Axiom 2.0 Assay 96-Array Format Automated Workflow€¦ · For Research Use Only. Not for use in diagnostic procedures. Axiom™ 2.0 Assay 96-Array Format Automated Workflow USER

Axiom™ 2.0 Assay 96-Array Format Automated WorkflowUSER GUIDE

for Biomek FXP (Windows® 7 and XP)

Publication Number 702963

Revision 5

For Research Use Only. Not for use in diagnostic procedures.

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

DISCLAIMER

TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES 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

Important Licensing Information

This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept the terms and conditions of all applicable Limited Use Label Licenses.

Corporate entity

Life Technologies | Carlsbad, CA 92008 USA | Toll Free in USA 1 800 955 6288

TRADEMARKS

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Jitterbug is a trademark of Boekel Scientific. Microsoft, Excel, and Windows are either registered trademarks or trademarks of Microsoft Corporation in the United States and/or other countries. QIAGEN and REPLI-g are registered or registration-pending trademarks of the QIAGEN Group. Beckman Coulter, and Biomek are either registered trademarks or trademarks of Beckman Coulter, Inc. TRobot is a trademark of Biometra GmbH. DNA Engine Tetrad, Bio-Rad, Microseal, and Hard-Shell are registered trademarks of Bio-Rad Laboratories, Inc.

©2018 Thermo Fisher Scientific Inc. All rights reserved.

Manufacturer:Affymetrix Pte Ltd 7 Gul Circle #2M-01Keppel Logistics Building Singapore 629563

Products:Axiom™ Array Plates

Manufacturer:Thermo Fisher Scientific Baltics UAB V.A. Graiciuno 8, LT-02241Vilnius, Lithuania

Products:Axiom™ 2.0 Reagent Kit

Table 1 Revision history of Pub. no. 702963

Revision Date Description

5 04 September 2018 Baseline for revision history.Updated to the current document template, with associated updates to trademarks, logos, licensing, and warranty.Updated to reflect that Axiom™ Reference gDNA 103 has been removed from the reagent kit and has been made available for purchase separately.

Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP

Contents

CHAPTER 1 The Axiom™ 2.0 Assay . . . . . . . . . . . . . . . . . . . . . . . . . . 10

About . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

What’s new . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Overview of the Axiom™ 2.0 Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Running multiple plate workflows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

CHAPTER 2 Genomic DNA preparation and requirements. . . . . . . . . 13

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Sources of genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

General requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Special requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Assessing the quality of genomic DNA using 1% Agarose E-gels . . . . . . . . . . . . . . . . . . 15

Genomic DNA extraction/purification methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Genomic DNA cleanup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Genomic DNA preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Equipment, consumables and reagents required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

1. Thaw samples and control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

2. Quantitate and dilute gDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

3. Aliquot the diluted samples and the control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

4. Freeze or proceed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

5. Create a Batch Registration file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

CHAPTER 3 Target preparation on the Biomek FXP with Windows® XP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Axiom™ 2.0 Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Before using the Biomek workstation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Seal, vortex, and centrifuge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Breaking the light curtain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Pipette tip usage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Plate centrifuge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Set the Biomek software default settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

3

Contents

Equipment, consumables, labware, and reagents required . . . . . . . . . . . . . . . . . . . . . . . . . 29

Labware and materials used on the Biomek™ workstation deck . . . . . . . . . . . . . . . . . . . 29

Proper tray alignment and loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Stain trays and covers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Loading tray consumables onto the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . 46

Reagent block template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Reservoir labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Related Biomek FXP Target Prep Express documentation . . . . . . . . . . . . . . . . . . . . . . . . 50

Stage 1: DNA Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

Equipment, consumables, labware, and reagents required . . . . . . . . . . . . . . . . . . . . . . . 51

1. Perform the pre-run checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

2. Thaw and prepare the reagents and Sample Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

3. Run the DNA Amplification step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Summary of DNA Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Stage 2: Fragmentation and Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65


1. Perform the pre-run checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

2. Thaw and prepare the amplified DNA samples and reagents . . . . . . . . . . . . . . . . . . . . 67

3. Run the Fragmentation step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

Summary of Fragmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

4. Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

5. Centrifuge and dry pellets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Stage 3: Resuspension and hybridization preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76


1. Preparing frozen pellets and Axiom Resusp Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77


3. Thaw and prepare the reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

4. Run the Resuspension and Hybridization Preparation step . . . . . . . . . . . . . . . . . . . . . 78

Summary of Resuspension and Hybridization Preparation . . . . . . . . . . . . . . . . . . . . . . . . 85

Stage 4: Preparation for the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

About Stage 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

Duration of GeneTitan™ MC Instrument reagent preparation and sample preparation . . . 93

Equipment and consumables required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94


2. Prepare the reagents for GeneTitan Reagent Tray Preparation . . . . . . . . . . . . . . . . . . . 96

3. Prepare the Sample Plate (if stored at –20°C) and the array plate . . . . . . . . . . . . . . . . 98

4. Prepare the GeneTitan™ MC Instrument. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

5. Run the Preparation for GeneTitan™ step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

4 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP

Contents

6a. Complete Stage 4: Preparation for GeneTitan™ - Hybridization Trays . . . . . . . . . . . . 106

6b. Complete Stage 4: Preparation for GeneTitan™ - Reagent Trays. . . . . . . . . . . . . . . . 107

6c. Complete Stage 4: Preparation for GeneTitan™ - multiple plate workflow . . . . . . . . 107

Summary of Preparation for GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . 109

CHAPTER 4 Target preparation on the Biomek FXP with Windows® 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

What's new for the Axiom™ 2.0 Target Preparation 96-Samples Biomek® FXP

Method for Windows® 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

New features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

Method changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

New off-deck step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

Axiom™ 2.0 Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

Before using the Biomek workstation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Seal, vortex and centrifuge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Breaking the light curtain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

Pipette tip usage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

Plate centrifuge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

Setting method preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

Equipment, consumables, labware, and reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

Labware and materials used on the Biomek workstation deck . . . . . . . . . . . . . . . . . . . . 124

Proper tray alignment and loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

Stain trays and covers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

Loading tray consumables onto the GeneTitan™ MC Instrument. . . . . . . . . . . . . . . . . . . 140

Reagent block template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

Reservoir stickers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

Related Biomek FXP Target Prep Express documentation . . . . . . . . . . . . . . . . . . . . . . . 144

Stage 1: DNA Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

Equipment, consumables, labware and reagents required . . . . . . . . . . . . . . . . . . . . . . . 145


2. Thaw and prepare the reagents and Sample Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

3. Run the DNA Amplification step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

Summary of DNA Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

Stage 2: Fragmentation and Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157


1. Perform the pre-run checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

2. Thaw and prepare the amplified DNA samples and reagents . . . . . . . . . . . . . . . . . . . 159

Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 5

Contents

3. Run the Fragmentation step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159

4. Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165

Summary of Fragmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166

Stage 3: Centrifugation and drying pellets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169

Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169

Equipment and consumables required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169

Stage 4: Resuspension, Hybridization Preparation, and QC . . . . . . . . . . . . . . . . . . . . . . . . 171

Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171


1. Preparing frozen pellets and Axiom Resusp Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . 173


3. Thaw and prepare the reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

4. Run the Resuspension, Hybridization Preparation, and QC step . . . . . . . . . . . . . . . . 173

Summary of Resuspension and Hybridization Preparation . . . . . . . . . . . . . . . . . . . . . . . 180

Stage 5: Preparation for the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . 184

About Stage 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184

Duration of GeneTitan™ MC Instrument reagent preparation and sample preparation . . 188

Equipment and consumables required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189


2. Prepare the reagents for GeneTitan Reagent Tray Preparation . . . . . . . . . . . . . . . . . . 191

3. Prepare the Sample Plate (if stored at –20°C) and the array plate . . . . . . . . . . . . . . . . 193

4. Prepare the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193

5. Run the Preparation for GeneTitan™ step. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195

6a. Complete Stage 4: Preparation for GeneTitan™ - Hybridization Trays . . . . . . . . . . . . 201

6b. Complete Stage 4: Preparation for GeneTitan™ - Reagent Trays . . . . . . . . . . . . . . . 202

6c. Complete Stage 4: Preparation for GeneTitan™ - Multiple Plate Workflow . . . . . . . . 202

Summary of Preparation for GeneTitan MC™ Instrument . . . . . . . . . . . . . . . . . . . . . . . . 204

CHAPTER 5 Array processing with the GeneTitan™ MC Instrument. 209

Before using the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

Proper tray alignment and loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

Stain trays and covers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212

Email and telephone notifications from the GeneTitan™ MC Instrument . . . . . . . . . . . . . 214

GeneTitan™ MC Instrument lamp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215

Setup options for array plate processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215

Aborting a process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218

Stage 1: Create and upload Batch Registration file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

Stage 2: Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220

Reagents required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220

Setup the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221


Contents

Load Axiom™ array plate and hybridization tray onto the GeneTitan™ MC Instrument . . 225

Load a second Axiom™ array plate and hybridization tray onto the

GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231

Status window prompts and actions required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233

Stage 3: Ligate, Wash, Stain, and Scan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

Equipment, consumables, and reagents required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

Proper installation of the GeneTitan™ tray consumables . . . . . . . . . . . . . . . . . . . . . . . . . 236

Load trays onto the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237

Continuing the workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244

Shutting down the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245

CHAPTER 6 Processing 8 Axiom™ array plates per week. . . . . . . . . 246

Overview of the 8-plate workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

Plate numbering scheme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

Week 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247

Week 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248

Thawing frozen plates of amplified DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

Automated target preparation for processing 8 Axiom™ array plates per week . . . . . . . . . 249

Initial target prep week—Biomek FXP Target Prep Express . . . . . . . . . . . . . . . . . . . . . . . . 250

Initial target prep week—Day 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250

Initial target prep week—Day 2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251

Initial target prep week—Day 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252



Simultaneous 8-plate workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257

Eight-plate workflow—Day 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257





CHAPTER 7 Processing 3 Axiom™ array plates per week. . . . . . . . . 271

Overview of the 3-plate workflow for automated target preparation . . . . . . . . . . . . . . . . . . 272

Thawing frozen plates of amplified DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274

Target prep and array processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275

Three plate/week workflow—Day 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275






Contents

CHAPTER 8 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281

Biomek FXP Target Prep Express . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281

GeneTitan™ Multi-Channel Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281

Miscellaneous messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282

Fluidic diagnostic messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284

Wash/Scan Resume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288

Aborting a run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288

APPENDIX A Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289

General safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290

Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291

APPENDIX B Fragmentation quality control gel protocol . . . . . . . . . 292

Protocol for running a fragmentation quality control gel . . . . . . . . . . . . . . . . . . . . . . . . . . . 292

Equipment required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292

E-Gels and reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292

Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292

Diluting the TrackIt™ Cyan/Orange Loading Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293

Fragmentation QC gel protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293

APPENDIX C Sample quantitation after resuspension . . . . . . . . . . . 295

Protocol for sample quantitation after resuspension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295

Equipment required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295

Quantitate the diluted samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295

Assess the OD readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296

Suggested protocol for OD quantitation using the DTX 880 . . . . . . . . . . . . . . . . . . . . . . . . 297

If performing sample quantitation on a plate reader other than the DTX880 . . . . . . . . . . . 303

APPENDIX D Registering samples in GeneChip™ Command Console™ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304

Creating a GeneTitan™ Array Plate Registration file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304


Contents

APPENDIX E Deionization procedure for GeneTitan™ trays and covers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307

Deionization procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308

Ion-indicator cap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310

APPENDIX F GeneTitan™ Multi-Channel Instrument Care . . . . . . . . 311

Cleaning and maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311

Monthly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311

Every 6 months . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311

Servicing the outer enclosure fan filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312

Cleaning schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312

Cleaning procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312

Replacing the bottle filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313

Removing and inspecting the filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314

Replacing the filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314

Replacing the xenon lamp in the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . 315

Lamp life/imaging device status notices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315

Removing the xenon lamp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316

Replacing the lamp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317

Resetting the lamp counter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319

Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320

Log files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320

GCC log files for GeneTitan™ MC Instrument Systems . . . . . . . . . . . . . . . . . . . . . . . . . . 321

Insufficient disk space notice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322

Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323

Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323

Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326


1 The Axiom™ 2.0 Assay

About

The first genome-wide association study (GWAS) was published in 2005 (Manolio and Collins) when individuals carrying particular haplotypes of SNP rs380390 were found to have increased risk of developing age-related macular degeneration, a study performed with the Applied Biosystems GeneChip Mapping 100K Array Set (Klein et al.).

As of September, 2009, there have been over 400 peer-reviewed GWAS publications and over 1774 SNPs have been implicated in human disease (Hindorff et al.). Initial GWAS studies focused on the “common disease, common variant” hypothesis (Manolio and Collins) that held that haplotypes with a minor allele frequency (MAF) 5% would show measurable contribution to human disease research.

Current research is shifting towards “complex disease, complex/rare variant” studies. As such, these research projects require a broader catalog of human variation, such as is being generated by the 1000 Genomes Project (http://www.1000genomes.org). This project focuses on identifying alleles with a MAF 5% across a broader spectrum of human ethnicities. In order to allow our customers to take advantage of this novel and rare content for genome association and candidate gene studies in a cost effective and timely manner, Thermo Fisher Scientific is introducing a new genotyping product line: the Axiom™ Genotyping Solution.

The Axiom Genotyping Solution introduces a new genotyping technology platform that includes novel assay biochemistry, array configuration and processing, and automated target preparation. This solution has applications in human disease research and basic and applied agriculture research.

For human disease research applications, Thermo Fisher Scientific conducted an empirical screen of genomic content from dbSNP (http://www.ncbi.nlm.nih.gove/projects/SNP/). The screen included markers from HapMap and the 1000 Genomes Project as well as other sources, using HapMap phase 3 samples and/or the original 270 HapMap samples. All of this information has gone into creating a proprietary Thermo Fisher Scientific database of validated markers that can be interrogated using the Axiom™ 2.0 Assay.

There are several arrays available for use with the Axiom 2.0 Assay which leverage the content of this proprietary Thermo Fisher Scientific database. For a complete list of supporting products visit www.thermofisher.com.


http://www.ncbi.nlm.nih.gove/projects/SNP/

http://www.ncbi.nlm.nih.gove/projects/SNP/

http://www.1000genomes.org

Chapter 1 The Axiom™ 2.0 AssayWhat’s new 1

The Axiom 2.0 Assay interrogates biallelic SNPs and simple indels (human only) in a single, fully automated assay workflow. Starting with genomic DNA, the samples are processed by performing either an automatic or manual target preparation protocol followed by automated processing of the array plates in the GeneTitan MC Instrument.

