Urinary Tract Microbiota Profiling Experiments Application Guide (Pub. No. MAN0017750 Rev. C.0)

Urinary Tract Microbiota Profiling Experiments

Thermo Fisher Scientific (29 March 2021)

Urinary Tract Microbiota Profiling Experiments

C.0 29 March 2021 Updated information for TaqMan™ Universal DNA Spike In Control. B.0 9 August 2018 • Added information for the TaqMan™ Array Urinary Tract Microbiota Comprehensive Plate. • Added references for additional user documentation. • Updated DNA isolation protocol per V&V testing. A.0 25 March 2018 New document.

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MAN0017750 UTMicrobiotaProfilingExp AG

Urinary Tract Microbiota Profiling Experiments
APPLICATION GUIDE TaqManTM Assays for urinary tract microbiota profiling experiments in TaqManTM OpenArrayTM Plate-format
for use with: TaqManTM Array Urinary Tract Microbiota Comprehensive Plate Custom TaqManTM OpenArrayTM Plates QuantStudioTM 12K Flex instrument with OpenArrayTM block (AccuFillTM System)
Catalog Numbers A39900
Publication Number MAN0017750 Revision C.0
For Research Use Only. Not for use in diagnostic procedures.

Life Technologies Corporation | 6055 Sunol Blvd | Pleasanton, California 94566 USA 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. MAN0017750

Revision

Date

C.0

29 March 2021

B.0

9 August 2018

Description Updated information for TaqManTM Universal DNA Spike In Control.
· Added information for the TaqManTM Array Urinary Tract Microbiota Comprehensive Plate. · Added references for additional user documentation. · Updated DNA isolation protocol per V&V testing.

A.0

25 March 2018

New document.

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. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license.
©2021 Thermo Fisher Scientific Inc. All rights reserved.

Contents

CHAPTER 1 Introduction and workflow overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Urinary tract microbiota profiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Workflow: TaqManTM urinary tract microbiota profiling experiments . . . . . . . . . . . . . . . . . . . . . 6
CHAPTER 2 Background and tools for assay selection . . . . . . . . . . . . . . . . . . . . . . . . . 7
TaqManTM Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 TaqManTM Assays for urinary tract microbiota profiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Optional controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
(Optional) TaqManTM Universal DNA Spike In Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 (Optional) Amplification control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 TaqManTM OpenArrayTM Plate products and formats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 TaqManTM Array Urinary Tract Microbiota Comprehensive Plate . . . . . . . . . . . . . . . . . . . 9 Custom TaqManTM OpenArrayTM Plate products and formats . . . . . . . . . . . . . . . . . . . . . 10 Configure and order custom TaqManTM OpenArrayTM plates . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
CHAPTER 3 Isolate DNA from urine research samples using the
MagMAXTM DNA Multi-Sample Ultra Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Compatible sample collection and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Download the KingFisherTM Flex program (if needed) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Before first use of the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Set up the sample layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Concentrate the samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Digest the samples with the Preliminary Digestion Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Digest the samples with Proteinase K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Set up the processing plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Add Multi-Sample DNA Lysis Buffer, Bead/RNase A Mix, and isopropanol . . . . . . . . . . . . 19 Process samples on the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Urinary Tract Microbiota Profiling Experiments Application Guide

Contents

CHAPTER 4 Prepare and run urinary tract microbiota profiling
experiments with OpenArrayTM plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Workflow: Microbiota profiling experiments with OpenArrayTM plates . . . . . . . . . . . . . . . . . . 23 Required materials for OpenArrayTM plate workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Generate 384well sample plate layouts in the OpenArrayTM Sample Tracker Software . . . 25 Set up the PCR reactions in an OpenArrayTM 384-well Sample Plate . . . . . . . . . . . . . . . . . . 26 Set up the AccuFillTM instrument and software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Transfer reactions to the OpenArrayTM plate using the AccuFillTM instrument . . . . . . . . . . . . 28 Seal the OpenArrayTM plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Run the OpenArrayTM plate on the QuantStudioTM 12K Flex Instrument . . . . . . . . . . . . . . . . . 30 Check the qualitycontrol images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 One-time procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Set up default folders and software preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Download TPF files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
CHAPTER 5 Export and review urinary tract microbiota profiling data . . . . . . 35
Export data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Prepare exported data for analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Review results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Fields for reviewing results with pivot tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
APPENDIX A Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Troubleshoot DNA isolation errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Troubleshoot unexpected Crt values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Troubleshoot with cycling and imaging run images (QC images) . . . . . . . . . . . . . . . . . . . . . . 39 AccuFillTM instrument plate loading errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 OpenArrayTM plate assembly and handling errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
APPENDIX B Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Symbols that may be displayed on the instrument, in the software, or in this guide . . . . . 46 Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Urinary Tract Microbiota Profiling Experiments Application Guide

Introduction and workflow overview

IMPORTANT! Before using this product, read and understand the information in the "Safety" appendix in this document.
This guide describes the OpenArrayTM plate high-throughput, sampletoresult workflow for urinary tract microbiota profiling. The workflow uses:
· MagMAXTM DNA Multi-Sample Ultra Kit for DNA isolation from urine samples · OpenArrayTM plates with TaqManTM Assays for urinary tract microbiota profiling · QuantStudioTM 12K Flex instrument with OpenArrayTM block (AccuFillTM System)
Urinary tract microbiota profiling
Microorganism-specific TaqManTM Assays offer a rapid and accurate approach to investigate and monitor urinary tract microbiome composition and dynamics. We offer a collection of qualified TaqManTM Assays that are optimized for detection of urinary tract microbes (see "TaqManTM Assays for urinary tract microbiota profiling" on page 8). The TaqManTM Assay design and their target sequences have undergone rigorous bioinformatics selection and analysis to ensure maximum strain coverage and minimal off-target cross-reactivity. Qualified TaqManTM Assays for urinary tract microbiota profiling demonstrate accurate, reproducible performance in multiple rounds of testing for sensitivity and specificity. The assays perform well with DNA isolated from urine samples using optimized MagMAXTM DNA Multi-Sample Ultra Kit protocols. Additional TaqManTM Assays for microbial targets are available from our predesigned assay collection. For Custom TaqManTM Assays contact QuantStudioFrontDesk@thermofisher.com.

Urinary Tract Microbiota Profiling Experiments Application Guide

Chapter 1 Introduction and workflow overview Workflow: TaqManTM urinary tract microbiota profiling experiments

Workflow: TaqManTM urinary tract microbiota profiling experiments
Collect urine sample (page 12)
Isolate DNA from urine research samples using the MagMAXTM DNA Multi-Sample Ultra Kit (page 12)
Prepare and run urinary tract microbiota profiling experiments with OpenArrayTM plates (page 22)
Export and review urinary tract microbiota profiling data (page 35)

Urinary Tract Microbiota Profiling Experiments Application Guide

Background and tools for assay selection

TaqManTM Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 TaqManTM Assays for urinary tract microbiota profiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Optional controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 TaqManTM OpenArrayTM Plate products and formats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Configure and order custom TaqManTM OpenArrayTM plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
TaqManTM Assays
TaqManTM Assays for urinary tract microbiota profiling consist of a pair of unlabeled PCR primers and a TaqManTM probe with a FAMTM dye label on the 5' end and minor groove binder (MGB) and nonfluorescent quencher (NFQ) on the 3' end. For more information about real-time PCR and TaqManTM Assays, visit thermofisher.com/ qpcreducation.

Urinary Tract Microbiota Profiling Experiments Application Guide

Chapter 2 Background and tools for assay selection TaqManTM Assays for urinary tract microbiota profiling

TaqManTM Assays for urinary tract microbiota profiling

All of the TaqManTM Assays for urinary tract microbiota profiling, including the controls, are included in the TaqManTM Array Urinary Tract Microbiota Comprehensive Plate. The assays can also be configured with Custom TaqManTM OpenArrayTM Plate (see "TaqManTM OpenArrayTM Plate products and formats" on page 9). For optional reference and controls, see "Optional controls" on page 9
For more information about available TaqManTM Assays for urinary tract microbiota profiling, visit thermofisher.com/utm.
Table 1 TaqManTM Assays for urinary tract microbiota profiling

Assay ID

Classification

Target organism name

Ba04932084_s1

Bacteria

Acinetobacter baumannii

Ba04932088_s1

Bacteria

Citrobacter freundii

Ba04932080_s1

Bacteria

Enterobacter aerogenes

Ba04932087_s1

Bacteria

Enterobacter cloacae

Ba04646247_s1

Bacteria

Enterococcus faecalis

Ba04932086_s1

Bacteria

Enterococcus faecium

Ba04646242_s1

Bacteria

Escherichia coli

Ba04932079_s1

Bacteria

Klebsiella oxytoca

Ba04932083_s1

Bacteria

Klebsiella pneumoniae

Ba04932078_s1

Bacteria

Morganella morganii

Ba04932076_s1

Bacteria

Proteus mirabilis

Ba04932082_s1

Bacteria

Proteus vulgaris

Ba04932077_s1

Bacteria

Providencia stuartii

Ba04932081_s1

Bacteria

Pseudomonas aeruginosa

Ba04932085_s1

Bacteria

Staphylococcus saprophyticus

Ba04646276_s1

Bacteria

Streptococcus agalactiae

Fn04646233_s1

Yeast

Candida albicans

(Optional) Control assays[1]

Hs04930436_g1

Control

Human RNase P RPPH1 gene[2]

Ac00010014_a1

Control

XenoTM[3]

[1] Included in the TaqManTM Array Urinary Tract Microbiota Comprehensive Plate. [2] Use to assess sample adequacy. [3] Use to control for nucleic acid recovery in sample preparation process.

