QuantStudio 6 and 7 Flex Real Time PCR Systems

Product Information – QuantStudioTM 6 and 7 Flex Real-Time PCR

Systems

This user guide provides maintenance and administration
instructions for the QuantStudioTM 6 and 7 Flex Real-Time PCR
Systems. It is intended for research use only and should not be
used for diagnostic procedures.

Publication Number: 4489821 Revision B

Manufacturer: Life Technologies Holdings Pte Ltd

Address: Block 33, Marsiling Industrial Estate Road 3, #07-06,
Singapore 739256

For descriptions of symbols on product labels or product
documents, visit thermofisher.com/symbols- definition

The information in this guide is subject to change without
notice.

Product Usage Instructions

Chapter 1 – Getting started

• Differences between the QuantStudioTM 6 and 7 Flex Real-Time
  PCR Systems

• About the QuantStudioTM 6 and 7 Flex Real-Time PCR Systems

Chapter 2 – Calibrate the instrument

This chapter provides instructions on how to calibrate the
QuantStudioTM 6 and 7 Flex Real-Time PCR Systems.

Appendix A – Manual instrument operation

• Overview of instrument touchscreen functions
• Operate the instrument from the touchscreen

Appendix B – Maintenance

• Replace the instrument fuses
• Materials required
• Replace the fuses
• Install the QuantStudioTM RealTime PCR Software
• Computer requirements
• Install the software

QuantStudioTM 6 and 7 Flex Real-Time PCR Systems
USER GUIDE Maintenance and Administration
for use with QuantStudioTM RealTime PCR Software v1.3
Publication Number 4489821 Revision B
For Research Use Only. Not for use in diagnostic procedures.

Life Technologies Holdings Pte Ltd | Block 33 | Marsiling Industrial Estate Road 3 | #07-06, Singapore 739256 For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Revision history: Pub. No. 4489821 Revision B

Date 22 August 2022

Description
· The branding was updated. · The statement covering limited use label licenses was updated. · The name of the software was updated to the QuantStudioTM RealTime
PCR Software and the base software does not require a license. · The firewall ports that must be open were added. · The TwisterTM Robot was removed. · The volume of deionized water to create a background plate was updated
(“Create a background plate” on page 153). · The volume to determine the optimum dye concentration for a custom dye
was updated (“Determine optimum dye concentration” on page 155). · The volume of dye was updated when creating a custom dye plate for
calibration (“Create a custom dye plate” on page 156). · The consumables and storage temperatures were updated (“Kits,
consumables, and accessories” on page 160). · The instructions to order from the website were removed. · The safety and electromagnetic compatibility standards were updated. · The environmental design standards were updated.

A

November 2013

New document for the QuantStudioTM 6 and 7 Flex Real-Time PCR Systems.

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses. TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Internet Explorer, Excel, Microsoft, and Windows are registered trademarks of Microsoft Corporation. Cy is a registered trademark of Global Life Sciences Solutions Germany GmbH. Safari, Apple, and Macintosh are registered trademarks of Apple, Inc. Mozilla and Firefox are registered trademarks of Mozilla Foundation. Adobe and Acrobat are registered trademarks of Adobe Systems, Inc. Twister is a registered trademark of Caliper Life Sciences, Inc. TaqMan is a trademark of Roche Molecular Systems, Inc., used under permission and license.

©2022 Thermo Fisher Scientific Inc. All rights reserved.

Contents

About this guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Audience . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Assumptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 User attention words . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Symbols on this instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Contents of this manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
CHAPTER 1 Getting started . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Differences between the QuantStudioTM 6 and 7 Flex Real-Time PCR Systems . . . . . . . . . 13 About the QuantStudioTM 6 and 7 Flex Real-Time PCR Systems . . . . . . . . . . . . . . . . . . . . . . 13
Data collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Instrument filters and supported dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Instrument hardware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Front . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Rear . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Barcode scanner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Electrical protective devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Instrument software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Computer requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Software installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Third-party software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Third-party software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Instrument consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Interchangeable sample blocks, heated covers, and plate retainers . . . . . . . . . . . . . . . . . . . 23
CHAPTER 2 Calibrate the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Calibration overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Calibration and maintenance schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Planned maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 About maintenance reminders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

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Calibration workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 View the calibration status of an instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Enable or change the calibration reminders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Requirements to email calibration reminders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Define the mail server settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Modify the notification settings for a monitored instrument . . . . . . . . . . . . . . . . . . . . . . 31 Guidelines for preparing calibration plates and array cards . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Preparing array cards for instrument calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Fill the calibration TaqManTM Array Cards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 ROI calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 When to perform the calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 About the calibration data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Preparing the calibration plate or array card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Prepare the ROI calibration plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Perform the ROI calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Background calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 When to perform the calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 About the calibration data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 Preparing the calibration plate or array card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 Prepare the background calibration plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 Perform the background calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Uniformity calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 When to perform the calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Preparing the calibration plate or array card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Prepare the ROI calibration plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Perform the uniformity calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 Dye calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 When to perform the calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 About the dye calibration data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Preparing the calibration plate or array card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Prepare the dye calibration plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Perform the dye calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Normalization calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 When to perform the calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 About the calibration data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Preparing the calibration plate or array card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Prepare the normalization calibration plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Perform the normalization calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 Verify the instrument performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 When to perform the test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 About the RNase P kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Analytical performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Installation specification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Prepare the RNase P plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
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Preparing an array card for instrument verification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 Run the RNase P plate or array card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 How to identify contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 Remove a plate stuck within the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
APPENDIX A Manual instrument operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
Overview of instrument touchscreen functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 Operate the instrument from the touchscreen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Create an experiment from a template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Run an experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 Transfer experiments, templates, and results data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Maintain the instrument from the touchscreen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 Back up and restore the instrument settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 Perform an instrument self test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Update the instrument firmware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Administrate the instrument from the touchscreen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 Define the date and time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Define the instrument settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Define the maintenance reminders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 Define the network settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 Define the system shortcuts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 Review the instrument statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Review the instrument version information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Enable or disable instrument security . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 View the instrument log . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
APPENDIX B Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Decontaminate the sample block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Materials required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 How to handle the sample block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 Clean the sample block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Replace the halogen lamp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 Materials required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Halogen lamp warnings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 View the lamp status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Replace the lamp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Replace the instrument fuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 Materials required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 Replace the fuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Install the QuantStudioTM RealTime PCR Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Computer requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Install the software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

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Software licenses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Activate a license . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Install a license file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Update the QuantStudioTM RealTime PCR Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 Update the software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Update the MicrosoftTM WindowsTM operating system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Change the sample block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 When to change the sample block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Materials required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 How to handle the sample block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 Change the sample block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Change the heated cover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 When to change the heated cover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 Materials required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 How to handle the heated cover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 Change the heated cover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Change the plate adapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 When to change the plate adapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 Materials required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 Change the plate adapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
APPENDIX C Networking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Networking overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 Controlling and monitoring networked QuantStudioTM 6 and 7 Flex Systems . . . . . . 114 About the ethernet port . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 Firewall ports that must be open . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 Example network layouts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 Networking guidelines and best practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Network setup workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Collect the required network information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Connect the QuantStudioTM 6 or 7 Flex System to the network . . . . . . . . . . . . . . . . . . . . . . 118 Materials required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 Define the instrument internet protocol settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Connect the computer to the network . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 Materials required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 Computer requirement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 Collect required information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 Set up the computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 Install the QuantStudioTM RealTime PCR Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Monitor the QuantStudioTM 6 or 7 Flex Real-Time PCR System . . . . . . . . . . . . . . . . . . . . . . 121 About remote monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 Monitor the status of a run from the QuantStudioTM RealTime PCR Software . . . . . 122 Download experiment files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 Enable or change the calibration reminders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

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Contents
APPENDIX D Power on or off, store, and move . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Place the QuantStudioTM 6 or 7 Flex Real-Time PCR System on standby . . . . . . . . . . . . . 125 Power on the QuantStudioTM 6 or 7 Flex Real-Time PCR System . . . . . . . . . . . . . . . . . . . . . 125 Power off the QuantStudioTM 6 or 7 Flex Real-Time PCR System . . . . . . . . . . . . . . . . . . . . 126 Store the QuantStudioTM 6 or 7 Flex System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Materials required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 Prepare the QuantStudioTM 6 or 7 Flex Real-Time PCR System . . . . . . . . . . . . . . . . . . 127 Move the QuantStudioTM 6 or 7 Flex System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 How to handle the sample block and heated cover . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Prepare the QuantStudioTM 6 or 7 Flex System components . . . . . . . . . . . . . . . . . . . . 129 Move the QuantStudioTM 6 or 7 Flex System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 Reinstall the QuantStudioTM 6 or 7 Flex System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
APPENDIX E Security, audit, and electronic signature . . . . . . . . . . . . . . . . . . . . . . . 132
Administrator overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132 Example applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Configure the security system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 Access the Security screen and enable or disable security . . . . . . . . . . . . . . . . . . . . . 133 Set account setup and security policies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 Set up messaging notifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
User account management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 Create or edit user accounts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 Determine the name of the logged-in user . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 Create or edit a user role . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 View or print a user report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
Audit trail management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 Access the audit screen and enable or disable auditing . . . . . . . . . . . . . . . . . . . . . . . . 140 Modify the auditing settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
Audit report generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 Viewing audit histories from the security settings dialog box . . . . . . . . . . . . . . . . . . . 141 Viewing audit histories from an experiment, template, or study . . . . . . . . . . . . . . . . . 144
Manage electronic signature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 Access the e-signature settings screen, and enable or disable the e-signature . . . 145 Configure the meanings of the electronic signatures . . . . . . . . . . . . . . . . . . . . . . . . . . 146 Configure the e-signature rights for user roles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 Select the actions that require signature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 How the software prompts electronic signature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
Generate e-signature reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

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Contents
Export and import user, security, audit, and e-signature settings . . . . . . . . . . . . . . . . . . . . 149 User overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
User account permissions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 Account suspension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 Session time-out . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 User security tasks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
APPENDIX F Calibration consumable preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
Create a background plate or array card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152 Required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152 Create a background plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 Create a background array card (QuantStudioTM 7 Flex System only) . . . . . . . . . . . . . 153
Create a custom dye plate for calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 Before you use custom dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 Required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 Determine optimum dye concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 Create a custom dye plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 Add the custom dye to the software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
APPENDIX G Parts and materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
How to order . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 How to order from the system software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
Kits, consumables, and accessories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 96-well 0.2-mL consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 96-well 0.1-mL consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 384-well consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 TaqManTM Array Card consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 QuantStudioTM 6 and 7 Flex Systems accessories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 QuantStudioTM 6 and 7 Flex Systems empty plates and array cards . . . . . . . . . . . . . . 164
General-use materials and consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
APPENDIX H Instrument specification and layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
QuantStudioTM 6 and 7 Flex Systems specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 Required clearances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 Electrical requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 Environmental requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168 Network requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
QuantStudioTM 6 and 7 Flex Systems layout and connections . . . . . . . . . . . . . . . . . . . . . . . 169

