click setup.exe to start the software

installation wizard.

Tip: In the installation wizard, click the Documentation button to find searchable copies of

the release notes, instrument manuals, and other documentation.

5. Follow the on-screen instructions to complete installation. When completed, the CFX manager

software icon will appear on the desktop of the computer.

6. After the installation completes, you can safely eject the CD.

Detecting Connected Instruments

During installation, the CFX Manager Dx software installer automatically installs the instrument

drivers onto the CFX Manager Dx computer. CFX Manager Dx detects connected instruments

when you start the software.

To detect connected instruments

1. If you have not yet done so, insert the square (male) end of the supplied USB Type B cable

into the USBType B port located on the back of the instrument’s base.

2. Insert the other (port) end into a USB port on the CFX Manager Dx computer.

Operation Manual | 33

Chapter 3 Installing CFX Manager Dx Software

3. If the instrument is not already running, press the power switch on the back of the instrument to

turn it on.

4. Start CFX Manager Dx.

The software automatically detects the connected instrument and displays its name in the Detected

Instruments pane on the Home window.

Note: If the instrument does not appear in the Detected Instruments pane, verify that the USB

cable is properly installed. To reinstall drivers, on the Home window in CFX Manager Dx

select Tools>Reinstall Instrument Drivers.

Software Files

Table 9 lists the CFX Manager Dx software file types.

Table 9. CFX Manager Dx software file types

File Type Extension Details

Protocol .prcl Contains protocol setup details to perform a PCR run.

Plate .pltd Contains plate setup details to perform a PCR run.

Data .pcrd Contains the results of an experiment run and PCR

analysis.

PrimePCR™ run .csv Contains protocol and plate layout for PrimePCR plates.

Gene study .mgxd Contains results of multiple PCR runs and gene

expression analyses.

LIMS .plrn Contains plate setup and protocol information required to

conduct a LIMS compatible run.

Recommended Cybersecurity Measures

Bio-Rad recommends you work with your IT department to implementcybersecurity measures for
the computer used with theCFX96 Dx system. For example:

n Install and configureappropriate virus protectionand firewall applications.

Important: Configure the virus scan to occur during off-hours or when the instrument is

not actively running. If a virus scan is initiated while CFX Manager Dx is running an

experiment, the run may be canceled and the data lost.

34 | CFX96 Dx and CFX96 Deep Well Dx Systems

Recommended CybersecurityMeasures

n CFX Manager Dx software does not have a user session inactivity timeout feature. Implement

Windows or third-parity user access security measures (for example, implement a screen

saver with log in required).

n Removable media security:

o Use passwords and encryption on your USB device to protect data.

o Disable the autorun and autoplay features for all removable media devices.

o Enforce USB scanning whenever a thumb drive is plugged in.

n Employ a backup utility to facilitate data recovery.

Operation Manual | 35

Chapter 3 Installing CFX Manager Dx Software

36 | CFX96 Dx and CFX96 Deep Well Dx Systems

Chapter 4 The Workspace

CFX Manager™ Dx software provides an interface for setting up plates, developing PCR protocols,

running them on CFX Dx instruments, and analyzing data from PCR runs.

CFX Manager Dx software presents five primary workspaces:

n The Home window

n The Startup Wizard

n The Protocol Editor window

n The Plate Editor window

n The Data Analysis window

Each workspace is shown and briefly described in this chapter.

Operation Manual | 37

Chapter 4 The Workspace

The Home Window

CFX Manager Dx software opens to the Home window and displays the Startup Wizard, from which

you can set up an experiment, perform or repeat a run, or analyze an existing run. From the Home

window you can also view application and instrument logs, create and manage users, and access

multiple useful tools. For more information, see Chapter 5, The Home Window.

38 | CFX96 Dx and CFX96 Deep Well Dx Systems

The Startup Wizard

Use the Startup Wizard to quickly set up and run user-defined experiments or select and run a

PrimePCR™ experiment. You can also use this wizard to repeat a run or analyze run data.

The Startup Wizard

Operation Manual | 39

Chapter 4 The Workspace

The Protocol Editor Window

In the Protocol Editor you can create, open, review, and edit a protocol. You can also modify the lid

temperature for the open protocol. Protocol Editor functionality is detailed in Chapter 6, Creating

Protocols.

40 | CFX96 Dx and CFX96 Deep Well Dx Systems

The Plate Editor Window

In the Plate Editor you can create, open, review, and edit a plate. Plate Editor functionality is

detailed in Chapter 7, Preparing Plates.

The Plate Editor Window

Operation Manual | 41

Chapter 4 The Workspace

The Data Analysis Window

In the Data Analysis window you can view and compare run data, perform statistical analyses,

export data, and create publication-ready reports. Data Analysis functionality is detailed in Chapter

9, Data Analysis Overview. See also Chapter 10, Data Analysis Details.

42 | CFX96 Dx and CFX96 Deep Well Dx Systems

Chapter 5 The Home Window

CFX Manager™ Dx software provides an interface for developing PCR protocols, running them on

CFX Dx systems, and analyzing PCR run data.

This chapter introduces the CFX Manager Dx software and describes the features accessible from

the Home window.

Operation Manual | 43

Chapter 5 The Home Window

The Home Window

CFX Manager Dx opens to the Home window and displays the Startup Wizard, from which you can

set up a run, perform or repeat a run, or analyze an existing run. From the Home window you can

also view application and instrument logs, create and manage users, and access multiple useful

tools.

LEGEND

1. The software title bar displays the name of the software and the logged in user.

2. The menu bar provides quick access to File, View, Users, Run, Tools, Window, and Help menu

commands.

3. Toolbar commands provide quick access to menu options.

4. The left pane displays the instruments connected to the CFX Manager Dx computer and

provides buttons from which you can operate the lid and view the status of the instruments.

5. The main pane displays the working window. The default working window on the Home screen

is the Startup Wizard.

6. The status bar displays names of the connected instruments and the logged in user.

44 | CFX96 Dx and CFX96 Deep Well Dx Systems

The Home Window

File Menu Commands

New — opens a dialog box from which you can choose to create a new protocol, plate, or gene study.

Open — opens a dialog box from which you can choose to navigate to and open an existing

protocol, plate, data file, gene study, LIMS file, or PrimePCR™ run file.

Recent Data Files — displays a list of recently opened PCR files.

Repeat a Run — opens Windows Explorer to the location of saved PCR files, in which you can

locate a run to repeat.

Exit — closes CFX Manager Dx.

View Menu Commands

Application Log — displays a software usage log from initial installation to the current day.

Run Reports — displays a list of run reports.

Startup Wizard — displays the Startup Wizard in the main pane.

Run Setup — displays the Run Setup window in the main pane.

Instrument Summary — displays the Instrument Summary window in the main pane.

Detected Instruments — toggles between displaying and not displaying connected instruments in

the left pane. By default, the software displays connected instruments in the left pane.

Toolbar — toggles between displaying and not displaying the toolbar on the top of the screen. By

default, the software displays the toolbar.

Status Bar — toggles between displaying and not displaying the status bar on the bottom of the

screen. By default, the software displays the status bar.

Show — opens a dialog box from which you can

n View or block the Status log.

n Open and view the CFX Manager Dx data folder.

n Open and view the user’s data folder.

n Open and view the LIMS file folder.

n Open and view the PrimePCR folder.

n View the run history.

n View the properties of all connected instruments.

Operation Manual | 45

Chapter 5 The Home Window

User Menu Commands

Select User — opens the Login screen on which you can select a user from the User Name

dropdown list and log in to the application.

Change Password — opens the Change Password dialog box, in which users can change their

CFX Manager Dx software password.