Urinary Tract Microbiota Profiling Experiments Application Guide

2 Chapter 2 Background and tools for assay selection Optional controls
Optional controls
(Optional) TaqManTM Universal DNA Spike In Control
The TaqManTM Universal DNA Spike In Control (Cat. No. A39175) is an exogenous XenoTM DNA process control that can be used to monitor the recovery efficiency for the DNA extraction and purification process. The control also indicates the presence of PCR inhibitors in molecular detection workflows. This control is of particular importance when working with urine samples that can have a higher frequency of inhibition. This product contains a sequence for AmpC betalactamase and may amplify for any assays designed to detect this antibiotic resistance target. For example, TaqManTM Assay ID Ba04646128_s1 will amplify in any sample that contains the TaqManTM Universal DNA Spike In Control. TaqManTM Universal DNA Spike In Control is supplied at a concentration of 200,000 copies/µL. The control is added during DNA isolation and is then carried through the remainder of the urinary tract microbiota profiling workflow. Coupled with the proprietary TaqManTM Assay for the XenoTM DNA control (Assay ID Ac00010014_a1), this verification layer helps ensure that PCR results are accurate, and it reduces the likelihood of false negatives. For information about TaqManTM Universal DNA Spike In Control, see TaqManTM Universal DNA Spike In Control Product Information Sheet (Pub. No. MAN0017852).
(Optional) Amplification control
The TaqManTM Urinary Tract Microbiota Amplification Control (Cat. No. A39174) contains a linearized multi-target plasmid with target sequences for each available urinary tract microbiota profiling assay. The plasmid also contains target sequences for Xeno DNA and human RNase P RPPH1 genes, for a general control for the sample preparation process. The TaqManTM Urinary Tract Microbiota Amplification Control can be included in urinary tract microbiota profiling experiments to verify assay performance and to help with troubleshooting. For information about the amplification control, see TaqManTM Urinary Tract Microbiota Amplification Control Product Information Sheet (Pub. No. MAN0017753).
TaqManTM OpenArrayTM Plate products and formats
TaqManTM Array Urinary Tract Microbiota Comprehensive Plate
The TaqManTM Array Urinary Tract Microbiota Comprehensive Plate (Cat. No. A39900 ) contains preplated, dried down TaqManTM Assays for urinary tract microbiota profiling. For the complete lists of assays included with the TaqManTM Array Urinary Tract Microbiota Comprehensive Plate, see Table 1.

Urinary Tract Microbiota Profiling Experiments Application Guide

Chapter 2 Background and tools for assay selection TaqManTM OpenArrayTM Plate products and formats

Contents and storage
Table 2 TaqManTM Array Urinary Tract Microbiota Comprehensive Plate (Cat. No. A39900 )

Component
TaqManTM Array Urinary Tract Microbiota Comprehensive Plate

Amount 1 plate

Storage 25°C to 15°C

Custom TaqManTM OpenArrayTM Plate products and formats

Custom TaqManTM OpenArrayTM Plates contain pre-plated, dried down TaqManTM Assays for urinary tract microbiota profiling.

Array format 18 56 112

Number of assays 18 56 112

Maximum number of samples 48 48 24

Note: We recommend at least three technical replicates of each reaction.

Urinary Tract Microbiota Profiling Experiments Application Guide

Chapter 2 Background and tools for assay selection Configure and order custom TaqManTM OpenArrayTM plates

Configure and order custom TaqManTM OpenArrayTM plates
1. Go to thermofisher.com/order/custom-array.
2. For array type, select TaqManTM OpenArrayTM Real-Time PCR Inventoried Assays Format.
3. In the table, click Select to configure a plate with the desired array format. The Custom Array Configurator screen displays.

4. Enter the custom array name in the Array name text field.
5. Click Import Your Assay List, then upload or copy-paste the assay information: · Under Upload a list of Assay IDs, click Choose File, then select a tab-delimited text file (TXT) containing Assay IDs. or · Under Enter a list of Assay IDs, paste the Assay IDs, then click Import Entered List.
6. Follow the onscreen instructions to configure the assays on the plate.
7. (Optional) Click Save Your Array at any time to save the array configuration to your Thermo Fisher Scientific account.
8. When the plate is configured, click Complete Your Design, then follow the onscreen instructions to complete the order.

Urinary Tract Microbiota Profiling Experiments Application Guide

Isolate DNA from urine research samples using the MagMAXTM DNA

Multi-Sample Ultra Kit

Compatible sample collection and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Before first use of the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Set up the sample layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Concentrate the samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Digest the samples with the Preliminary Digestion Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Digest the samples with Proteinase K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Set up the processing plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Add Multi-Sample DNA Lysis Buffer, Bead/RNase A Mix, and isopropanol . . . . . . . . . . . . . . . . . . . 19 Process samples on the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Compatible sample collection and storage
Collect urine samples using BDTM Vacutainer® urine collection cups and tubes. · Compatible urine samples: Unstabilized urine that is collected in sterile containers (BDTM Cat. No. 364975) Urine that is collected and stored in Urine Analysis (UA) tubes (BDTM Cat. No. 364992) Urine that is collected and stored in Culture and Sensitivity (C&S) tubes (BDTM Cat. No. 364951) · (Optional) Store samples according to the instructions provided with the collection container, or use the following storage conditions: Store at 4°C for up to one week. Store at 80°C for long-term storage. We recommend storing samples in smaller volumes to prevent multiple freeze/thaw cycles.

Urinary Tract Microbiota Profiling Experiments Application Guide

Chapter 3 Isolate DNA from urine research samples using the MagMAXTM DNA Multi-Sample Ultra Kit Contents and storage

Contents and storage

Table 3 MagMAXTM DNA Multi-Sample Ultra Kit

Contents

Cat. No. A25597 (500 rxns)

Cat. No. A25598 (2,500 rxns)

Storage

Proteinase K

4 mL

5 × 4 mL

25°C to 15°C

PK Buffer Multi-Sample DNA Lysis Buffer

96 mL 100 mL

5 × 96 mL 5 × 100 mL

15°C to 30°C

DNA Binding Beads

8 mL

5 × 8 mL

2°C to 8°C

RNase A

2 × 1.25 mL

10 × 1.25 mL

25°C to 15°C

Nuclease-free Water

100 mL

5 × 100 mL

Wash Solution 1 Concentrate[1]

80 mL

5 × 80 mL

Wash Solution 2 Concentrate[1]

162 mL

5 × 162 mL

15°C to 30°C

DNA Elution Buffer 1

25 mL

5 × 25 mL

DNA Elution Buffer 2

25 mL

5 × 25 mL

[1] Before use of the kit, prepare all applicable wash solutions as described on their bottles and in this protocol.

Required materials not supplied

Unless otherwise indicated, all materials are available through thermofisher.com. "MLS" indicates that the material is available from fisherscientific.com or another major laboratory supplier.