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QuantStudioTM 6 and 7 Flex Real-Time PCR Systems (v1.3) Maintenance and Administration Guide

Contents
APPENDIX I Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
Symbols on this instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 Standard safety symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 Additional safety symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 Location of safety labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174 Control and connection symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 Conformity symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Safety information for instruments not manufactured by Thermo Fisher Scientific . . . . . 176 Instrument safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 Physical injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 Electrical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 Cleaning and decontamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 Instrument component and accessory disposal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 Laser safety–barcode scanner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 Workstation safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 Safety and electromagnetic compatibility (EMC) standards . . . . . . . . . . . . . . . . . . . . . . . . . 179 Safety standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 EMC standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 Environmental design standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181 Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
APPENDIX J Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

QuantStudioTM 6 and 7 Flex Real-Time PCR Systems (v1.3) Maintenance and Administration Guide

9

About this guide
Purpose
This user guide provides reference information for the QuantStudioTM 6 and 7 Flex Real-Time PCR Systems and describes how to prepare, maintain, and troubleshoot the systems.
WARNING! The protection provided by the equipment may be impaired if the instrument is operated outside the environment and use specifications, the user provides inadequate maintenance, or the equipment is used in a manner not specified by the manufacturer (Thermo Fisher Scientific).
Note: For information and instructions on performing experiments on the QuantStudioTM 6 and 7 Flex Real-Time PCR Systems, see the following documents:
· QuantStudioTM RealTime PCR Software Getting Started Guide (Pub. No. 4489822) · High Resolution Melt Module for QuantStudioTM 6 and 7 Flex Real-Time PCR System Software Getting
Started Guide (Pub. No. 4489823)
· QuantStudioTM 6 and 7 Flex Real-Time PCR Systems Quick Reference (Pub. No. 4489826)
Audience
This user guide is written for laboratory staff who operate and maintain the QuantStudioTM 6 and 7 Flex Real-Time PCR Systems.
Assumptions
This guide assumes that your QuantStudioTM 6 and/or 7 Flex System has been installed by a Thermo Fisher Scientific service representative. This guide also assumes that you have:
· Familiarity with MicrosoftTM WindowsTM operating system. · Knowledge of techniques for handling and preparing nucleic acid samples for PCR. · A general understanding of data storage, file transfers, and copying and pasting.

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QuantStudioTM 6 and 7 Flex Real-Time PCR Systems (v1.3) Maintenance and Administration Guide

About this guide User attention words

User attention words
The following user attention words might appear in this document. Each word implies a particular level of observation or action as described below: Note: Provides information that may be of interest or help but is not critical to the use of the product.
IMPORTANT! Provides information that is necessary for proper instrument operation or accurate chemistry kit use.
Safety alert words appear in this document with an open triangle figure that contains a hazard symbol. These hazard symbols are identical to the hazard symbols that are affixed to the instrument. For descriptions of the safety symbols, see Appendix I, “Safety”.

Symbols on this instrument

Symbols may be found on the instrument to warn against potential hazards or convey important safety information. In this document, the symbol is used along with the user attention words to highlight important safety information. The following table gives the meaning of these symbols.

Symbol

English

Français

Caution, risk of danger
Consult the manual for further safety information.

Attention, risque de danger
Consulter le manuel pour d’autres renseignements de sécurité.

Contents of this manual

This guide is divided into the following chapters and appendixes:

Chapter/appendix

Content

Chapter 1, “Getting started”

Introduces the QuantStudioTM 6 and 7 Flex Systems, including descriptions of all related hardware and software.

Chapter 2, “Calibrate the instrument”

Describes how to maintain a QuantStudioTM 6 or 7 Flex Real-Time PCR System, including calibration and performance verification.

Appendix A, “Manual instrument operation”

Describes how to operate the QuantStudioTM 6 and 7 Flex Systems manually using the touchscreen interface.

Appendix B, “Maintenance”

Describes how to replace the user-serviceable parts of the QuantStudioTM 6 and 7 Flex Systems and how to resolve infrequent problems that can occur during normal use.

Appendix C, “Networking”

Describes how to install the QuantStudioTM 6 and 7 Flex Systems to a local area network for remote monitoring and control.

QuantStudioTM 6 and 7 Flex Real-Time PCR Systems (v1.3) Maintenance and Administration Guide

11

About this guide Contents of this manual

(continued) Chapter/appendix

Content

Appendix D, “Power on or Describes how to store, move, and reinstall the components of the QuantStudioTM 6

off, store, and move”

and 7 Flex Systems.

Appendix E, “Security, audit, and electronic signature”

Describes how to configure the security, audit, and electronic signature functions of the QuantStudioTM RealTime PCR Software.

Appendix F, “Calibration consumable preparation”

Describes how to prepare consumables for custom dye calibration.

Appendix G, “Parts and materials”

Describes how to order parts, accessories, and consumables for the QuantStudioTM 6 and 7 Flex Systems.

Appendix H, “Instrument specification and layout”

Describes the QuantStudioTM 6 and 7 Flex System specifications, requirements, layout, and connections.

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QuantStudioTM 6 and 7 Flex Real-Time PCR Systems (v1.3) Maintenance and Administration Guide

1

Getting started

Differences between the QuantStudioTM 6 and 7 Flex Real-Time PCR Systems . . . . . . . . . . . . . . . 13 About the QuantStudioTM 6 and 7 Flex Real-Time PCR Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Instrument hardware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Instrument software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Instrument consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Interchangeable sample blocks, heated covers, and plate retainers . . . . . . . . . . . . . . . . . . . . . . . . . 23

Differences between the QuantStudioTM 6 and 7 Flex RealTime PCR Systems

The following table displays the differences between the QuantStudioTM 6 Flex System and the QuantStudioTM 7 Flex System.

QuantStudioTM 6 Flex System

QuantStudioTM 7 Flex System

Does not support the TaqManTM Array Card block

Supports the TaqManTM Array Card block

Features a coupled five-color filter set

Features a decoupled six-by-six-color filter set

About the QuantStudioTM 6 and 7 Flex Real-Time PCR Systems
The QuantStudioTM 6 and 7 Flex Real-Time PCR Systems use fluorescent-based polymerase chain reaction (PCR) reagents to provide:
· Quantitative research detection of target nucleic acid sequences (targets) using real-time analysis. · Qualitative research detection of targets using post-PCR (endpoint) analysis. · Qualitative analysis of the PCR product (achieved by melt curve analysis that occurs post-PCR).

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Data collection

The QuantStudioTM 6 and 7 Flex Systems collect raw fluorescence data at different points during a PCR, depending on the type of run that the QuantStudioTM 6 and 7 Flex Systems perform:
Table 1

Run type

Real-time runs Standard curve

Relative standard curve

Comparative Ct (Ct) Melting curve

Post-PCR (endpoint) runs

Genotyping Presence/absence

Data collection point The QuantStudioTM 6 and 7 Flex Systems collect data following each extension step of the PCR.
The QuantStudioTM 6 and 7 Flex Systems collect data: 1. Before the PCR. (For presence/absence experiments, data collection before the PCR is optional, but recommended.) 2. (Optional) During the PCR. The QuantStudioTM 6 and 7 Flex System can collect data during the run (realtime); collecting data during the run can be helpful for troubleshooting. 3. After the PCR.

The instrument collects raw fluorescence data following each extension step of the PCR. A data collection point, or read, on the QuantStudioTM 6 and 7 Flex System consists of three phases:

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1

1. Excitation: The instrument illuminates all wells of the plate or array card, within the instrument, exciting the fluorophores in each reaction.
2. Emission: The instrument optics collect the residual fluorescence emitted from the wells of the plate or array card. The resulting image collected by the device consists only of light that corresponds to the range of emission wavelengths.
3. Collection: The instrument assembles a digital representation of the residual fluorescence collected over a fixed time interval. The software stores the raw fluorescent image for analysis.

After a run, the software uses calibration data (ROI, background, uniformity, dye, and normalization) to determine the location and intensity of the fluorescent signals in each read, the dye associated with each fluorescent signal, and the significance of the signal.

Instrument filters and supported dyes

System dyes

The QuantStudioTM 6 Flex System features a coupled five-color filter set while the QuantStudioTM 7 Flex System features a decoupled six-by-six-color filter set that supports the dyes shown in the following table. For more information about the spectral dye calibration kits available for the QuantStudioTM 6 and 7 Flex Systems, contact Thermo Fisher Scientific.

Filter set

Color

Filter wavelength[1]

Excitation

Emission

Supported dye

x1-m1

Blue

470 ± 15 nm

520 ± 15 nm FAMTM dye and SYBRTM Green dye

x2-m2

Green

520 ± 10 nm

558 ± 12 nm

JOETM dye, HEXTM dye, TETTM dye, and VICTM dye[2]

x3-m3

Yellow

549.5 ± 10 nm 586.5 ± 10 nm

CyTM 3 dye, NEDTM dye, and TAMRATM dye

x4-m4

Orange

580 ± 10 nm

623 ± 14 nm

ROXTM dye and Texas RedTM dye

x5-m5

Red

640 ± 10 nm 682 ± 14 nm

CyTM 5 dye and LIZTM dye[2]

x6-m6[3]

Deep red

662 ± 10 nm 711 ± 12 nm

None[4] [1] The central wavelengths are the optimized wavelengths. [2] The HEXTM dye, TETTM dye, and LIZTM dye from Thermo Fisher Scientific fall within the emission wavelength range of the system,
therefore they can be added and adapted for use in experiments on the system. To add any of these dyes to the Dye Library, perform a custom dye calibration. For more information, see Appendix F, “Calibration consumable preparation”. [3] QuantStudioTM 7 Flex System only. [4] This filter set currently does not support any dyes supplied by Thermo Fisher Scientific.

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Wavelength (nm)

Emission Spectra

x1-m1 x2-m2 x3-m3 x4-m4 x5-m5 x6-m6

12

34 5

Filters

1 FAMTM dye and SYBRTM Green 2 VICTM dye and JOETM dye 3 CyTM 3 dye, NEDTM dye, and TAMRATM dye 4 ROXTM dye and Texas RedTM dye 5 CyTM 5 dye

Custom dyes
The QuantStudioTM 6 and 7 Flex Systems can run assays designed with custom dyes (dyes not supplied by Thermo Fisher Scientific) that are excited between 455 to 672 nm and read between 505 to 723 nm. To add a custom dye to the Dye Library, perform a custom dye calibration. For more information, see Appendix F, “Calibration consumable preparation”.

Instrument hardware
The QuantStudioTM 6 and 7 Flex Systems consist of the components shown in the following figure and described in the subsequent table.