• Target preparation uses methods including DNA amplification, fragmentation, purification and resuspension of the target in hybridization cocktail.

• The hybridization-ready targets are then transferred to the GeneTitan™ Multi-Channel (MC) Instrument for automated, hands-free processing including hybridization, staining, washing and imaging.

Cel files generated by the GeneTitan MC Instrument are processed using the Axiom™ Genotyping Algorithm version 1 (Axiom GT1) available through Applied Biosystems™ Microarray Power Tools or Genotyping Console™ v4.1.

In summary, the Axiom Genotyping Solution is a product line that provides catalog arrays that:

• Are optimized for high genetic coverage of their population in question.

• Provide highly automated, reproducible results suitable for GWAS.

What’s new

In this version of the Axiom™ 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP User Guide instructions are provided for running the Biomek FXP Target Prep Instrument using either Windows® XP or Windows 7 operating system. Instructions for running Windows XP are provided in Chapter 3 and instructions for running Windows 7 are provided in Chapter 4.

Overview of the Axiom™ 2.0 Assay

Running the Axiom 2.0 Assay requires the following sets of steps:

1. Genomic DNA preparation—Resulting in samples that meet requirements spelled out in Chapter 2, ʺGenomic DNA preparation and requirementsʺ on page 13.

2. Target Preparation of the samples:

• Chapter 3, ʺTarget preparation on the Biomek FXP with Windows® XPʺ on page 22, or

• Chapter 4, ʺTarget preparation on the Biomek FXP with Windows® 7ʺ on page 114

3. Array processing, done with:

• GeneTitan MC Instrument

• GeneTitan Instrument Control software

• Applied Biosystems™ GeneChip™ Command Console Portal software

See Chapter 5, ʺArray processing with the GeneTitan™ MC Instrumentʺ on page 209.

A list of the required equipment and supplies for running the Axiom 2.0 Assay using the Biomek FXP for automated target preparation can be found in the Axiom™ 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP Site Preparation Guide, Pub. No. 702984.


Chapter 1 The Axiom™ 2.0 AssayOverview of the Axiom™ 2.0 Assay1

Running multiple plate workflows

Thermo Fisher Scientific provides workflows that allow you to run a set of samples and array plates through the protocol using a minimum of personnel and a forty-hour week. The timing of steps is critical because of the following constraints:

• Incubation after DNA Amplification is 23 hours.

• Hybridization in the GeneTitan MC Instrument is 23.5 hours.

• Reagent trays for wash/stain/imaging must be prepared as Hybridization finishes.

• Limits to when a second hybridization tray and array plate can be loaded into the GeneTitan MC Instrument.

These limitations require careful timing. The details are covered in:

• Chapter 6, ʺProcessing 8 Axiom™ array plates per weekʺ on page 246.

• Chapter 7, ʺProcessing 3 Axiom™ array plates per weekʺ on page 271.


2 Genomic DNA preparation andrequirements

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Sources of genomic DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

General requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Genomic DNA extraction/purification methods . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Genomic DNA cleanup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Genomic DNA preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Overview

The general requirements for genomic DNA (gDNA) sources and extraction methods are described in this chapter. The success of this assay requires uniform amplification of the genome starting with relatively intact gDNA. To achieve this, the gDNA must be of high quality, and must be free of contaminants that may affect the enzymatic reactions to be performed.

For this protocol, you use the Axiom™ 2.0 Reagent Kit (96 reaction, Cat. No. 901758). Axiom Reference Genomic DNA 103 (Cat. No. 951957) meets the requirements outlined below, and is used as a control. The size and purity of sample gDNA can be compared with those of the control DNA to assess sample quality. The control DNA should also be used routinely as an experimental positive control and for troubleshooting purposes.

Assay performance may vary for gDNA samples that do not meet the general requirements described below. However, the reliability of any given result should be assessed in the context of overall experimental design and goals.


Chapter 2 Genomic DNA preparation and requirementsSources of genomic DNA2

Sources of genomic DNA

The following sources of human gDNA have been successfully tested in the laboratories at Thermo Fisher Scientific and meet the requirements outlined under ʺGeneral requirementsʺ on page 15.

• Blood

• Saliva

• Cell line

• WGA pre-amplified DNA: Genomic DNA amplified with the REPLI-g® Kit (a whole genome amplification kit; QIAGEN, Cat. No. 150025) has been tested successfully with the Axiom 2.0 Genome-Wide Human Reagent Kit Assay. The REPLI-g Kit was used to amplify 20 ng genomic DNA, and the resulting yields were quantitated by a PicoGreen® assay. The amplified products (either 100 or 200 ng amplified DNA as required according to the Axiom array type) were used (without purification) as the input DNA sample in the subsequent Axiom 2.0 Assay steps. The stability of this amplified product to storage and repeated cycles of freeze/thaw have not been evaluated by Thermo Fisher Scientific.

The following sources of animal gDNA have been successfully tested in the laboratories at Thermo Fisher Scientific and meet the requirements outlined under ʺGeneral requirementsʺ on page 15:

• Blood

• Semen

• Nasal swab

• Hair bulbs

• Ear punch tissue

The following sources of plant gDNA have been successfully tested in the laboratories at Thermo Fisher Scientific and meet the above requirements:

• Seeds

• Leaves

Note: DNA derived from formalin-fixed paraffin-embedded (FFPE) blocks should not be used with this assay.

Success with other types of samples will depend on quality (degree of degradation, level of purity, etc.) and quantity of gDNA extracted.


Chapter 2 Genomic DNA preparation and requirementsGeneral requirements 2

General requirements

• Starting DNA must be double-stranded for the purpose of accurate concentration determination.

• DNA must be of high purity.DNA should be free of DNA polymerase inhibitors. Examples of inhibitors include high concentrations of heme (from blood) and high concentrations of chelating agents (i.e., EDTA). The gDNA extraction/ purification method should render DNA that is generally salt-free because high concentrations of particular salts can also inhibit enzyme reactions. DNA purity is indicated by OD260/OD280

and OD260/OD230 ratios. The OD260/OD280 ratio should be between 1.8 and 2.0 and the OD260/OD230 ratio should be greater than 1.5. We recommend that DNA samples that do not meet these criteria be cleaned up as described under ʺGenomic DNA cleanupʺ on page 17.

• DNA must not be degraded.The approximate average size of gDNA may be assessed on a 1% agarose gel using an appropriate size standard control. Approximately 90% of the DNA must be greater than 10 Kb in size. Control DNA can be run on the same gel for side-by-side comparison.

Special requirements

Preamplification areaPrecautions are required when manipulating genomic DNA to avoid contamination with foreign DNA amplified in other reactions and procedures. It is recommended that genomic DNA manipulations are performed in a dedicated preamplification room or area separate from the main laboratory.

This preamplification area should have a dedicated set of pipettes and plasticware. If no dedicated area is available, use of a dedicated bench or a dedicated biosafety hood and dedicated pipettes is suggested. If no dedicated bench or biosafety hood is available, a set of dedicated pipettes is recommended.

Assessing the quality of genomic DNA using 1% Agarose E-gels

We recommend this quality control step to assess the quality of the gDNA prior to starting the assay.

Equipment and reagents recommended

Table 1 E-Gel® and reagents required

Item Cat. No

Mother E-Base Device EB-M03

Daughter E-Base Device EB-D03

E-Gel® 48 1% agarose gels G8008-01

RediLoad™ 750026

E-Gel® 96 High Range DNA Marker 12352-019


Chapter 2 Genomic DNA preparation and requirementsGeneral requirements2

Guidelines for preparing the Genomic DNA Plate for gel analysis• Loading a DNA mass of 10 ng to 20 ng per well is recommended. If lower amounts

are loaded, omission of the loading dye is recommended in order to improve visualization. Loading 25 ng gDNA per well can improve the image.

• Add 3 µL of 0.1X of RediLoad dye to each sample.

• Bring each sample to a total volume of 20 µL using H2O (for example, if the volume of genomic DNA is 5 µL, add 3 µL of RediLoad, and bring to 20 µL total by adding 12 µL of H2O).

• Seal, vortex and centrifuge.

Run a 48-lane 1% agarose e-gel

1. Power on for E-Base (red light).

2. Push the Power/Prg button to ensure that the program is at EG mode (not EP).

3. Insert the two 48-well 1% agarose e-gels into the slots.

4. Remove 2 combs.

5. Load 20 µL from the above plate onto two 48-well 1% agarose e-gels.

6. Load 15 µL of diluted High Range DNA Marker (1:3 dilution or ~0.34 X from stock) into all marker wells (as needed).

7. Fill all empty wells with water.

8. Adjust the run time to ~27 minutes.

9. Push the Power/Prg button again (it will change from red to green).

When run time is reached (the ladder band reaches the end of the lane), the system will automatically shut off. The gel is then ready for imaging.

Figure 1 shows gel images of intact gDNA (that is suitable for use in the Axiom™ 2.0 Assay) and degraded gDNA samples. Customers whose gDNA is degraded (similar to the image in Figure 1) should perform a test experiment to investigate the performance of their samples in the Axiom Genotyping Assay prior to beginning any large scale genotyping projects.

Figure 1 Gel images showing intact gDNA and degraded gDNA.


Chapter 2 Genomic DNA preparation and requirementsGenomic DNA extraction/purification methods 2

Genomic DNA extraction/purification methods

Genomic DNA extraction and purification methods that meet the general requirements outlined above should yield successful results. Methods that include boiling or strong denaturants are not acceptable because the DNA would be rendered single-stranded and can no longer be accurately quantitated using a PicoGreen-based assay.

Genomic DNA cleanup

If a gDNA preparation is suspected to contain inhibitors, the following cleanup procedure can be used:

1. Add 0.5 volumes of 7.5 M NH4OAc, 2.5 volumes of absolute ethanol (stored at –20°C), to gDNA.

2. Vortex and incubate at –20°C for 1 hour.

3. Centrifuge at 12,000 x g in a microcentrifuge at room temperature for 20 minutes.

4. Remove supernatant and wash pellet with 80% ethanol.

5. Centrifuge at 12,000 x g at room temperature for 5 minutes.

6. Remove the 80% ethanol and repeat the 80% ethanol wash 1 more time.

7. Resuspend the pellet in reduced EDTA TE Buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA).

(See the Axiom™ Assay 96-Array Format Automated Workflow for Biomek FXP Site Preparation Guide, Pub. No. 702984 for reagents, equipment, labware and consumables for Axiom 2.0 Assay).

Genomic DNA preparation

This step needs to be done before proceeding with the DNA amplification stages for automated target preparation.

The genomic DNA (gDNA) you will process using the Axiom 2.0 Assay should meet the general requirements listed earlier in this chapter. The amount of gDNA depends on which Axiom array will be used in the downstream protocol. All human Axiom arrays (except the Axiom™ Genome-Wide Pan-African Array Set) require a total of 100 ng. The Axiom Genome-Wide Pan-African Array Set requires a total of 300 ng, or 100 ng per array (there are 3 arrays in the Axiom Genome-Wide Pan-African Array Set). Diploid plants and animals require 150 ng per array and polyploid plants and animals require 200 ng per array.

Table 2 Input requirements for Axiom™ 2.0 Assay

Sample type Volume per well

Input mass per well

gDNA concentration

Human 20 μL 100 ng 5 ng/μL

Diploid Plants and Animals 20 μL 150 ng 7.5 ng/μL

Polyploid Plants and Animals 20 μL 200 ng 10 ng/μL


Chapter 2 Genomic DNA preparation and requirementsGenomic DNA preparation2

To prepare gDNA

ʺ1. Thaw samples and controlʺ

ʺ2. Quantitate and dilute gDNAʺ.

ʺ3. Aliquot the diluted samples and the controlʺ

ʺ4. Freeze or proceedʺ

ʺ5. Create a Batch Registration fileʺ

Duration Thirty minutes to an hour for reagents to thaw and half an hour for setup.

Equipment, consumables and reagents required

Equipment and consumablesThe equipment and consumables listed in Table 3 are required for this stage.

Table 3 Equipment and consumables required for genomic DNA preparation

Quantity Item

As required Adhesive seals for plates

1 Ice bucket, filled with ice

1 each Pipettes:• Single channel P10 or P20 • Optional: multichannel P10 or P20

As required Pipette tips

1 Plate, deep well: Beckman Deep Well Titer, polypropylene; Cat. No. 2670071

1 Note that a different deep-well plate is used for each manual (96, 24, and Mini 96) and automated(NIMBUS 96 and Biomek FXP 384HT) workflow versions of the Axiom 2.0 Assay. See Chapter 2 of thespecific Axiom™ 2.0 Assay user guide for each workflow for further details.

1 Plate centrifuge

1 Plate spectrophotometer (required only if no OD measurements available for samples)

1 Vortexer


Chapter 2 Genomic DNA preparation and requirementsGenomic DNA preparation 2

ReagentsThe reagents listed in Table 4 are required for this stage.

1. Thaw samples and control

Thaw the components listed below to room temperature:

• gDNA samples

• Axiom Reference Genomic DNA 103

To thaw, either:

• Place items on benchtop for 1 hour

• Thaw in a water bath:

a. Fill a small plastic dish with Millipore water. Do not overfill as the level of the water should not overflow when the sample tubes or plates are placed in the bath.

b. Thaw the sealed Sample Plate and reference sample for 30 minutes.

c. Remove the Sample Plate and/or sample tube from the water bath and wipe dry using laboratory tissues. Ensure the outside is completely dry before opening the Sample Plate or tube to minimize any contamination, which can lead to reaction failure.

2. Quantitate and dilute gDNA

1. Gently vortex (50% maximum) and centrifuge the gDNA and Reference Genomic DNA 103.

2. Recommendation: quantitate each sample (e.g., using the Quant-iT™ PicoGreen® dsDNA Kit).

3. Using reduced EDTA TE buffer, dilute each sample to a concentration of:

• 5 ng/µL for Human DNA samples

• 7.5 ng/µL for diploid plant and animal DNA samples

• 10 ng/µL for polyploid plant and animal DNA samples

4. Seal, vortex and centrifuge.

Note: Do not dilute the Reference Genomic DNA 103 control. It is already at a working concentration.

Table 4 Reagents required for genomic DNA preparation

Reagent Cat. No.

Axiom Reference Genomic DNA 103, –20°C(use as a positive control if genotyping human samples)

951957

Reduced EDTA TE Buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) 75793

Positive control gDNA (if genotyping non-human samples)


Chapter 2 Genomic DNA preparation and requirementsGenomic DNA preparation2

3. Aliquot the diluted samples and the control

Next, the samples and control are placed in the following deep well plate for target preparation:

• For automated target preparation: Beckman Deep Well Titer, polypropylene; Cat. No. 267007

1. 20 µL of each diluted gDNA sample (this should be the equivalent of 100 ng to 200 ng of gDNA, as required by the sample type).