Table 4 Required materials and equipment not included with the kit

Item KingFisherTM Flex Magnetic Particle Processor Equipment Plate shaker, capable of shaking plates at a minimum of 900 rpm
Analog Vortex Mixer
Adjustable micropipettors Multi-channel micropipettors (Optional) Magnetic Stand-96

Source 5400630
88880023 Fisher Scientific
02-215-365 MLS MLS
AM10027

Urinary Tract Microbiota Profiling Experiments Application Guide

Chapter 3 Isolate DNA from urine research samples using the MagMAXTM DNA Multi-Sample Ultra Kit Required materials not supplied

Item Plates and combs Deep Well Plates, one of the following: KingFisherTM Flex Microtiter Deep-Well 96 plates, sterile MagMAXTM Express-96 Deep Well Plates Standard Well Plates, one of the following: KingFisherTM 96 KF microplates MagMAXTM Express-96 Standard Plates Tip Combs, one of the following: KingFisherTM 96 tip comb for DW magnets MagMAXTM Express-96 Deep Well Tip Combs Other consumables MicroAmpTM Clear Adhesive Film RNase-free Microfuge Tubes (2.0 mL) Conical tubes (15 mL) Conical tubes (50 mL) Aerosol-resistant pipette tips Reagent reservoirs (Optional) Paraffin film Reagents Ethanol, 200 proof (absolute) Isopropanol, 100% (molecular grade or higher)

Source
95040460 4388476
97002540 4388475
97002534 4388487
4306311 AM12425 AM12500 AM12502
MLS MLS MLS
MLS MLS

Table 5 Additional materials and equipment required for processing urine samples

Item
Centrifuge, capable of spinning deep-well plates at 2,250 × g
Laboratory incubator with slatted shelves, capable of maintaining 65°C MagMAXTM Wash Solution 1 Concentrate B-PERTM Bacterial Protein Extraction Reagent Lysozyme Solution

Source Fisher Scientific
75-412-452 MLS
AM8504 78243 90082

Urinary Tract Microbiota Profiling Experiments Application Guide

Chapter 3 Isolate DNA from urine research samples using the MagMAXTM DNA Multi-Sample Ultra Kit Procedural guidelines

Table 5 Only for urinary tract (UTM)Additional materials and equipment required for processing urine samples (continued)

Item

Source

Zymolyase

Fisher Scientific 50-444-504

(Optional) TaqManTM Universal DNA Spike In Control (XenoTM DNA control)

A39175

Download the KingFisherTM Flex program (if needed)

The program required for this protocol is not pre-installed on the KingFisherTM Flex Magnetic Particle Processor.
1. On the MagMAXTM DNA Multi-Sample Ultra Kit web page, scroll down to the Product Literature section.
2. Click A25597_UTM to download the program to your computer.
3. See Thermo ScientificTM KingFisherTM Flex User Manual (Cat. No. N07669) and BindItTM Software User Manual (Cat. No. N07974) for instructions for installing the program on the instrument.

Procedural guidelines
IMPORTANT! Arrange plates in the incubator to allow adequate flow around the plate wells, to ensure that samples quickly reach and maintain the incubation temperature.
· Perform all steps at room temperature (2025°C) unless otherwise noted. · Preheat an incubator to 65°C before each use of the kit. · Use the KingFisherTM Flex Magnetic Particle Processor with 96 Deep-Well Head and 96-well
standard heat block. · When mixing samples by pipetting up and down, avoid creating bubbles. · Cover the plate during the incubation and shaking steps to prevent spill-over and cross-
contamination. The same MicroAmpTM Clear Adhesive Film can be used throughout the procedure, unless it becomes contaminated. · If you use a plate shaker other than the recommended shaker, verify that:
The plate fits securely on your plate shaker. The recommended speeds are compatible with your plate shaker. Ideal shaker speeds allow
for thorough mixing without splashing. · To prevent evaporation and contamination, cover the prepared processing plates with paraffin film
until they are loaded into the instrument. · Per-plate volumes for reagent mixes are sufficient for one plate plus overage. To calculate volumes
for other sample numbers, refer to the per-well volume and add 5% overage. · For convenience, you can extend the Proteinase K digestion to 30 minutes.

Urinary Tract Microbiota Profiling Experiments Application Guide

Chapter 3 Isolate DNA from urine research samples using the MagMAXTM DNA Multi-Sample Ultra Kit Before first use of the kit

Before first use of the kit
· Prepare the Wash Solutions from the concentrates: Add 25 mL of isopropanol to Wash Solution 1 Concentrate (from the kit). Mix the isopropanol and concentrate, then store at room temperature. Add 132 mL of ethanol to Wash Solution 2 Concentrate (from the kit). Mix the ethanol and concentrate, then store at room temperature. Add 70 mL of isopropanol to Wash Solution 1 Concentrate (separately purchased bottle, Cat. No. AM8504). Mix the isopropanol and concentrate, then store at room temperature.
· Reconstitute the zymolyase with 500 µL of the provided storage buffer (final concentration of 4 U/µL), vortex to mix, then store at 20°C. For more information, see the documentation provided with the zymolyase.

Set up the sample layout

The sample plate layout provides sample tracking from the 96well plate used for DNA isolation to the 96well sample plate CSV file used for import into the OpenArrayTM Sample Tracker Software. Set up the sample plate layout using the CSV file described in the following table.
Note: We recommend at least three technical replicates of each reaction.

Tool
96-well Sample Plate 1.csv template

Source
On the computer on which the OpenArrayTM Sample Tracker Software is installed:
C:\Program Files\Applied Biosytems\Sample Tracking Utility\examples

Description
Contains a sample layout tab.

Urinary Tract Microbiota Profiling Experiments Application Guide

Chapter 3 Isolate DNA from urine research samples using the MagMAXTM DNA Multi-Sample Ultra Kit Concentrate the samples

Concentrate the samples
1. Gently invert, shake, or swirl the sample contents to ensure thorough mixing of the sample.
2. Following the sample layout, transfer 1 mL of sample to the wells of a deep-well plate.
3. Seal the plate with a clear adhesive film, then centrifuge the plate at 2,250 × g for 15 minutes to concentrate the samples.
4. IMPORTANT! There may not be an obvious pellet. If a pellet is visible, be careful not to disturb the pellet.
After centrifugation, carefully remove, then discard the supernatant. a. Set a P1000 pipette (or similar) to 900 L.
Note: We recommend using a manual P1000 multichannel pipette. An electronic multichannel pipette can also be used at low speeds (<6 on Ranin pipettes).
b. Angle the pipette so that the pipette tips sit at the bend from square to conical in the plate well.
c. Carefully remove supernatant, then discard.
d. Repeat substep 4b and substep 4c.
e. Visually inspect the samples to ensure that all urine has been removed. If > 30 µL of urine remains, repeat substep 4b and substep 4c.

Digest the samples with the Preliminary Digestion Mix

1. Prepare sufficient Preliminary Digestion Mix according to the following table.
IMPORTANT! Prepare the Preliminary Digestion Mix no more than 30 minutes before use and store on ice. Prolonged storage at room temperature can reduce its efficiency.

Component B-PERTM Bacterial Protein Extraction Reagent Lysozyme Solution Zymolyase solution (4 U/µL) Total Preliminary Digestion Mix

Volume per well 185 µL 10 µL 5 µL 200 µL

Volume per plate 18.5 mL 1 mL 0.5 mL 20 mL

2. Add 200 µL of Preliminary Digestion Mix to each sample well. (Optional) If large pellets are present, disperse by pipetting up and down 1020 times.
3. Seal the plate with a clear adhesive film, then shake at 1,050 rpm for 3 minutes.

Urinary Tract Microbiota Profiling Experiments Application Guide

Chapter 3 Isolate DNA from urine research samples using the MagMAXTM DNA Multi-Sample Ultra Kit Digest the samples with Proteinase K

4. Incubate the plate for 15 minutes at 65°C. IMPORTANT! Arrange plates in the incubator to allow adequate flow around the plate wells, to ensure that samples quickly reach and maintain the incubation temperature.
During the incubation, prepare the PK Mix (next section).

Digest the samples with Proteinase K

1. Prepare sufficient PK Mix according to the following table, then invert several times to thoroughly mix components.
IMPORTANT! Prepare the PK Mix no more than 30 minutes before use and store at room temperature. Do not place PK Buffer or PK Mix on ice, to avoid precipitation.

Proteinase K PK Buffer Total PK Mix

Component

Volume per well 8 µL 42 µL 50 µL

Volume per plate 0.8 mL 4.2 mL 5.0 mL

2. When the incubation with Preliminary Digestion Mix is complete, add 50 µL of PK Mix to each sample well of the plate.
3. Seal the plate with a clear adhesive film, then shake the sealed plate at 1,050 rpm for 3 minutes.
4. Incubate for 15 minutes at 65°C. IMPORTANT! Arrange plates in the incubator to allow adequate flow around the plate wells, to ensure that samples quickly reach and maintain the incubation temperature.

Urinary Tract Microbiota Profiling Experiments Application Guide

Chapter 3 Isolate DNA from urine research samples using the MagMAXTM DNA Multi-Sample Ultra Kit Set up the processing plates

Set up the processing plates

1. While the samples are incubating at 65°C, set up the Wash, Elution, and Tip Comb Plates outside the instrument as described in the following table.