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Front
1

1 Chapter 1 Getting started Instrument hardware 4

7 2
8
3 9

Component 1 Touchscreen
2 USB ports
3 Access door 4 Lamp 5 Heated cover 6 Sample block 7 Side door 8 Plate adapter 9 Tray arm

5

6

Description
Provides access to the instrument functions. Can be used to run experiment, transfer data, and operate the instrument functions without the use of the computer.
Provide USB communication with the instrument. Can be used to transfer data to and from the instrument. Note: If multiple USB drives are plugged into the instrument, the instrument mounts only the first drive that is installed, regardless of the USB port used.
Provides access to the instrument lamp, the heated cover, and the sample block.
Illuminates the plate or array card during a run.
Covers the plate or array card during a run to prevent condensation and leakage through the cover.
Heats the plate or array card during a run.
Opens to allow extension of the tray arm.
Secures the plate or array card to the tray arm.
Conveys the plate or array card to and from the sample block in the interior of the instrument.

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1 Chapter 1 Getting started Instrument hardware
Rear
4

1

2

3

Ethernet

USB 3

USB

RS232

4

Component 1 Ethernet port 2 USB ports
3 RS232 port 4 Instrument fans 5 On/Off switch

Ethernet U1SB

U2SB

RS232

PWR F1 F2

5
PWR

F1

6

F2
7

Description An RJ45 port that provides Ethernet (gigabit) communication with the instrument.[1] Provide USB communication with the instrument. USB ports can be used to transfer data from the instrument. Note: If multiple USB drives are plugged into the instrument, the instrument mounts only the first drive that is installed, regardless of the USB port used.
Provides serial communication between the instrument and the computer.
IMPORTANT! The serial port is for Thermo Fisher Scientific use only.
Cool the interior of the instrument.
IMPORTANT! The fans must be unobstructed to ensure adequate cooling and proper function of the instrument.
Power switch for the instrument, where the states are on ( | ) or off ( O ).

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(continued) Component

Description

6 Fuse cover

Dual 12.5A, Time-Lag T, 250 VAC, 5 × 20mm electrical fuses that protect the instrument from excessive electrical current.
For AU/NZ only – 6.3A, Time-Lag, 250VAC, 5 × 20mm.

7 Power port

The 100­240 VAC port that provides power to the instrument.

[1] Use the Ethernet cable supplied with the instrument to connect the instrument (Ethernet port) to the network interface card in the computer.

Barcode scanner

The QuantStudioTM 6 and 7 Flex Systems include an optional Handheld Barcode Scanner (Cat. No. 4453271) that you can purchase to assist you with data entry. The barcode scanner uses a 670 nm Class II laser to scan plates or array cards (for only the QuantStudioTM 7 Flex System) and it is capable of reading Code 128 (alphanumeric), which supports 128 ASCII character barcodes.

WARNING! LASER. Exposure to direct or reflected laser light can burn the retina and leave permanent blind spots. Never look into the laser beam. Remove jewelry and anything else that can reflect the beam into your eyes. Protect others from exposure to the beam.

The optional hand-held barcode scanner functions as an extension of the keyboard. You can use the scanner to scan barcodes into the instrument software.
To scan a barcode using the hand-held barcode scanner:
1. Select the field in the instrument software where you want to enter the barcode.
2. Hold the hand-held barcode scanner 20­30 cm away from a plate or array card and aim at the center of the barcode, then press the trigger. Slowly move the scanning beam across the barcode until the scanner emits a high-pitched tone.
When the scanner scans a barcode, it automatically: · Transmits the alphanumeric equivalent of the barcode to the instrument software. The software enters the barcode text wherever the cursor is active. · Transmits a carriage-return character (the equivalent of pressing the Enter key).
For more information on the hand-held barcode scanner, see the user documentation shipped with the barcode scanner.

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Electrical protective devices
We recommend several protective devices to protect the instrument in environments with large voltage and power fluctuations.
Power line regulator
We recommend the use of a 1.5kVA power line regulator in areas where the supplied power fluctuates in excess of ± 10% of the normal voltage. Power fluctuations can adversely affect the function of the instrument and the computer.
Note: A power line regulator monitors the input current and adjusts the power supplied to the instrument or computer. It does not protect against a power surge or failure.
Uninterruptible power supply (UPS)
We recommend the use of a 1.5kVA uninterruptible power supply (UPS), especially in areas prone to power failure. Power failures and other events that abruptly terminate the function of the instrument and the computer can corrupt data and possibly damage the system.
WARNING! PHYSICAL INJURY HAZARD. Do not attempt to lift the UPS unit without assistance (minimum of two people). Improper lifting can cause painful and permanent back injury. Refer to the UPS manufacturer user guide for more information.
IMPORTANT! UPSs provide power for a limited time. They are meant to delay the effects of a power outage, not to serve as replacement power sources. In the event of a power loss, power off the instrument and the computer, unless you expect to regain power within the battery life of the UPS.
Surge protector
We recommend the use of a 10kVA surge protector (line conditioner) in areas with frequent electrical storms or near devices that are electrically noisy, such as refrigerators, air conditioners, or centrifuges. Short-duration, high- voltage power fluctuations can abruptly terminate the function of, and thereby damage the components of, the computer and the instrument.
Note: A dedicated line and ground between the instrument/computer and the building’s main electrical service can also prevent problems caused by power fluctuations.
Instrument software
The QuantStudioTM 6 and 7 Flex Systems include a suite of software applications that can be used to calibrate, run, automate, and integrate the instrument into a laboratory workflow. The installation of the software contains the components described in the following sections; however, additional software may be available for the QuantStudioTM 6 and 7 Flex Systems. Go to thermofisher.com/qpcrsoftware for a complete list of compatible software, and for updates and patches for the system software and firmware.

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Computer requirements

The requirements for the computer used to operate the QuantStudioTM 6 and 7 Flex Systems can vary depending on the version of the QuantStudioTM RealTime PCR Software that you are running. To determine the computer requirements for your system, check the software release notes at thermofisher.com/qpcrsoftware. The release notes are on the same page as the link for the software download.

Software installation

The default installation of the instrument partitions the computer hard drive to create the logical drives as explained below.

Drive

Software

Description

C drive MicrosoftTM WindowsTM OS[1]

Operating system files.

QuantStudioTM RealTime PCR Software C or D drive

Used to calibrate and perform experiments on the QuantStudioTM 6 and 7 Flex System.
Stores the installation files, application data files, and user files (experiments, exports, and imports).

QuantStudioTM 6 and 7 Flex System Command Line Utility
[1] We recommend that you do not install programs to the C drive.

Used to automate the creation of new experiments and the export of existing experiments.

Third-party software

Before you install third-party software to the computer running the QuantStudioTM RealTime PCR Software, confirm that the software will not restrict Ethernet communication or interfere with the software operation.

Third-party software
1. Install the third-party software to the computer that contains the QuantStudioTM RealTime PCR Software.
2. Perform several test experiments using plates that do not contain reagents.
Note: The goal of the test experiments is to run plates under conditions that match normal instrument operation. Therefore, the characteristics of the test experiments (plate layout and run method) must closely resemble your actual experiments.

3. Confirm that the QuantStudioTM 6 and 7 Flex Systems perform each test experiment without producing errors.
If the systems perform the tests successfully, perform experiments normally. If the systems encounter errors during the test runs, the software may not be compatible with the QuantStudioTM RealTime PCR Software.

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1 Chapter 1 Getting started Instrument consumables

Instrument consumables
The QuantStudioTM 6 and 7 Flex Systems support a series of specialized consumables through interchangeable sample blocks. Use the consumables appropriate for the sample block of your QuantStudioTM 6 or 7 Flex Systems.
Note: The QuantStudioTM 6 Flex System does not support the Array Card block.

WARNING! Use the flat caps for 0.2 mL tubes and 0.1 mL tubes. Rounded caps can damage the heated cover.

Sample block 96-well plate, 0.2 mL

Consumable
· MicroAmpTM Optical 8-Cap Strip · MicroAmpTM 8-Tube Strips (0.2mL) · MicroAmpTM Reaction Tubes without Caps
(0.2mL) · MicroAmpTM 96-Well Tray/Retainer Set

Reaction volume
100 µL

· MicroAmpTM Optical Adhesive Film
· MicroAmpTM Optical 96-Well Reaction Plate with Bar Code

100 µL

96-well plate, 0.1 mL

· MicroAmpTM Optical Adhesive Film
· MicroAmpTM Optical 96-Well Fast Reaction Plate with Bar Code

50 µL

384-well plate

· MicroAmpTM Optical Adhesive Film
· MicroAmpTM Optical 384-Well Reaction Plate with Bar Code

20 µL

TaqManTM Array Card[1]

TaqManTM Array Card

1 µL

[1] QuantStudioTM 7 Flex System only.

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Interchangeable sample blocks, heated covers, and plate retainers
The QuantStudioTM 6 and 7 Flex Systems feature a set of interchangeable sample blocks, heated covers, and plate retainers that allow the instruments to support multiple consumable formats. All of the hardware is user-installable. Replacement can be done without the need for recalibration of the instrument, provided that the calibrations for the new format have not expired. See Appendix B, “Maintenance” for more information about changing the sample block, heated cover, and plate retainer.

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2

Calibrate the instrument

Calibration overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Enable or change the calibration reminders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Guidelines for preparing calibration plates and array cards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Preparing array cards for instrument calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 ROI calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Background calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 Uniformity calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Dye calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Normalization calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Verify the instrument performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
IMPORTANT! Perform the procedures for preparing array cards only if you are verifying the performance of a QuantStudioTM 7 Flex System with an array card sample block. The QuantStudioTM 6 Flex System does not support the array card sample block.

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2 Chapter 2 Calibrate the instrument Calibration overview

Calibration overview
Calibration and maintenance schedule
The QuantStudioTM 6 and 7 Flex Systems require regular calibration and maintenance for proper operation. To ensure proper operation of your instrument, perform weekly, monthly, and semiannual maintenance as indicated in the following table.
IMPORTANT! Perform calibrations and run experiments under the environmental conditions specified in Appendix H, “Instrument specification and layout”. Exposure to extreme temperatures can have adverse effects on the run results, as well as shortening the life span of the instrument components.
IMPORTANT! Do not use organic solvents to clean the QuantStudioTM 6 and 7 Flex Systems.

Frequency

User-performed maintenance task

Check the computer disk space. If necessary, archive or back up your experiment files and instrument settings.

Weekly

Power off the computer that controls the instrument, then power on the computer after 30 seconds.

Clean the surface of the instrument with a lint-free cloth. Perform a background calibration.[1]

Monthly

Run disk cleanup and disk defragmentation.

Perform an instrument self test.

Semi-annually (every 6 months)

Perform an ROI calibration. Perform a background calibration.[1] Perform a uniformity calibration. Perform a dye calibration.

Perform a normalization calibration.

As needed

Perform an RNase P instrument verification run. Replace the instrument lamp.[2] [1] You can perform a background calibration to check for contamination. [2] After replacing the instrument lamp, perform all calibrations and an RNase P instrument verification run.