User Preferences — opens the User Preferences dialog box, in which users can change the

default settings for

n Sending and receiving email notification upon run completion

n Saving data files

n Creating protocols via the Protocol Editor or the Protocol AutoWriter

n Creating plates

n Analyzing data

n Performing gene expression analysis

n Determining the quality of the data

n Exporting CFX Dx instrument data

User Administration — opens the User Administration dialog box, in which administrators can

create users, modify role permissions, and assign roles to users.

Bio-Rad Service Login — for Bio-Rad technical service staff use only. Do not select this command.

Run Menu Commands

User-defined Run — opens the Run Setup window, in which you can set up a user-defined

protocol and plate, and then run a PCR experiment on selected instruments.

PrimePCR Run — opens the Start Run tab in the Run Setup window with the default PrimePCR

protocol and plate layout loaded based on the selected instrument.

End-Point Only Run — opens the Start Run tab in the Run Setup window with the default end-

point protocol and plate layout loaded based on the selected instrument.

Qualification Run — opens the Start Run tab in the Run Setup window with the default Bio-Rad

qualification protocol and plate layout loaded for the selected instrument.

46 | CFX96 Dx and CFX96 Deep Well Dx Systems

The Home Window

Tools Menu Commands

Master Mix Calculator — opens the Master Mix Calculator, in which you can create a reaction

mixture and print the calculations.

Protocol AutoWriter — opens the Protocol AutoWriter dialog box, in which you can easily create a

new protocol.

TaCalculator — opens the TaCalculator, in which you can easily calculate the annealing

temperature of primers.

Dye Calibration Wizard — opens the Dye Calibration wizard, in which you can calibrate an

instrument for a new fluorophore.

Reinstall Instrument Drivers — reinstalls the drivers that control communication with Bio-Rad’s

real-time PCR systems.

Zip Data and Log Files — opens a dialog box in which you can select files to condense and save

in a zipped file for storage or to email.

Batch Analysis — opens the Batch Analysis dialog box, in which you can set parameters for

analyzing more than one data file at a time.

Options — opens a dialog box in which you can

n Configure your email server settings.

n Configure export settings for LIMS and other data files.

Help Menu Commands

Tip: The Help menu is available on the menu bar in all CFX Manager Dx software windows.

Open Operation Manual — opens a PDF of this manual.

Gene Expression Gateway Web Site — opens the Bio-Rad home page for the CFX Dx system.

PCR Reagents Web Site — opens Bio-Rad’s PCR reagents web site, from which you can order

PCR reagents, supermixes, dyes, and kits.

PCR Plastic Consumables Web Site — opens Bio-Rad’s PCR Plastics and Consumables web

site, from which you can order PCR plates, plate seals, tubes and caps, and other plastics

accessories.

Software Web Site — opens Bio-Rad’s PCR Analysis Software web site, from which you can order

updated versions of Bio-Rad’s CFX Manager Dx software.

About — displays CFX Manager Dx copyright and version information.

Operation Manual | 47

Chapter 5 The Home Window

Toolbar Commands

— opens Windows Explorer, in which you can navigate to and open a data file or a gene

study file.

— opens the Master Mix Calculator.

— opens the Run Setup window.

— opens the Run Setup window with the default PrimePCR protocol and plate layout loaded

based on the selected instrument.

— opens the Startup Wizard.

48 | CFX96 Dx and CFX96 Deep Well Dx Systems

The Home Window

The Startup Wizard

When CFX Manager Dx starts, the working pane displays the Startup Wizard. From the Startup

Wizard you can

n Select an instrument from the detected instruments and set up a user-defined or PrimePCR

run.

n Open and repeat a run.

n Open a data file to analyze results from a single run or a gene study file for results from

multiple gene expression runs.

These tasks are explained in detail in the chapters that follow.

Status Bar

The left side of the status bar at the bottom of the main software window displays the current status

of the detected instruments. The right side of the status bar displays name of the current user and

the date and time.

Operation Manual | 49

Chapter 5 The Home Window

Detected Instruments Pane

The Detected Instruments pane displays each instrument that is connected to the CFX Manager Dx

computer. By default, each instrument appears as an icon, and its serial number appears as its name.

For example, the following image displays four detected instruments:

n Two C1000™ thermal cyclers with CFX96™ Deep Well reaction modules (CFX96DW1 and

CFX96DW2)

n Two C1000™ thermal cyclers with CFX96™ reaction modules (CFX96l01 and CFX96l02)

From this pane you can do the following:

n View the properties and calibrated dyes for a selected instrument.

For information about instrument properties, see Viewing the Properties of an Instrument on

page 53.

n View the status of a connected instrument.

n Open the motorized lid on the selected instrument.

n Close the motorized lid on the selected instrument.

n View the status of all connected instruments.

To view the status of a connected instrument

u In the Detected Instruments pane, select the target instrument and do one of the following:

n Click View Status in the Selected Instrument section.

n Right-click and select View Status on the menu that appears.

50 | CFX96 Dx and CFX96 Deep Well Dx Systems

The Run Details dialog box appears displaying the Run Status tab. The status of the selected

instrument appears below the run status pane, for example:

To open or close the lid of an instrument

u In the Detected Instruments pane, select target instrument and do one of the following:

n Click Open Lid or Close Lid in the Selected Instrument section.

The Home Window

n Right-click and select the appropriate action on the menu that appears.

n Open the Run Details dialog box, select Run Status tab, and click Open Lid or Close Lid.

To view the status of all detected instruments

u Do one of the following:

n In the All Instruments section in the Detected Instruments pane, click View Summary.

n On the menu bar, select View > Instrument Summary.

The Instrument Summary dialog box appears:

Tip: If the system detects only one connected instrument, the All Instruments section does

not appear in the Detected Instruments pane. To view the instrument summary for a single

instrument, select View > Instrument Summary.

Operation Manual | 51

Chapter 5 The Home Window

Instrument Summary Toolbar Controls

Table 10 lists the controls and functions in the Instrument Summary toolbar.

Table 10. Instrument Summary Toolbar Controls

Button Button Name Function

Create a new Run Creates a run on the selected block by

opening the Run Setup window.

Stop Stops the current run on selected blocks.

Pause Pauses the current run on selected blocks.

Resume Resumes the run on selected blocks.

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

selected blocks.

Open Lid Opens the selected block’s motorized l id.

Close Lid Closes the selected block’s 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 blocks

that are running with the current user, or all

running blocks

52 | CFX96 Dx and CFX96 Deep Well Dx Systems

The Home Window

Viewing the Properties of an Instrument

From the Detected Instruments pane you can view details about a selected instrument, including its

properties, the status of its shipping screw, and a list of its calibrated dyes (fluorophores).

To view instrument properties

u In the Detected Instruments pane, right-click the target instrument and select Properties on the

menu that appears.

Properties Tab

The Properties tab lists technical details about the selected instrument including the model, serial

numbers of its components, and firmware versions. The default name of the instrument (its serial

number) appears in many locations, including the Detected Instruments pane and in the header

bar of the Instrument Properties dialog box. You can rename the instrument in order to more easily

identify it.

To rename an instrument

u In the Instrument Properties tab, type a name in the Rename box at the top of the Properties

tab and click Rename.

The new name appears in the Nickname row in the Properties tab as well as in the Instrument

Properties header bar and the Detected Instruments pane.

Operation Manual | 53

Chapter 5 The Home Window

Shipping Screw Tab

The Shipping Screw tab displays the current status of the shipping screw for the selected

instrument (Removed or Installed). The tab also includes instructions for installing or removing the

red shipping screw.

Tip: If the software detects the shipping screw, the Instrument Properties dialog box

automatically displays the Shipping Screw tab. Follow the instructions to remove the screw.