Plate ID Wash Plate 1 Wash Plate 2 Wash Plate 3 Wash Plate 4 Elution Plate[2]
Tip Comb

Plate position[1] 2 3 4 5 6 7

Plate type Deep Well Deep Well Deep Well Deep Well Standard Deep Well

Reagent

Volume per well

Wash Solution 1

300 µL

Wash Solution 1

300 µL

Wash Solution 2

150 µL

Wash Solution 2

150 µL

DNA Elution Buffer 1

30 µL

Place a tip comb in the plate.

[1] Position on the instrument [2] The instrument prompts the user to add DNA Elution Buffer 2 to the Elution Plate, after incubation with DNA Elution Buffer 1.

2. (Optional) To prevent evaporation and contamination, cover the prepared processing plates with paraffin film until they are loaded into the instrument.

Add Multi-Sample DNA Lysis Buffer, Bead/RNase A Mix, and isopropanol

1. (Optional) If condensation is present at the end of the 65°C incubation, briefly centrifuge the plate at 1,500 × g for 12 minutes.
2. Prepare sufficient Bead/RNase A Mix according to the following table.
IMPORTANT! Prepare the Bead/RNase A Mix no more than 1 hour before use and store on ice. Prolonged storage at room temperature can reduce its efficiency.
Vortex the DNA Binding Beads at moderate speed to form a uniform suspension before preparing the Bead/RNase A Mix.

Component DNA Binding Beads RNase A Nuclease-free Water Total Bead/RNase A Mix

Volume per well 16 µL 5 µL 19 µL 40 µL

Volume per plate 1.6 mL 0.5 mL 1.9 mL 4.0 mL

3. Add 125 µL of Multi-Sample DNA Lysis Buffer to each sample.

Urinary Tract Microbiota Profiling Experiments Application Guide

Chapter 3 Isolate DNA from urine research samples using the MagMAXTM DNA Multi-Sample Ultra Kit Add Multi-Sample DNA Lysis Buffer, Bead/RNase A Mix, and isopropanol

4. (Optional) Add 10 µL of TaqManTM Universal DNA Spike In Control (XenoTM DNA control) to each sample.
Note: For more information about the XenoTM DNA control, see TaqManTM Universal DNA Spike In Control Product Information Sheet (Pub. No. MAN0017852).
5. Vortex the Bead/RNase A Mix at moderate speed to ensure thorough mixing, then add 40 µL to each sample. If you see that the beads in the Bead/RNase A Mix are settling, vortex the mix again briefly before continuing to pipette.
6. Add 200 µL of isopropanol to each sample, then proceed immediately to process the samples on the instrument (next section).

Urinary Tract Microbiota Profiling Experiments Application Guide

Chapter 3 Isolate DNA from urine research samples using the MagMAXTM DNA Multi-Sample Ultra Kit Process samples on the instrument

Process samples on the instrument
1. Select the program on the instrument. · KingFisherTM Flex Magnetic Particle Processor: A25597_UTM
2. Start the run, remove the temporary paraffin plate seals (if present), then load the prepared processing plates in their positions when prompted by the instrument.
3. Load the sample plate (containing lysate, isopropanol, and Bead/RNase A Mix) at position 1 when prompted by the instrument.
4. When prompted by the instrument (approximately 30 minutes after initial start): a. Remove the Elution Plate from the instrument.
b. Add 30 µL of DNA Elution Buffer 2 to each sample well. IMPORTANT! Add DNA Elution Buffer 2 immediately after the prompt, to prevent excessive drying of any beads that are still captured on the Tip Comb.
c. Load the Elution Plate back onto the instrument, then press Start.
5. At the end of the run (approximately 5 minutes after the addition of DNA Elution Buffer 2), remove the Elution Plate from the instrument and seal immediately with a new clear adhesive film. · (Optional) Eluates can be transferred to a new storage plate after collection. · If you see excessive bead residue in the wells, place the Elution Plate on the Magnetic Stand-96 to capture any residue prior to downstream use of the DNA.
IMPORTANT! Do not allow the purified samples to sit uncovered at room temperature for more than 10 minutes, to prevent evaporation and contamination.
The purified samples are ready for immediate use. Alternatively, store the covered Elution Plate: · At 26°C for up to 24 hours. · At 20°C or 80°C for long-term storage.

Urinary Tract Microbiota Profiling Experiments Application Guide

Prepare and run urinary tract microbiota profiling experiments

with OpenArrayTM plates

Workflow: Microbiota profiling experiments with OpenArrayTM plates . . . . . . . . . . . . . . . . . . . . . . . . 23 Required materials for OpenArrayTM plate workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Generate 384well sample plate layouts in the OpenArrayTM Sample Tracker Software . . . . . . . . . 25 Set up the PCR reactions in an OpenArrayTM 384-well Sample Plate . . . . . . . . . . . . . . . . . . . . . . . . 26 Set up the AccuFillTM instrument and software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Transfer reactions to the OpenArrayTM plate using the AccuFillTM instrument . . . . . . . . . . . . . . . . . . 28 Seal the OpenArrayTM plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Run the OpenArrayTM plate on the QuantStudioTM 12K Flex Instrument . . . . . . . . . . . . . . . . . . . . . . 30 Check the qualitycontrol images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 One-time procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

This chapter describes how to run profiling experiments with custom OpenArrayTM plates using a QuantStudioTM 12K Flex RealTime PCR System. This chapter contains brief procedures. For detailed procedures, see the following documentation.

Document
QuantStudioTM 12K Flex RealTime PCR System: OpenArrayTM Experiments User Guide
OpenArrayTM Sample Tracker Software Quick Reference
OpenArrayTM AccuFillTM System User Guide

Pub. No. 4470935 4460657 4456986

Urinary Tract Microbiota Profiling Experiments Application Guide

Chapter 4 Prepare and run urinary tract microbiota profiling experiments with OpenArrayTM plates Workflow: Microbiota profiling experiments with OpenArrayTM plates

Workflow: Microbiota profiling experiments with OpenArrayTM plates

Genomic DNA (page 12)

Generate 384well sample plate layouts in the OpenArrayTM Sample Tracker Software (page 25)

Set up the PCR reactions in an OpenArrayTM 384-well Sample Plate (page 26)

Set up the AccuFillTM instrument and software (page 27)

Transfer reactions to the OpenArrayTM plate using the AccuFillTM instrument (page 28)

Seal the OpenArrayTM plate (page 29)

Run the OpenArrayTM plate on the QuantStudioTM 12K Flex Instrument (page 30)

Check the qualitycontrol images (page 31)

Export and review urinary tract microbiota profiling data (page 35)

Urinary Tract Microbiota Profiling Experiments Application Guide

Chapter 4 Prepare and run urinary tract microbiota profiling experiments with OpenArrayTM plates Required materials for OpenArrayTM plate workflow

Required materials for OpenArrayTM plate workflow

Unless otherwise indicated, all materials are available through thermofisher.com.
"MLS" indicates that the material is available from fisherscientific.com or another major laboratory supplier.

Item Instruments, software, and equipment OpenArrayTM Sample Tracker Software QuantStudioTM 12K Flex OpenArrayTM Plate Press 2.0 QuantStudioTM 12K Flex instrument with OpenArrayTM block (AccuFillTM System) Centrifuge, capable of spinning sample plates at 1,000 rpm Plates and accessories OpenArrayTM 384-well Sample Plates, black BiomekTM Seal and Sample Foil Lids (for pre-plating step) OpenArrayTM AccuFillTM System Tips QuantStudioTM 12K Flex OpenArrayTM Accessories Kit[2] Forceps Reagents Genomic DNA (Optional) TaqManTM Urinary Tract Microbiota Amplification Control
OpenArrayTM plates with TaqManTM Assays

Source
--[1] A24945 4471090
MLS
4482221 Beckman CoulterTM
538619 4458107 4469576
MLS
See page 12 A39174
· A39900 · Custom ordered[3]

TaqManTM OpenArrayTM Real-Time PCR Master Mix

4462164

Ethanol

MLS

[1] Included with the QuantStudioTM 12K Flex Software. [2] Each kit contains the items needed to assemble up to 10 plates: 12 lids and plugs, 12 immersion fluid syringes, and 2 carriers. Each
custom OpenArrayTM plate order is shipped with accessories kits. [3] See "Configure and order custom TaqManTM OpenArrayTM plates" on page 11.