Note: Normalization calibration is required for all block types with QuantStudioTM RealTime PCR Software v1.3.

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2 Chapter 2 Calibrate the instrument Calibration overview
Planned maintenance
The instrument requires regular planned maintenance by Thermo Fisher Scientific service personnel to ensure that the instrument is operating within the installation specification. The planned maintenance service can help you maximize your productivity by minimizing downtime, lowering the cost of ownership by addressing potential problems before they manifest (lost data, samples, or reagents), and increasing the quality of your results by maintaining your instrument at optimal performance. For more information, contact your Thermo Fisher Scientific sales representative.
About maintenance reminders
The QuantStudioTM RealTime PCR Software features an automated notification system that alerts you of required calibrations. The feature will also prevent you from operating an instrument that is out of calibration. For more information on setting the maintenance reminders, see “Enable or change the calibration reminders” on page 29.
Calibration workflow
Whether you are performing all calibrations or just a subset, perform calibrations for the QuantStudioTM 6 and 7 Flex Systems in the following sequence.
Start
Perform an ROI calibration (page 37)
Perform a background calibration (page 42)
Perform a uniformity calibration (page 46)
Perform dye calibrations (page 49)
Perform a normalization calibration (page 55)
Perform an RNase P instrument verification test (page 59)
Finish
Note: Normalization calibration is required for all block types with QuantStudioTM RealTime PCR Software v1.3.

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View the calibration status of an instrument
You can use the Instrument Console of the QuantStudioTM RealTime PCR Software to view the calibration status of an instrument.
1. In the QuantStudioTM RealTime PCR Software Home tab, click Instrument Console. 2. From the Instrument Console open the Instrument
Manager. a. Select your instrument icon from the My Instruments group.
b. Click Manage Instrument.
Note: If the Manage Instrument option is inactive, connect your instrument to the My Instruments group.
3. From the Instrument Manager, click Monitor, then click Information to open the instrument monitor. The instrument monitor screen displays the following information: a. The Instrument type (the QuantStudioTM 6 Flex System or the QuantStudioTM 7 Flex System)
b. The status of all calibrations, including the date and time of each calibration
c. The maintenance status of the instrument
d. The properties of the instrument

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Note: While viewing the Monitor screen, you can generate reports of the calibration history and status for the instrument. The following options are available from the Instrument Manager:
· Print Calibration Log–Prints the calibration history of the instrument. · Print Maintenance Summary–Prints a summary of the current instrument calibrations.
Note: The details for an instrument in the My Preferred list can be exported even if the network connection has been interrupted. The exported details from the disconnected instrument would contain the data most recently downloaded from the instrument before the interruption.

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Enable or change the calibration reminders
Enable or change the calibration reminders
The calibration reminder settings allow you to configure the QuantStudioTM RealTime PCR Software to alert you by email when the QuantStudioTM 6 or 7 Flex Real-Time PCR System requires calibration. The notifications settings feature is optional, and it does not affect instrument performance.
IMPORTANT! The calibration reminders feature requires a connection between the QuantStudioTM 6 or 7 Flex Real-Time PCR System computer and a computer network. If you choose to set up the feature as described below, ensure that your network has appropriate security measures in place to protect data from threats such as computer viruses.
IMPORTANT! The QuantStudioTM RealTime PCR Software transmits emails only while the QuantStudioTM 6 or 7 Flex Real-Time PCR System is monitored. If the network connection is interrupted, the software will stop transmitting updates.
Requirements to email calibration reminders
The QuantStudioTM RealTime PCR Software requires access to a Simple Mail Transfer Protocol (SMTP) server to email calibration reminders. Contact your systems administrator or information technology department for the following information:
· Network address of an SMTP server. · A user name and password for the server, if required for access. · The Secure Sockets Layer (SSL) setting of the server (on or off).
Define the mail server settings
1. In the QuantStudioTM RealTime PCR Software, select Tools4Preferences.
2. From the Preferences dialog box, select the SMTP Settings tab.
3. Define the settings for the SMTP server: a. From the Outgoing Mail Server (SMTP) field, enter the network address of a Simple Mail Transfer Protocol (SMTP) server. Optionally, you can specify the transmission control protocol (TCP) port for the server by appending the port number to the server name, separating the two using a colon (:). For example: smtp.mycompany.com:2023
Note: If a TCP port is not specified, the software uses the default port number (25).
b. Select Encryption Required if the mail server has SSL enabled.
c. Select Authentication Required if the mail server requires a user name and password.

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2 Chapter 2 Calibrate the instrument Enable or change the calibration reminders
d. If the mail server requires authentication, enter the user name and password provided by your systems administrator.

4. Click OK.
5. Test the mail server settings: a. In the Home tab of the QuantStudioTM RealTime PCR Software, select any experiment in the Set Up panel, then click Set Up to create a new experiment.
b. In the experiment document, click Run in the Experiment Menu, then click Notification Setting to view the notification settings for the experiment.
c. In the Notification Settings of the experiment document, select Yes to enable the Enable Notifications setting.
d. From the Enter e-mail addresses for notifications field, enter the email address that you want to receive the test emails.

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Enable or change the calibration reminders
e. Click Test Configuration to test the mail server settings. Confirm that the email account you specified in substep 5d received the test messages from the QuantStudioTM RealTime PCR Software. If not, consult your network administrator to confirm that the mail server settings are defined correctly.

Modify the notification settings for a monitored instrument
1. Open the Calibration Reminders screen for the QuantStudioTM 6 or 7 Flex Real-Time PCR System: a. In the QuantStudioTM RealTime PCR Software Home tab, click Instrument Console.
b. In the Instrument Console, select the icon for your instrument, then click Manage Instrument.
Note: If the Manage Instrument button is inactive, add your instrument to the My Instruments group.
c. In the Instrument Manager, click Maintenance, then click Calibration Reminders.
2. In the Calibration Reminders Setting table, configure the notification settings for the calibrations in interest. For each calibration that you want to monitor: a. In the Send a Reminder column, select the check box to configure the QuantStudioTM RealTime PCR Software to email a reminder to perform the calibration.
b. In the Reminder Interval column, enter the number of days before the calibration expires that the software should send a reminder email.
3. In the Enter e-mail addresses for notifications field, enter the email address(es) that you want to receive notifications. Separate multiple email addresses with commas ( , ).
4. Click Apply to change the notification settings.

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2 Chapter 2 Calibrate the instrument Guidelines for preparing calibration plates and array cards

Guidelines for preparing calibration plates and array cards

Observe the following guidelines when preparing calibration plates and array cards: · Wear appropriate protective eyewear, clothing, and gloves. · Prepare and run calibration plates and array cards within the recommended time limits listed below.

Consumable

Time to thaw

After thawed, run within…

All 96-/384-well calibration consumables[1] All array card calibration consumables[2]

30 minutes 30 minutes

120 minutes 120 minutes

RNase P plate (96-/384-well) RNase P array card[3]

5 minutes 15 minutes

30 minutes 60 minutes

[1] All 96-well plates and 384-well plates used in the calibration of the instrument (ROI, background, uniformity, dye, and normalization).
[2] All array cards used in the calibration of the QuantStudioTM 7 Flex System only (ROI, background, uniformity, dye, and normalization).
[3] For use with QuantStudioTM 7 Flex System only.
· (Plates only) Do not discard the packaging for the calibration plates. Each plate can be used to calibrate the QuantStudioTM 6 and 7 Flex Systems 3 times for up to 6 months if it is stored in its packing sleeve.
· Store calibration plates or array cards in a dark place until you are ready to use them. The fluorescent dyes in the wells of calibration consumables are photosensitive. Prolonged exposure to light can diminish the fluorescence of the dyes.

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Chapter 2 Calibrate the instrument Preparing array cards for instrument calibration

2

· Do not allow the bottoms of the plates or array cards to become dirty. Fluids and other contaminants that adhere to the bottoms of the consumables can contaminate the sample block and cause an abnormally high background signal.
· Confirm that your centrifuge is clean. Before centrifugation, wipe down the bucket(s) using a tissue. · Vortex and centrifuge all calibration plates to ensure complete mixing and that all reagents are
contained at the bottom of the wells. The calibration plates must be well mixed and centrifuged before use.
Preparing array cards for instrument calibration
IMPORTANT! Perform the following procedure only if you are verifying the performance of a QuantStudioTM 7 Flex System with an array card sample block. The QuantStudioTM 6 Flex System does not support the array card sample block.
Required materials
· ViiATM 7 Array Card Spectral Calibration Kit ­ Empty TaqManTM Array Cards ­ FAMTM dye ­ VICTM dye ­ ROXTM dye ­ ROI dye mix ­ Background buffer ­ FAMTM/ROXTM dye ­ VICTM/ROXTM dye
· TaqManTM Array Card Sealer · Centrifuge with array card buckets and array card carrier clips · Permanent marker or pen · Pipettor, 200-µL (with pipette tips) · Powder-free gloves · Safety glasses

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2 Chapter 2 Calibrate the instrument Preparing array cards for instrument calibration

Fill the calibration TaqManTM Array Cards

IMPORTANT! Wear powder-free gloves while creating the calibration array cards.

This procedure explains how to create all of the array cards required to calibrate the QuantStudioTM 7 Flex System, but not all of them are required for a monthly maintenance. Before preparing array cards for calibration, determine which calibrations are required (see “Calibration and maintenance schedule” on page 25).
Note: For more information on the array card loading procedure, contact Technical Support.

1. Remove the tubes of calibration solutions from the freezer, allow them to thaw, then vortex the tubes to mix the contents well.
2. Remove the array cards from their box and place them on a clean, dry surface.
3. Mark the side of the empty array cards with: Background, FAMTM, ROI, ROXTM, VICTM, FAMTM/ROXTM, and VICTM/ROXTM.

4. For each array card, pipet 100 µL of the appropriate

calibration solution into each of the eight reservoirs in

the array card:

a. Place the array card on a lab bench, with the foil

1

side down.

1 Mark here

b. Load 100 µL of the calibration solution into a pipette.

c. Hold the pipette in an angled position (approximately 45 degrees) and place the tip into the fill port. The fill port is the larger of the two holes on the left side of the fill reservoir.

1

2

1 Fill port 2 Vent port

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Preparing array cards for instrument calibration
d. Dispense the fluid so that it sweeps in and around the fill reservoir toward the vent port. Pipet the entire 100 µL into the fill reservoir, but do not go past the first stop of pipettor plunger or you may blow the solution out of the port. IMPORTANT! Do not allow the tip to contact and possibly damage the coated foil beneath the fill port.
5. Repeat step 4 to fill the remaining array card with the appropriate calibration reagents.
6. Centrifuge the array card(s): a. Place the filled array card(s) into a centrifuge array card carrier clip and place empty array cards in the remaining slots. Confirm that the labels on the buckets and clips are oriented in the same direction.
b. Place the filled carrier clips into the centrifuge buckets. Make sure that the array card fill reservoirs and bucket and clip labels face outward when loaded into the centrifuge. IMPORTANT! You must run the centrifuge with all four buckets in place and each of the two carriers filled with array cards. Place empty array cards into unfilled slots.
IMPORTANT! Balance the loads in opposite buckets in the centrifuge.

c. Close the centrifuge cover, then spin the array card(s) for 1 minute at 1200 rpm.
d. When the run is finished, stop the centrifuge, then spin the array card(s) again for 1 minute at 1200 rpm. IMPORTANT! Do not try to save time by doing one spin for 2 minutes. The two sets of ramps are important for a good fill into the array card.