2. 20 µL of control DNA.

We recommend including at least 1 positive control on each plate.

3. Seal and centrifuge.

Note: For samples to be processed on the Axiom Genome-Wide Pan-African Array Set, 3 identical deep well plates of 100 ng gDNA per well should be made.

4. Freeze or proceed

At this point you can:

• Store the Sample Plate at –20°C, or

• Proceed to DNA amplification (see Chapter 3, ̋ Target preparation on the Biomek FXP with Windows® XPʺ on page 22).

Note: You can leave the gDNA Sample Plate at room temperature if proceeding immediately to DNA Amplification.

5. Create a Batch Registration file

GeneTitan Array Plate Registration files contain information that is critical for:

• Data file generation during imaging.

• Tracking the experimental results for each sample loaded onto an array plate.

Detailed instructions for creating this file are located in Appendix D, ʺRegistering samples in GeneChip™ Command Console™ʺ on page 304. See also Figure 2 for a screen shot showing an example of a batch registration file.

1. Open GCC Portal Samples, and select:

a. GeneTitan Array Plate Registration.

b. The array plate format.

c. Click Download.

2. Enter a unique name for each sample and any additional information.

3. Save the file.

The array plate barcode will not be scanned until you are ready to load the array plate and samples onto the GeneTitan MC Instrument for processing.

IMPORTANT! It is very important to create and upload a GeneTitan™ Array Plate Registration file with your sample information prior to loading the array plate and hybridization tray into the GeneTitan MC Instrument. We recommend that you create (but not upload) this file at the same time you prepare your plate of genomic DNA. When your samples are ready for hybridization, you will scan the array plate barcode and upload the file to Applied Biosystems™ GeneChip™ Command Console (GCC).


Chapter 2 Genomic DNA preparation and requirementsGenomic DNA preparation 2

Figure 2 Example of a Batch Registration file


3 Target preparation on the BiomekFXP with Windows® XP

This chapter is intended for users running the Axiom 2.0 automated target preparation method using the Biomek FXP Target Prep Express software on Windows XP. If you are currently using Windows® 7, see Chapter 4, ̋ Target preparation on the Biomek FXP with Windows® 7ʺ on page 114.

Axiom™ 2.0 Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Before using the Biomek workstation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Equipment, consumables, labware, and reagents required . . . . . . . . . . . . . . . . . 29

Stage 1: DNA Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

Stage 2: Fragmentation and Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Stage 3: Resuspension and hybridization preparation . . . . . . . . . . . . . . . . . . . . . 76

Stage 4: Preparation for the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . 89

Axiom™ 2.0 Assay

The Axiom 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP instrument with Windows XP is designed for processing 96 samples at a time. The protocol is performed in 2 parts:

• Part 1: Target Preparation is performed on the Biomek FXP Target Prep Express

• Part 2: Array Processing is performed on the GeneTitan™ Multi-Channel (MC) Instrument

A list of all equipment and resources for the Axiom 2.0 Assay with Automated Target Preparation is in the Axiom™ 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP Site Preparation Guide, Pub. No. 702984.

This chapter includes instructions for Part 1: Target Preparation.

IMPORTANT! Before proceeding to DNA Amplification, do the gDNA preparation described in Chapter 2, ʺGenomic DNA preparation and requirementsʺ on page 13.


Chapter 3 Target preparation on the Biomek FXP with Windows® XPBefore using the Biomek workstation 3

Before using the Biomek workstation

Seal, vortex, and centrifuge

Unless otherwise stated in the protocol, follow the guidelines below when instructed to seal, vortex and centrifuge plates or reagent tubes for the Biomek FXP Target Prep Express portion of this assay.

• Seal plates—we recommend using MicroAmp™ Clear Adhesive Films to seal your plates.

• Vortex:

– Reagents 3 times, 1 second each time at the maximum setting.

– Plates 1 second each corner, and 1 second in the center at the maximum setting.

• Centrifuge: When instructed to centrifuge plates or reagent vials, follow these guidelines unless otherwise instructed (for example, when centrifuging and drying pellets, ʺ5. Centrifuge and dry pelletsʺ on page 75).

– Plates:

• Centrifuge at 1,000 rpm for 1 minute at room temperature.

• Do not centrifuge for more than 1 minute.

– Reagent Vials: 3 seconds

IMPORTANT! Always ensure that your plates are tightly sealed. A tight seal will prevent sample loss and cross-well contamination, particularly when plates are being vortexed. Never reuse a seal. always use a new seal.

Figure 3 Vortex plates at the corners and center


Chapter 3 Target preparation on the Biomek FXP with Windows® XPBefore using the Biomek workstation3

Breaking the light curtain

For your safety, the Biomek FXP Target Prep Express is designed to immediately halt all movement when the light curtain is broken.

Light curtain broken while running the assayFor your safety, the Biomek FXP Target Prep Express is equipped with a light curtain (Figure 4). The light curtain senses when an object (such as a hand or an arm) enters the space surrounding the deck. When this curtain is broken, all movement on the deck halts until the user either clicks OK to resume the activity that was taking place, or aborts the activity. Incubation timers are not interrupted.

Pipette tip usage

Figure 4 Prompt displayed when the light curtain is broken during the process referred to as Homing All Axes.

The light curtain covers the front of the instrument. The sides are protected by Plexiglas. Both areas are shown as red in this figure. Any penetration of the light curtain from outside the deck halts all movement of the workstation. To resume activity on the deck, click OK. To abort the step or other activity, click Stop on the toolbar.

Table 5 Pipette tip usage for 1 full run of the Axiom™ 2.0 Assay on the Biomek workstation

StepMultichannel

P50, pink96 tips, Cat. No. A21586

Multichannel AP96 P250,

aqua96 tips,

Cat. No. 717253

Span-8 Span P250,

green96 tips,

Cat. No. 379503

Span-8 Span P1000,

yellow96 tips,

Cat. No. 987925

96 rxns 96 rxns 96 rxns 96 rxns

DNA Amplification — 96 tips — 33 tips

Fragmentation — 96 tips 24 tips 20 tips

Resuspension and Hybridization Preparation 96 tips 96 tips 17 tips 23 tips

Preparation for the GeneTitan™ MC Instrument • Denature samples• Transfer denatured samples to hyb tray• Prepare GeneTitan™ reagent trays

— 96 tips 26 tips 96 tips

Total 96 tips 384 tips 67 tips 172 tips



Plate centrifuge One plate centrifuge is required for the Axiom™ 2.0 Assay. See the Assay 96-Array Format Automated Workflow for Biomek FXP Site Preparation Guide, Pub. No. 702984, for an appropriate plate centrifuge that can be used with the Axiom Genotyping Solution. When centrifuging and drying pellets as instructed under ʺ5. Centrifuge and dry pelletsʺ on page 75, the centrifuge must be able to spin down plates at:

• rcf: 3,200 x g (4,000 rpm for the Eppendorf 5810R with the rotor configuration described in the Axiom™ 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP Site Preparation Guide)

• temperature: 4°C

In addition, the bottom of the rotor buckets should be soft rubber to ensure that the deep well plates do not crack. Do not centrifuge plates in metal or hard plastic buckets.

Set the Biomek software default settings

Typically you will select the default settings for the Biomek software once. The settings you select will:

• Determine the default step selected when the user prompt is displayed at the start of each run.

• Determine which process controls will be run during stages 2 and 3 (Fragmentation and Resuspension). The process controls include:

– Highly recommended: Preparation of sample dilution plates for OD and gel analysis during resuspension and hybridization preparation. The dilution plates are taken off-deck. One is used for OD quantitation to evaluate DNA mass; the other is used to check fragment size.

– If desired: Prompt you to manually take samples for DNA quantitation prior to fragmentation.

• Select deck configuration options for the type of integrated thermal cycler used.

To select Biomek software default settings:

1. Launch the Biomek Software.

2. Open Project Open Project Axiom 2.0 Target Prep and click OK (Figure 5).

3. Open File Open to display the Open Method window (Figure 6).

Or click the Open Method icon

4. Select Axiom 2.0 Target Prep and click OK.

Figure 5 Opening the Axiom 2.0 target preparation project



.

5. In the left pane of the window, select Axiom Target Prep (Figure 7).

The default settings window is displayed on the right.

Figure 6 Opening the Axiom target preparation method

The Axiom 2.0 Target Prep project folder must be displayed in this menu.

Figure 7 Click Startup Dialog to open the Default Settings window

The selections made in this box, Choose default settings, are displayed each time this window appears.

The options in the Select preferences box must be selected prior to starting a run.

You are not prompted to select any preferences at start up.



6. Select your default settings.

Choose defaults settings—these settings can be changed at the start of each step

• Which step do you want to run?

The step that you choose will be the default setting for the runtime prompt. The actual step selected when the runtime prompt is shown reflects the state of the last completed run of the Axiom target preparation method. For example, if DNA Amplification was the step completed in the last run, then the step chosen in the subsequent run would be Fragmentation (the next step in the process). If there is no record of the previous run, then the default choice specified is selected.

Select preferences—These settings are displayed in this window only. You must select/deselect here.

• QC check points

• Prompt for manual DNA quantitation before fragmentation—the workstation will pause following inactivation of the DNA amplification reaction to allow you to manually remove an aliquot of each sample for off-line (manual) DNA quantitation. This extra quality control step is available for troubleshooting the DNA amplification reaction.

• Prepare plates for gel QC and OD after resuspension—the workstation will prepare 2 plates of resuspended samples properly diluted for the fragmentation gel QC and OD quantitation process control checks. See Appendix B, ̋ Fragmentation quality control gel protocolʺ on page 292 and Appendix C, ʺSample quantitation after resuspensionʺ on page 295 for instructions and result assessment guidelines.

• Custom run options

• Run method in test mode—select this option to skip all of the incubation timers in a step. If selected, a prompt is displayed asking you to confirm that you want to run a step in test mode (Figure 8). Use this option to perform runs using deionized water only, not actual reagents or samples.

IMPORTANT! For troubleshooting and support purposes, we strongly recommend that you perform the gel QC and OD quantitation process controls after resuspension.

CAUTION! Never process samples in test mode. The assay will fail; all of your samples and reagents will be lost.

Figure 8 Prompt displayed when the custom run option, Run method in test mode, is selected.



• Deck configuration options

• TRobot—select this option to perform the denaturation of the Axiom Hyb Ready Plate on the integrated Biometra TRobot 96 thermal cycler

• PTC—select this option to perform the denaturation of the Axiom Hyb Ready Plate on the integrated Bio-Rad PTC-200 thermal cycler

• No integrated thermal cycler—select this option to perform the denaturation of the Hyb Ready Plate on an off-deck thermal cycler or if your Biomek does not have an integrated thermal cycler. A list of thermal cyclers that have been verified with the assay can be found below. When selecting this option, select the appropriate plate type that should be used for the Hyb Ready Plate.

– Select the Bio-Rad Skirted option when using the HSP-9631 plate for the PTC-200 or the Bio-Rad Tetrad 0240G thermal cycler.

– Select the Bio-Rad Semi-Skirted on Costar Round U-Bottom option for the Applied Biosystems 9700 or the Applied Biosystems 2720 thermal cycler. The Bio-Rad HSS-9601 plate must be stacked onto the Costar Round Bottom plate from Corning (VWR International Cat. No. 29442-392, E&K Scientific: EK 680568, Corning Mfg. Cat. No. 3795) for the Biomek FXP Target Prep Express to prepare the Hyb Ready Plate.

• Controllers—select the appropriate Peltier controller used by the Biomek FXP Target Prep Express.

– Black TEC Control

– Blue Single TEC Control

We have verified the performance of this assay using the Bio-Rad PTC-200/PTC-200G and Biometra TRobot 96 on the Beckman Biomek Target Prep Express liquid handler. We have also verified the performance of this assay using the following off-deck thermal cyclers, with 96-well blocks:

• Bio-Rad PTC-200G

• Biometra TRobot 96

• Applied Biosystems 9700 with a gold, silver or aluminum block

• Applied Biosystems 2720

• Bio-Rad / MJ Tetrad® 2 PTC-0240G

The performance of this assay has not been verified with other thermal cyclers.

Use of other thermal cyclers may result in assay failure and may violate the Axiom Array and Reagent replacement policy.

The thermocycler needs to be programmed with the Axiom Denature protocol:

a. 95°C 10 minutes

b. 48°C 3 minutes

c. 48°C hold

Use the heated lid option when setting up or running the protocol.

WARNING! Evaporation during denaturation can negatively impact assay performance. Use the recommended thermal cycler consumables and sealing film to eliminate condensation and evaporation. The arched, auto-sealing metal plate with P pads as shown in Table 6 on page 29 should be replaced as per the manufacturer’s recommendation.


Chapter 3 Target preparation on the Biomek FXP with Windows® XPEquipment, consumables, labware, and reagents required 3

7. Click Start at the top of the left pane to close the default settings window.

Equipment, consumables, labware, and reagents required

Labware and materials used on the Biomek™ workstation deck

IMPORTANT! The default settings you select here will be displayed each time the Axiom target preparation window is displayed.

The options displayed in the “Select preferences” section of the Startup dialog box as shown in Figure 7 on page 26 must be selected prior to starting a run. These settings are not prompted for at runtime.

Table 6 Labware and materials used on the Biomek™ workstation deck

Labware Supplier and Cat. No.

Labware image

Biomek AP96 – P250 Pipette Tips (aqua box; pre-sterile, barrier)

Beckman CoulterCat. No. 717253

Biomek Span P250 Pipette Tips (green box; pre-sterile, barrier)



Chapter 3 Target preparation on the Biomek FXP with Windows® XPEquipment, consumables, labware, and reagents required3

Biomek Span P1000 Pipette Tips (yellow box; pre-sterile, barrier, conductive)


Biomek Span P50 Pipette Tips (pink box; pre-sterile, barrier)

Beckman CoulterCat. No. A21586

Bio-Rad Hard-Shell 96-well Plate(available in multiple colors)

Bio-RadCat. No. HSP-9631

96 Deep-Well Square Plate Thermo ScientificAB-0932

Table 6 Labware and materials used on the Biomek™ workstation deck (Continued)


Labware image



OD Plate, UV E & K ScientificEK-25801

Lid, metal(arched, auto-sealing with P pads)

Bio-RadCat. No. MSL-2032 andP Pad Cat. No. MSP-1003

Hard-Shell Full-Height 96-Well Semi-Skirted PCR Plate • This consumable is required only if

using off-deck Applied Biosystems 2720 or Applied Biosystems 9700 Thermal cyclers

Bio-RadCat. No. HSS-9601



Labware image

Front view

Side view



Plate, Costar Brand Serocluster Round Bottom Plate from Corning• Note: this consumable is required

only if using an off-deck Applied Biosystems 9700 or Applied Biosystems 2720 thermal cycle

VWR International Cat. No. 29442-392E&K Scientific Cat. No. EK 680568Corning Mfg Cat. No. 3795

The Applied Biosystems 9700 and the Applied Biosystems 2720 use the semi-skirted 96-well plates (Cat. No. HSS-9601). For use on the Biomek deck, the semi-skirted PCR plate must be stacked on a Costar brand Serocluster 96-well Round Bottom Microtitration plate as shown in the figure to the right.