Urinary Tract Microbiota Profiling Experiments Application Guide

Chapter 4 Prepare and run urinary tract microbiota profiling experiments with OpenArrayTM plates Generate 384well sample plate layouts in the OpenArrayTM Sample Tracker Software

Generate 384well sample plate layouts in the OpenArrayTM Sample Tracker Software

Before generating 384well sample plate layouts, see "One-time procedures" on page 32 to complete the following tasks:
· Set up optimized folder locations and software preferences. · Download the TPF files for the OpenArrayTM plates into the TPF Files folder.
1. Using a spreadsheet program, create a 96well sample CSV file. a. Navigate to the following folder, then open the 96-Well Sample Plate 1.csv template that is provided with the OpenArrayTM Sample Tracker Software. <drive>:\Program Files (x86)\Applied Biosystems\OpenArray Sample Tracker\examples

b. Save As the template as a new 96well sample CSV file. Save your 96well sample CSV file in the Sample Tracker 96-well Input folder.

c. Enter or copy the sample names into your 96well sample CSV file.

2. Open the OpenArrayTM Sample Tracker Software.

3. In the Properties screen, select Gene Expression for Experiment Type, then select the appropriate settings for OpenArrayTM Plate and Pipettor.

4. In the Samples screen, click you created in step 1.

Import, then select and import your 96well sample CSV file that

5. In the Sample Mapping screen, confirm that the samples for a single OpenArrayTM plate are assigned to one color.

Note: If necessary, correct the OpenArrayTM Plate and Pipettor settings in the Properties screen.

6. In the Sample Mapping screen, click the 384-Well Plate tab, then click Export4Export *.csv.
7. Select 384-Well Plate (for AccuFill), enter a file name, then save the exported file.
Plate layouts for the 384-well sample plates are saved to individual CSV files in the Sample Tracker 384-well CSV Files folder.

Urinary Tract Microbiota Profiling Experiments Application Guide

Chapter 4 Prepare and run urinary tract microbiota profiling experiments with OpenArrayTM plates Set up the PCR reactions in an OpenArrayTM 384-well Sample Plate

Set up the PCR reactions in an OpenArrayTM 384-well Sample Plate

IMPORTANT! The 4 × 12 area(s) of the OpenArrayTM 384-well Sample Plate being filled must match the area(s) designated in the OpenArrayTM Sample Tracker Software for that set of samples.

1. Remove an OpenArrayTM plate from the freezer and set it aside. Allow it to come to room temperature in its unopened sleeve (~15 minutes).
The OpenArrayTM plate must be completely thawed before transferring reactions to it from the OpenArrayTM 384-well Sample Plate created in this section.

2. Gently swirl the contents of the TaqManTM OpenArrayTM Real-Time PCR Master Mix to thoroughly mix. Do not invert the bottle.

3. Following the plate layout designated in the OpenArrayTM Sample Tracker Software, add master mix, then DNA samples, to the wells of an OpenArrayTM 384-well Sample Plate.

Component[1]
TaqManTM OpenArrayTM Real-Time PCR Master Mix DNA sample Total reaction volume

OpenArrayTM Plate Format

Volume per well

2.5 µL

2.5 µL 5.0 µL

[1] (Optional) Include the TaqManTM Urinary Tract Microbiota Amplification Control. For information about the amplification control, contact CustomControls@thermofisher.com.

4. Thoroughly mix each PCR reaction by pipetting up and down or by using the "mix" function on a multi-channel pipette.

5. Seal the OpenArrayTM 384-well Sample Plate with an aluminum foil seal, remove the foil flap, then mark the edges of the filled 4 × 12 area with a pen.

6. Centrifuge the plate at 1,000 rpm for 1 minute.

7. Score the foil along the lines that were marked before centrifuging. Do not remove the foil from the scored area at this time.

If you make a sample layout error before the AccuFillTM procedure Repeat "Generate 384well sample plate layouts in the OpenArrayTM Sample Tracker Software" on page 25 with a corrected 96well sample CSV file.

Urinary Tract Microbiota Profiling Experiments Application Guide

Chapter 4 Prepare and run urinary tract microbiota profiling experiments with OpenArrayTM plates Set up the AccuFillTM instrument and software

Set up the AccuFillTM instrument and software
IMPORTANT! Do not use OpenArrayTM AccuFillTM System Tips that exceed the expiration date (shown on the outer box that contains the tip trays).
1. In the OpenArrayTM AccuFillTM software, click Setup and Load. The Setup Load Information window appears.

1 Use Sample Integration checkbox; select to integrate TPF files and the 384-well sample plate CSV file. 2 Browse button; click to locate and select the 384-well sample plate CSV file. The button is displayed only if Use
Sample Integration is selected. 3 Browse buttons; click to locate and select the TPF files for the OpenArrayTM plates that will be placed in the
corresponding Plate Holder Position on the deck of the AccuFillTM instrument. The buttons are displayed only if Use Sample Integration is selected. 4 Plate Holder Position corresponding to the position of the OpenArrayTM plate on the deck of the AccuFillTM instrument.
2. Configure the Loading Information pane for sample integration using the 384-well sample plate CSV file and TPF files. a. In the Loading Information pane (top section of the window), ensure that the Use Sample Integration checkbox is selected.
b. Click Browse to the right of the Sample Plate field, then select the 384-well sample plate CSV file that you generated with the OpenArrayTM Sample Tracker Software in the Sample Tracker 384-well CSV Files folder.
c. Click Browse to the right of the Plate Holder Position of the OpenArrayTM plate, then select the TPF file for the OpenArrayTM plate in the TPF Files folder.

Urinary Tract Microbiota Profiling Experiments Application Guide

Chapter 4 Prepare and run urinary tract microbiota profiling experiments with OpenArrayTM plates Transfer reactions to the OpenArrayTM plate using the AccuFillTM instrument

3. In the Select Samples to Load pane (bottom section of the window), click the corresponding 4 × 12 area of the 384-well sample plate image, then click Next. The Setup Deck window is displayed.
4. In the AccuFillTM instrument, ensure that: · Tip boxes and tips are loaded as shown in the Setup Deck window. · The lids are removed from the tip boxes. · The waste bin in the instrument is emptied.
5. In the Setup Deck window, confirm that the deck is ready: · Select The tips are configured as shown above. · Select The Waste Bin is empty.

Transfer reactions to the OpenArrayTM plate using the AccuFillTM instrument
IMPORTANT! Ensure that the OpenArrayTM plate is thawed and that the entire plate is at room temperature.
1. Prepare the items needed to seal the loaded OpenArrayTM plate (next section).
Note: The OpenArrayTM plate must be sealed promptly after being loaded with the reactions, as described here.
· Ensure that the QuantStudioTM 12K Flex OpenArrayTM Plate Press 2.0 is ready. · Gather and remove from their packaging the following: an OpenArrayTM Lid, plug, syringe with
OpenArrayTM Immersion Fluid, and syringe tip. · Attach the syringe tip to the syringe, carefully push some of the fluid through the tip to remove
air bubbles, then lay the syringe aside.
2. Load the OpenArrayTM plate and the OpenArrayTM 384-well Sample Plate into the AccuFillTM instrument. · OpenArrayTM plate--Remove the plate from its sleeve, then place the plate in the appropriate plate holder position in the instrument. Ensure that the barcode on the OpenArrayTM plate is facing left and the serial number is facing right. · OpenArrayTM 384-well Sample Plate--Place the 384-well sample plate onto the deck of the instrument, then use forceps to peel the foil from the filled area of the plate.
3. Close the door of the AccuFillTM instrument.
4. In the AccuFillTM software Setup Deck window, select the following confirmations: · The OpenArray Plate is in the Plate Holder · Remove foil from the highlighted section of the Sample Plate

Urinary Tract Microbiota Profiling Experiments Application Guide

Chapter 4 Prepare and run urinary tract microbiota profiling experiments with OpenArrayTM plates Seal the OpenArrayTM plate

5. Click Load.
6. As soon as the Remove OpenArray Plate window appears, open the instrument door and remove the loaded OpenArrayTM plate.
7. Proceed immediately to seal the OpenArrayTM plate (next section).
Note: For best results, seal the OpenArrayTM plate within 90 seconds of completion of loading to prevent evaporation.

Seal the OpenArrayTM plate

IMPORTANT! Throughout this procedure, handle the OpenArrayTM plate and the OpenArrayTM Case only by the edges.

Note: The OpenArrayTM Case consists of the sealed OpenArrayTM plate and the OpenArrayTM Lid.
1. Place the newly loaded OpenArrayTM plate in the QuantStudioTM 12K Flex OpenArrayTM Plate Press 2.0. Ensure that the barcode is facing left and the serial number is facing right.
2. From the OpenArrayTM Lid, remove the clear protective film from the inside of the lid and the red adhesive-protective strip from around the edge of the lid.