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2 Chapter 2 Calibrate the instrument Preparing array cards for instrument calibration

7. When the second run is finished, open the centrifuge and check that the fluid levels in the reservoirs of each array card have decreased by the same amount. Also, check all wells for bubbles and note the locations as possible problems.

Correct fill

Incorrect/partial fill

If necessary, centrifuge the array cards for an additional minute to fill any unfilled wells. Do not exceed three 1minute runs or centrifuge the array card for longer than 1 minute at a time. 8. Seal the array card(s):
a. With the carriage (roller assembly) of the TaqManTM Array Card Sealer in the Start position, place a filled array card into the fixture with the foil side up so that the fill reservoirs are the farthest away from the carriage.
b. Press down on all four corners of the array card to ensure that it is fully seated within the fixture.
c. Use the two alignment pins in the fixture to position the array card correctly.

d. Seal the array card by running the carriage slowly over it. Run the carriage over the array card in one direction only. Do not apply downward force on the carriage as you move it forward over the card.

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e. Remove the sealed array card from the fixture and trim the fill reservoirs from the array card assembly using scissors. Trim the foil array card so that the edge is even with the plastic carrier.
IMPORTANT! Completely remove the fill reservoirs from the array card so that the edge is free of residual plastic. The plastic from the fill reservoirs that extends beyond the edge of the card can prevent the array card from seating properly on the sample block and can affect amplification.
Correct trim

Incorrect trim

9. Repeat step 8 to seal the remaining array cards.
IMPORTANT! If an array card is sealed improperly, it can leak and contaminate the sample block and/or it can cause the associated calibration or RNase P test to fail.
IMPORTANT! As you seal the remaining filled array cards, store them in a dark place. Do not expose the array cards to light until you are ready to use them. The dyes in the array cards are photosensitive. Prolonged exposure to light can diminish the fluorescence of the dye.
ROI calibration
A region of interest (ROI) calibration maps the positions of the wells on the sample block of the instrument. The software uses the ROI calibration data to associate increases in fluorescence during a run with the wells on the plate or array card. The instrument uses a set of optical filters to distinguish the fluorescence emissions gathered during runs. The software generates a calibration image for each filter to account for minor differences in the optical path.
When to perform the calibration
Perform the ROI calibration once every 6 months.
IMPORTANT! After every ROI calibration, you must perform (in order): a background calibration, uniformity calibration, dye calibration, and a normalization calibration.

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2 Chapter 2 Calibrate the instrument ROI calibration

About the calibration data

During the ROI calibration, the instrument software captures images of the ROI calibration plate or array card at each instrument filter. An ROI calibration passes if the collected image for each filter distinguishes all wells of the ROI plate or array card. Each well in the image must be distinct and visible at the same luminosity relative to the other wells in the image.
Note: You can review the ROI calibration image for each filter set by selecting the desired filter combination from the Filter Set menu of the ROI tab in the Instrument Manager.

Passing image

Failing image

Green circles around all wells indicate that the wells calibrated successfully. Each green circle indicates that the region of interest for the well position is sufficiently bright.

Red circles around some of the wells indicate that the wells did not calibrate. The absence of a circle indicates that the region of interest for the well position is not sufficiently bright.

Preparing the calibration plate or array card

IMPORTANT! Wear powder-free gloves and safety glasses when you prepare plates or array cards.

Prepare the calibration consumable appropriate for your QuantStudioTM 6 or 7 Flex System: · “Prepare the ROI calibration plate” on page 38 · “Preparing array cards for instrument calibration” on page 33
Prepare the ROI calibration plate
Materials required
· Region of Interest (ROI) Plate · Centrifuge with plate adapter · Powder-free gloves · Safety goggles

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Prepare the plate
IMPORTANT! Wear powder-free gloves and safety glasses when you prepare the plate.
1. Remove the ROI calibration plate from the freezer, then thaw it at room temperature for approximately 30 minutes. IMPORTANT! Use the calibration plate within 2 hours of defrosting it. Until you are ready to run the plate, store it in the dark and at ambient temperature (15­30). Do not remove the calibration plate from its packaging until you are ready to run it. The fluorescent dyes in the wells of the plate are photosensitive. Prolonged exposure to light can diminish the fluorescence of the dyes.
2. Remove the ROI plate from its packaging. Do not remove the optical film. 3. Vortex and centrifuge the plate:
a. Vortex the ROI calibration plate for 5 seconds. b. Centrifuge the plate for 2 minutes at < 1500 rpm.
IMPORTANT! The ROI calibration plate must be well mixed and centrifuged.
c. Confirm that the liquid in each well of the ROI calibration plate is at the bottom of the well. If not, centrifuge the plate again at a higher rpm and for a longer period of time.

Perform the ROI calibration
1. In the QuantStudioTM RealTime PCR Software Home tab, click Instrument Console. 2. In the Instrument Console, select the icon for your instrument, then click Manage Instrument.
Note: If the Manage Instrument button is inactive, add your system to the My Instruments group.
3. In the Instrument Manager, start the calibration: a. Click Maintenance, then click ROI.
b. In the ROI Calibration screen, click Start Calibration. 4. Click Next, then prepare for the calibration as instructed.

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2 Chapter 2 Calibrate the instrument ROI calibration 5. From the bottom of the Setup tab, enter the reagent information for the ROI plate or array card that you are using.
6. Load the calibration plate or array card into the instrument: a. From the instrument touchscreen, tap to eject the instrument tray. b. Load the plate or array card into the plate holder so that: · Well A1 of the plate or array card is in the top-left corner of the plate adapter. · The barcode faces the front of the instrument.

IMPORTANT! Plates and array cards should be loaded and unloaded by operators who have been warned of the moving parts hazard and have been adequately trained.
c. From the instrument touchscreen, tap to close the instrument tray.
7. After loading the plate or array card, start the calibration: a. At the bottom of the Setup tab, select Check the box when the ROI calibration plate has been loaded, then click Next.
b. In the Run screen, click Start run.

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IMPORTANT! Do not attempt to open the access door during the run. The door is locked while the instrument is in operation.

Note: Before starting the calibration, the instrument may pause (up to 10 minutes) to allow the heated cover to reach the required temperature.

8. When the run is complete and the software displays the Analysis screen, select each filter from the Filter Set drop-down list, then confirm that the corresponding ROI Image displays a green circle
around each well area.

9. After you inspect all ROI images, confirm the status of the calibration, where passed indicates that the run produced viable calibration data, and failed indicates that the run did not produce data or that the data it collected is unusable.

Analysis status Passed Failed

Action Click Next. Troubleshoot the failed calibration (see “Troubleshooting” on page 71).

10. Unload the calibration plate or array card: a. After the calibration, tap on the touchscreen to eject the plate or array card.
Note: If the instrument does not eject the plate or array card, remove it as explained in “Troubleshooting” on page 71.

b. Remove the ROI calibration plate or array card from the instrument tray and store it in the dark at room temperature until you are ready to perform the uniformity calibration.

WARNING! PHYSICAL INJURY HAZARD. During instrument operation, the plate or array card temperature can reach 100°C. Allow the consumable to cool to room temperature before removing.
c. Tap on the instrument touchscreen to close the instrument tray.
11. In the ROI Calibration screen, click Finish to complete the calibration, then click Yes when prompted to save the results.
Note: If the QuantStudioTM RealTime PCR Software displays the warning Saving ROI calibration will invalidate other existing calibrations except HRM Calibrations, click OK to proceed.
Note: HRM (High Resolution Melt) is a separately licensed module. If your instrument requires this module, contact Thermo Fisher Scientific.
12. (Optional) Click Print Report in the upper right corner of the screen to print a summary of the calibration results for your records.

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2 Chapter 2 Calibrate the instrument Background calibration
Background calibration
The software uses the background calibration data during subsequent experiments to remove background fluorescence from the experiment data.
When to perform the calibration
Perform the background calibration monthly.
About the calibration data
During the background calibration, the software captures a series of images of the background plate or array card using each instrument filter. The software compares the fluorescence from each well to the average for the plate or array card. A background calibration passes if the collected images for all filters are free of abnormal fluorescence.
Background fluorescence
Fluorescence data collected by the instrument includes a fluorescence signal inherent to the instrument, referred to as “background fluorescence.” Background fluorescence is a composite signal found in all spectral data that consists of fluorescence from several sources, including:
· Background electronic signal · Contaminants in the sample block · The plastic consumable (plate or array card)
Preparing the calibration plate or array card
IMPORTANT! Wear powder-free gloves and safety glasses when you prepare plates or array cards.
Prepare the calibration consumable appropriate for your QuantStudioTM 6 or 7 Flex System: · “Prepare the background calibration plate” on page 42 · “Preparing array cards for instrument calibration” on page 33
Prepare the background calibration plate
Materials required
· Background Plate · Centrifuge with plate adapter · Powder-free gloves · Safety goggles

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Prepare the plate
IMPORTANT! Wear powder-free gloves and safety glasses when you prepare the plate.
1. Remove the background calibration plate from the freezer, then thaw it at room temperature for approximately 30 minutes. IMPORTANT! Use the calibration plate within 2 hours of defrosting it. Until you are ready to run the plate, store it in the dark and at ambient temperature (15­30°C). Do not remove the plate from its packaging until you are ready to run it.
2. Remove the background calibration plate from its packaging. Do not remove the optical film. 3. Vortex and centrifuge the plate:
a. Vortex the background calibration plate for 5 seconds.
b. Centrifuge the plate for 2 minutes at <1500 rpm. IMPORTANT! The background calibration plate must be well mixed and centrifuged.
c. Confirm that the liquid in each well of the background calibration plate is at the bottom of the well. If not, centrifuge the plate again at a higher rpm and for a longer period of time. IMPORTANT! Do not allow the bottom of the plate to become dirty. Fluids and other contaminants that adhere to the plate bottom can contaminate the sample block and cause an abnormally high background signal.

Perform the background calibration
1. In the QuantStudioTM RealTime PCR Software Home tab, click Instrument Console. 2. In the Instrument Console, select the icon for your instrument, then click Manage Instrument.
Note: If the Manage Instrument button is inactive, add your instrument to the My Instruments group.
3. In the Instrument Manager, start the calibration: a. Click Maintenance, then click Background.