Note: You must remove the shipping screw before you can use the instrument. For more

information, see Removing the Shipping Screw on page 26.

Calibrated Dyes Tab

The Calibrated Dyes tab displays the calibrated fluorophores and plates for the selected

instrument.

To see detailed information about a calibration, click its Info button in the Detail column.

54 | CFX96 Dx and CFX96 Deep Well Dx Systems

Before You Begin

Setting User Preferences

Tip: It is not required to perform these tasks in order to use CFX Manager Dx software. You

can safely skip this section or perform these tasks at any time.

In CFX Manager Dx you can customize your working environment. If your administrator created

software users, each user can customize his or her working environment. If your administrator did

not create users, preference changes apply to everyone who logs in to CFX Manager Dx. (For

information about creating CFX Manager Dx users, see Appendix B, Managing CFX Manager Dx

Users and Roles.)

For example, in the Users > User Preferences menu, you can do the following:

n Set up email notification of run completion.

n Change the default settings for

Before You Begin

o The location in which to save files

o The run setup files

o The file naming prefix

n Set the default parameters to use when creating a new protocol and plate.

n Set the default data analysis and gene expression parameters.

n Customize the default quality control parameters.

n Customize data export data parameters.

In the Tools menu, you can do the following:

n Create a master mix.

n Calibrate dyes for a specific instrument.

Note: The master mix and dye calibration are available to anyone who logs in to CFX

Manager Dx.

This section explains how to perform these tasks in detail.

Setting Up Email Notification

You can connect CFX Manager Dx to your outgoing email server to send email notification of run

completion to a list of users. You can also choose to attach a data file and an analysis report to the

Operation Manual | 55

Chapter 5 The Home Window

list of users. To set up the connection between CFX Manager Dx and your SMTP server, see

Connecting CFX Manager Dx to an SMTP Server on page 57.

Note: A user's ability to access email setup features depends on the user group and

permissions assigned by the administrator. For details on managing users and their roles, see

Managing Users on page 233.

To set up email notifications

1. Select Users > User Preferences to open the User Preferences dialog box.

The User Preferences dialog appears displaying the Email tab.

Note: You are informed if the system detects that you have not set up a valid SMTP server

for CFX Manager Dx. Click Configure Outgoing Email to open the Options dialog box and

configure the email SMTP server. For more information, see Connecting CFX Manager Dx

to an SMTP Server on page 57.

2. In the To text box, type the email address of each person who you plan to inform of run

completion. All recipients will receive email after the run completes.

Note: You must enter each email address on a separate line. Press Enter or Return after

each address.

3. (Optional) In the cc text box, type the email address of any recipient to whom you plan to send

a copy of each email notification.

4. (Optional) By default, all recipients receive a copy of the data file as an attachment. Clear this

checkbox if you do not want to attach a copy the data file.

5. (Optional) Select Attach Analysis Report to attach a PDF of the analysis report to the email.

6. Click OK to save the changes and close the User Preferences dialog box.

56 | CFX96 Dx and CFX96 Deep Well Dx Systems

Before You Begin

To edit a recipient’s email address

u Modify the email address as necessary and click OK.

To remove an email recipient

1. Select the email recipient and press the Delete key.

2. Click OK to save the changes and close the dialog box.

Important: Clicking Restore Defaults in the User Preferences dialog box resets all

preferences on all tabs to the original factory settings. Take care when clicking this button.

Connecting CFX Manager Dx to an SMTP Server

Important: Some commercial webmail service providers (such as Yahoo! and Gmail) have

increased email security. If you use these accounts, you must enable the setting Allow less

secure apps in their account settings to enable CFX Manager Dx to send email. See the

security information for your webmail service provider for more information.

You must establish a connection from CFX Manager Dx to your email server before the software

can send email notification.

To connect CFX Manager Dx to an email server

1. Do one of the following:

n Select User > User Preferences and click Configure Outgoing Email on the Email tab.

n Select Tools > Options.

The Options dialog box appears displaying the Email tab.

Operation Manual | 57

Chapter 5 The Home Window

2. Provide the following information for your company:

n SMTP Server Name — the name of the outgoing email server at your company.

n Port — the port number of your SMTP server. This is usually 25.

n Use SSL — Secure Sockets Layer (SSL) option. Some SMTP servers require this setting.

If it is not required at your company, clear this checkbox.

n Use Default “From” Address — the name of the email server at your company. Some

SMTP servers require all sent email to have a “from” address that is from a certain
domain, for example, [e-mail]. If that is the case, clear this checkbox
and provide a valid email address.

n Authentication Required — if your site requires account authentication, verify that this

checkbox is selected.

n User Name — the name of the authenticated account. This is required only if

Authentication Required is selected.

n Password — the password for the authenticated account. This is required only if

Authentication Required is selected.

3. To verify that the SMTP server settings are correct, enter a valid email address in the Test

Email Address text box and click Test Email.

Note: Some SMTP servers do not allow attachments and others allow attachments only

up to a specific size. If you plan to email data files and/or reports using CFX Manager Dx,

select Test Attachment and set Attachment Size in MB to 5 megabytes (MB) or more.

4. Click OK to save the changes and close the dialog box.

Changing the Default File Settings

In the Files tab on the User Preference dialog box, you can change the following:

n The default location in which to save CFX Manager Dx files

n The default files for run setup

n The default file naming parameters

To change the default file settings

1. Select Users > User Preferences to open the User Preferences dialog box.

2. In the User Preferences dialog box, select the Files tab.

58 | CFX96 Dx and CFX96 Deep Well Dx Systems

Before You Begin

3. In the Default Folder for File Creation section, navigate to and select a default folder in which

you want to save new files. You can select a different location for each file type:

n Protocol

n Plate

n Data File

n Gene Study

4. In the File Selection for Run Setup section, navigate to and select the target protocol and plate

files to appear when you open the Experiment Setup window.

5. In the Data File section, define the prefix and/or suffix for data files. For any part, select a new

value from its dropdown list. You can also provide custom prefix and suffix values in the Prefix

and Suffix text boxes.

CFX Manager Dx displays a preview of the file name below the selection boxes.

6. Click OK to save the changes and close the dialog box.

Important: Clicking Restore Defaults in the User Preferences dialog box resets all

preferences on all tabs to the original factory settings. Take care when clicking this button.

Operation Manual | 59

Chapter 5 The Home Window

Setting the Default Protocol Parameters

To set default protocol parameters for the Protocol Editor and Protocol AutoWriter

1. Select Users > User Preferences to open the User Preferences dialog box.

2. In the User Preferences dialog box, select the Protocol tab.

3. In the Protocol Editor section, specify values for the following settings that appear in the

Protocol Editor:

n Sample volume — the volume of each sample in the wells (in µl).

n Lid Shutoff temperature — the temperature in ºC at which the lid heater turns off during a

run.

4. In the Protocol AutoWriter section, specify values for the following settings that appear in the

Protocol AutoWriter:

n Annealing temperature — the temperature in ºC for experiments that use iProof™ DNA

polymerase, iTaq™ DNA polymerase, or other polymerases.

n Amplicon length — the length of the amplicon in bp.

5. Click OK to save the changes and close the dialog box.

Important: Clicking Restore Defaults in the User Preferences dialog box resets all

preferences on all tabs to the original factory settings. Take care when clicking this button.

60 | CFX96 Dx and CFX96 Deep Well Dx Systems

Before You Begin

Setting Default Plate Parameters

Changes that you make to the Plate tab are available to all users of the software. Changes that you

make during plate setup are available to users after you save and close the plate file.

In the User Preferences dialog box you can do the following:

n Set default plate parameters.

n Add new target and sample names to their respective libraries.

n Delete target and sample names from their respective libraries.