Reagent Block Template(designed specifically for use with the Axiom 2.0 Reagent Kit)

Contact Thermo Fisher Scientific



Labware image

Template on reagent block. Metal posts on block circled in red.



Beckman Deep Well Titer Plate(polypropylene)


Frame for reservoirs Beckman CoulterCat. No. 372795

Half reservoirHalf module, 75 mL capacity




Labware image

Frame



Reagent block, chilled to 4°C Beckman CoulterCat. No. A83054

Quarter Reservoirs• Quarter module, 40 mL capacity• Quarter module divided by width,

19 mL capacity each receptacle

Beckman Coulter

Cat. No. 372790 (40 mL)Cat. No. 372792 (19 mL)

24-Position Tube Rackwith one 11 mm tube insert in position A6.

Beckman CoulterCat. No. 373661 (rack)Cat. No. 373696 (insert)



Labware image

Metal posts on block circled in red.A1

Divided by width19 mL capacity

Undivided40 mL capacity

Tube insert A6



Adaptor, Deep Well Plate (installed on the Shaking Peltier)

This adaptor is typically installed by a Beckman Coulter field service technician during new system installation or a system upgrade. Ensure that you have one of these adaptors on the deck prior to running this assay.


Zerostat Anti-static Gunand Ion-Indicator Cap

Milty Zerostat, Thermo Fisher Scientific Cat. No. 74-0014



Labware image

The metal block is the adaptor.



GeneTitan MC Instrument consumablesAll consumables for the GeneTitan MC Instrument are provided by Thermo Fisher Scientific. The following table provides guidance on the consumables that are shipped with the array plate.

IMPORTANT! All covers must have barcodes. Discard any cover without a barcode.

Table 7 Axiom™ GeneTitan™ MC Instrument consumables (Axiom 96-array plate)

Item Part number Image Information

Axiom 96-array plate, various designs

Varies, depending on array design.

Axiom 96-Array plate:• Comprised of 3 parts:

clear plastic cover, array plate, and blue array plate protective base.

• The clear plastic cover for the array plate protects the array plate during transport. Discard after opening pouch.

• The array plate must always be kept in the blue array plate protective base at all times.

• The blue array plate protective base in the package holds the array and protects it from damage or exposure to dust.

Note: Array plate is not included in the Axiom GeneTitan Consumables Kit.

Clear tray shipping cover (to be discarded)Array plate protective baseArray plate

1

2

3

1

2

3



Table 8 Axiom™ GeneTitan™ MC Instrument Consumables (from the Axiom™ Array GeneTitan™ Consumables Kit, Cat. No. 901606)


Scan Tray 900746 Box501006 Pouch

96-Plate Scan Tray:• Comprised of 3

parts: scan tray, black protective base, and a scan tray cover.

• The black scan tray protective base in the package protects the glass bottom of the scan tray from damage before it is loaded into the GeneTitan MC Instrument.

• The scan tray cover protects the contents in the scan tray and must be deionized before used. See Appendix E, "Deionization procedure for GeneTitan™ trays and covers" on page 307.

• Remove the black scan tray protective base before loading the scan tray with the scan tray cover into the GeneTitan MC Instrument.

• The Scan Tray must be loaded into the GeneTitan Instrument with the Scan Tray Cover only.

• Do not load the Scan Tray with the protective base still on.



Black Scan Tray Protective Base, shown without the Scan Tray with cover

• The black scan tray protective base in the package is used to protect the bottom of the scan tray glass from damage. The black scan tray is distinct from the blue array plate protective base and must not be used with the array plate.

• Remove and set aside the protective base from the scan tray before loading.

Scan Tray with cover, shown without the black protective base

• The GeneTitan scan tray must be loaded with the scan tray cover into the GeneTitan MC Instrument.

• Do not load the scan tray with the protective base.

Table 8 Axiom™ GeneTitan™ MC Instrument Consumables (from the Axiom™ Array GeneTitan™ Consumables Kit, Cat. No. 901606) (Continued)




GeneTitan 5 Stain Trays Kit

4249910 Kit501025 Tray

• The GeneTitan Stain Tray Kit comes with 5 stain trays packaged in zip-top bags to keep them free of dust.

• The GeneTitan stain trays are barcoded and the trays have separator walls that are flush with the frame of the stain tray, as shown by the yellow line and the yellow oval in the lower photo.

GeneTitan™ Stain and Scan Tray Cover

202757 • The GeneTitan stain and scan tray covers prevent evaporation of the stains in stain trays and the array holding buffer in the scan tray.

• All stain and scan trays must be placed in the GeneTitan MC Instrument with the GeneTitan stain tray cover.

• All tray covers must be deionized to remove static electricity prior to placing the cover on the tray.

• See the section "Deionization procedure for GeneTitan™ trays and covers" on page 307 for the anti-static procedure.





Proper tray alignment and loading

Proper alignment and loading of plates, covers and trays is critical when using the GeneTitan MC Instrument. Each plate, cover and tray has 1 notched corner. The notched corner of plates, trays, covers and bases must be in vertical alignment with each other, and placed in position A1 per the Tray Alignment guide inside each GeneTitan MC Instrument drawer (Figure 9 and Figure 10 on page 42).

Note: Tip: Mark the notched corner of each plate, cover and tray with permanent marker to help ensure proper alignment and loading onto the GeneTitan MC Instrument.

Note: The instrument control software will display a warning if it detects a problem during the fluid dispense operations. The filters in the GeneTitan Wash A, Wash B and Rinse bottles should be replaced if the software displays such a warning. See Appendix F, ʺGeneTitan™ Multi-Channel Instrument Careʺ on page 313 for the

GeneTitan stain tray shown with the stain tray cover

Tray 501025Cover 202757

Hybridization Tray

900747 • After aliquoting the denatured hybridization ready samples into the hybridization tray, the tray should be immediately loaded into the GeneTitan MC Instrument with the barcode facing away from the operator, i.e., Barcode should be on the back side.



IMPORTANT! When running a multi-plate workflow, you must pay careful attention to the software prompts that tell you which side of the drawer to place or remove a plate/tray.

CAUTION! Take care not to damage the consumables or bend the blue cover posts or scan tray posts.



message displayed to the user and the procedure for replacing the filters.

Figure 9 Proper alignment and loading of plates, covers and trays in the GeneTitan MC Instrument

Plates and trays must be seated in this rectangular recess.

The notched corner of all plates, bases, and covers and must be seated in this corner of the drawer per

Tip: Mark the notched corner of each plate, cover and tray with permanent marker to help ensure proper alignment and loading.

Notched corner of array plate aligned with notched corner of blue base.

IMPORTANT! Remove the plastic protective shipping tray cover. 1

2

3


1

2

344

5

5

6

7

7

6



Figure 10 Array plate with protective blue base and the hybridization tray aligned and properly loaded into drawer 6

Array plate with protective blue base

Hybridization tray

1

2

1 2

IMPORTANT! When you install the consumables, ensure that the fingers are retracted. Do not lay the consumables on top of the drawer fingers—this indicates that the instrument is not functioning correctly. Notify your field service engineer if the fingers do not retract automatically. You should place the trays into the instrument drawers when a drawer is fully extended by the instrument. The fingers are retracted when the drawer is open and are extended when the drawer is closed in order to restrain the consumable. See Figure 11 for an image showing the location of the tabs.

Figure 11 Photo identifying the location of drawer tabs

2

1

Drawer tab, or “finger” in back.

Drawer tab, or “finger” on right side.

1

2



Stain trays and covers

Labeling GeneTitan hybridization and reagent traysWhen preparing the hybridization and reagent trays to be loaded onto the GeneTitan MC Instrument, you will need to mark each tray in a way that identifies its contents.

Proper labeling for hybridization trays and reagent trays is described in:

• ʺLabeling for hybridization traysʺ, below

• ʺLabeling for stain traysʺ on page 44

Labeling for hybridization trays

You may label the hybridization tray on the front part of the short side of the tray, next to the notch at the left, as shown in Figure 13. The proper section for labeling is closest to the notched corner, corresponding to the A1 and B1 wells.

IMPORTANT! Always place the flat side of the cover against the stain tray.

Figure 12 Placement of covers on trays

Correct placement of cover on stain tray. Incorrect placement of cover on stain tray.

IMPORTANT! It is critical that you write only on the proper locations of the proper sides of hybridization and stain trays. Do NOT write in any other location, as this can interfere with sensors inside the GeneTitan MC Instrument and result in experiment failure. To ensure proper placement of lids onto stain trays, and trays onto the GeneTitan MC Instrument, you can also mark the notched corner of the trays and lids.



Labeling for stain trays

You may label the stain trays on the left side of the front of the tray as shown in Figure 14. The correct side is closest to the notched corner, corresponding to the A1 through C1 wells.

Figure 13 Labeling GeneTitan hybridization trays

CAUTION! Writing on the wrong side of the hybridization tray may interfere with the operation of sensors in the GeneTitan MC Instrument.

IMPORTANT! Do not confuse hybridization trays with stain trays.

Do NOT label trays on the long side of the tray.

Notched corner of the hybridization tray should face the front.

Label the hybridization tray in this area.

1

2

3

1 2 3



Figure 14 Labeling GeneTitan stain tray (stain tray shown with lid)


Notched corner of the stain tray should face the front.

Label the stain tray here.

1 2 3

1

2

3



Loading tray consumables onto the GeneTitan™ MC Instrument

Loading, or installing, the trays and plates onto the GeneTitan MC Instrument is a simple procedure, but there are certain precautions that you must take in order to ensure an error-free procedure. The following figures show you how to do it. See Figure 15 through Figure 18.

Figure 15 Scan tray with cover loaded in drawer 2

IMPORTANT! When you load the plates, or trays, insert them under the tabs, or fingers, that may protrude into the stage. Confirm that the tray is not resting on these fingers. See Figure 108 on page 211.

Do NOT load the protective black base packaged with the scan tray.



Figure 16 Stain 1 tray and Ligation tray loaded in drawer 3

Figure 17 Stain 2 tray and Stabilization tray loaded in drawer 4



Reagent block template

The Axiom™ 2.0 Reagent Kit template fits precisely onto the top of the chilled reagent block, and is held in place by the metal posts on the block (Figure 19). Using this template will help ensure the proper placement of reagent tubes onto the block for each method.

Reservoir labels The reservoir labels are stick-on labels for the modular reagent reservoir frames for the different stages of the Automated workflow. These stick-on labels are color-coded to match the colors found on the caps of the reagent tubes in the Axiom 2.0 Reagent Kit. Using these labels helps ensure the proper placement of reservoirs and reagents for each method.

There are 4 reservoir holders used in the Axiom 2.0 method. Three of these will have templates on 2 sides and the remaining reservoir holder will have a template on 1 side for a total of 7 templates.

Figure 18 Stain 1 tray loaded in drawer 5

Figure 19 Axiom 2.0 Reagent Template for the reagent block



Remove the protective surface from the back of the label and place on the reservoir frames as directed in the Figure 20 through Figure 23.

Reservoir frame 1

Reservoir frame 2

Figure 20 Reservoir frame 1 for Windows® XP users


Side A

Side B

Side A

Side B



Reservoir frame 3

Reservoir frame 4

Related Biomek FXP Target Prep Express documentation

The following user manuals are installed at the same time as the Biomek FXP Target Prep Express software (Start All Programs Beckman Coulter Manuals). See these for troubleshooting the Biomek workstation.

• Biomek® Liquid Handler User’s Manual, Beckman Coulter Pub. No. 987834

• Biomek® Software User’s Manual, Beckman Coulter Pub. No. 987835



Side A

Side B

Side A


Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 1: DNA Amplification 3

Stage 1: DNA Amplification

Note: For this protocol, the term samples includes the positive control.

Duration Note: A 22–24 hours incubation is required at the end of this stage.


Equipment and labware required


Table 9 Time required for "Stage 1: DNA Amplification"

Activity Time

Hands-on time ~30 minutes

Biomek FXP Target Prep Express ~19 minutes

Incubation 23 hours

Total time ~24 hours

Table 10 Equipment and labware required

Quantity Item


1 Benchtop cooler, chilled to –20°C

As required Kimwipes laboratory tissues

1 Marker, fine point, permanent

1 Mini microcentrifuge (microfuge with microtube rotor)

1 Oven (must maintain a constant temperature of 37°C for at least 23 hours with a temperature accuracy of 1°C)• >3 array plates per week—we recommend using the BINDER ED 56• 3 array plates per week—OK to use the GeneChip Hybridization Oven

or the BINDER ED 56

1 Plate centrifuge

1 Vortex

Biomek workstation labware

1 box of each Barrier pipette tips:• Biomek Span P1000 (yellow)• Biomek AP96, P250 (aqua)

2 Plate, Bio-Rad Hard-Shell PCR 96-well

2 Plate, deep well titer


Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 1: DNA Amplification3

Reagents required

1 Reagent block, chilled to 4°C

3 Reservoir, quarter module (40 mL)

2 Reservoir, half module (75 mL)

Table 11 Reagents required for DNA Amplification

Axiom 2.0 Reagent Kit Module

Axiom 2.0 Denat Soln 10X

Module 1, –20°C

Axiom 2.0 Neutral Soln

Axiom 2.0 Amp Soln

Axiom 2.0 Amp Enzyme

Axiom Water

IMPORTANT! The Axiom 2.0 Assay is compatible with only reagents from an Axiom Reagent Kit. These reagents are not interchangeable with reagents from other Applied Biosystems reagent kits, such as SNP 6.0, DMET Plus, etc. The new Axiom 2.0 Reagent Kit contains a different Module 1 than the original Axiom Reagent Kit; however, Modules 2, 3, and 4 are identical between both versions and may be used from either kit for this assay. Only use new Module 1 from the Axiom 2.0 Reagent Kit for the Axiom 2.0 Assay.

Table 10 Equipment and labware required (Continued)

Quantity Item



1. Perform the pre-run checklist

Check the water and waste containers

1. If necessary, fill the system water container using deionized water (no need for ultra-pure water).

2. If necessary, empty the system waste bottle.

Turn on the Biomek FXP Target Prep Express

1. Power on the workstation.

2. Ensure that all of the peripherals are powered on.

• Watlow temperature controllers

Control the Static Peltier (Pelt_1) and the Shaking Peltier (SPelt_96); no additional power supply.