OpenArrayTM Lid 1 Protective film on inside of the lid (remove before sealing) 2 Red adhesive-protective strip (remove before sealing) 3 Protective film on the outside of the lid (remove before running) 4 Notched end (align with serial number on plate)
3. Seat the lid on the OpenArrayTM plate with the lid adhesive against the plate.
4. Engage the press mechanism until the green flashing light changes to a steady green light (approximately 20 seconds).
5. Disengage the press and remove the OpenArrayTM Case.

Urinary Tract Microbiota Profiling Experiments Application Guide

Chapter 4 Prepare and run urinary tract microbiota profiling experiments with OpenArrayTM plates Run the OpenArrayTM plate on the QuantStudioTM 12K Flex Instrument

6. While holding the case by its edges, insert the prepared syringe tip into the port in the case, then carefully inject OpenArrayTM Immersion Fluid until the case is filled.
Note:
· Minimize creation of air bubbles when you dispense the fluid. · Leave a small bubble at the fill point to prevent fluid leaks during the instrument run.

The syringe tip must be in front of the array when filling the case with immersion fluid.
7. While holding the case vertically, remove the syringe tip, insert the screw end of the OpenArrayTM plug into the port of the case, then rotate clockwise until the black handle breaks off.
IMPORTANT! To avoid leaking of immersion fluid, hold the case vertically and rotate the plug slowly.
If the plug handle breaks off prematurely, use a Phillips #0 screwdriver to complete this step.
8. If needed, clean the case with a laboratory wipe that has been thoroughly sprayed with ethanol, then dry the case with a clean laboratory wipe.

Run the OpenArrayTM plate on the QuantStudioTM 12K Flex Instrument

You can run up to four OpenArrayTM plates at one time on the QuantStudioTM 12K Flex Instrument.

1. On the QuantStudioTM 12K Flex Instrument touchscreen, touch arm.

to extend the instrument tray

2. Remove the clear protective film from the outside of the OpenArrayTM case (sealed plate + lid).

3. Place the OpenArrayTM case on the tray arm plate adapter.
· Support the case from underneath the tray arm to prevent the case from slipping through the adapter.
· Ensure that the plate barcode and serial number are facing the front of the instrument.

4. Touch to retract the instrument tray arm.

Urinary Tract Microbiota Profiling Experiments Application Guide

Chapter 4 Prepare and run urinary tract microbiota profiling experiments with OpenArrayTM plates Check the qualitycontrol images

5. In the Home screen of the QuantStudioTM 12K Flex Software, in the Run pane, click OpenArray.
6. In the Select Instrument pane, select your instrument.
7. Click Get Plate IDs to import the barcode of the OpenArrayTM plate. Once the OpenArrayTM serial number appears, the loaded TPF file corresponding to the plate should appear in the Setup File field. If the TPF file does not appear, click Browse, then select the correct loaded TPF file from the Loaded TPF folder.
8. (Optional) Click Browse to change the Experiment File Location.
9. (Optional) Change the software-determined Experiment File Name.
10. Click Start Run.
Note: The instrument pauses at 41 or 42 seconds prior to the end of the run. Wait for the system to complete the run before opening the EDS file.
11. Transfer the EDS file from the instrument to an accessible location for analysis.
12. Check the QC images for loading issues or leaks.

Check the qualitycontrol images

Check the qualitycontrol (QC) images before analysis. Images can be viewed using ImageJ, an opensource software available from the NIH at imagej.nih.gov/ig. For additional information, see Appendix A, "Troubleshooting".

1. In the QuantStudioTM 12K Flex Software named folder for the QC images export.

Export screen, click Browse, then create a uniquely

IMPORTANT! Create a new folder for images each time. Exporting a second run to the same folder overwrites the images.

2. Click Export QC Images at the bottom of the screen.
3. View the following ROXTM image to check for loading quality issues: · POST-READ_CHANNEL_4.tiff
4. Check the following spotfinding images for leaks or other displaced sample issues. · s02_c001_t03_p0001_m1_x2_e1_cp#_spotfind.tiff · s02_c040_t03_p0001_m1_x2_e1_cp#_spotfind.tiff
Note: The "cp#" in the image file name refers to array positions 1 through 4 within the instrument.

Urinary Tract Microbiota Profiling Experiments Application Guide

Chapter 4 Prepare and run urinary tract microbiota profiling experiments with OpenArrayTM plates One-time procedures

5. If a problem is found, view the following pre-run spotfinding image to determine whether the issue existed before cycling: · s00_c001_t01_p0001_m2_x3_e1_cp#_spotfind.tiff
6. View the following FAMTM images to check for fluorescent abnormalities and to confirm any problem seen in the spotfinding images: · STAGE2_CYCLE1_CHANNEL_1.tiff · STAGE2_CYCLE40_CHANNEL_1.tiff
7. Note any abnormalities found, as well as all other potentially relevant information related to the setup of the run.

One-time procedures
Set up default folders and software preferences
This procedure simplifies the file locations used in the AccuFillTM software. Set up the default file locations and preferences before using the OpenArrayTM AccuFillTM system for the first time. You must be logged in as an administrator.
1. Create the following four folders in a convenient location on the same computer drive as the AccuFillTM software: · TPF Files · Sample Tracker 96-well Input · Sample Tracker 384-well CSV Files · Loaded TPF Files
2. (Optional) Copy a template file into the OpenArrayTM Sample Tracker Software folder. · Navigate to this folder on your computer: <drive>:\Program Files (x86)\Applied Biosystems\OpenArray Sample Tracker\examples. · Copy the 96Well Sample Plate 1.csv template file, which is provided with the OpenArrayTM Sample Tracker Software. · Paste the template file into the Sample Tracker 96well Input folder.

Urinary Tract Microbiota Profiling Experiments Application Guide

Chapter 4 Prepare and run urinary tract microbiota profiling experiments with OpenArrayTM plates One-time procedures

3. In the OpenArrayTM Sample Tracker Software, select View4Preferences, then enter the following preferences:

Field Experiment Type OpenArrayTM Plate
Pipettor Import Data Directory Export Data Directory

Selection Gene Expression Select the OpenArrayTM format that will be run most often, such as Gene Expression 56. Fixed or Adjustable tip spacing Sample Tracker 96-well Input Sample Tracker 384-well CSV Files

4. In the AccuFillTM software, select Instrument4Edit Preferences, then: a. Select Require Sample Integration.

b. Select the folders indicated in this table:

AccuFillTM folder
OpenArrayTM Plate File Input Folder

Default folder TPF Files

Sample Plate File Folder
Loaded OpenArrayTM Plate File Folder

Sample Tracker 384-well CSV Files
Loaded TPF Files

Folder contents
TPF files for the OpenArrayTM plates, with assay name and location
CSV 384-well sample plate layout files
Integrated TPF files generated during processing with the AccuFillTM software.

5. In the QuantStudioTM 12K Flex Software, select Tools4Preferences4OpenArray, then select the Loaded TPF Files folder for the software Setup Folder.
Note: If the QuantStudioTM 12K Flex Software is not on the same computer as the AccuFillTM software, transfer the loaded TPF files to the computer running the QuantStudioTM 12K Flex Software.

Urinary Tract Microbiota Profiling Experiments Application Guide

Chapter 4 Prepare and run urinary tract microbiota profiling experiments with OpenArrayTM plates One-time procedures

Download TPF files
Set up the optimized folder locations and software preferences before downloading TPF files. See "Set up default folders and software preferences" on page 32. To download TPF files for custom OpenArrayTM plates, you need the Lot# and the Serial# from the packaging of each OpenArrayTM plate.
1. Go to thermofisher.com/OA-platefiles.
2. From the Select Your Product dropdown list, select TaqManTM OpenArrayTM Custom Gene Expression/Genotyping Plates.
3. Select the desired option for downloading either only the TPF files or both the TPF files and the AIF files.
4. Enter the Lot# and the Serial#, then click Submit.
Note: The Serial# is case-sensitive.
5. Save the TPF files to the desktop TPF Files folder.
Note: Do not create sub-folders in the TPF Files folder. The software cannot access sub-folders.

Urinary Tract Microbiota Profiling Experiments Application Guide

Export and review urinary tract microbiota profiling data

Export data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Prepare exported data for analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Review results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Export data
1. Open an EDS file in the QuantStudioTM 12K Flex Software. 2. In the Experiment Menu pane, click Export. 3. Click Load Export Set (bottom of the screen), select GE_export_setting, then click OK. 4. Select .xlsx from the File Type dropdown list (top-right of the screen). 5. (Optional) Perform any of the following actions to customize the file export.
· Click Browse to select a new Export File Location. · Enter a new file name in the Export File Name text field. · Click the Results tab, then select the content to export. 6. Click Start Export (bottom of the screen). If Open file(s) when export is complete is selected, then the file automatically opens. If the option is not selected, navigate to and open the exported XLSX file.
Prepare exported data for analysis
1. Open the exported XLSX data file. 2. Ensure that the barcode, run conditions, and all selected data columns were exported correctly. 3. Scroll down to the data rows, select the headers and data, then copy-paste into a new worksheet. 4. Rename the new worksheet Data Table_Run File Name. 5. (Optional) To combine data from multiple OpenArrayTM plates:
a. Insert a Barcode column in the Data Table worksheet to track OpenArrayTM barcodes.