To set the default plate parameters

1. Select Users > User Preferences to open the User Preferences dialog box.

2. In the User Preferences dialog box, select the Plate tab.

3. Specify values for the followings settings for a new plate file. These values appear in the Plate

Editor window:

n Plate type

n Plate size

n Units — the concentration of the starting template for wells that contain standards.

CFX Manager Dx uses these units to create a standard curve in the Data Analysis

Quantification tab.

Operation Manual | 61

Chapter 5 The Home Window

n Scientific notation — when selected, CFX Manager Dx displays the concentration units in

scientific notation.

n Scan mode — the number or type of channels to scan during a run.

n Fluorophores — the default fluorophores that appear in the Plate Editor well loading

controls.

n Libraries — the target and sample names that you typically use in your experiments:

o Target names — the names of target genes and sequences.

o Sample names — the names of experiment samples or an identifying characteristic

for the samples (for example, Mouse1, Mouse2, Mouse3).

4. Click OK to save the changes and close the dialog box.

To add a new target or sample name

u In the appropriate library box, type the name for the target or sample and click OK.

To delete a target or sample name

u In the appropriate library box, select the name and press the Delete key and then click OK.

Important: Names that you remove from the library are removed from the software and are no

longer available to users. To restore the default CFX Manager Dx names, click Restore

Defaults. Clicking Restore Defaults in the User Preferences dialog box resets all preferences

on all tabs to the original factory settings. Take care when deleting default CFX Manager Dx

names and when clicking this button.

62 | CFX96 Dx and CFX96 Deep Well Dx Systems

Setting Default Data Analysis Parameters

To set default Data Analysis parameters

1. Select Users > User Preferences to open the User Preferences dialog box.

2. In the User Preferences dialog box, select the Data Analysis tab.

Before You Begin

3. In the Analysis Mode section, select the mode in which to analyze the data (either Fluorophore

or Target).

4. In the PCR Quantitation section, set default parameters for the following options:

n Baseline Setting — the baseline method for analysis mode.

n Cq Determination Mode — the mode in which Cqvalues are calculated for each

fluorescence trace (either regression or single threshold).

n Threshold Calc. Mode — the end-point target amount.

The default is Auto. That is, the software automatically calculates the end-point target. To

set a specific threshold, clear the Auto checkbox and enter your end-point amount,

calculated in relative fluorescence units (or RFU). The maximum value is 65000.00 RFUs.

Data files for subsequent runs will use this threshold setting.

Operation Manual | 63

Chapter 5 The Home Window

n Baseline Calc. Mode — the baseline value for all traces.

The default is Auto. That is, the software automatically calculates the baseline for all

traces. To set a specific baseline value, clear the Auto checkbox and enter minimum and

maximum values for the cycle range (1 to 9999). Data files for subsequent runs will use

this cycle range.

n Log View — determines how the software displays the amplification data:

o On — the amplification data are displayed in a semilogarithmic graph.

o Off — (the default) the amplification data are displayed in a linear graph.

5. In the End Point section, select the number of end cycles to average when calculating the end-

point calculations:

n PCR — the number of end cycles to average for quantification data (default is 5).

n End Point Only run — the number of end cycles to average for end-point data (default is

2).

6. In the Melt Curve section, select the peak type to detect (either positive or negative).

7. In the Well Selector section, select how to display well labels (by sample type, target name, or

sample name).

8. Click OK to save the changes and close the dialog box.

Important: Clicking Restore Defaults in the User Preferences dialog box resets all

preferences on all tabs to the original factory settings. Take care when clicking this button.

64 | CFX96 Dx and CFX96 Deep Well Dx Systems

Setting Default Gene Expression Data File Parameters

To set the default parameters for a new gene expression data file

1. Select Users > User Preferences to open the User Preferences dialog box.

2. In the User Preferences dialog box, select the Gene Expression tab.

Before You Begin

3. Specify the values for the following settings:

n Relative to — graphs the gene expression data relative either to a control (originating at

1) or to zero:

o Zero — the software ignores the control. This is the default when no control sample is

assigned in the Experiment Settings window.

o Control — the software calculates the data relative to the control sample assigned in

the Experiment Setup window.

n X-axis — graphs the sample or the target on the x-axis.

n Y-axis — graphs linear, log2, or log10 scale on the y-axis.

n Scaling — the scaling option for the graph (the default option is unscaled):

o Highest — the software scales the graph to the highest data point.

Operation Manual | 65

Chapter 5 The Home Window

o Lowest — the software scales the graph to the lowest data point.

o Unscaled — the software presents the data unscaled in the graph.

n Mode — the analysis mode, either relative quantity (∆C

(∆∆C

).

q

) or normalized expression

q

n Error Bar — the data variability presented as either the standard deviation (Std. Dev.) or

the standard error of the mean (Std. Error Mean).

n Error Bar Multiplier — the standard deviation multiplier used to graph the error bars

(defaultis1).

You can increase the multiplier to either 2 or 3.

n Sample Types to Exclude — the sample types to exclude from the analysis.

You can select one or more sample to exclude from the analysis. To exclude all sample

types, clear the checkboxes of any selected sample types.

4. Click OK to save the changes and close the dialog box.

Important: Clicking Restore Defaults in the User Preferences dialog box resets all

preferences on all tabs to the original factory settings. Take care when clicking this button.

Customizing Quality Control Rules

In CFX Manager Dx, you can set quality control rules, which are applied to data in the Data

Analysis window. The software validates the data against the rules that you set.

Note: By default, all quality control rules are enabled.

Tip: You can easily exclude wells that fail a QC parameter from analysis in the QC module of

the Data Analysis window.

To customize quality control rules

1. Select Users > User Preferences to open the User Preferences dialog box.

2. In the User Preferences dialog box, select the QC tab.

66 | CFX96 Dx and CFX96 Deep Well Dx Systems

where:

n NTC — no template control

Before You Begin

n NRT — no reverse transcriptase control

n Efficiency — reaction efficiency

n Std Curve R^2 — R square value for the standard curve

n Replicate group Cq Std Dev — standard deviation calculated for each replicate group

3. For each QC rule, do one of the following:

n To use its default value, do nothing.

n To change its value, click its Value text box, type a new value, and press the Enter key.

n To disable the rule, clear its Use checkbox.

4. Click OK to save the changes and close the dialog box.

Important: Clicking Restore Defaults in the User Preferences dialog box resets all

preferences on all tabs to the original factory settings. Take care when clicking this button.

Operation Manual | 67

Chapter 5 The Home Window

Customizing Data Export Parameters

You can export CFX Manager Dx data in the following formats:

n Text (.txt)

n CSV (.csv)

n Excel 2007 (.xlsx)

n Excel 2003 (.xls)

n XML (.xml)

n HTML (.html)

You can specify the type of data to export and customize the output of the exported data.

To customize data export parameters

1. Select Users > User Preferences to open the User Preferences dialog box.

2. In the User Preferences dialog box, select the Custom Export tab.

3. On the Export Format dropdown list, select a format in which to export the data.

68 | CFX96 Dx and CFX96 Deep Well Dx Systems

Before You Begin

4. In the Data to Export section, select or clear the checkboxes for the type of data to export. The

selected items appear in the Exported Columns list box.

Note: By default, the run information is included in the header. Clear this checkbox if you

do not want the run information included.

5. You can change the output display order of the selected items.

In the Exported Columns list box, highlight the item and then click the arrow buttons to the left

of the list to move it up or down.

6. Optionally, you can change the output column names of the selected items:

a. Click Customize Column Names.

The Column Name Customizer dialog box appears.

b. For each default column name that you want to change, type the new name in its Custom

Name field.

c. Do one of the following:

n Click OK to save the changes and return to the Custom Export tab. The new name

appears in parentheses beside the default column name in the Exported Columns list

box.

n Click Cancel to clear the changes and return to the Custom Export tab.

