CFX Maestro Software User Guide

1 Real-Time PCRCFX Maestro SoftwareUser Guide V e r s i o n 1. 0 CFX Maestro SoftwareUser GuideVersion Technical Support DepartmentThe Bio-Rad Technical Support department in the United States is open Monday through Friday, 5:00 AM to 5:00 PM, Pacific time. Go to for worldwide technical :1-800-424-6723, option (United States/Canada only)For technical assistance outside the United States and Canada, contact your local technical support 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-RadBio-Rad reserves the right to modify its products and services at any time. This user 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 following are trademarks of Bio-RadLaboratories, Inc.

2 : Bio-Rad, CFX Maestro , CFX384, CFX384 Touch, CFX96 Touch, CFX Connect, and C1000 Touch and iProof and iTaq. Adobe, Acrobat, and Reader are trademarks of Adobe Systems Incorporated. Cy is a trademark of GE Healthcare Group companies. CAL Fluor and Quasar are trademarks of Biosearch Technologies, Inc. SYBR and Texas Red are trademarks of Life Technologies Corporation. Bio-Rad Laboratories, Inc. is licensed by Life Technologies Corporation to sell reagents containing SYBR Green for use in real-time PCR, for research purposes only. Excel, Microsoft, PowerPoint, and Windows are trademarks of Microsoft Corporation. Gmail is a trademark of Google, Inc. Yahoo! is a trademark of Yahoo! Inc. Pentium is a trademark of Intel Corporation. EvaGreen is a trademark of Biotium, Inc. FAM, ROX, and VIC are trademarks of Applera Corporation. Bio-Rad Laboratories, Inc. is licensed by Appelera Corporation to sell reagents containing EvaGreen dye for use in real-time PCR, for research purposes thermal cyclers and real-time systems are covered by one or more of the following patents or their foreign counterparts owned by Eppendorf AG: Patent Numbers 6,767,512 and 7,074,367.

3 Hard-Shell plates are covered by one or more of the following patents or their foreign counterparts owned by Eppendorf AG: Patent Numbers 7,347,977; 6,340,589; and 6,528, 2017 by Bio-Rad Laboratories. All rights reserved. User Guide | iiiTable of ContentsSafety and Regulatory Compliance .. 11 Safety Warning Labels .. 11 Standard Safety Warnings ..11 Instrument Safety Warnings ..12 Safe Use Specifications and Compliance.. 13 Regulatory Compliance.. 13 Hazards .. 14 Biohazards .. 14 Chemical Hazards .. 15 Explosive or Flammability Hazards .. 15 Electrical Hazards .. 15 Transport.. 16 Storage .. 16 Disposal .. 16 Chapter 1 Introduction .. 17 Main Features of CFX Maestro Software .. 18 Finding Out More .. 18 Chapter 2 Installing CFX Maestro Software .. 19 System Requirements.. 20 Installing CFX Maestro Software ..21 Detecting Connected Instruments .. 22 Software Files .. 23 Chapter 3 The Workspace.

4 25 The Home Window .. 26 The Startup Wizard .. 27 The Protocol Editor Window .. 28 The Plate Editor Window .. 29 The Data Analysis Window .. 30 Chapter 4 The Home Window .. 31 The Home Window .. 32 File Menu Commands ..33 View Menu Commands ..33 User Menu Commands ..34iv | CFX Maestro SoftwareTable of ContentsRun Menu Commands ..34 Tools Menu Commands ..35 Help Menu Commands ..35 Toolbar Commands .. 36 The Startup Wizard .. 37 Status Bar .. 37 Detected Instruments Pane ..38 Viewing the Properties of an Instrument .. 42 Before You Begin .. 44 Performing Software and Instrument Updates .. 44 Setting User Preferences .. 45 Creating a Reaction Master Mix .. 59 Calibrating New Dyes .. 61 Chapter 5 Creating Protocols .. 65 Protocol Editor Window .. 66 File Menu Commands ..66 Settings Menu Command .. 66 Tools Menu Commands ..67 Toolbar Commands .. 67 Protocol Editing Controls .. 68 Creating a Protocol in the Protocol Editor.

5 70 Opening a New Protocol File in the Protocol Editor .. 70 Opening an Existing Protocol in the Protocol Editor .. 71 Setting Up a New Protocol ..72 Adding Steps to a Protocol..74 Inserting a Gradient Step .. 74 Inserting a GOTO Step .. 75 Inserting a Melt Curve Step ..76 Adding or Removing a Plate Read Step .. 77 Changing Step Options.. 77 Deleting a Step .. 77 Copying, Exporting, or Printing a Protocol .. 78 Creating a Protocol with the Protocol AutoWriter .. 79 Using the Ta Calculator.. 82 About the Ta Calculator .. 82 Chapter 6 Preparing Plates .. 87 Plate Editor Window .. 88 File Menu Commands ..88 Edit Menu Commands ..88 Settings Menu Commands .. 89 Editing Tools Menu Commands .. 89 User Guide | vTable of ContentsPlate Editor Toolbar Commands.. 89 Creating a Plate File Using the Plate Editor .. 91 Opening a New Plate File in the Plate Editor .. 91 Opening an Existing Plate File in the Plate Editor.. 92 Setting Up a New Plate File.