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2 Chapter 2 Calibrate the instrument Background calibration b. In the Background Calibration screen, click Start Calibration. 4. Click Next, then prepare for the calibration as instructed. 5. At the bottom of the Setup tab, enter the reagent information for the background plate or array card that you are using.
6. Load the calibration plate or array card into the instrument: a. From the instrument touchscreen, tap to eject the instrument tray. b. Load the calibration plate or array card into the plate holder so that: · Well A1 of the plate or array card is in the top-left corner of the plate adapter. · The barcode faces the front of the instrument.

IMPORTANT! Plates or array cards should be loaded and unloaded by operators who have been warned of the moving parts hazard and have been adequately trained.
c. From the instrument touchscreen, tap to close the instrument tray.
7. After loading the plate or array card, start the calibration: a. In the Setup tab, select Check the box when the background calibration plate has been loaded, then click Next.

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b. From the Run screen, click Start Run.

IMPORTANT! Do not attempt to open the access door during the run. The door is locked while the instrument is in operation.

Note: Before starting the calibration, the instrument may pause (up to 10 minutes) to allow the heated cover to reach the required temperature.

8. When the run is complete and the QuantStudioTM RealTime PCR Software displays the Analysis screen, confirm the status of the calibration. The analysis status indicates the success of the calibration, where passed indicates that the run produced viable calibration data, and failed indicates that the run did not produce data or that the data it collected is unusable.

Analysis status

Action

Passed

Click Next.

Failed

Troubleshoot the failed calibration (see “Troubleshooting” on page 71).

Note: Abnormal spectra or abnormally high background fluorescence can indicate the presence of contamination on the plate or array card or sample block, which can cause the calibration to fail.
9. Select the QC tab to review the quality check summary. The QC status indicates the quality of the calibration data, where Passed indicates that all wells produced data that passed the quality check, and Failed indicates that one or more wells produced spectra that deviate significantly from the other wells on the plate or array card.
Note: If the calibration failed, the QC tab can provide information regarding the cause of the failure.
10. Unload the calibration plate or array card: a. After the calibration, tap on the touchscreen to eject the plate or array card.
Note: If the instrument does not eject the plate or array card, remove it as explained in “Troubleshooting” on page 71.
b. Remove the plate or array card from the instrument tray and discard it.

WARNING! PHYSICAL INJURY HAZARD. During instrument operation, the plate or array card temperature can reach 100°C. Allow the consumable to cool to room temperature before removing.
c. Tap on the instrument touchscreen to close the instrument tray.
11. From the Background Calibration screen, click Finish to complete the calibration, then click Yes when prompted to save the results.
12. (Optional) Click Print Report in the upper right corner of the screen to print a summary of the calibration results for your records.

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2 Chapter 2 Calibrate the instrument Uniformity calibration
Uniformity calibration
The uniformity calibration generates data that allows the software to compensate for the physical effects of the instrument filters.
When to perform the calibration
Perform a uniformity calibration once every 6 months.
Preparing the calibration plate or array card
IMPORTANT! Wear powder-free gloves and safety glasses when you prepare plates or array cards.
Prepare the calibration consumable appropriate for your QuantStudioTM 6 or 7 Flex System: · “Prepare the ROI calibration plate” on page 46 · “Preparing array cards for instrument calibration” on page 33
Prepare the ROI calibration plate
Materials required
Note: The uniformity calibration requires the same materials used to perform the ROI calibration. · Region of Interest (ROI) Plate · Centrifuge with plate adapter · Powder-free gloves · Safety goggles
Prepare the plate
IMPORTANT! Wear powder-free gloves and safety glasses when you prepare the plate.
If you have a prepared ROI calibration plate from a recent ROI calibration, go to step 3b; otherwise, prepare the ROI calibration plate as follows:
1. Remove the ROI calibration plate from the freezer, then thaw it at room temperature for approximately 30 minutes. IMPORTANT! Use the calibration plate within 2 hours of defrosting it. Until you are ready to run the plate, store it in the dark and at ambient temperature (15­30°C). Do not remove the plate from its packaging until you are ready to run it.
2. Remove the ROI plate from its packaging. Do not remove the optical film. 3. Vortex and centrifuge the plate:
a. Vortex the ROI calibration plate for 5 seconds.

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b. Centrifuge the plate for 2 minutes at < 1500 rpm. IMPORTANT! The ROI calibration plate must be well mixed and centrifuged.
c. Confirm that the liquid in each well of the ROI calibration plate is at the bottom of the well. If not, centrifuge the plate again at a higher rpm and for a longer period of time.

Perform the uniformity calibration
1. In the QuantStudioTM RealTime PCR Software Home tab, click Instrument Console. 2. In the Instrument Console, select the icon for your instrument, then click Manage Instrument.
Note: If the Manage Instrument button is inactive, add your instrument to the My Instruments group.
3. From the Instrument Manager, start the calibration: a. Click Maintenance, then click Uniformity.
b. From the Uniformity Calibration screen, click Start Calibration. 4. Click Next, then prepare for the calibration as instructed. 5. At the bottom of the Setup tab, enter the reagent information for the ROI plate or array card that
you are using.

6. Load the calibration plate or array card into the instrument: a. From the instrument touchscreen, tap to eject the instrument tray.

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2 Chapter 2 Calibrate the instrument Uniformity calibration
b. Load the calibration plate or array card into the plate holder so that: · Well A1 of the plate or array card is in the top-left corner of the plate adapter. · The barcode faces the front of the instrument.

IMPORTANT! Plates or array cards should be loaded and unloaded by operators who have been warned of the moving parts hazard and have been adequately trained.

c. From the instrument touchscreen, tap to close the instrument tray.
7. After loading the plate or array card, start the calibration: a. In the Setup tab, select Check the box when the ROI calibration plate has been loaded, then click Next.
b. In the Run screen, click Start Run.

IMPORTANT! Do not attempt to open the access door during the run. The door is locked while the instrument is in operation.

Note: Before starting the calibration, the instrument may pause (up to 10 minutes) to allow the heated cover to reach the required temperature.

8. When the run is complete and the Analysis screen displays, confirm the status of the calibration. The analysis status indicates the success of the calibration, where passed indicates that the run produced viable calibration data, and failed indicates that the run did not produce data or that the data it collected is unusable.

Analysis status

Action

Passed

Click Next.

Failed

Troubleshoot the failed calibration (see “Troubleshooting” on page 71).

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9. Select the QC tab to review the quality check summary.
Note: The QC tab can provide information on the cause of a failed calibration.
10. Unload the calibration plate or array card: a. After the calibration, tap on the touchscreen to eject the plate or array card.
Note: If the instrument does not eject the plate or array card, remove it as explained in “Troubleshooting” on page 71.
b. Remove the calibration plate or array card from the instrument tray and discard it.
WARNING! PHYSICAL INJURY HAZARD. During instrument operation, the plate or array card temperature can reach 100°C. Allow the consumable to cool to room temperature before removing.
c. Tap on the instrument touchscreen to close the instrument tray. 11. From the Uniformity Calibration screen, click Finish to complete the calibration, then click Yes
when prompted to save the results. 12. (Optional) Click Print Report in the upper right corner of the screen to print a summary of the
calibration results for your records.
Dye calibration
The software uses the calibration data collected from the dye standards to characterize and distinguish the individual contribution of each dye in the total fluorescence collected by the instrument. After each run, the instrument software receives data in the form of a raw spectra signal for each reading. The software determines the contribution of the fluorescent dyes used in each reaction by comparing the raw spectra to the pure spectra calibration data. When you save an experiment after analysis, the instrument software stores the pure spectra with the collected fluorescence data for that experiment.
When to perform the calibration
Perform a dye calibration once every 6 months.
IMPORTANT! Perform a background calibration before every series of dye calibrations. Because the age and use of instrument components can affect spectra readings, you must perform a dye calibration at least every 6 months.

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2 Chapter 2 Calibrate the instrument Dye calibration

About the dye calibration data

The product of a dye calibration is a collection of spectral profiles that represent the fluorescence signature of each dye standard. Each profile consists of a set of spectra that correspond to the fluorescence collected from the wells of the calibration plate or array card. Following the calibration of each dye, the software “extracts” a spectral profile for each dye. The software plots the resulting data for each profile in a graph of fluorescence versus filter.
When the software extracts the dye calibration data, it evaluates the fluorescence signal generated by each well in terms of the collective spectra for the entire calibration plate or array card. Dye spectra are generally acceptable if they peak within the same filter as their group, but diverge slightly at other wavelengths (see below).

Acceptable spectra

Unacceptable spectra

Spectra peak at the same wavelength and do not diverge significantly

Spectra peak at the different wavelengths

Note: Because the wells of a calibration plate or array card contain identical concentrations of a dye, the resulting signals for the wells should be similar. Variations in spectral position and peak position are caused by minor differences in the optical properties and excitation energy between the individual wells.
Preparing the calibration plate or array card
IMPORTANT! Wear powder-free gloves and safety glasses when you prepare plates or array cards.
Prepare the calibration consumable appropriate for your QuantStudioTM 6 or 7 Flex System: · “Prepare the dye calibration plates” on page 51 · “Preparing array cards for instrument calibration” on page 33

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Prepare the dye calibration plates
Materials required
· Spectral calibration plates ­ Spectral Calibration Plate with FAMTM Dye ­ Spectral Calibration Plate with VICTM Dye ­ Spectral Calibration Plate with ROXTM Dye ­ Spectral Calibration Plate with NEDTM Dye ­ Spectral Calibration Plate with TAMRATM Dye ­ Spectral Calibration Plates as required by the experiment that you intend to run
· Centrifuge with plate adapter · Powder-free gloves · Safety goggles
Prepare the plate
IMPORTANT! Before performing a dye calibration, you must perform an ROI calibration, a background calibration, and a uniformity calibration.
IMPORTANT! Wear powder-free gloves and safety glasses when you prepare the plate.
1. Remove the dye calibration plate from the freezer, then thaw it at room temperature for approximately 30 minutes. IMPORTANT! Use the calibration plates within 2 hours of defrosting them. Until you are ready to run the plates, store them in the dark and at ambient temperature (15­30). Do not remove the plates from their packaging until you are ready to run them.
2. Before using each dye plate, vortex the plate for 5 seconds, centrifuge it for 2 minutes at < 1500 rpm, then confirm that the liquid in each dye plate is at the bottom of the wells. If not, centrifuge the plate again at a higher rpm and for a longer period of time.

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2 Chapter 2 Calibrate the instrument Dye calibration
Perform the dye calibration
IMPORTANT! The software guides you through the calibration of each dye separately. You must set up, run, and analyze each dye independently.
1. In the QuantStudioTM RealTime PCR Software Home tab, click Instrument Console. 2. In the Instrument Console, select the icon for your instrument, then click Manage Instrument.
Note: If the Manage Instrument button is inactive, add your system to the My Instruments group. 3. In the Instrument Manager, start the calibration:
a. Click Maintenance, then click Dye. b. In the Dye Calibration screen, select System Dye Calibration, then click Start Calibration. 4. In the Dye Calibration screen, select the dye to calibrate from the Dye Name drop-down list, then perform the calibration as instructed. 5. At the bottom of the Setup tab, enter the reagent information for the dye plate or array card that you are using.
6. Load the dye plate or array card into the instrument: a. From the instrument touchscreen, tap to eject the instrument tray.