• Thermal cycler:

Bio-Rad PTC-200 or Biometra TRobot 96 if present on the Biomek workstation deck. Otherwise, a stand-alone thermal cycler can be used.

3. Launch the Biomek Software by double-clicking the Biomek Software icon on the desktop .

You can also open Start All Programs Beckman Coulter Biomek Software.

Close the thermal cycler (on deck thermal cycler)If your Biomek workstation includes a PTC200 or a Biometra TRobot 96, the lid may remain open upon startup. You must close the thermal cycler lid prior to homing the axes or starting a method.

The lid of the PTC-200 opens automatically when the device is powered up. While the lid of the TRobot does not power up automatically, it is possible that the lid may be open from a previous run or at the beginning of the next run.

Note: See page 12 of the setup guide and user’s manual for Biometra TRobot on the Biomek FXP. After following the instructions to close the thermal cycler lid, proceed to ʺHome All Axesʺ on page 56.

CAUTION! Close the thermal cycler lid:

• If it remains open after you have powered on the workstation.

• If the lid is up before you home the axes or before you begin a method.

If not closed, the MC Pod may collide with the thermal cycler lid and damage the instrument.



Closing the thermal cycler lid on the PTC-200

1. Open Instrument Device Editor.

2. Open the Device drop-down menu and select PTC200Left.

3. Click Action Commands.

4. Select the following (Figure 24 on page 55):

a. In the Actions box, select Close.

b. In the Open/Close box, select Without plate.

5. Click Close Lid.

A Status window is displayed while the lid is being closed (Figure 25).

6. Click OK when the Command executed prompt is displayed (Figure 25).

7. Click Cancel; then click Close.

IMPORTANT! It is critical that you select Without plate in the Open/Close box. If a plate is present, remove it now.



Figure 24 Closing the thermal cycler lid

Figure 25 Closing PTC lid prompts.

1

3

2

1

2

3

Select Close in the Actions box.

Select Without plate in the Open/Close box. This is very IMPORTANT to avoid damage to the thermal cycler!

Click Close Lid.



Home All AxesThis procedure will home all axes and prime the fluidics lines.

1. Open Instrument Home All Axes.

2. Ensure all conditions in the Warning prompt Figure 26-A are met, then click OK.

An icon instructing you to stop and wait while the instrument homes is displayed (Figure 26-B).

3. When:

a. the Warning prompt in Figure 27-A is displayed, confirm that no tips are loaded in the Span-8 Pod, and click OK.

The lines for the Span-8 tips are primed and the next prompt shown in Figure 27-B is displayed.

b. the intake (syringes and tubing) for the Span-8 tips is clear of bubbles, click OK.

Figure 26 Homing All Axes

Figure 27 Prompts displayed for priming the Span-8 pod fluidic lines

A B

A B



2. Thaw and prepare the reagents and Sample Plate

Thaw and prepare the reagents and Sample Plate

To thaw and prepare the reagents:

1. Thaw the Sample Plate (containing 200 ng of gDNA at 10 ng/µL or 100 ng of gDNA at 5 ng/µL depending on the Axiom array plate to be used) on the benchtop at room temperature and centrifuge.

Note: Do not place a frozen Sample Plate directly on the workstation deck.

2. Thaw the following reagents on the benchtop at room temperature.

• Axiom 2.0 Denat Soln 10X

• Axiom 2.0 Neutral Soln

• Axiom 2.0 Amp Soln

• Axiom Water

Leave the Axiom 2.0 Amp Enzyme in the freezer until ready to use.

Note: Allow ~1 hour for Axiom 2.0 Amp Soln to thaw on the benchtop at room temperature. If the solution is not completely thawed after 1 hour, vortex briefly and return to the benchtop to complete thawing. The bottles can also be thawed in a dish with Millipore water. The Axiom 2.0 Amp Soln must be thoroughly mixed before use.

3. Vortex all reagents (except Axiom 2.0 Amp Enzyme), then place at room temperature.

• Vortex the Axiom 2.0 Amp Soln and Axiom 2.0 Neutral Soln for 30 seconds to thoroughly mix.

• Vortex and centrifuge the Axiom 2.0 Denat Soln 10X before placing on the deck.

• For the Axiom 2.0 Amp Enzyme, just before placing on the deck gently flick the tube 3 times to mix and centrifuge.

4. Preheat the Oven to 37°C.

We recommend using one of these ovens:

• BINDER ED 56

• GeneChip™ Hybridization Oven (turn rotation on to 15 rpm)

3. Run the DNA Amplification step

To run the DNA Amplification step:

1. Open Project Open Project Axiom 2.0 Target Prep and click OK.



2. Open File Open to display the Open Method window.

Or click the Open Method icon .


4. At the top of the main window (Figure 28-A), click the Run button to open the Axiom™ Target Prep window.

5. In the Axiom Target Prep window (Figure 28-B):

a. Select DNA Amplification.

b. Click OK.

The Deck Layout for DNA Amplification is displayed (Figure 29 on page 59).


1

1

Figure 28 Opening the Axiom Target Preparation methods window

A B



6. Place the labware and reagents on the deck as directed in the following figures and table:

• Figure 29 on page 59—deck layout

• Figure 30 on page 60—location names of empty deck positions

• Table 12 on page 60—table detailing the labware, reagent and modular reservoir placement for the deck layout

• Figure 31 on page 61—reagent block

IMPORTANT! Axiom 2.0 Amp Enzyme—Immediately prior to placing on the reagent block, gently flick the tube with your finger 2 to 3 times to mix; then centrifuge. Do not vortex.

Figure 29 Deck layout window for the DNA Amplification method

IMPORTANT: No tips should be loaded onto the movable arms referred to as the Span-8 (right) and the Multi-Channel (MC; left) pods.

MC pod uses the pipette tips in this position.

Span-8 pod uses the pipette tips in this position.



Figure 30 Empty deck positions

Table 12 Labware and reagent locations on the deck for the DNA Amplification method

Position on deck

Labware Reagent or samples

TL1 Biomek AP96 – P250 Pipette Tips (aqua) —

P1 Beckman Deep Well Titer Plate gDNA samples

P4 Bio-Rad Hard-Shell 96-well plate (any color) —

P10 Reservoirs in frame:• Quarter module (1)• Quarter module (2)• Quarter module (3)

Pour Axiom 2.0 Neutral Solution into Reservoir 2

P11 Reservoirs in frame:• Half module (1)• Half module (2)

• Pour Axiom Water into reservoir 1• Pour Axiom 2.0 Amp Soln into reservoir 2

Pelt_1 Reagent block, chilled to 4°C See Figure 31.

P14 Biomek Span P1000 Pipette Tips (yellow) —

P15 Bio-Rad Hard-Shell 96-well plate (any color)

SPelt96_1 Beckman Deep Well Titer Plate —

1Empty

Reservoir

2Axiom

2.0 Neutral

Soln

3Empty

Reservoir

1

Axiom Water

2

Axiom 2.0 Amp Soln



7. Check the deck layout to ensure that all labware and reagents are in the proper locations.

Note: If the physical deck does not match the Deck Layout and Confirmation window exactly (Figure 29 on page 59), either modify the physical deck to match exactly or choose Abort in the Deck Layout and Confirmation window.

8. Click OK.

The system flushes the Span-8 fluidics system. Observe the lines and syringes for air bubbles.

Figure 31 Placement of reagents on chilled reagent block for the DNA Amplification step

Important:

• Position all plates and the chilled reagent block with A1 in the upper left corner of the frame.• Centrifuge all reagent tubes prior to placing in block to avoid loss of solution volume to the cap and sides of the tube.• Press reagent tubes into the block to ensure that they are fully seated.

Axiom 2.0Amp

Enzyme

Axiom 2.0 Denat Soln

10X

Reagent block with template.

Only the positions used for this method are shown.

Flick and centrifuge the Amp Enzyme before placing in block.

Diagram of reagent block without template

A1

A1



9. At the prompt to repeat the Span-8 fluidics system flush (Figure 32):

• Click No if no air bubbles are present.

• Click Yes if air bubbles are present. Repeat the flush until no air bubbles are present.

The DNA Amplification step runs until the Amplification Master Mix has been added to the Sample Plate. Once complete, the instructions and prompt shown in Figure 33 are displayed.

• Follow the instructions for the Sample Plate (see also ʺUser interventionʺ below).

• Follow the instructions for clearing the workstation deck.

User intervention

1. Remove the Sample Plate.

2. Blot the top of the plate with a Kimwipes laboratory tissue to remove any droplets that may be present.

3. Tightly seal the plate.

4. Vortex and centrifuge the plate.

5. Place in a preheated oven and incubate at 37°C for 23 1 hour.

Note: If using a GeneChip™ Hybridization Oven, place the plate on the bottom of the oven. Plates do not rotate.

6. After 22–24 hours of incubation, do one of the following:

• Proceed directly to ʺStage 2: Fragmentation and Purificationʺ on page 65.

• Tightly seal and store the amplified samples at –20°C.

Figure 32 Flushing the Span-8 fluidics system to purge air bubbles

Figure 33 Instructions for clearing the workstation deck

IMPORTANT:

• Seal the Sample Plate before placing in the oven.• Always discard the used multichannel pipette tips in position P3.• Always store the reagent block at 4°C.



Summary of DNA Amplification

Stage 1: Amplification

Stage 1: Amplification Page 1

Transfer Denat Soln 1X to Sample Plate

Prepare Denat Soln 1X & Distribution Plate

Span-8

Incubate on Deck10 mins @ RT

Denat Soln 10X Axiom Water

[96] 488 μL 96] 4392 μL

(4°C) (RT)D1 Reservoir

D1 Plate

30 μL/well

D1 Plate Sample Plate

20 μL/well

MC

Continued on AmplificationPage 2

Fill Neutral Soln Plate

N1 Reservoir N1 Plate

140 μL/well

[96] FORMAT TRANSFER MIX MOVE



Span-8MC

Continued from AmplificationPage 1

Prepare Amplification Master Mix & Distribution Plate

Amp Soln Amp Enzyme

[96] 26374 μL [96] 586 μL

(RT) (4°C)MM Reservoir

Amp Mastermix Plate

255 μL/well

Stage 1: Amplification Page 2

Stage 1: Amplification

Transfer Neutral Soln to Sample Plate

N1 Plate Sample Plate

130 μL/well

Incubate Sample PlateOffline for 23 ±1hrs @ 37°C

Transfer Amplification Master Mix to Sample Plate

Amp Mastermix Plate Sample Plate

230 μL/well



Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 2: Fragmentation and Purification 3

Stage 2: Fragmentation and Purification

Note: Purification is done by precipitation (See Step ʺ4. Precipitationʺ on page 75).

Duration



Table 13 Time required for "Stage 2: Fragmentation and Purification"

Activity Time

Hands-on time ~25 minutes~50 minutes if frozen DNA from Step 1

Biomek FXP Target Prep Express• Deactivation incubation—20 minutes to deactivate the

amplification reaction and 20 minutes to equilibrate to the fragmentation temperature

• Fragmentation incubation—30 minutes

~1:35 hours

Total time 2:00 to 2:25 hours

Table 14 Equipment, consumables, and labware required

Quantity Item



1 Freezer, –20°C





1 Oven, preheated to 37°C

1 Plate centrifuge

1 Vortex (for plates and microtubes)


1 box of each Barrier pipette tips:• Biomek AP96, P250 (aqua)• Biomek Span P250 (green)• Biomek Span P1000 (yellow)


1 96 Deep-Well Square Plate


Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 2: Fragmentation and Purification3

Reagents required


The following actions are the same as described under ʺ1. Perform the pre-run checklistʺ on page 53. Refer back to this step for details. Some or all of these steps may not be required depending upon the current state of the Biomek workstation.

To perform the pre-run checklist:

1. Power on the Biomek FXP Target Prep Express and all peripherals.

2. Check the water and waste containers; replenish or empty as required.


4. If applicable, close the thermal cycler lid (on page 53).

5. Home all axes (on page 56).


3 Reservoir, quarter module

1 Reservoir, half module

Table 15 Reagents required for the Fragmentation method

Reagent Module

From the Axiom 2.0 Reagent Kit

Axiom Frag Enzyme (leave at –20°C until ready to use)Module 2-1, –20°C

Axiom 10X Frag Buffer

Axiom Precip Soln 2

Axiom Frag DiluentModule 2-2, 2–8°C

Axiom Frag Rxn Stop

Axiom Precip Soln 1

User-supplied

Isopropanol, 99.5% 96 samples: 65 mL

Table 14 Equipment, consumables, and labware required (Continued)

Quantity Item



2. Thaw and prepare the amplified DNA samples and reagents

Thaw and prepare the amplified DNA Sample Plate

If the plate of amplified samples is frozen:

1. Place the deep well plate in a small water bath.

For example, pour Millipore water into a small tray. Place the frozen plate on the water in the tray.

2. Leave the plate in the water bath for ~50 minutes until all wells have thawed.

3. Centrifuge at 1,000 rpm for 30 seconds.

4. To avoid cross-contamination of wells during vortexing:

a. Remove the seal and blot the top of the plate with a Kimwipes laboratory tissue.

b. Tightly reseal the plate with a fresh seal.

5. Vortex the plate for 30 seconds to thoroughly mix.


Thaw and prepare the reagents


• Axiom 10X Frag Buffer

• Axiom Precip Soln 2

2. Vortex all reagents (except Axiom Frag Enzyme), then place on ice.

• Vortex and centrifuge Axiom Precip Soln 2 before placing onto deck.

• For the Axiom Frag Enzyme: Leave at –20°C until ready to use. Just before placing on the deck gently flick the tube 3 times to mix and centrifuge.

3. Run the Fragmentation step

To open the Target Preparation methods window:


2. Click the Open Method icon, select Axiom 2.0 Target Prep, and click OK.

3. Click the Run button.

4. Select Fragmentation, then click OK.

The deck layout for Fragmentation is displayed (Figure 34).







• Figure 36 on page 70—reagent cold block

IMPORTANT! Remove the seal from the Sample Plate before placing on the deck.

Figure 34 Deck layout for the Fragmentation method




Table 16 Labware and reagent locations on the deck for the Fragmentation step

Position on deck



P4 Bio-Rad Hard-Shell 96-well plate —



P7 Beckman Deep Well Titer Plate Amplified gDNA

P8 96 Deep-Well Square Plate —

P10 Reservoirs in frame:• Quarter module (1)• Quarter module (2)

• Pour Axiom Frag Rxn Stop into reservoir 1• Pour Axiom 10X Frag Buffer into reservoir 2

P11 Reservoirs in frame:• Half module (1)• Quarter module (2)

• Pour Isopropanol into reservoir 1• Pour Axiom Precip Soln 1 into reservoir 2

Pelt_1 Reagent block, chilled to 4°C See Figure 36 on page 70.