7. Click OK to save the changes and close the dialog box.

Important: Clicking Restore Defaults in the User Preferences dialog box resets all

preferences on all tabs to the original factory settings. Take care when clicking this button.

Creating a Reaction Master Mix

Using CFX Manager Dx’s Master Mix Calculator, you can easily calculate the required volume of

each component in your master mix. You can print the master mix calculation table to your default

printer, and save the calculations for each target for later use.

To create a reaction master mix using the Master Mix Calculator

1. To open the Master Mix Calculator, do one of the following:

n Select Tools > Master Mix Calculator.

n Click Master Mix Calculator on the toolbar.

The Master Mix Calculator appears.

Operation Manual | 69

Chapter 5 The Home Window

2. In the Reaction section, select a detection method:

n SYBR®Green/EvaGreen

n Probes

3. To create a new target, in the Target section click Create New. A new target name appears in

the target dropdown list.

4. (Optional) To change the default target name:

a. Highlight the target’s name in the dropdown target list.

b. Type a new target name in the Target box.

c. Press the Enter key.

5. Adjust the starting and final concentrations for the forward and reverse primers and any

probes.

70 | CFX96 Dx and CFX96 Deep Well Dx Systems

Before You Begin

6. In the Master Mix Setup section, adjust the values for

n Number of reactions to run

n Reaction volume per well

n Template volume per well

n Supermix concentration per well

n Excess reaction volume per well

7. (Optional) Perform steps 2–6 for as many targets as necessary.

8. In the Choose Target to Calculate section, select the target to calculate.

Tip: You can calculate only one or several or all targets at the same time.

The calculated volumes of the required components for each selected target appear in the

master mix table.

9. Click Set as Default to set the quantities input in the Target and Master Mix Setup sections as

new defaults.

10. Click OK to save the contents of the Master Mix Calculator dialog box.

To print the master mix calculations table

u To print a master mix calculations table, click Print.

The calculations table prints to your default printer.

To save the master mix calculations table as a PDF

u Change your default printer to a PDF driver and click Print on the Master Mix Calculator.

To delete targets

u Select the target using the dropdown target list and click Remove.

Important: Removing a target from the target list also removes it from any master mix

calculations it is used in. Take care when deleting a target.

Operation Manual | 71

Chapter 5 The Home Window

Calibrating New Dyes

The CFX96™ Dx systems are factory calibrated for commonly used fluorophores in white-well and

clear-well plates. Table 11 lists the fluorophores and channel for which each instrument is calibrated.

Note: The CFX96 systems also include a channel dedicated to FRET chemistry. This channel

does not require calibration for specific dyes.

Table 11. Factory calibrated fluorophores and channels

Fluorophores Channel Excitation, nm

FAM, SYBR® Green I 1 450–490 515–530

Detection,

nm

VIC, HEX, CAL Fluor Gold 540, Cal Fluor

Orange 560

ROX, Texas Red, CALFluor Red 610, TEX

615

CY5, Quasar 670 4 620–650 675–690

Quasar 705, Cy5.5 5 672–684 705–730

2 515–535 560–580

3 560–590 610–650

To calibrate new dyes for CFX systems

1. In the Home window, select a target instrument in the Detected Instruments pane.

2. Select Tools > Calibration Wizard to open the Dye Calibration wizard.

Fluorophores already calibrated for the target instrument appear in the Calibrated

Fluorophores table.

72 | CFX96 Dx and CFX96 Deep Well Dx Systems

Before You Begin

3. In the Calibrate New or Existing Fluorophores section, select the fluorophore to calibrate from

the dropdown list.

If the fluorophore name is not included in the list, type its name in the text box to add it to the

list.

4. Select the plate type for the fluorophore.

If the plate type is not included in the list, type the name in the text box to add it to the list.

5. Select a channel for the fluorophore.

6. Select a plate column for the fluorophore.

7. (Optional) Type a color to associate with the fluorophore.

8. Click Add to Plate to add the fluorophore.

9. (Optional) Repeat steps 3—8 to add each fluorophore you plan to calibrate for the plate.

10. When you finish adding fluorophores, click View Plate to open the Pure Dye Plate Display

window.

Use this window as a guide for loading dyes into the plate.

11. Prepare a 96-well plate for dye calibration:

a. Pipette dye solution into each well, following the pattern shown in the Pure Dye Plate

Display.

b. For each fluorophore, fill four wells with 50µl (96-well plate) of 300 nM dye solution.

Notice that at least half the plate contains blank wells.

c. Seal the plate using the sealing method you will use in your experiment.

12. Place the calibration plate in the block and close the lid.

13. In the Dye Calibration wizard, click Calibrate and then OK to confirm that the plate is in the

block.

14. When CFX Manager Dx software completes the calibration run, a dialog box appears. Click

Yes to finish calibration and open the Dye Calibration Viewer.

15. Click OK to close the window.

Operation Manual | 73

Chapter 5 The Home Window

74 | CFX96 Dx and CFX96 Deep Well Dx Systems

Chapter 6 Creating Protocols

A protocol is set of steps that are executed in a specific sequence. In CFX Manager™ Dx software,

all of the steps are associated with options on the instrument. For example, the steps instruct the

instrument to control block and lid temperature, apply a temperature difference across the block,

take a plate read, or perform a melt curve analysis. Each option is specified for different plate and

run types.

CFX Manager Dx provides two options for creating protocols: Protocol Editor and Protocol

AutoWriter.

The Protocol Editor features include the following:

n Standard protocol controls to quickly create protocols

n Ability to quickly calculate a gradient for the selected number of rows

n Ability to quickly calculate run time for the selected plate type

n Ability to edit protocol steps

n Ability to save protocols for reuse

n Ability to print the protocol to a default printer

The Protocol AutoWriter automatically generates a customized PCR protocol with hot start, initial

denaturation, annealing, and extension steps using parameters that you provide. You can then

view a graphical representation of the suggested protocol and edit, run, or save the protocol.

Operation Manual | 75

Chapter 6 Cr eating Protocols

Protocol Editor Window

Use the Protocol Editor to create, open, review, and edit a protocol. By default, the Protocol Editor

opens displaying a generic real-time 2-step protocol for a 96-well plate.

LEGEND

1. The menu bar provides quick access to the File, Settings, and Tools menu commands.

2. The toolbar provides quick access to save and print the protocol, determine where to insert a

step, set sample volume, and view estimated protocol run time.

3. The main pane displays a graphical representation of the protocol.

4. The lower pane displays the protocol outline.

5. The left pane displays the protocol controls that you can add to customize the protocol.

File Menu Commands

Save — saves the current protocol.

Save As — saves the current protocol with a new name or in a new location.

Close — closes the Protocol Editor.

76 | CFX96 Dx and CFX96 Deep Well Dx Systems

Protocol Editor Window

Settings Menu Command

Lid Settings — opens the Lid Setting dialog box in which you can change or set the lid

temperature.

Tools Menu Commands

Gradient Calculator — opens a dialog box from which you can select the block type for a gradient

step. The default is 96 wells.

Run time Calculator — opens a dialog box from which you can select the plate type and scan

mode in order to calculate the estimated run time in the Run Setup window. The default is 96 wells,

all channels.

Toolbar Commands

— saves the current protocol file.

— prints the selected window.

— use this command to select where to insert steps relative to the currently

selected step.

— use this command to enter a sample volume in µl. Sample volumes

differ depending on the type of block:

n For a 96–deep well block the range is 0–125 µl.

n For a 96–well block the range is 0–50 µl.