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b. Confirm that the dye plate or array card matches the dye selected in the software, then load it into the plate holder. Load the plate or array card so that: · Well A1 of the plate or array card is in the top-left corner of the plate adapter. · The barcode faces the front of the instrument.

Note: The name of the dye contained by the plate or array card is next to the barcode on the front of it.
IMPORTANT! Plates or array cards should be loaded and unloaded by operators who have been warned of the moving parts hazard and have been adequately trained.
c. From the instrument touchscreen, tap to close the instrument tray.
7. After loading the plate or array card, start the calibration: a. In the Dye Calibration screen, select Check the box when the dye calibration plate has been loaded, then click Next.
b. In the Run screen, click Start Run.
IMPORTANT! Do not attempt to open the access door during the run. The door is locked while the instrument is in operation.
Note: Before starting the calibration, the instrument may pause (up to 10 minutes) to allow the heated cover to reach the required temperature.

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2 Chapter 2 Calibrate the instrument Dye calibration

8. When the run is complete and the software displays the Analysis screen, confirm the grouping of the dye spectra: a. Select the Plate Layout tab, then review the raw data. For each spectrum, confirm that the peak is:
· Within the detectable range for the QuantStudioTM 6 and 7 Flex Systems. · Free of irregular spectral peaks. · Present at the correct filter for the dye (see “Instrument filters and supported dyes” on
page 15).
Note: Among wells containing the same dye, variations in spectral position and peak position are caused by minor differences in the optical properties and excitation energy between the individual wells.

b. Select the QC tab, then review the summary of wells that failed the quality check (QC).

9. After you inspect the dye spectra, confirm the analysis status of the calibration. The analysis status indicates the success of the calibration, where passed indicates that the run produced viable calibration data, and failed indicates that the run did not produce data or that the data it collected is unusable.

Analysis status

Action

Passed

1. Click Next. 2. Enter comments in the Comments field, click Finish, then click Yes
when prompted to save the results.

Failed

Troubleshoot the failed calibration (see “Troubleshooting” on page 71).

10. Select the QC tab to review the quality check summary. The QC status indicates the quality of the calibration data, where passed indicates that all wells produced data that passed the quality check, and failed indicates that one or more wells produced dye spectra that differ significantly from the other wells on the plate or array card.
Note: If the calibration failed, the QC tab can provide information regarding the cause of the failure.

11. Unload the calibration plate or array card: a. After the calibration, tap on the touchscreen to eject the plate or array card.
Note: If the instrument does not eject the plate or array card, remove it as explained in “Troubleshooting” on page 71.

b. Remove the calibration plate or array card from the instrument tray and discard it.

WARNING! PHYSICAL INJURY HAZARD. During instrument operation, the plate or array card temperature can reach 100°C. Allow the consumable to cool to room temperature before removing.

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c. Touch on the instrument touchscreen to close the instrument tray. 12. Repeat the calibration to calibrate the instrument for the remaining dyes that are present in the
experiments that you will be running. 13. (Optional) Click Print Report in the upper right corner of the screen to print a summary of the
calibration results for your records.
Normalization calibration
During the normalization calibration, the instrument collects and stores the data from the normalization standards.
When to perform the calibration
Perform a normalization calibration once every 6 months. Note: Normalization calibration is required for all block types with QuantStudioTM RealTime PCR Software v1.3.
About the calibration data
During the normalization calibration, the software captures a series of images of each normalization plate or array card using each instrument filter. The normalization calibration yields a “Pass” or “Fail” result for each normalization consumable used.
Preparing the calibration plate or array card
IMPORTANT! Wear powder-free gloves and safety glasses when you prepare plates or array cards.
Prepare the calibration consumable appropriate for your QuantStudioTM 6 or 7 Flex System: · “Prepare the normalization calibration plates” on page 55 · “Preparing array cards for instrument calibration” on page 33
Prepare the normalization calibration plates
Materials required
· Normalization Plate with FAMTM/ROXTM dyes · Normalization Plate with VICTM/ROXTM dyes · Centrifuge with plate adapter · Powder-free gloves · Safety goggles

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Prepare the plate
IMPORTANT! Before performing a normalization calibration, you must perform an ROI calibration, a background calibration, a uniformity calibration, and a dye calibration.
IMPORTANT! Wear powder-free gloves and safety glasses when you prepare the plate.
1. Remove the normalization calibration plates from the freezer, then allow them to warm to room temperature (approximately 30 minutes). IMPORTANT! Use the calibration plates within 2 hours of defrosting them. Until you are ready to run the plates, store them in the dark and at ambient temperature (15­30). Do not remove the plates from their packaging until you are ready to run them.
2. Go to “Perform the normalization calibration” on page 56. Before using each normalization plate, vortex the plate for 5 seconds, centrifuge it for 2 minutes at < 1500 rpm, then confirm that the liquid in each dye plate is at the bottom of the wells. If not, centrifuge the plate again at a higher rpm and for a longer period of time.

Perform the normalization calibration
1. In the QuantStudioTM RealTime PCR Software Home tab, click Instrument Console. 2. In the Instrument Console, select the icon for your instrument, then click Manage Instrument.
Note: If the Manage Instrument button is inactive, add your system to the My Instruments group.
3. In the Instrument Manager, start the calibration: a. Click Maintenance, then click Normalization.
b. In the Normalization Calibration screen, click Start Calibration.
4. In the Normalization Calibration screen, select the reporter and passive dye combination that you want to calibrate, then perform the calibration as instructed.

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5. From the bottom of the Setup tab, enter the reagent information for the normalization plate or array card that you are using.
6. Load the calibration plate or array card into the instrument: a. From the instrument touchscreen, tap to eject the instrument tray. b. Confirm that the normalization plate or array card matches the selection in the software, then load it into the plate holder so that: · Well A1 of the plate or array card is in the top-left corner of the plate adapter. · The barcode faces the front of the instrument.

Note: The dyes contained in each plate or array card appear next to the barcode on the front of it.
IMPORTANT! Plates or array cards should be loaded and unloaded by operators who have been warned of the moving parts hazard and have been adequately trained.
c. From the instrument touchscreen, touch to close the instrument tray.
7. After loading the array card, start the calibration: a. In the Normalization Calibration screen tab, select Check the box when the normalization calibration plate has been loaded, then click Next.

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b. In the Run screen, click Start Run.
IMPORTANT! Do not attempt to open the access door during the run. The door is locked while the instrument is in operation.

Note: Before starting the calibration, the instrument may pause (up to 10 minutes) to allow the heated cover to reach the required temperature.

8. When the run is complete and the QuantStudioTM RealTime PCR Software displays the Analysis screen, confirm the status of the calibration. The analysis status indicates the success of the calibration, where passed indicates that the run produced viable calibration data, and failed indicates that the run did not produce data or that the data it collected is unusable.

Analysis status

Action

Passed

Enter comments in the Comments field, click Next.

Failed

Troubleshoot the failed calibration (see “Troubleshooting” on page 71).

9. Select the QC tab to review the quality check summary. The QC status indicates the quality of the calibration data, where passed indicates that all wells produced data that passed the quality check, and failed indicates that one or more wells produced unusable data.
Note: If the calibration failed, the QC tab can provide information regarding the cause of the failure.

10. Unload the calibration plate or array card: a. After the calibration, tap on the touchscreen to eject the plate or array card.
Note: If the instrument does not eject the plate or array card, remove it as explained in “Troubleshooting” on page 71.

b. Remove the calibration plate or array card from the instrument tray and discard it.

WARNING! PHYSICAL INJURY HAZARD. During instrument operation, the plate or array card temperature can reach 100. Allow the consumable to cool to room temperature before removing.
c. Tap on the instrument touchscreen to close the instrument tray.
11. In the Normalization Calibration screen, click Finish to complete the calibration, then click Yes when prompted to save the results.
12. Repeat the calibration procedure to perform the remaining normalization calibration.
13. (Optional) Click Print Report in the upper right corner of the screen to print a summary of the calibration results for your records.

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Verify the instrument performance
When necessary, you can perform the RNase P instrument verification test to confirm the performance of the QuantStudioTM 6 and 7 Flex Systems.
When to perform the test
We recommend performing an RNase P instrument verification test: · After moving the instrument to another location. · As needed to confirm the function of the instrument.
About the RNase P kits
The RNase P instrument verification test uses instrument verification kits available from thermofisher.com. The kits differ only in the consumable format for which they are designed: an RNase P Instrument Verification Plate for QuantStudioTM 6 and 7 Flex Systems with 96/384-well sample blocks and an Array Card RNase P Kit for the QuantStudioTM 7 Flex System with array card sample blocks.
RNase P instrument verification plates
The RNase P plate is preloaded with the reagents necessary for the detection and quantitation of genomic copies of the human RNase P gene (a single-copy gene encoding the RNase moiety of the RNase P enzyme). Each well contains: PCR master mix, RNase P primers, FAMTM dye-labeled probe, and a known concentration of human genomic DNA template. Figure 1 illustrates the arrangement of the standard and unknown populations on a 96-well and Fast 96-well RNase P plate. The RNase P plate contains five replicate groups of standards (1250, 2500, 5000, 10000, and 20000 copies), two unknown populations (5,000 and 10,000 copies), and a no template control (NTC).

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Figure 1 96-well and Fast 96-well RNase P plate layout
Figure 2 illustrates the arrangement of the standard and unknown populations on a 384-well RNase P plate. The RNase P plate contains five replicate groups of standards (1250, 2500, 5000, 10000, and 20000 copies), two unknown populations (5,000 and 10,000 copies), and a no template control (NTC).

Figure 2 384-well RNase P plate layout

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Array card RNase P kits
The RNase P Kits include one empty array card and eight tubes of solution. Each tube contains reaction mix (PCR master mix, RNase P primers, and FAMTM- MGB dye-labeled probe) and a known concentration of human genomic DNA template.
To perform an instrument verification run, each solution is loaded into the empty array card in the arrangement shown in Figure 3. When complete, the array card contains five replicate groups of standards (200, 400, 800, 1600, and 3200 copies), two of unknown populations (800 and 1600 copies), and one that serves as a no template control (NTC).

1 2 3 4 5 6 7 8

Figure 3 RNase P standards array card layout
1 No Template Control 2 Unknown A (800) 3 Unknown B (1600) 4 Standard 200

5 Standard 400 6 Standard 800 7 Standard 1600 8 Standard 3200

Analytical performance

The software performs the same analysis of data from an instrument verification run regardless of the type of consumable used. After the run, the software:
1. Generates a standard curve from the averaged threshold cycle (Ct) values of the replicate groups of standards.
2. Calculates the concentration of the two unknown populations using the standard curve.
3. Calculates the following to assess the QuantStudioTM 6 or 7 Flex System performance: [(CopyUnk2) ­ 3(CopyUnk2)] > [(CopyUnk1) + 3(CopyUnk1)]

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where: · CopyUnk1 = Average copy number of unknown population A · CopyUnk1 = Standard deviation of unknown population A · CopyUnk2 = Average copy number of unknown population B · CopyUnk2 = Standard deviation of unknown population B
Note: Unknown population A refers to the 5,000-copy population and unknown population B refers to the 10,000-copy population in the RNase P plate.