Urinary Tract Microbiota Profiling Experiments Application Guide

5 Chapter 5 Export and review urinary tract microbiota profiling data Review results

b. Copy-paste the barcode numbers to the appropriate cells in the new Barcode column.
6. Find-replace all "Undetermined" values with an empty cell (no value) in the Crt column. This step ensures an exact count of Crt values.
7. Delete rows that do not contain run data.

Review results

Note: These guidelines apply to results from experiments that included three or more technical replicates.

Note: We encourage testing and establishing your own Crt cut-off value for each assay to achieve high sensitivity and specificity.

1. Review the exported data for through-hole results that may require special attention.

2. Consider filtering out from analysis through-holes with the following values:

Parameter to examine
Crt
Cq Confidence

Consider filtering out through-holes if...
Crt 31 Cq Conf < 0.8
· RNase P (Hs04930436_g1) -- acceptable range is 0.7 1.0

Amp Score

Amp Score < 1.2

Note: Through-holes with unexpected Crt values can also be identified by reviewing the Amplification Plot (see page 38). 3. Review through-holes with Crt > 28 and ensure that the Crt values are reproducible in all technical replicates. Note: Crt = 28 is approximately equal to 1 copy of the target sequence in a reaction. 4. Take note of technical replicates with mean Crt 25 and a high standard deviation (> 0.5). The data from these through-holes might require further review. 5. Ensure that at least half of the replicates amplified adequately and pass your review specifications. 6. Use your preferred method to analyze the data.

Urinary Tract Microbiota Profiling Experiments Application Guide

5 Chapter 5 Export and review urinary tract microbiota profiling data Review results

Fields for reviewing results with pivot tables
To review results using the pivot table feature of a spreadsheet program, you can use the following settings.
Note: For the "Average of" and "StdDev of" summarizations, use the appropriate source field (Crt, Amp Score, or Cq Conf), then choose the calculation type.

Area of pivot table

Fields to add

Target-oriented view

Sample-oriented view

Filters

Sample Name[1]

Columns

Sample Name

Rows

Target Name

Values

Average of Crt StdDev of Crt[2]
Count of Crt --

Average of Crt StdDev of Crt[2]
Count of Crt Average of Amp Score

Average of Cq Conf

[1] To see individual sample results, select the sample from the dropdown list next to the Sample Name header. [2] A Values field will automatically appear in the Column Labels area.

Urinary Tract Microbiota Profiling Experiments Application Guide

Troubleshooting

Troubleshoot DNA isolation errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Troubleshoot unexpected Crt values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Troubleshoot with cycling and imaging run images (QC images) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 AccuFillTM instrument plate loading errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 OpenArrayTM plate assembly and handling errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Troubleshoot DNA isolation errors

Troubleshoot unexpected Crt values

Observation Unexpected Crt values in the amplification plot
1 2
1 Expected Crt value (noted in most replicates) 2 Unexpected Crt value (too low)

Possible cause Unexpectedly low Crt values (< 10) -- Signal variation or noise in early PCR cycles
Unexpectedly high Crt values ( < 28) -- Sporadic amplification

Recommended action
Review amplification curves, AmpScore, and Cq Confidence values.
Consider filtering Crt values from analysis.
Compare replicates, if available.
Repeat the experiment.
Review amplification curves, AmpScore, and Cq Confidence values.
Consider filtering Crt values from analysis if < 50% of replicates amplified at similar levels.
Repeat the experiment.

Urinary Tract Microbiota Profiling Experiments Application Guide

Appendix A Troubleshooting Troubleshoot with cycling and imaging run images (QC images)

Troubleshoot with cycling and imaging run images (QC images)

Many problems with OpenArrayTM results can be diagnosed by examining the quality control (QC) images taken at various points during a cycling/imaging run.
The QC images are fluorescent or reflected light images taken before, during, and after cycling. They may require adjustment to make image features visible. To view the images, we recommend that you install the free software program ImageJ, which allows you to easily manipulate the images in ways that other image viewers cannot.
1. In the QuantStudioTM 12K Flex Software Export screen : a. Click Browse to select a uniquely-named folder for the QC images export.
b. Click Export QC Images (bottom of screen).

IMPORTANT! Select a new folder for images each time; exporting a second run to the same folder overwrites the images.

2. Use ImageJ to view the images of interest.

To...

View image...

Image description

Confirm the identity of BARCODE IMAGE.tiff images within a folder

Reflected light image of the entire OpenArrayTM plate

Evaluate the loading quality

PRE-READ_CHANNEL_4.tiff POST-READ_CHANNEL_4.tiff

Pre- and post-ROXTM dye images

Check for existing contamination on the case and/or heated cover

s00_c001_t01_p0001_m2_x3_e1_cp#_spotfind.tiff[1] Pre-run reflected light spotfinding image (used by the software to determine the location of the holes)

Identify potential leaks s02_c001_t03_p0001_m1_x2_e1_cp#_spotfind.tiff[1] Mid-run reflected light spotfinding

or other contamination

image

s02_c040_t03_p0001_m1_x2_e1_cp#_spotfind.tiff[1] Post-run reflected light spotfinding image

Look at patterns in the fluorescent data (for example, gradients)

STAGEx_CYCLEy_CHANNEL_1.tiff

FAMTM images at a particular cycle (y) of a particular stage (x) of the run.

[1] The "cp#" in the image file name refers to the array position (14) within the QuantStudioTM 12K Flex Real-Time PCR Instrument.

3. (Optional) Adjust the images for brightness and/or contrast to make image features visible. a. Open the image in ImageJ.

b. Select Image4Adjust Brightness/Contrast (or press Ctrl+Shift+C).

c. Click Auto or adjust the sliders until the features of interest in the image are visible.

Urinary Tract Microbiota Profiling Experiments Application Guide

Appendix A Troubleshooting AccuFillTM instrument plate loading errors

AccuFillTM instrument plate loading errors

Observation There are empty through-holes
Turn-holes are repeatedly missed

Possible cause

Recommended action

Insufficient sample was added to the 384-well Sample Plate.

Use proper pipetting techniques. Ensure that there are no air bubbles in the pipette tips after sample aspiration.

Reaction mix (sample + master mix) is not at the bottom of the 384-well Sample Plate.
AccuFillTM instrument is aligned too far to the left or to the right.

Centrifuge the plate at 1,000 rpm for 60 seconds.
Contact your local field service engineer.

Systematic loading problems can occur with the AccuFillTM instrument, which indicates a need for service. For example, when turn-holes are repeatedly missed across multiple subarrays, service is required. Turnholes are where the AccuFillTM instrument changes direction during sample loading.

Entire subarrays are missing
40

Turn holes Start points Stop points

The sample/master mix was not added to particular wells in the 384-well Sample Plate.
Stuck tip mandrel on AccuFillTM instrument may need cleaning.

Visually inspect the plate to ensure that the wells have sample/master mix.
Contact your local field service engineer.

Urinary Tract Microbiota Profiling Experiments Application Guide

Observation Entire subarrays are missing

Appendix A Troubleshooting OpenArrayTM plate assembly and handling errors

Possible cause
Pipette tip was not loaded on mandrel.

Recommended action
Contact your local field service engineer for frequent occurrences (infrequent occurrences can be due to a poorly molded tip).

(continued)

OpenArrayTM plate assembly and handling errors

Observation Case leaks and bubbles inside the case
Improper sealing of the OpenArrayTM plate in the OpenArrayTM Case can lead to immersion fluid leaks or bubble formation inside the case, leading to uneven heating and imaging throughout PCR and to poor quality data. The images above are examples of OpenArrayTM plates that have been affected by immersion fluid leaks. The images show where leaked fluid has condensed on the underside of the heated cover

Possible cause Plate press was not engaged for at least 20 seconds. Damaged lid adhesive.
Damaged fill port screw gasket.
Damaged fill port screw assembly. Breaks off too easily. Oily lid or case from immersion fluid overflow.
Immersion fluid was exposed to air for too long.