— displays the estimated run time based on the protocol steps, ramp rate,

and the type of block selected.

— displays Help information about protocols.

Operation Manual | 77

Chapter 6 Cr eating Protocols

Protocol Editing Controls

The left pane of the Protocol Editor window comprises controls that you can use to create

protocols.

Each control consists of a set of parameters that represent a step in the protocol. You can modify

each parameter and add or remove them to customize your protocol. This section describes the

options in each control.

repetitions of steps 2–4. After the final repetition, the software will have performed steps 2–4 a
total of 40 times. You can edit the return-to (GOTO) step and the number of cycles either in the
graphical display or in the protocol outline.

n Insert Step — inserts a step before or after the selected step. You

can edit the temperature and hold time values either in the
protocol’s graphical display or in the protocol outline.

n Insert Gradient — inserts a gradient step based on the type of well

block selected in the gradient calculator. You can edit the gradient
range in the Gradient pane that appears when a gradient step is
inserted.

n Insert GOTO — inserts a cycling (loop) step, which informs the

software to repeat specific steps in sequence for a specified
number of cycles. The repetitions begin after the first cycle is
complete. For example, you can inform the software to perform 39

n Insert Melt Curve — inserts a melt curve read step.

n Insert Plate Read to Step — adds a plate read command to the selected step. A plate read

measures the amount of fluorescence at the end of a cycle. The plate read step is generally
the last step in a GOTO loop.

Tip: After you add a plate read command to a step, the button changes to Remove Plate

Read when you select the step.

n Remove Plate Read — removes a plate read command from the selected step.

Tip: After you remove a plate read command from a step, the button changes to Add Plate

Read to Step when you select the step.

n Step Options — opens the Step Options dialog box and displays the options available for the

selected step. See Step Options on page 79 for detailed information about the step options.

Tip: You can also access the Step Options by right-clicking the step in the graphic display.

n Delete Step — deletes the selected step from the protocol.

78 | CFX96 Dx and CFX96 Deep Well Dx Systems

Protocol Editor Window

Step Options

Open the Step Options dialog box to view the options you can add, change, or remove from a step.

n Plate Read — when selected, adds a plate read to the step.

n Temperature — sets the target temperature for the selected step.

n Gradient — sets the gradient range for the step; the range is 1–24°C.

Note: A gradient runs with the lowest temperature in the front of the block (in this image,

row H) and the highest temperature in the back of the block (in this image, row A).

n Increment — the amount to increase (or decrease) the temperature of the selected step; this

value amount is added to the target temperature with each cycle. The range is ±0.1–10°C.

Note: To decrease the temperature, type a minus sign (–) before the numerical value

(forexample, –5°C).

n Ramp Rate — the ramp rate for the selected step; the range depends on the block size.

n Time — the hold time for the selected step.

n Extend — the amount of time (in sec) to extend or decrease the selected step; this option is

added to the hold time in each cycle; the range is 1–60 sec.

n Beep — when selected, a beep sounds 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.

Operation Manual | 79

Chapter 6 Cr eating Protocols

Creating a Protocol in the Protocol Editor

Using the Protocol Editor, you can create custom protocol files. You can also edit and save

previously saved protocol files or sample protocol files shipped with CFX Manager Dx software.

To create a new protocol file, perform the following:

n Open a protocol file in the Protocol Editor.

Tip: You can open a new or existing protocol in the Protocol Editor.

n Set up the new protocol.

n Add steps to the protocol from the protocol controls pane.

n Edit the properties of the steps.

n Save the protocol.

Tip: To create a new protocol from a previously saved or sample protocol file, see Opening an

Existing Protocol in the Protocol Editor on page 81.

Opening a New Protocol File in the Protocol Editor

CFX Manager Dx offers multiple options to open a new protocol file:

n From the Home window

n From the Startup Wizard dialog box

n From the Run Setup dialog box

To open a new protocol file from the Home window

u Select File > New > Protocol.

The Protocol Editor window opens, displaying the default protocol file.

Tip: For information about setting your default protocol, see Changing the Default File

Settings on page 58.

To open a new protocol file from the Startup Wizard

1. In the Home window, do one of the following to open the Startup Wizard if it is not in view:

n Select View > Startup Wizard.

n Click Startup Wizard on the toolbar.

By default, the Startup Wizard displays the Run setup tab with the CFX96™ instrument type

selected.

80 | CFX96 Dx and CFX96 Deep Well Dx Systems

Creating a Protocol in the ProtocolEditor

2. If necessary, select the instrument type from the dropdown list.

3. Click User-defined as the run type.

The Run Setup dialog box opens to the Protocol tab and displays the default protocol file.

4. Click Create New.

The Protocol Editor window opens, displaying the default real-time protocol.

To open a new protocol from the Run Setup dialog box

1. In the Home window, do one of the following to open the Run Setup dialog box:

n Select Run > User-defined Run.

n Click User-defined Run Setup on the toolbar.

The Run Setup dialog box opens to the Protocol tab and displays your default protocol file.

2. Click Create New.

The Protocol Editor window opens, displaying the default real-time protocol.

Opening an Existing Protocol in the Protocol Editor

CFX Manager Dx provides sample protocol files that you can edit and save as custom new

protocols. You can also create a new protocol from an existing custom protocol.

To open a sample protocol file

1. In the Home window, select File > Open > Protocol.

By default, Windows Explorer opens to the location of the CFX Manager Dx Sample files

folder.

2. Open the Sample files folder. You see the following folders:

n ConventionalProtocols — contains example protocol files for traditional PCR analysis.

n DataFiles — contains example data files that you can use to explore CFX Manager Dx’s

features.

n MeltCalibration — contains example protocol files for use with Bio-Rad’s Precision Melt

Analysis software.

n Plates — contains example plate files.

n RealTimeProtocols – contains example protocol files for real-time PCR analysis.

Operation Manual | 81

Chapter 6 Cr eating Protocols

3. Open the protocol folder for the type of run you plan to perform, either ConventionalProtocols

or RealTimeProtocols.

4. Select the protocol of choice and click Open.

The sample protocol opens in the Protocol Editor window.

5. Select File > Save As and save the protocol with a new name or in a new folder.

To open an existing protocol

1. In the Home window, do one of the following:

n Select File > Open > Protocol, navigate to and select the target protocol, and click Open.

n Open the Startup Wizard and do one of the following:

o To edit the displayed protocol, click Edit Selected.

o To edit another existing protocol, click Select Existing and navigate to the target file.

The protocol opens in the Protocol Editor window.

2. Select File > Save As and save the protocol with a new name or in a new folder.

Setting Up a New Protocol

Tip: If your protocol file includes the required parameters (for example, if you are editing an

existing plate file) you can skip this section. Proceed to Adding Steps to a Protocol on page 84.

New protocol files require the following parameters:

n Block type

n Scan mode for the chosen block type

n Lid temperature

n Sample volume

Setting the Block Type

CFX Manager Dx automatically calculates temperature increments for gradient steps based on the

block type.

Note: The plate type set in the Protocol Editor must be the same as the plate in the reaction

module.

82 | CFX96 Dx and CFX96 Deep Well Dx Systems

Creating a Protocol in the ProtocolEditor

To set the block type

u In the Protocol Editor window, select Tools > Gradient Calculator and choose the appropriate

plate type in the dropdown list that appears.

Selecting the Scan Mode for the Chosen Block Type

To determine the run time for the protocol, select the target block type and scan mode.

To select the block type and scan mode

u In the Protocol Editor window, select Tools > Run time Calculator and choose the appropriate

plate type and scan mode in the dropdown list that appears.

Adjusting the Lid Temperature

CFX Manager Dx sets the default lid temperature to 105.0°C.