P13 Biomek Span P250 Pipette Tips (green)


1AxiomFragRxnStop

2Axiom10X Frag

Buffer

1

Isopropanol

2AxiomPrecipSol 1



6. Check the deck layout to ensure that the labware, reagents and samples are in the proper locations.


7. Click OK to continue.

The system will automatically flush the Span-8 fluidics system. Observe the lines and syringes for air bubbles.

8. At the prompt to repeat the Span-8 fluidics system flush:



Figure 36 Placement of reagents on chilled reagent block for the Fragmentation step

IMPORTANT:

• Always position the chilled reagent block with A1 in the upper left corner of the frame.• Centrifuge all reagent tubes prior to placing in block to avoid loss of solution volume to the cap and sides of the tube.• Press reagent tubes into the block to ensure that they are fully seated.

Axiom Precip Soln 2

Axiom Frag

Diluent

Axiom Frag

Enzyme




Axiom Frag Enzyme:

Flick to mix 3 tomes and centrifuge immediately prior to placing on the chilled reagent block.

A1

A1



The Fragmentation step begins. The Sample Plate is incubated at 65°C to inactivate amplification.

9. If you selected Prompt for manual DNA quantitation in the default software settings, you will then be prompted to remove an aliquot of each sample for a optional quantitation process control. The plate will remain at 40°C until the aliquots have been collected. Remove 4 µL aliquots from each well into a 96 well PCR plate and set aside for later quantitation (e.g., using the Quant-iT™ PicoGreen® dsDNA Kit from Life Technologies). Immediately continue sample processing by placing the amplification reactions back onto the shaking peltier and clicking OK at the prompt shown in Figure 37.

If Prompt for manual DNA quantitation was not selected, the box in Figure 37 will not appear.

Note: Remain near the Biomek FXP Target Prep Express if you are going to remove aliquots for quantitation. Avoid leaving the samples at 40°C for a long period of time.

10. Once the Fragmentation Complete message box appears (Figure 38). Follow the instructions in the message box discarding used labware and tips, storing unused tips and storing the cold block at 4°C. Click OK when ready to continue and proceed to Step ʺ4. Precipitationʺ on page 75.

Figure 37 Prompt for manual DNA quantitation

Figure 38 Fragmentation complete message



Summary of Fragmentation

Stage 2: Fragmentation

Span-8MC

Stage 2: Fragmentation Page 1

Inactivate DNA Amplification Rxn

Sample Plate

Incubate:

1) 20 mins @ 65°C

2) 20 mins @ 40°C

SPelt

Fill Fragmentation Buffer Plate

FragB Reservoir FragB Plate

56 μL/well

Transfer Fragmentation Buffer to Sample Plate

Sample Plate

45.7 μL/well

FragB Plate

Prepare Fragmentation Reagent & Distribution Plate

Frag Di luent Frag Enzyme

[96] 1551.4 μL [96] 150.6 μL

(4 oC)

(4°C)Frag Enzyme Tube

FragR Plate

16.5 μL/well

((((((((((((((((((((((((((((4 444444 44444 444 ooooooCCCCCCCCCCCCCC))))))))))))))))))oCCC))))))

Continued on FragmentationPage 2

(4°C)






Span-8MC

Continued from FragmentationPage 1

Incubate on Deck

30 mins @ 40oC

Transfer Fragmentation Reagent to Sample Plate

Sample Plate

11.3 μL/well

FragR Plate

Fill Stop Solution Plate

Stop Reservoir Stop Plate

29 μL/well

Prepare Prepitation Soln & Distribution Plate

Precip Soln 2 Precip Soln 1

[96] 192 μL

(4°C)

Precip Plate

240 μL/well






Span-8MC



Transfer Stop Solution to Sample Plate

Stop Plate Sample Plate

19 μL/well

Transfer Precipitation Solution to Sample Plate

Precip Plate Sample Plate

Plate Contents

Transfer Isopropanol to Precipitation Plate

Iso Reservoir Precip Plate

600 μL/well

Offline PrecipitationSee user guide

Transfer Precipitation Solution to Sample Plate

Sample Plate Precip Plate

Plate Contents




4. Precipitation To freeze the samples:

1. Remove the Precipitation Plate from the deck.

2. Blot the top of the plate with a Kimwipes laboratory tissue and seal tightly.

3. Place the plate in a –20°C freezer overnight to precipitate.

4. Return to the Biomek workstation and clear the deck.

5. Centrifuge and dry pellets

Note: Keep the centrifuge ready at 4°C.

To centrifuge and dry the pellets:

1. Turn the oven on and preheat to 37°C.

Use an oven that can sustain a constant temperature of 37°C and has a temperature accuracy of 1°C (we recommend the BINDER ED 56). If processing 3 or fewer array plates, you can use a GeneChip Hybridization Oven.

2. Centrifuge the plate for 40 minutes at 4°C at 3,200 x g (4,000 rpm for the Eppendorf 5810R centrifuge with the rotor configuration described in the Axiom™ 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP Site Preparation Guide).

3. Immediately after the 40 minutes centrifugation period, empty the liquid from the plate as follows:

a. Remove the seal.

b. Invert the plate over a waste container and allow the liquid to drain.

c. While still inverted, gently press the plate on a pile of Kimwipes laboratory tissues on a bench and leave it for 5 minutes.

4. Turn the plate right side up and place in an oven for 20 minutes at 37°C to dry.

Note: If using a GeneChip Hybridization Oven, place the plate on the bottom of the oven. Plates do not rotate.

5. Do one of the following:

• Proceed directly to ʺStage 3: Resuspension and hybridization preparationʺ on page 76, even if some droplets of liquid remain. Leave the Sample Plate at room temperature. It is helpful to begin preparing reagents for Stage 3 while centrifuging and drying pellets.

• Tightly seal the plate and store at –20°C.

CAUTION! During this step, handle the plate gently to avoid disturbing the pellets. Do not bump or bang the plate.

WARNING! Use rotor buckets with a soft rubber bottom to ensure that the deep well plates do not crack. Do not use buckets where the plates sit directly on a metal or hard plastic bottom, such as the A-4-62 rotor with a WO-15 plate carrier (hard bottom) for the Eppendorf 5810R centrifuge. Use of hard bottom plate carriers may result in cracked plates, loss of sample, unbalanced centrifugation, damage to the instrument and possible physical injury.


Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 3: Resuspension and hybridization preparation3

Stage 3: Resuspension and hybridization preparation

Duration Resuspension and hybridization preparation



Table 17 Time required for Resuspension

Activity Time

Hands-on time 15 minutes

Frozen pellet equilibration to room temperature 1.5 hours

Biomek FXP Target Prep Express 40 minutes

Table 18 Equipment, consumables, and labware required

Quantity Item






1 Plate centrifuge, at room temperature

1 Vortex


1 box of each Barrier pipette tips:• Biomek Span P50 (pink)• Biomek AP96, P250 (aqua)• Biomek Span P250 (green)• Biomek Span P1000 (yellow)

5 platesor:

Bio-Rad Hard-Shell PCR 96-well (HSP-9631)For on-deck thermal cycling or when using the PTC-0240/PTC-200 off-deck thermal cycler

6 plates for off deck

cycling with Applied

Biosystems thermal cyclers

4 Bio-Rad (HSP-9631) Hard-Shell PCR 96-well Plate and 1 HSS-9601 Hard-Shell Full-Height 96-Well Semi- Skirted PCR Plate1 Plate, Costar Brand Serocluster round bottom plate

For off-deck thermal cycling using the Applied Biosystems 9700 or Applied Biosystems 2720 thermal cycler.Note: The HSS-9601 plate stacked on the Costar brand serocluster round-bottom plate should only be used on position P2 on the Biomek FXP deck. See Figure 46 on page 92.

1 Plate, OD


Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 3: Resuspension and hybridization preparation 3

Reagents required

1. Preparing frozen pellets and Axiom Resusp Buffer

The equilibration of resuspension buffer and pellets to room temperature (18°C to 25°C) is very critical for the success of the Axiom 2.0 assay. When either is cooler than room temperature, pellets may not resuspend completely. This may result in compromised assay performance. Note following guidelines on how to work with plates with fresh, cold or frozen pellets:

Pellets:

• Plates with fresh pellets can be kept at room temperature for up to 1 hour if proceeding with the resuspension and hybridization protocol within an hour.

• Plates with fresh pellets that are not processed within an hour can be transferred to a refrigerator (2-8°C) for few hours only if processed within a day. However, it is critical to equilibrate the plate to room temperature for at least 30 minutes before proceeding with the resuspension and hybridization protocol.

• Plates with frozen pellets (e.g., on Day-5 of 8-plate workflow) must be pre-equilibrated at room temperature for at least 1.5 hour before proceeding with the resuspension and hybridization protocol.


1 Reservoir, 19 mL (quarter module, divided)

3 Reservoir, 40 mL (quarter module)

Table 19 Reagents required for Resuspension and Hybridization

Reagent Module


Axiom Hyb Buffer Module 2-1, –20°C

Axiom Hyb Soln 1

Axiom Resusp Buffer Module 2-2, 2–8°C

Axiom Hyb Soln 2

Other reagents required

Nuclease-free Water, ultrapure MB grade (Thermo Fisher Scientific, Cat. No. 71786; for OD and gel plate preparation)

To fill line of divided reservoir

TrackIt Gel Loading Buffer, diluted 1,000-fold(see Appendix B, "Fragmentation quality control gel protocol" on page 292 for dilution instructions.)

13 mL

Table 18 Equipment, consumables, and labware required (Continued)

Quantity Item

IMPORTANT! The pellets and the resuspension buffer must be at room temperature before proceeding with this step.



Resuspension Buffer:

• Resuspension buffer when pulled out from Module 2-2 at 2-8°C, needs at least 1 hour to equilibrate to room temperature.






4. If applicable, close the thermal cycler lid (on page 53).


3. Thaw and prepare the reagents


1. Thaw Axiom Hyb Soln 1 and warm Axiom Resusp Buffer on the benchtop at room temperature.

2. Vortex the Axiom Resusp Buffer and the Axiom Hyb Buffer.

3. Vortex and centrifuge Axiom Hyb Soln 1 and Axiom Hyb Soln 2.

4. Run the Resuspension and Hybridization Preparation step

Note: We strongly recommend that you run 2 quality process controls during this step:

• A gel to verify successful fragmentation

• An OD quantitation of each resuspended sample

The Biomek FXP Target Prep Express can be set to prepare fragmentation and OD plates that are ready for processing. These process controls must be selected as a run preference prior to starting a run. See ʺSet the Biomek software default settingsʺ on page 25 for instructions.



3. Click the Run button.The Axiom 2.0 Method selection dialog box appears (Figure 39).



4. Select Resuspension and Hybridization Preparation and click OK.The deck layout for this method is displayed (Figure 40 on page 80).






6. Label the Bio-Rad plates placed on the deck in positions P2 and P11. For example:

• P2—Hyb Ready + <sample description>

• P11—Gel QC

Note: Verify the appropriate plastic consumables are being used on the deck for the Hyb Reaction Plate when using the Applied Biosystems 9700 or Applied Biosystems 2720 thermal cycler.

Figure 39 Selecting the Resuspension and Hybridization Preparation method



Figure 40 Deck layout for the Resuspension and Hybridization Preparation method

If Prepare plates for gel QC and OD after resuspension is selected in your run preferences, the following labware is required on the deck:

• Multichannel P50 pipette tips• Dilution QC plate• Gel QC plate• OD QC plate (do NOT touch bottom of

plate)If not selected, these plates will not appear in the deck layout.

See "Set the Biomek software default settings" on page 25 for more information.




Table 20 Labware and reagent locations on the deck for Resuspension and Hybridization Preparation

Position on deck



P1 ABgene 96 Square Well Storage plate Pelleted samples

P2 Bio-Rad Hard-Shell 96-well plate(Hyb Reaction Plate).• Bio-Rad Hard-Shell 96-well plate (HSP-9631) for on-deck or

off-deck thermal cycling using the TRobot 96, PTC-200 or PTC 0240. OR

• Bio-Rad Hard-Shell Full-Height 96-Well Semi-Skirted PCR Plate (HSS-9601) stacked on a Costar Round Bottom plate for off-deck thermal cycling with the Applied Biosystems 9700 or 2720 thermal cycler

WARNING: When using the Applied Biosystems 9700 or the Applied Biosystems 2720 off-deck thermal cycler for denaturing the Hyb Ready Plate, the Bio-Rad HSS-9601 Hard-Shell Full-Height 96-Well Semi-Skirted PCR Plate must be stacked on a CoStar Brand Serocluster Round Bottom plate (29442-392 from VWR International or EK-680568 from E&K Scientific) as shown in Table 6 on page 29.The HSS-9601 and HSP-9631 PCR plates are not interchangeable on the Biomek FXP deck.

P3 Biomek Span P50 Pipette Tip (pink) —


P8 Bio-Rad Hard-Shell 96-well plate




P10 Reservoirs in frame:• Quarter module (1)• Quarter module (2)• Quarter module (3)• Quarter module, divided (4 and 5)

• Pour Axiom Resus Buffer into reservoir 1• Pour Axiom Hyb Buffer into reservoir 2• Leave reservoir 3 empty• Pour diluted gel loading buffer into reservoir 4: 13 mL• Pour nuclease-free water into reservoir 5 to fill line

P11 Bio-Rad Hard-Shell 96-well plate (for Gel QC) —

P12 OD plate, 96-well UV —


P13 Biomek Span P250 Pipette Tips (green) —


Table 20 Labware and reagent locations on the deck for Resuspension and Hybridization Preparation

Position on deck


1Axiom

ResuspBuffer

2AxiomHyb

Buffer

3Empty

Reservoir

4Gel Load

Buffer

5Water




Note: If the physical deck does not match the Deck Layout window exactly (Figure 40 on page 80), either modify the physical deck to match exactly or choose Abort in the Deck Layout window.

8. Click OK to continue the method.





Figure 42 Placement of reagents on chilled reagent block for Resuspension and Hybridization Preparation

IMPORTANT:


Axiom Hyb

Soln 2

Axiom Hyb

Soln 1




A1

A1



10. Run the fragmentation gel and OD quantitation process controls.

For instructions and guidelines on assessing gel and OD results, see:

• Appendix B, ʺFragmentation quality control gel protocolʺ on page 292.

• Appendix C, ʺSample quantitation after resuspensionʺ on page 295.


• If the GeneTitan MC Instrument is free, and if the gel and OD quantitation results are good:

• Discard used labware and reagents.

• Discard used tips.

• Store unused Span-8 tips.

• Click OK in the Resuspension and Hyb Prep complete dialog box and proceed to ʺStage 4: Preparation for the GeneTitan™ MC Instrumentʺ on page 89.