6 93 Assigning Optional Parameters to the Plate File .. 99 Assigning a Target to Wells ..99 Assigning a Sample Name to Wells .. 101 Assigning Biological Groups to Wells .. 102 Assigning Technical Replicate Numbers to Wells.. 104 Assigning a Dilution Series to Standard Sample Types .. 106 Copying Well Contents into Another Well.. 107 Adding a Note to a Well .. 107 Clearing Wells of All Content ..108 Changing Experiment Settings ..109 Creating Well Groups .. 111 Changing Trace Styles .. 113 Viewing the Plate in Spreadsheet Format.. 114 Creating a Plate Layout Using the Plate Setup Wizard .. 116 Using the Plate Setup Wizard .. 116 Chapter 7 Running Experiments.. 119 The Run Setup Window .. 120 Accessing the Run Setup Window .. 121 Protocol Tab .. 121 Plate Tab .. 123 Start Run Tab .. 125 Running an Experiment.. 126 Run Details Dialog Box .. 128 Run Status Tab .. 128 Real-time Status Tab .. 130 Time Status Tab .. 132 Performing PrimePCR Experiments.

7 133 Transferring Stand-Alone Data for Analysis .. 134 Transferring Data Through CFX Maestro Software .. 134 Transferring Data Through Email .. 134 Transferring Data Using a USB Drive .. 134 Creating a Data File.. 135 Chapter 8 Data Analysis Overview .. 137 Data Analysis Window .. 137 Data Analysis Toolbar .. 138 Data Analysis Menu Bar .. 138 Tab Details .. 140 Step Number Selector .. 141 Viewing Well Groups in Data Analysis .. 141vi | CFX Maestro SoftwareTable of ContentsChanging Well Contents after a Run.. 141 Data Analysis Settings .. 142 Adjusting the Threshold .. 142 Baseline Settings .. 142 Analysis Mode.. 143 Cycles to Analyze .. 144 Well Selector .. 145 Well Selector Right-Click Menu Items .. 146 Temporarily Excluding Wells from Analysis .. 146 Charts .. 147 Chart Tools .. 147 Magnifying an Area in the Chart .. 153 Copying Charts into a Microsoft File .. 154 Common Right-Click Menu Items for Charts.

8 154 Spreadsheets .. 155 Common Right-Click Menu Items for Spreadsheets .. 155 Export .. 156 Exporting All Data Sheets to Excel .. 156 Exporting RDML Files .. 156 Creating Custom Export Files .. 157 Exporting to a LIMS Folder ..157 Chapter 9 Data Analysis Details .. 159 Quantification Tab.. 160 Fluorophore Options .. 160 Trace Styles .. 161 Log Scale Option .. 162 Standard Curve Chart .. 163 Amplification Chart Menu Options .. 164 Quantification Tab Spreadsheet .. 164 Quantification Data Tab .. 165 Results Spreadsheet .. 165 Standard Curve Results Spreadsheet.. 166 Plate Spreadsheet.. 167 RFU Spreadsheet .. 168 Melt Curve Tab .. 169 Adjusting Melt Curve Data ..170 Melt Curve Data Tab .. 171 Melt Peaks Spreadsheet ..171 Plate Spreadsheet.. 172 RFU Spreadsheet .. 173-d(RFU)/dT Spreadsheet ..173 End Point Tab .. 174 Results Data .. 175 User Guide | viiTable of ContentsAdjusting the End Point Data Analysis .. 175 RFU Spreadsheet for End Point Analysis.

9 176 Allelic Discrimination Tab .. 177 Adjusting Data for Allelic Discrimination .. 178 Chart Menu Options .. 179 Allelic Discrimination Spreadsheet .. 179 Custom Data View Tab .. 180 Creating a Custom Data View .. 180QC Tab .. 182 Changing QC Criteria .. 182 Excluding Wells That Fail QC .. 183 Run Information Tab .. 183 Data Analysis Reports.. 184 Data Analysis Report Categories .. 185 Creating a Data Analysis Report .. 187 Creating Well Group Reports .. 188 Appendix A Gene Expression Analysis.. 189 Plate Setup for Gene Expression Analysis .. 190 Guided Plate Setup.. 190 Gene Expression Charts .. 191 Graphing .. 192 Changing and Annotating the Chart View.. 194 Adjusting Gene Expression Data .. 199 Experiment Settings .. 202 Right-Click Menu Options..203 Data Spreadsheet .. 204 Show Details Option .. 205 Clustergram .. 206 Settings .. 206 Right-Click Menu Options..206 Data Spreadsheet .. 206 Scatter Plot .. 207 Settings.

10 207 Right-Click Menu Options..207 Data Spreadsheet .. 207 Volcano Plot .. 208 Settings .. 208 Right-Click Menu Options..208 Data Spreadsheet .. 208 Heat Map .. 209 Settings .. 209 Right-Click Menu Options..209 Data Spreadsheet .. 209 Well Results Spreadsheet .. 210 ANOVA Calculations .. 211viii | CFX Maestro SoftwareTable of ContentsSettings .. 212 Results Table .. 213 Right-Click Menu Options..215 Gene Study .. 216 Inter-Run Calibration.. 216 Gene Study Dialog Box.. 217 Study Setup Tab .. 217 Preparing a Gene Study .. 218 Study Analysis Tab .. 218 Gene Study Reports .. 220 Appendix B Data Analysis Calculations .. 223P-Value .. 223 Differences in P-Value Results .. 224 Reaction Efficiency .. 225 Relative Quantity.. 225 Relative Quantity When a Control Is Selected .. 226 Standard Deviation of Relative Quantity .. 226 Efficiency Corrected Cq (CqE).. 226 Mean Efficiency Corrected Cq (MCqE) .. 227 Normalization Factor.