You can change the default settings or turn off the lid heater as necessary for the protocol.

Tip: You can change the default lid temperature in the User Preferences dialog box. See

Setting the Default Protocol Parameters on page 60.

To adjust the lid temperature

1. In the Plate Editor window, select Settings > Lid Settings.

The Lid Settings dialog box appears.

2. Do one of the following:

n Select User Defined and enter a temperature value in the text box.

n Select Turn Off Lid Heater.

3. Click OK to accept the changes and close the dialog box

Setting the Sample Volume

By default, CFX Manager Dx sets the sample volume for each well to 25 µl. However, the CFX Dx

system range is 0–125 µl.

The instrument uses one of two temperature control modes to determine when the sample reaches

the target temperature in a protocol:

n Calculated mode — when the sample volume is set to a volume appropriate for the block, the

instrument calculates the sample temperature based on the sample volume. This is the

standard mode.

Operation Manual | 83

Chapter 6 Cr eating Protocols

n Block mode — when the sample volume is set to zero (0) µl, the instrument records the

sample temperature as the same as the measured block temperature.

To set the sample volume for a specific block

u In the Plate Editor window, type the correct value in the Sample Volume text box on the

toolbar.

Tip: You can change the default sample volume in the User Preferences dialog box. See

Changing the Default File Settings on page 58.

Adding Steps to a Protocol

To add a step to a protocol

1. Open the protocol in the Protocol Editor window.

2. Determine where to insert the new step. On the toolbar, select Before or After in the Step

dropdown list.

3. On the graph, select the step before or after which you plan to insert the new step.

4. In the left pane, click Insert Step.

5. To change the temperature or hold time, click the default value on the graph or the protocol

outline and type a new value.

6. (Optional) In the left pane, click Step Options to display the Step Options dialog box and

modify the available options for the selected step.

Tip: You can access the Step Options dialog box on the right-click menu in either the

graph pane or the protocol outline pane.

7. Click OK and then click Yes to save changes to the protocol.

The Save As dialog box appears

8. In the Save As dialog box, type a name for the new protocol file and click Save.

Inserting a Gradient Step

To insert a gradient step

1. Verify that the plate size for the gradient is the same as the block type of the instrument, 96-

well.

2. If you have not yet done so, select the plate size for the gradient:

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Creating a Protocol in the ProtocolEditor

Select Tools > Gradient Calculator and choose the appropriate well type from the dropdown

list.

3. On the toolbar, select either Before or After from the Insert Step dropdown list.

4. In the graph or outline pane, select the step before or after which you plan to insert the

gradient step.

5. In the left pane, click Insert Gradient. The new gradient step is highlighted in the graph and the

outline pane, for example:

The temperature of each row in the gradient appears in the Gradient table in the right pane.

6. To edit the gradient temperature range, do one of the following:

n Click the default temperature in the graph or outline pane and enter a new temperature.

n Click Step Options to enter the gradient range in the Step Options window.

n Change the Range value in the Gradient table.

7. To edit the hold time, click the default time in the graphic or text view and enter a new time.

8. Click OK and then Yes to save the changes.

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Chapter 6 Cr eating Protocols

Inserting a GOTO Step

Note: You cannot insert a GOTO step within a GOTO set; you cannot create nested GOTO

loops.

To insert a GOTO step

1. On the toolbar, select Before or After from the Insert Step dropdown list.

2. In the graph, select the step before or after which you plan to insert the GOTO step.

3. In the left pane, click Insert GOTO.

4. To edit the GOTO step number or number of GOTO repeats, select the default number in the

graph or outline pane and enter a new value.

5. Click OK and then Yes to save the changes.

Inserting a Melt Curve Step

Tip: You cannot insert a melt curve step inside a GOTO loop.

Note: The melt curve step includes a 30 sec hold at the beginning of the step that is not shown

in the protocol.

To insert a melt curve step

1. On the toolbar, select Before or After from the Insert Step dropdown list.

2. In the graph, select the step before or after which you plan to insert the melt curve step.

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Creating a Protocol in the ProtocolEditor

3. In the left pane, click Insert Melt Curve. The new melt curve step is highlighted in the graph

and the outline pane, for example:

4. To edit the melt temperature range or increment time, select the default number in the graph or

outline pane and enter a new value.

5. Click OK and then Yes to save the changes.

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Chapter 6 Cr eating Protocols

Adding or Removing a Plate Read Step

Tip: After you add a plate read command to a step, the button changes to Remove Plate Read

when you select the step.

To add a plate read to a step

1. On the toolbar, select Before or After from the Insert Step dropdown list.

2. On the graph, select the step before or after which you plan to insert the plate read step.

3. In the left pane, click Add Plate Read to Step to add a plate read to the selected step.

4. Click OK and then Yes to save the changes.

To remove a plate read from a step

u On the graph, select the step that contains the plate read and click Remove Plate Read in the

left pane.

Changing Step Options

To change step options for a selected step

1. Select the target step in the graph or outline pane.

2. In the left pane, click Step Options to open the Step Options dialog box.

Alternatively, right-click the target step in either pane and select Step Options in the menu that

appears.

3. To add, modify, or remove options:

n Enter a value in the appropriate text box.

n Edit a value in the specific text box.

n Select or clear a checkbox.

4. Click OK to save the changes and close the Step Options dialog box.

5. Click OK and then Yes to save the protocol.

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Deleting a Step

To delete a step in the protocol

1. Select the step in the graph or outline pane.

2. In the left pane, click Delete Step to delete the selected step.

3. Click OK and then Yes to save the protocol.

Copying, Exporting, or Printing a Protocol

To copy a protocol

u Right-click the protocol outline and select Copy Protocol.

You can paste the outline into a .txt, .xls, .doc, or .ppt file.

To export a protocol

1. Right-click the protocol outline and select Export Protocol.

Creating a Protocol in the ProtocolEditor

The Save As dialog box appears.

2. (Optional) In Windows Explorer, navigate to a folder in which to save the protocol file.

3. In File name, type a name for the exported protocol file.

4. Click Save.

To print a protocol

u Right-click the protocol outline and select Print.

You can print the protocol outline to your default printer.

Operation Manual | 89

Chapter 6 Cr eating Protocols

Creating a Protocol with the Protocol AutoWriter

Important: Bio-Rad does not guarantee that running a protocol created with the Protocol

AutoWriter will always result in a real-time PCR product.

CFX Manager Dx’s Protocol AutoWriter automatically generates cycling protocols based on the

following input parameters:

n Amplicon length — the expected length of the PCR product

n Annealing temperature — the reaction Tafor the primers being used

If the Tais unknown, you can use the Tacalculator to automatically calculate it based on your

primer sequences.

Note: The Tais adjusted from the primer melting temperature (Tm) information that is

based on the selected enzyme and the protocol speed.

n Enzyme type — the DNA polymerase enzyme (iTaq™, iProof™ DNA polymerase, or Other)

If you use an enzyme other than iTaq or iProof DNA polymerase, you can enter additional

information, including the gradient range, hot-start activation time (in sec), and the final

extension time (in sec).

n Run speed — the reaction speed (standard, fast, or ultrafast)

The Protocol AutoWriter optimizes the protocol depending on the selected speed setting. The

total run time is determined 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.

Using parameters that you enter and standard PCR guidelines, the Protocol AutoWriter

automatically generates a customized PCR protocol with hot start, initial denaturation, annealing,

and extension steps. You can then view a graphical representation of the suggested protocol and

edit, run, or save the protocol.

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Creating a Protocol with the Protocol AutoWriter

To create a new protocol using CFX Manager Dx’s Protocol AutoWriter

1. In the Home window, select Tools > Protocol AutoWriter.

The Protocol AutoWriter dialog box appears.