Recommended action
Fully engage the plate press for at least 20 seconds.
Remove the liner and visually inspect the lid adhesives for defects. Ensure that adhesive is not damaged or warped.
Visually inspect the screw to ensure that the orange gasket is present and not damaged.
The screw may be misthreaded: unscrew it and use a new screw assembly.
Wipe off excess overflow of immersion fluid from the lid, case bottom, and crevices with 70% isopropyl alcohol, using a lint-free cloth (the cloth included with the OpenArrayTM plate is acceptable).
Do not remove the immersion fluid syringe cap or draw air bubbles into the syringe until you are ready to load.

Urinary Tract Microbiota Profiling Experiments Application Guide

Appendix A Troubleshooting OpenArrayTM plate assembly and handling errors

Observation windows and obscured the view of the through- holes. The best image in which to detect leaks is the s02_c001_t03_p0001_m1_x2_e1_cp#_spotfind.tiff image. This image is taken at the start of cycling, which is where most leaks occur. See "Troubleshoot with cycling and imaging run images (QC images)" on page 39.
Sample blow-out during the addition of immersion fluid
Evaporation of reaction mixture in through-holes

Possible cause Too large of a bubble inside the OpenArrayTM Case after sealing.
Damaged plate press, leading to uneven pressure.
The reactions in A12 were compromised during the addition of immersion fluid. Injecting the immersion fluid too quickly can purge the sample out of the throughholes near the fill port. Often this is caused by the user not purging the syringe slightly before use. Too much time elapsed before the plate was sealed with lid and immersion fluid. In this example, the top half of each subarray was intentionally left open to the environment to demonstrate the effect of evaporation. "Donuts" are a result of the evaporated fluid in the though-holes.

Recommended action
Minimize the size of the bubble by tilting the OpenArrayTM Case so that the fill port is at the highest point. Slowly fill the case with immersion fluid until only a small air bubble remains. Attach the screw and wipe off any excess oil that may have spilled onto the case. Contact your field service engineer if you suspect that your plate press may be damaged. Dispense a small amount of immersion fluid onto a paper towel before use to ensure smooth operation of the syringe.
Add immersion fluid as soon as the case is removed from the plate press to minimize the likelihood of evaporation, then seal the case with the lid.

Urinary Tract Microbiota Profiling Experiments Application Guide

Safety

WARNING! GENERAL SAFETY. Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device. Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document.
· Before using an instrument or device, read and understand the safety information provided in the
user documentation provided by the manufacturer of the instrument or device.
· Before handling chemicals, read and understand all applicable Safety Data Sheets (SDSs) and use
appropriate personal protective equipment (gloves, gowns, eye protection, and so on). To obtain SDSs, see the "Documentation and Support" section in this document.

Urinary Tract Microbiota Profiling Experiments Application Guide

B Appendix B Safety Chemical safety
Chemical safety
WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel read and practice the general safety guidelines for chemical usage, storage, and waste provided below. Consult the relevant SDS for specific precautions and instructions:
· Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer
before you store, handle, or work with any chemicals or hazardous materials. To obtain SDSs, see the "Documentation and Support" section in this document.
· Minimize contact with chemicals. Wear appropriate personal protective equipment when handling
chemicals (for example, safety glasses, gloves, or protective clothing).
· Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with
sufficient ventilation (for example, fume hood).
· Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer
cleanup procedures as recommended in the SDS.
· Handle chemical wastes in a fume hood. · Ensure use of primary and secondary waste containers. (A primary waste container holds the
immediate waste. A secondary container contains spills or leaks from the primary container. Both containers must be compatible with the waste material and meet federal, state, and local requirements for container storage.)
· After emptying a waste container, seal it with the cap provided. · Characterize (by analysis if needed) the waste generated by the particular applications, reagents,
and substrates used in your laboratory.
· Ensure that the waste is stored, transferred, transported, and disposed of according to all local,
state/provincial, and/or national regulations.
· IMPORTANT! Radioactive or biohazardous materials may require special handling, and disposal
limitations may apply.
WARNING! HAZARDOUS WASTE (from instruments). Waste produced by the instrument is potentially hazardous. Follow the guidelines noted in the preceding General Chemical Handling warning.
WARNING! 4L Reagent and Waste Bottle Safety. Four-liter reagent and waste bottles can crack and leak. Each 4-liter bottle should be secured in a low-density polyethylene safety container with the cover fastened and the handles locked in the upright position.

Urinary Tract Microbiota Profiling Experiments Application Guide

Biological hazard safety

B Appendix B Safety
Biological hazard safety

WARNING! Potential Biohazard. Depending on the samples used on this instrument, the surface may be considered a biohazard. Use appropriate decontamination methods when working with biohazards.

WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other animals have the potential to transmit infectious diseases. Conduct all work in properly equipped facilities with the appropriate safety equipment (for example, physical containment devices). Safety equipment can also include items for personal protection, such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, or goggles. Individuals should be trained according to applicable regulatory and company/ institution requirements before working with potentially biohazardous materials. Follow all applicable local, state/provincial, and/or national regulations. The following references provide general guidelines when handling biological samples in laboratory environment.
· U.S. Department of Health and Human Services, Biosafety in Microbiological and Biomedical
Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC) 21-1112, Revised December 2009; found at:
https://www.cdc.gov/labs/pdf/CDC-BiosafetymicrobiologicalBiomedicalLaboratories-2009P.pdf
· World Health Organization, Laboratory Biosafety Manual, 3rd Edition,
WHO/CDS/CSR/LYO/2004.11; found at:
www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Urinary Tract Microbiota Profiling Experiments Application Guide

Documentation and support

Related documentation
Document Urinary Tract Microbiota Profiling Experiments Application Guide DNA isolation for Urinary Tract Microbiota Profiling Experiments Quick Reference OpenArrayTM Urinary Tract Microbiota Profiling Experiments Quick Reference TaqManTM Urinary Tract Microbiota Amplification Control Product Information Sheet TaqManTM Universal DNA Spike In Control Product Information Sheet QuantStudioTM 12K Flex RealTime PCR System: OpenArrayTM Experiments User Guide OpenArrayTM Sample Tracker Software Quick Reference OpenArrayTM AccuFillTM System User Guide

Publication Number MAN0017750 MAN0017751 MAN0017752 MAN0017753 MAN0017852 4470935 4460657 4456986

Symbols that may be displayed on the instrument, in the software, or in this guide

Symbol

Description

MANUFACTURER

CATALOG NUMBER

CONSULT INSTRUCTIONS FOR USE[1]
[1] "Documentation and support" on page 46

Symbol

Description

DATE OF MANUFACTURE

SERIAL NUMBER

CAUTION[1]

Urinary Tract Microbiota Profiling Experiments Application Guide

Documentation and support Customer and technical support
Customer and technical support
Visit thermofisher.com/support for the latest service and support information. · Worldwide contact telephone numbers · Product support information Product FAQs Software, patches, and updates Training for many applications and instruments · Order and web support · Product documentation User guides, manuals, and protocols Certificates of Analysis Safety Data Sheets (SDSs; also known as MSDSs)
Note: For SDSs for reagents and chemicals from other manufacturers, contact the manufacturer.
Limited product warranty
Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of Sale at www.thermofisher.com/us/en/home/ global/terms-and-conditions.html. If you have any questions, please contact Life Technologies at www.thermofisher.com/support.

Urinary Tract Microbiota Profiling Experiments Application Guide

Index

96-well Sample Plate 1.csv template 16
A
Accufill instrument, set up 27 Accufill software, set up 27 amplification control 9 assays, complete list 8
B
before first use of MagMAX kit 16 biohazard safety 45
C
concentrate samples, preliminary 17 contents 9, 10 create sample plate layouts 25
D
data, export 35 data review guidelines 36 default folders 32 digest samples
preliminary 17 Proteinase K 18 documentation, related 46 download KingFisher program 15 TPF files 34
E
export data 35
K
KingFisher program 15 kit contents 13 kit storage 13
48

L
limited product warranty 47
M
materials required DNA isolation 13 OpenArray plate workflow 24
O
OpenArray plate, run 30 OpenArray plates
order 11 workflow 23 order, OpenArray plates 11
P
PCR reactions, set up 26 Preliminary Digestion Mix, prepare 17 processing plates, set up 19 Proteinase K digest 18
Q
QC images 31, 39
R
related documentation 46 run, OpenArray plate 30
S
safety, biohazard 45 sample collection 12 sample tracking 16 search for assays 11 software preferences 32 storage 9, 10 support, customer and technical 47 symbols 46
Urinary Tract Microbiota Profiling Experiments Application Guide

T
terms and conditions 47 TPF files, download 34 troubleshooting
Accufill 40 cycling and imaging run images 39 OpenArray 41
U
Universal DNA Spike In 9

W
warranty 47 workflow, OpenArray plates 6, 23
X
Xeno DNA control 9

Index

Urinary Tract Microbiota Profiling Experiments Application Guide

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