• If the GeneTitan MC Instrument is not free, then follow the instructions shown in Figure 43:

• Tightly seal the Hyb Rxn plate and store at –20°C.

• Discard used labware and reagents.

• Discard used tips.

• Store unused Span-8 tips.

• Store cold block at 4°C.

• Click OK when complete.

Figure 43 Instructions to follow if the GeneTitan MC Instrument is not free



Summary of Resuspension and Hybridization Preparation

Stage 3: Resuspension & Hybridization Prep

Span-8MC

Stage 3: Resuspension & Hybridization Prep Page 1

Fill Resuspension Buffer Plate

ResB Reservoir ResB Plate

45 μL/well

Resuspend Pellet

ResB Plate Precip Plate

35 μL/well

Prepare Hybridization Master Mix & Distribution Plate

Hyb Soln 2 Hyb Soln 1

[96] 1197 μL [96] 66.5 μL

(4°C) (4°C)MM Reservoir

Hyb Plate

90 μL/well

Hyb Buffer

(RT)

[96] 9376.5 μL

Continued on ResuspensionPage 2





Span-8MC


Continued from ResuspensionPage 1

Transfer Hybridization Master Mix to Precipitation Plate

Hyb Plate Precip Plate

80 μL/well

See QC flowchart if performing analysis after resuspension

Transfer Hybridization Mix to Hybridization Plate

Precip Plate Hyb Rxn Plate

115 μL/well

Store HybridizationReaction Plate




Stage 3: QC

Span-8MC

Stage 3: QC Page 1

Fill Dilution QC Plate with Water

Water Reservoir Dilution QC Plate

55 μL/well

Fill OD QC Plate with Water

Water Reservoir OD QC Plate

90 μL/well

Fill Gel QC Plate with Dye

Dye Reservoir Gel QC Plate

120 μL/well

Continued on QCPage 2

Transfer Hybridization Master Mix to Dilution Plate

Hyb Rxn Plate Dilution QC Plate

5 μL/well




Perform Offline Analysis


Span-8MC

Stage 3: QC Page 2

Transfer Diluted Sample to OD QC Plate

Dilution QC Plate OD QC Plate

10 μL/well

Continued from QCPage 1

Transfer Diluted Sample to Gel QC Plate

Dilution QC Plate Gel QC Plate

3 μL/well



Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 4: Preparation for the GeneTitan™ MC Instrument 3

Stage 4: Preparation for the GeneTitan™ MC Instrument

About Stage 4 You will proceed to Stage 4 in 1 of 2 ways:

• Directly from Stage 3 without interruption.

• With samples that were stored at –20°C after Stage 3.

This stage, Preparation for GeneTitan, can include any combination of the options shown in Figure 44. The first 2 options complete target preparation on the Biomek FXP Target Prep Express.

The options are:

Option 1

• Denature samples—the Hyb Rxn plate is placed on the thermal cycler and the samples are denatured. At this step, you must also select Transfer denatured samples to hybridization tray. After the denature program has completed, the block will hold temperature until the user has dismissed the prompt, indicating that they are ready to continue the method to transfer the samples to the hybridization tray and then carry it to the GeneTitan MC Instrument. Do not leave samples on the thermal cycler for a long period of time.

Option 2

• Transfer denatured samples to hyb tray—the denatured samples are transferred from the Hyb Rxn plate to the hybridization tray, and are ready to load onto the GeneTitan MC Instrument.

Note: When using an Applied Biosystems 9700 or the Applied Biosystems 2720 thermal cycler for off-deck denaturation, the Bio-Rad Hard-Shell Full-Height 96-Well Semi-Skirted PCR Plate (Hyb Reaction Plate) must be stacked on a Costar brand Serocluster Round Bottom plate from Corning (Corning Mfg Cat. No. 3795).

Figure 44 Software setup for Preparation for GeneTitan

You can run one step, or a combination of steps.


Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 4: Preparation for the GeneTitan™ MC Instrument3

Option 3

• Prepare GeneTitan™ reagent trays - the solutions required for the fluidics stage of array processing on the GeneTitan MC Instrument are prepared and aliquoted to the appropriate trays (3 stain trays, 1 ligation tray, 1 stabilization tray and 1 scan tray with Holding Buffer).

When performing a 1 plate workflow, select 2 options (option 1 and option 2).

• Denature samples and Transfer denatured samples to hyb tray when you are preparing the samples to begin hybridization in the GeneTitan MC Instrument.

- or select 1 option (option 3)

• Prepare GeneTitan™ reagent trays to prepare reagents for loading onto the GeneTitan MC Instrument the following day when the plate is finished with the hybridization period and about to begin GeneTitan fluidics processing.

Options for performing a multi-plate workflow

When performing a multi-plate workflow (see Chapter 6, ʺProcessing 8 Axiom™ array plates per weekʺ on page 246 for a description of the 8 plate workflow), the need to carry out preparation of reagents for 1 plate while simultaneously hybridizing a second plate will arise. In this case, all 3 options in the Biomek FXP method can be carried out concurrently. Select all 3 options when using an on-deck thermal cycler.

• Denature samples and Transfer denatured samples to hyb tray will prepare samples for the new plate going into the GeneTitan MC Instrument to begin hybridization.

-and-

• Prepare GeneTitan™ reagent trays will prepare reagents for the plate that is already in the GeneTitan MC Instrument hybridization oven and is ready to go into the Wash, Ligation, Stain, and Scan phases of GeneTitan array processing.

Note: In the 8 plate workflow, all 3 options will only be selected for middle days of the workflow. On the first day of the workflow, only the Denature samples and Transfer denatured samples to hyb tray options will be used. On the last day of the workflow, only the Prepare GeneTitan reagent trays option will be used.

Note: When using an off-deck thermal cycler, avoid letting the samples sit a room temperature for an extended period of time after denaturation. Do not begin denaturation at the same time as the GeneTitan reagent preparation.

Off-deck thermal cycler option

The steps for performing a simultaneous preparation of GeneTitan reagent trays and hybridization of a second plate required in the course of a multi-plate workflow are modified slightly when the off-deck thermal cycler option is selected. The reagent trays to be loaded into the GeneTitan MC instrument are prepared in an initial run of the method.

Denaturation of the hybridization ready samples in the off-deck thermal cycler begins mid way through the GeneTitan reagent prep on the Biomek deck.

After loading the reagent trays into the GeneTitan MC instrument, you must perform a second run of the Biomek method to transfer the denatured samples to the hybridization tray for loading into the GeneTitan MC instrument. You will need a timer for this modified step.



The modified steps are:

1. Select Preparation for GeneTitan step with only the Prepare GeneTitan™ reagent trays box checked, click OK.

2. Prepare deck as shown in Figure 45.

3. Click OK.

4. Start timer. Wait 25–30 minutes, then begin denaturation of the hybridization ready samples using the off-deck thermal cycler.

5. Upon completion of the GeneTitan reagent tray preparation, load reagent trays and scan tray into the GeneTitan MC instrument

6. Once the reagents are loaded into the GeneTitan MC instrument and the denaturation method on the thermal cycler is complete, retrieve the denatured hybridization ready samples from the thermal cycler

7. Return to Biomek FXP and begin a new method, select Preparation for GeneTitan step with only the Transfer denatured samples to hyb tray box checked, click OK.

8. Prepare deck as shown in Figure 46 (note that a spacer must be used with HSS-9601 plates for Applied Biosystems thermal cyclers).

Figure 45 Deck layout for off-deck thermal cycler option



9. Click OK.

10. After the denatured samples have been transferred to the GeneTitan hybridization tray, load hybridization tray into the GeneTitan MC Instrument.

Figure 46 Deck layout for off-deck thermal cycler option and position P2

-

Bio-Rad Hard-Shell Full-Height 96-Well Semi-Skirted

PCR Plate (HSS-9601) stacked on a Costar Round

Bottom plate for off-deck thermal cycling with the

Applied Biosystems 9700 or 2720 thermal cycler

The Hyb Reaction Plate in deck position P2 must be

one of the following:

Bio-Rad Hard-Shell 96-well plate (HSP-9631) for

on-deck or off-deck thermal cycling using the

TRobot 96, PTC-200 or PTC 0240

OR

--



If a plate is already in the hybridization ovenFor the plate that is already in the GeneTitan hybridization oven and that is ready to go into Ligation, Wash-Stain and Scan, select option 3, Prepare GeneTitan™ reagent trays. The solutions required for the fluidics stage of array processing on the GeneTitan MC Instrument are prepared and aliquoted to the appropriate trays (3 stain trays, 1 ligation tray, 1 stabilization tray and 1 scan tray with Holding Buffer).

Duration of GeneTitan™ MC Instrument reagent preparation and sample preparation

Sample denaturation

Denatured sample transfer to hybridization tray

IMPORTANT! The reagent trays prepared in the third sub-step, Prepare GeneTitan™ reagent trays:

• Are NOT for use with the hybridization tray currently being prepared on the Biomek workstation.

• Are for the continued processing of an Axiom array plate that is:

– already on the GeneTitan MC Instrument.

– has completed the hybridization stage.

– is ready for transfer to the fluidics area.

The reagent trays for the fluidics stage on the GeneTitan MC Instrument CANNOT be prepared in advance. Do not prepare these plates if there is no array plate ready for the fluidics stage. Once prepared, these plates must be loaded onto the instrument as soon as possible and cannot be stored.

Table 21 Time required to denature samples

Activity Time

Hands-on time: ~3 minutes


Total time: ~20 minutes

Table 22 Time required to transfer denatured samples to the hybridization tray

Activity Time



Total time ~4 minutes



Preparation of GeneTitan reagent trays

Note: When you select Denaturation and Preparation of the GeneTitan Reagent Trays, the on-deck processes run concurrently.

Equipment and consumables required

Sample Denaturation

Transfer to hybridization tray

Table 23 Time required to prepare reagent trays for the GeneTitan MC Instrument

Activity Time

Prepare reagents (thaw and organize reagents)

~30 minutes




Table 24 Consumable required for denaturing samples on-deck

Item Quantity

Lid, arched metal 1

Table 25 Consumables required for transferring denatured samples to a hybridization tray

Item Quantity

Item required from the GeneTitan™ Consumable Kit, Cat. No. 901606:• Hybridization Tray 1

Pipette tips, Biomek AP96 – P250 (aqua) 1 full box

If using off deck Applied Biosystems 9700 or Applied Biosystems 2720 thermal cyclers: Costar brand Serocluster Round Bottom plate from Corning Mfg. Cat. No. 3795) to be used for stacking under the HSS-9601 semi-skirted plate (Hyb Reaction Plate).

1



Prepare GeneTitan™ Reagent Trays

Note: See Table 7 on page 36 for GeneTitan MC Instrument consumable catalog numbers.







4. If open, close the thermal cycler lid (on page 53).


Table 26 Consumables and other equipment required

Item Quantity

Items required from the GeneTitan™ Consumable Kit, Cat. No. 901606: • Scan tray (with cover and protective base)• Stain trays• Covers for stain trays

155


Pipette tips, Biomek Span P1000 (yellow) 1 full box

Pipette tips, Biomek Span P250 (green) 1 full box

Reagent block, chilled to 4°C 1

Reservoirs, quarter module divided 3

Reservoirs, quarter module 3

Tube rack, 24 position with insert (room temperature rack) 1

Zerostat Anti-Static Gun 1



2. Prepare the reagents for GeneTitan Reagent Tray Preparation

Note: Ligation Buffer and Ligation Solution 2 require approximately 30 to 40 minutes to thaw on the benchtop at room temperature.

Reagents required

Preparing Axiom Wash A and Axiom Stabilize DiluentDuring storage of the Axiom Wash A and Axiom Stabilize Diluent (in Module 4-2 stored at 4°C), precipitation in the form of clear crystals can sometimes occur. Therefore, follow the procedure below to ensure that any precipitate is returned to solution prior to use.

Note: The presence of some precipitate is OK and will not adversely impact assay performance. Follow the instructions below to resuspend any precipitate before use.

Table 27 Reagents required for GeneTitan MC Instrument Reagent Tray Preparation

Module Reagent Thaw on benchtop, then place on ice

Place on ice Place on benchtop at room temperature

Module 3Room

Temperature

Axiom Wash Buffer A

Axiom Wash Buffer B

Axiom Water

Module 4-1–20°C

Axiom Ligate Buffer - for 30 minutes

Axiom Ligate Enzyme Keep at –20°C until ready to use

Axiom Ligate Soln 1

Axiom Probe Mix 1

Axiom Stain Buffer

Axiom Stabilize Soln

Module 4-22 to 8°C

Axiom Ligate Soln 2 - for 30 to 40 minutes

Axiom Probe Mix 21

Axiom Wash A - for 30 minutes

Axiom Stain 1-A1

Axiom Stain 1-B1

Axiom Stain 2-A1

Axiom Stain 2-B1

Axiom Stabilize Diluent

Axiom Water

Axiom Hold Buffer1

1 These solutions are light sensitive. Keep tubes out of direct light for a prolonged period of time.



To prepare the Axiom Wash A:

1. Vortex the bottle for 30 seconds.

2. Place on the benchtop at room temperature for 30 minutes.

3. Examine the reagent for precipitate (look into the top of the bottle).

4. If precipitate is still present, vortex again for 30 seconds.

5. Pour Axiom Wash A into the appropriate reagent reservoir and leave on the benchtop until ready to use.

To prepare the Stabilize Diluent:

If crystals are observed in the Axiom Stabilize Diluent:

1. Vortex and centrifuge.

2. Look for precipitate.

If any: Warm tube to room temperature and vortex again.

Preparing Axiom Ligate BufferWhite precipitate is sometimes observed when the Axiom Ligate Buffer is thawed.

Note: The presence of some precipitate is okay and will not adversely impact assay performance. Follow the instructions below to attempt to resuspend a majority of precipitate before use.

To prepare the Axiom Ligate Buffer:

1. Place on the benchtop at room temperature for 30 minutes. This bottle can also be thawed in a dish with room temperature Millipore water.

2. Examine the buffer for precipitate (look into the top of the bottle).

3. If precipitate is present, vortex the bottle for 30 seconds.

4. Re-examine the buffer for precipitate.

5. If precipitate is still present, warm the bottle with your hands and vortex again for 30 seconds.

6. If precipitate is still present after hand warming proceed with the protocol below.

7. Pour the Axiom Ligate Buffer into the appropriate reagent reservoir and leave on the benchtop until ready to use.



Prepare the remaining reagents

1. Leave the Axiom Ligate Enzyme at –20°C until ready to use.

2. Thaw the following reagents from Module 4-1 at –20°C on the benchtop at room temperature, then vortex, centrifuge and place on ice:

• Axiom Ligate Soln 1

• Axiom Probe Mix 1

• Axiom Stabilize Soln

• Axiom Stain Buffer

3. Prepare the remaining reagents from Module 4-2 as follows:

a. Gently flick each tube 2 to 3 times to mix, then centrifuge.

b. Place reagents on ice, except for the Axiom Hold Buffer, Axiom Ligate Soln 2 and Axiom Water— leave these reagents on the benchtop at room temperature until ready to use.