2. In the Enter Target Values/Enzyme section, do the following:

n Enter the annealing temperature (Ta) for the primers, if known.

Tip: Using the Ta Calculator on page 92 for more information.

Note: For information about the calculations used in the TaCalculator, see Breslauer

et al. 1986.

n Enter the amplicon length in base pairs (bp).

n Select an enzyme type from the list of options (iTaq™ DNA polymerase, iProof™ DNA

polymerase, or Other).

Tip: If you select Other as the enzyme type, the parameters in the Additional

Parameters (Optional) section become active.

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Chapter 6 Cr eating Protocols

3. If you selected Other as the enzyme type, you can add any or all of the following parameters to

the protocol:

n Gradient range

n Hot start activation temperature

n Final extension time

4. In the Type section, move the sliding bar to select a protocol speed (Standard, Fast, or

Ultrafast). CFX Manager Dx adjusts the total run time.

5. Select the type of PCR to perform (Real-time PCR is the default).

With real-time PCR, CFX Manager Dx adds a plate read step to collect fluorescence data.

6. In the Preview section, review the protocol. You can make changes as needed.

7. Do one of the following:

n Click OK to save the new protocol. After saving it, the protocol opens in the Startup

Wizard. Click Edit Selected to make any changes to the protocol. For example, you might

need to change the lid temperature and sample volume.

n Click Cancel to close the window without saving the protocol.

Using the TaCalculator

When the annealing temperature for the primer is unknown, you can use the TaCalculator to

calculate the value. You can use the value in the Protocol AutoWriter or in the Protocol Editor to

create your protocol.

About the TaCalculator

The TaCalculator calculates the Tmvalue for each primer as well as the Tavalue for the protocol at

standard speed.

The Tafor the protocol is based on the average primer Tmvalues with the following rules applied:

n If the difference between the primer Tmvalues is >4°C, the Ta= (lower of the two primer T

values + 2) – 4°C

n If the difference between the T

4°C

values is ≤4°C, the T

m

= (average of the primer Tmvalues) –

a

m

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Using the Ta Calculator

Base Pair Counting Method

For each primer, the TaCalculator uses the base pair counting method for sequences of 14 base

pairs (bp) or fewer.

Tm= ((w*A + x*T) * 2) + ((y*G + z*C) * 4)

where w, x, y, and z are the numbers of the bases A, T, G, and C in the sequence, respectively.

Nearest Neighbor Method

For sequences longer than 14 bp, the nearest neighbor method is used. In the nearest neighbor

method, the melting temperature calculations are based on the thermodynamic relationship

between entropy (order or a measure of the randomness of the oligonucleotide), enthalpy (heat

released or absorbed by the oligonucleotide), free energy, and temperature.

∆H = ∆G + T* ∆S

where:

n ∆H = Enthalpy value, Cal/Mole*K

n T = temperature, Kelvin

n ∆S = Entropy value, Cal/Mole*K
n ∆G = Gibbs free energy in Cal/Mole*K

The change in entropy and enthalpy is directly calculated by summing the values for nucleotide

pairs shown in Table 12 (Breslauer et al. 1986).

The relationship between the free energy and the concentration of reactants and products at

equilibrium is given by:

∆G = R*T*ln ((DNA * Primer)/(DNA + Primer))

where R is the gas constant (1.986 Cal/Mole*K).

Substituting G in the two equations and solving for T gives

T = ∆H/(∆S + R*Ln((DNA * Primer)/(DNA + Primer)))

assuming that the concentration of DNA and DNA-primer complex are equal.

It has been determined empirically that there is a 5 kcal free energy (3.4 kcal) (Sugimoto et al.

1996) change during the transition from single-stranded to B-form DNA. This is presumably helix

initiation energy. Finally, adding an adjustment for salt gives the equation that the Tacalculator

uses:

T = (∆H – 5(KCal/K*Mole))/(∆S + (R * ln(1/(primer)))) + 16.6 log

(SaltMolarity)

10

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Chapter 6 Cr eating Protocols

No adjustment constant for salt concentration is needed, since the various parameters were

determined at 1 M NaCl, and the log10of 1 is zero.

The thermodynamic calculations assume that annealing occurs at pH 7.0. The Tmcalculations

assume that the sequences are not symmetrical and contain at least one G or C.

The oligonucleotide sequence should be at least 14 bases long to give reasonable Tmvalues.

Less than 14 bases uses the base pair counting method (see Table 12 that follows).

Table 12. Breslauer interaction constants

Interaction

AA TT 9.1 24 1.5

AT TA 8.6 23.9 1.5

AC TG 6.5 17.3 1.3

AG TC 7.8 20.8 1.6

TA AT 6 16.9 0.9

TT AA 9.1 24 1.9

TC AG 5.6 13.5 1.6

TG AC 5.8 12.9 1.9

CA GT 5.8 12.9 1.9

CT GA 7.8 20.8 1.6

CC GG 11 26.6 3.1

CG GC 11.9 27.8 3.6

GA CT 5.6 13.5 1.6

∆H ∆S ∆G

GT CA 6.5 17.3 1.3

GC CG 11.1 26.7 3.1

GG CC 11 26.6 3.1

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Using the TaCalculator

To use the TaCalculator

1. To open the TaCalculator, do one of the following:

n If you are currently in the Protocol AutoWriter, click TaCalculator.

n In the Home window, select Tools > TaCalculator.

The TaCalculator dialog box appears.

Using the Ta Calculator

2. In the Forward Primer text box, type or paste the forward primer sequence.

Tip: You can also use the A, T, G, C buttons on the left side of the dialog box to enter the

sequence.

3. Type or paste the reverse primer sequence in the Reverse Primer text box.

4. Click Calculate.

The TaCalculator calculates and displays the Tmof each primer and the average Tmand T

values, for example:

a

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Chapter 6 Cr eating Protocols

If the primer Tmvalues are more than 4°C apart, the Protocol AutoWriter uses the lower primer

Tmvalue + 2°C as a basis for calculating the Tavalue, which you can further modify by

changing the enzyme and reaction speed.

The TaCalculator generates an annealing temperature for standard speed with iTaq DNA

polymerase. When using a different enzyme, the speed settings automatically adjust the Ta.

5. Do one of the following:

n If you opened the TaCalculator from the Protocol AutoWriter, click OK. You return to the

Protocol AutoWriter. The annealing temperature is automatically modified.

n If you opened the TaCalculator from the Tools menu, record the calculations and click

Cancel to close the calculator.

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Chapter 7 Preparing Plates

A plate file contains information about run parameters such as scan mode, fluorophores, and well

contents. After the run, CFX Manager™ Dx software links the well contents to the fluorescence data

collected during the run 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.

CFX Manager Dx software provides two options for creating plates: The Plate Editor for real-time

PCR runs and the Setup Wizard for normalized gene expression analysis.

The Plate Editor includes the following features:

n Standard fluorophores and sample types to assign to plate wells

n Ability to set reference target and control sample for gene expression analysis

n Ability to edit plate setup before, during, or after a run

n Ability to save plate files for reuse

n Ability to print the plate file to a default printer

The Setup Wizard guides you through creating a plate layout for normalized gene expression

analysis. You can use the Setup Wizard before, during, or after a run.

Operation Manual | 97

Chapter 7 Preparing Plates

Plate Editor Window

You use the Plate Editor to create custom plates or modify existing plates.

LEGEND

1. The menu bar provides quick access to File and Settings menu commands as well as plate

editing tools options.

2. The toolbar provides quick access to important plate loading functions.

3. The main pane displays the plate outline and the plate options as you apply them.

4. The right pane displays options that you use to customize your plate.

5. The bottom pane displays the plate type and provides quick access to viewing options.

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