Installation specification

The QuantStudioTM 6 or 7 Flex System passes the installation specification if the inequality holds and the instrument successfully distinguishes between unknown populations A and B with a statistical confidence level of 99.7%.
As shown in the following table, you can omit a limited number of outlier wells from the unknown populations to meet the installation specification.

Sample block

Maximum number of outlier wells that can be removed

Unknown population A[1]

Unknown population B[2]

Standards (STD)[3]

No template controls (NTC)

96-well plate[4]

6

6

1

0

384-well plate

10

10

2

0

Array card[5]

4

4

4

0

[1] 5,000-copy population for 384-well plates; 800-copy population for array cards. [2] 10,000-copy population for 384-well plates; 1,600-copy population for array cards. [3] Maximum number of wells that can be removed from each standard population. [4] Standard 96-well plates or Fast 96-well plates [5] QuantStudioTM 7 Flex System only.

Prepare the RNase P plate

Materials required
· RNase P Instrument Verification Plate · Centrifuge with plate adapter · Powder-free gloves · Safety goggles

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Prepare the plate
IMPORTANT! When performing the RNase P instrument verification test:
· Perform all calibrations beforehand. · Run the RNase P plate soon after you allow the plate to thaw. Minimizing the time that the prepared
plate or array card sits on the bench ensures optimal performance.
· Wear powder-free gloves and safety glasses when you prepare plates.
1. Obtain the RNase P Instrument Verification Plate from the freezer, then allow the plate to warm to room temperature (for approximately 5 minutes). IMPORTANT! Use the RNase P plate within 30 minutes of defrosting it. Until you are ready to run the plate, store it in the dark and at ambient temperature (15­30). Do not remove the plate from its packaging until you are ready to run it.
2. Remove the RNase P plate from its packaging.
3. Briefly vortex and centrifuge the RNase P plate: a. Vortex the plate for 5 seconds.
b. Centrifuge the reaction plate for 2 minutes at < 1500 rpm. IMPORTANT! The reaction plate must be well mixed and centrifuged.
c. Confirm that the liquid is at the bottom of each well of the reaction plate. If not, centrifuge the reaction plate again at a greater rpm and for a longer period of time. IMPORTANT! Do not allow the bottom of the plate to become dirty. Fluids and other contaminants that adhere to the bottom of the reaction plate can contaminate the sample block and cause an abnormally high background.

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Preparing an array card for instrument verification
IMPORTANT! Perform the following procedure only if you are verifying the performance of a QuantStudioTM 7 Flex System with an array card sample block.
Required materials
· TaqManTM Array Card Sealer · Centrifuge with centrifuge array card carrier clips and buckets · Powder-free gloves · Safety goggles · Pipettor, 200-µL (with pipette tips) · ViiATM 7 Array Card RNaseP Verification Kit, includes the following components:
­ TaqManTM Array Card ­ Eight (8) vials of RNase P detection reagents, one for each port in the TaqManTM Array Card.
Each vial contains the following reagents: ­ PCR master mix ­ RNase P primers ­ A FAMTM dye-labeled probe ­ A known concentration of human genomic DNA template
Preparing the RNase P array card
IMPORTANT! Wear powder-free gloves while preparing the array card.
1. Remove the Array Card RNase P Kit from the freezer, then allow it to warm to room temperature (for approximately 15 minutes). IMPORTANT! You must fill and run the RNase P array card within 60 minutes of thawing the Array Card RNase P Kit.
2. Remove an array card from its box and place it on a clean, dry surface.
3. Using a permanent marker, mark the side of the empty array card with RNase P.

1 1 Mark here

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4. Transfer 100 µL of each solution into the appropriate port of the array card:

1 2 3 4 5 6 7 8

1 No Template Control

5 Standard 400

2 Unknown A (800)

6 Standard 800

3 Unknown B (1600)

7 Standard 1600

4 Standard 200

8 Standard 3200

For each transfer:

a. Place the array card on a lab bench, with the foil side down.

b. Load 100 µL of fluid into a pipette.

c. Hold the pipette in an angled position (45 degrees) and place the tip into the fill port. The fill port is the larger of the two holes on the left side of the fill reservoir.

1

2

1 Fill port 2 Vent port

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d. Dispense the fluid so that it sweeps in and around the fill reservoir toward the vent port. Pipet the entire 100 µL into the reservoir, but do not go past the first stop of pipettor plunger or you may blow the solution out of the port. IMPORTANT! Do not allow the tip to contact and possibly damage the coated foil beneath the fill port.
5. Centrifuge and seal the array card as explained in step 6 through step 8 in “Fill the calibration TaqManTM Array Cards” on page 34.
6. Run the prepared array card as soon as possible after filling it. IMPORTANT! Store the array card in a dark place until you are ready to run it. The fluorescent dyes in the array card are photosensitive. Prolonged exposure to light can diminish the fluorescence of the dye.
Run the RNase P plate or array card
1. In the QuantStudioTM RealTime PCR Software Home tab, click Instrument Console. 2. In the Instrument Console, select the icon for your instrument, then click Manage Instrument.
Note: If the Manage Instrument button is inactive, add your system to the My Instruments group. 3. In the Instrument Manager, start the RNase P run:
a. Click Maintenance, then click RNase P Run. b. In the RNase P Run screen, click Start RNase P Run. 4. Click Next, then prepare for the instrument verification test as instructed.

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5. At the bottom of the Setup tab, enter the reagent information for the RNase P plate or array card that you are using.
6. Load the RNase P plate or array card into the instrument: a. From the instrument touchscreen, tap to eject the instrument tray. b. Load the plate or array card into the plate holder so that: · Well A1 of the plate or array card is in the top-left corner of the adapter. · The barcode faces the front of the instrument.

IMPORTANT! Plates and array cards should be loaded and unloaded by operators who have been warned of the moving parts hazard and have been adequately trained.
c. From the instrument touchscreen, touch to close the instrument tray.
7. After loading the plate or array card, start the instrument verification test: a. In the Overview screen, select Check the box when the RNase P plate has been loaded, then click Next.
b. In the Run screen, click Start Run to start the test.

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IMPORTANT! Do not attempt to open the access door during the run. The door is locked while the instrument is in operation.

Note: Before starting the instrument verification test, the instrument may pause (up to 10 minutes) to allow the heated cover to reach the required temperature.

8. When the run is complete and the software displays the Analysis screen, confirm the status of the run.

Analysis status

Action

Passed

The system passed the RNase P run.

Failed

The system failed the RNase P run. Review the data for outliers.
If the run fails, the software may have included outliers that caused the initial analysis to fail. Error may cause some wells to be amplified insufficiently or not at all. These wells typically produce Ct values that differ significantly from the average for the associated replicate wells. If included in the calculations, these outlying data (outliers) can result in erroneous measurements.

9. In the Amplification Plot, select Ct vs. Well from the Plot Type menu.
10. Confirm the uniformity of each replicate population (controls, standards, and unknowns) on the plate or array card by comparing the groupings of Ct values: a. In the Plate Layout, select the wells containing Unknown Population A:
b. From the plot, confirm that the Ct values of the replicate population are equivalent.
Note: The numbers on the X-axis of the plot correspond to the wells of the plate or array card. Beginning with well A1, the wells are numbered from left- to-right, and top-to-bottom.

c. If an outlier is present in the selected population, select the corresponding well of the plate layout, then click Omit to remove the well from the analysis. If the total number of outliers for the replicate population exceeds the limit in the table below, repeat the test using another RNase P plate or array card.

Sample block

Maximum number of outlier wells that can be removed

Unknown population A[1]

Unknown population B[2]

Standards (STD)[3]

No template controls (NTC)

96-well plate[4]

6

6

1

0

384-well plate

10

10

2

0

TaqManTM Array Card[5]

4

4

4

0

[1] 5,000-copy population for 384-well plates; 800-copy population for array cards. [2] 10,000-copy population for 384-well plates; 1,600-copy population for array cards. [3] Maximum number of wells that can be removed from each standard population. [4] Standard 96-well plates or Fast 96-well plates [5] QuantStudioTM 7 Flex System only.

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11. Repeat step 10 for each replicate population (unknowns, standards, and no template controls) on the plate or array card.
12. Review the Results Table for quality flags generated by the test: a. Select the Results Table tab.
b. Review the Flag column for wells that generated quality flags.
c. Troubleshoot each well that generated a flag as explained in “Troubleshooting” on page 71. · AMPNC–Amplification in negative control · BADROX–Bad passive reference signal · BLFAIL–Baseline algorithm failed · CTFAIL–Ct algorithm failed · EX

References

• Lab Equipment and Lab Supplies | Fisher Scientific
• Thermo Fisher Scientific - AU
• Thermo Fisher Scientific - US
• Contact Us | Thermo Fisher Scientific - US
• Software Downloads | Thermo Fisher Scientific - US
• Help and Support | Thermo Fisher Scientific - UK
• Symbols | Thermo Fisher Scientific - UK
• Thermo Fisher Scientific - US
• Software Downloads | Thermo Fisher Scientific - US
• Help and Support | Thermo Fisher Scientific - UK
• Thermo Fisher Scientific - US
• Terms and Conditions of Sale | Thermo Fisher Scientific - US
• RoHS Certificates | Thermo Fisher Scientific - US
• Laboratory biosafety manual, 4th edition
• 4331770 | Thermo Fisher Scientific
• TaqMan™ RNase P Instrument Verification Plate, Fast 96-well (for 0.1 mL block)
• Array Card Calibration Install Kit
• FAM™ Dye Spectral Calibration Plate, 384-well
• VIC™ Dye Spectral Calibration Plate, 384-well
• ROX™ Dye Spectral Calibration Plate, 384-well
• TAMRA™ Dye Spectral Calibration Plate, 384-well
• FAM™/ROX™ and VIC™/ROX™ Dye Normalization Plates, 384-well
• Region of Interest (ROI) and Background Plates, 384-well
• 4432327 | Thermo Fisher Scientific
• 4432334 | Thermo Fisher Scientific
• 4432340 | Thermo Fisher Scientific
• 4432352 | Thermo Fisher Scientific
• 4432364 | Thermo Fisher Scientific
• 4432370 | Thermo Fisher Scientific
• 4432382 | Thermo Fisher Scientific
• FAM™ Dye Spectral Calibration Plate, Fast 96-well
• VIC™ Dye Spectral Calibration Plate, Fast 96-well
• 4432402 | Thermo Fisher Scientific
• TAMRA™ Dye Spectral Calibration Plate, Fast 96-well
• Region of Interest (ROI) and Background Plates, Fast 96-well

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