3. Prepare the Sample Plate (if stored at –20°C) and the array plate

To prepare samples that were stored at –20°C:

1. Warm up the Hyb Ready Plate at room temperature for 5 minutes. It is not necessary to equilibrate the plate for longer duration.

2. Ensure that the Hyb Ready Plate is sealed well. If the plate is not sealed well:

a. Centrifuge the plate and carefully remove the old seal.

b. If there is condensation on the top of the plate, blot dry gently with a Kimwipes laboratory tissue.

c. Use a fresh seal and tightly reseal the plate.

3. Vortex the Hyb Ready Plate briefly, then centrifuge to 1,000 rpm.

4. Place the Hyb Ready Plate at room temperature until ready for use.

To prepare the array plate:

1. Warm up the array plate on the benchtop before setting up hybridization on the GeneTitan MC Instrument.

a. Leave the array plate in the pouch at room temperature, for a minimum of 25 minutes, before opening and loading on the GeneTitan MC Instrument to allow the plate to come to room temperature.

b. At the end of the array warm up time, open the pouch and scan the array plate barcode into the Batch Registration file (see ̋ Stage 1: Create and upload Batch Registration fileʺ on page 219).

WARNING! Do not remove the array plate from the protective base or touch the surface of any arrays.



4. Prepare the GeneTitan™ MC Instrument

Before you denature your hybridization ready samples, ensure that the GeneTitan MC Instrument is ready for use.

One or more of the following steps may need to be performed:

1. Launch Applied Biosystems™ GeneChip™ Command Console and select GCC GeneTitan Control.

2. Upload your sample registration file now, if you have not done so (see ʺStage 1: Create and upload Batch Registration fileʺ on page 219).

If you do not upload your samples before scanning the array plate barcode, the software will assign names to your samples.

3. On the GCC GeneTitan Instrument Control, select the System Setup tab (Figure 47).

4. Configure the software as follows:

a. Setup Option: Hyb-Wash-Scan

Other options available are described under ʺSetup options for array plate processingʺ on page 215.

b. Click Next.

c. Plate Information:

• Barcode: Scan or manually enter the array plate barcode and click Next.

• Protocol Name: Select the protocol name and click Next.

5. Fill the Wash A, Wash B and Rinse bottles.

6. Empty the Waste bottle.

After preparing the GeneTitan MC Instrument, perform steps 6a, 6b or 6c as appropriate for your workflow:

• Go to Step 6a on page 106: if you are denaturing the samples and transferring them to the hybridization tray.

• Go to Step 6b on page 107: if you are preparing reagents for the Ligation-Wash-Stain.

• Go To Step 6c on page 107: if you are performing a 2-8 plate workflow, and you are denaturing samples for the 2nd plate and are preparing ligation reagents for the 1st plate. The samples for the 2nd Axiom array plate will be denatured and transferred into the hybridization tray. The hybridization tray will be placed in the GeneTitan MC Instrument with the array plate in preparation for



hybridization. The Biomek FXP. Target Prep Express will also prepare reagents for Ligation-Wash-Stain for the first array plate that was placed in the GeneTitan MC Instrument hybridization oven 24 hours ago.

5. Run the Preparation for GeneTitan™ step

To run the Preparation for GeneTitan step:



3. Click the Run button.The Axiom 2.0 Target Prep run options window appears (Figure 48).

4. Select Preparation for GeneTitan™ and the sub-steps that you wish to run; then click OK.

Based upon your choices, the appropriate deck layout is displayed (Figure 49).

Figure 47 Setup Options for processing array plates

System Setup tab

Select Hyb-Wash-Scan option


You can run each step individually, or any combination of steps. If you do not select Prepare GeneTitan™ reagent trays, the deck layout will be different than the one shown in Figure 49.

• Select Denature Samples and Transfer denatured samples to hybridization tray– if you are preparing to load the array plate into the GeneTitan

Instrument for Hybridization.• Select Prepare GeneTitan™ Reagent Trays

– if you are preparing to load the GeneTitan Instrument for Wash-Stain and Imaging.

• Select Denature Samples and Transfer denatured samples to hyb tray and Prepare GeneTitan™ Reagent trays

– if you are running an 8-plate workflow then you will need to select all 3 check boxes in some instances in preparation for the plate that will go into the GeneTitan Instrument for Hybridization.

• Select Prepare GeneTitan™ Reagent trays for the array plate that has completed hybridization and is ready to be processed in the GeneTitan Instrument for Ligation- Wash-Stain and Imaging.



5. Deionize the GeneTitan stain trays. See Appendix E, section entitled ʺDeionization procedure for GeneTitan™ trays and coversʺ on page 307 for the deionization procedure.






• Figure 52 on page 105—tube block

Note: Prior to placing the arched metal lid on the deck, be sure to clean the attached Microseal® P pad with 70% ethanol and dry it. See the product information sheet for this product for further information on cleaning and replacement.

IMPORTANT! It is important to deionize the GeneTitan MC Instrument trays to remove any static electricity on the trays. Static attraction by the trays may prevent the tray cover from being lifted up by the instrument.

Figure 49 Deck layout for Preparation for GeneTitan—denature, transfer to hybridization tray, and GeneTitan plate preparation.

The deck layout displayed is based upon the selections you make for this step.

IMPORTANT: Destatic the stain trays prior to placing them on the deck. See the section "Deionization procedure for GeneTitan™ trays and covers" on page 307.

Included in deck layout if:

• Denature samples• Transfer denatured samples to

Hyb Tray are selected.

Included in deck layout if Prepare GeneTitan™ reagent trays is selected.




Table 28 Labware and reagent locations on the deck for GeneTitan Reagent Preparation

Position on deck


If Denature samples and Transfer denatured samples to Hyb Tray is selected, (no reagent tray preparation), only the labware listed below is required:


P1 Lid, arched metalClean before use (70% ethanol).

—

P2 Hyb Reaction Plate1 Hyb-ready samples

P3 Hybridization tray2 —

If Prepare GeneTitan™ reagent trays, the labware listed below is required:

P4 Stain 12, 3 (second of 2 trays with Stain 1) —

P5 Stain 22, 3 —

P6 Lig2, 3 —

P7 Scan Tray on protective base* —

P8 Stbl2, 3 —

P9 Stain 12, 3 (first of 2 trays with Stain 1) —

P10 Tube block with 1 insert, room temperature See Figure 52 on page 105.

P11 Reservoirs in frame:• Quarter module, divided (1 and 2)• Quarter module, divided (3 and 4)• Quarter module, divided (5 and 6)

• Pour Axiom Water into reservoir 1• Pour Axiom Ligation Buffer into reservoir 5

1Axiom Water

3Empty

5Axiom LigateBuffer

2Empty

4Empty

6Empty





• Pour Axiom Hold Buffer into reservoir 1• Pour Axiom Wash A into reservoir 2• Leave reservoir 3 empty

Pelt_1 Reagent block, chilled to 4°C See Figure 51 on page 104

P13 Biomek Span P250 Pipette Tips (green) —


1 Bio-Rad Hard-Shell 96-well plate (HSP-9631) for on-deck or off-deck thermal cycling using the TRobot 96, PTC-200 or PTC 0240. Bio-Rad Hard-Shell Full-Height 96-Well Semi-Skirted PCR Plate (HSS-9601) stacked on a Costar Round Bottom plate for off-deck thermalcycling with the Applied Biosystems 9700 or 2720 thermal cycler.

2 These trays are included in Axiom Genome-Wide and Custom myDesign™ Array Plate Kits.3 Label each of these stain trays as described above as described in "Labeling GeneTitan hybridization and reagent trays". For example,

label the Stain tray to be placed in P6 with the word Lig. This tray will contain the Ligation master mix.

Table 28 Labware and reagent locations on the deck for GeneTitan Reagent Preparation (Continued)

Position on deck


1Axiom Hold

Buffer

2Axiom

Wash A

3Empty

Reservoir

IMPORTANT! It is critical that you write only on the proper location of the hybridization tray (on the edge in front of wells A1 and B1) as illustrated in Figure 13 on page 44 or on the proper location of the stain/reagent trays (on the edge in front of wells A1 to C1) as illustrated in Figure 14 on page 45. Do NOT write on any other side, as this can interfere with sensors inside of the GeneTitan MC Instrument and result in experiment failure. To ensure proper placement of lids onto stain trays, and trays onto the GeneTitan MC Instrument, you can also mark the notched corner of the trays and lids.




Figure 51 Placement of reagents on chilled reagent block for GeneTitan Reagent Tray Preparation.

IMPORTANT:


Axiom Ligate

Enzyme

Axiom Probe Mix 1

Axiom Probe Mix 2

Axiom Ligate Soln 1

Axiom Stain 2-B

Axiom Stain Buffer

Axiom Stabilize

Soln


Axiom Stain 2-A

Axiom Stain 1-B

Axiom Stain 1-A




A1

A1





8. Click OK to continue the step.




Click Yes if air bubbles are present. Repeat the flush until no air bubbles are present.

If the “Denature Samples” option is selected, the Biomek FXP Target Prep Express places the samples onto the thermal cycler and runs the denaturation program. If Prepare GeneTitan™ reagent trays is also selected, reagent tray preparation for the GeneTitan MC Instrument starts while the samples are being denatured.

Figure 52 Placement of reagents on room temperature tube block.

AxiomLigationSoln 2

A1



6a. Complete Stage 4: Preparation for GeneTitan™ - Hybridization Trays

If you selected Denature samples and Transfer denatured samples to Hyb tray in Step 4 (See Step ʺ5. Run the Preparation for GeneTitan™ stepʺ on page 100 and Figure 48 on page 100), use the following instructions:

1. Once the GeneTitan MC Instrument is ready, return to the Biomek FXP Target Prep Express click OK (prompt shown in Figure 53 above).

The denatured samples are then taken off the thermal cycler and are transferred to the hybridization tray.

• Ensure that there are no air bubbles present in the hybridization tray. Puncture any air bubbles that you see using a pipette tip.

2. Transfer the hybridization tray to the GeneTitan MC Instrument and load. See the section ʺArray processing with the GeneTitan™ MC Instrumentʺ on page 209 for the proper way of loading the array plate and the hybridization tray.

3. Return to the Biomek FXP Target Prep Express and clear the deck (Figure 54).

• Always discard the used multichannel pipette tips in position P9.

• Always store the reagent block at 4°C.

• Clean the Microseal P Pad by wiping with 70% EtOH and dry.

See the product information sheet for this product for further information on cleaning and replacement.

Figure 53 Prompt indicating denaturation is complete.

IMPORTANT! Immediately load the array plate and hybridization tray into the GeneTitan MC Instrument.

Figure 54 Instructions displayed at the end of Step 4.



6b. Complete Stage 4: Preparation for GeneTitan™ - Reagent Trays

If you selected Prepare GeneTitan™ Reagent trays in Step 4 (See Step ʺ5. Run the Preparation for GeneTitan™ stepʺ on page 100 and Figure 48 on page 100), use the following instructions:

1. Once the prompt shown in Figure 54 appears, remove the reagent trays and scan tray with Hold Buffer from the deck and cover with the appropriate lids.

2. Examine each tray to ensure that:

• All of the wells have been filled. If any wells do not contain reagents, then manually add reagents to these wells.

• There are no air bubbles present. Puncture any air bubbles that you see using a pipette tip.

j

3. Transfer the reagent trays, scan tray to the GeneTitan MC Instrument and load. See the section ʺStage 3: Ligate, Wash, Stain, and Scanʺ on page 235 to continue the process on the GeneTitan MC instrument.

4. Return to the Biomek FXP Target Prep Express and clear the deck (See Figure 47 on page 100).




See the package insert for this product for further information on cleaning and replacement.

6c. Complete Stage 4: Preparation for GeneTitan™ - multiple plate workflow

If you selected all 3 options in Step 4 (see Step ʺ5. Run the Preparation for GeneTitan™ stepʺ on page 100 and Figure 48 on page 100) and are preparing hybridization tray and reagent trays in a 2+ plate workflow, use the following instructions:

The hybridization tray is prepared for a new Axiom array plate that will be loaded into the GeneTitan MC Instrument. The reagent trays are prepared for the Axiom array plate that is in the hybridization oven in the GeneTitan MC Instrument and is ready to move to the next stage of the process - Ligation, Wash-Stain and Scan.

Note: When using an off-deck thermal cycler see the instructions for the ʺOff-deck thermal cycler optionʺ on page 90.

1. Once the reagent trays are prepared and sample denaturation is complete, the prompt in Figure 53 on page 106 is displayed (do not click OK yet). Remove the reagent trays and scan tray with Hold Buffer from the deck and cover with the appropriate lids.




IMPORTANT! Immediately load the reagent trays onto the GeneTitan MC Instrument. Do not leave denatured samples or reagent trays at room temperature for any length of time.



3. Transfer the reagent trays, scan tray and array plate to the GeneTitan MC Instrument and load.

4. Return to the Biomek FXP Target Prep Express click OK at the prompt shown in Figure 53 on page 106.

The denatured samples are then taken off the thermal cycler and are transferred to the hybridization tray.

5. Transfer the hybridization tray to the GeneTitan MC Instrument and load. See the section ʺArray processing with the GeneTitan™ MC Instrumentʺ on page 209 for the proper way of loading.

6. Transfer the reagent trays, scan tray to the GeneTitan MC Instrument and load. See the section ʺArray processing with the GeneTitan™ MC Instrumentʺ on page 209 to continue the process on the GeneTitan MC Instrument.

7. Return to the Biomek FXP Target Prep Express and clear the deck (Figure 54 on page 106).



• Clean the Microseal® P Pad by wiping with 70% EtOH and dry.


IMPORTANT! Immediately load the reagent trays and the hybridization tray onto the GeneTitan MC Instrument. Then load the array plate and hybridization tray. Do not leave denatured samples or reagent trays at room temperature for any length of time.



Summary of Preparation for GeneTitan™ MC Instrument

Stage 4: Preparation for the GeneTitan Instrument

Span-8MC

Stage 4: Preparation for the GeneTitan Instrument Page 1

Move Hyb Lid onto Hyb Rxn Plate

Hyb Lid Hyb Rxn Plate

Fill Scan Tray with Hold Buffer

Hold Buffer Reservoir Hold Buffer Plate

150 μL/well

Continued onGeneTitan Preparation - Page 2


Prepare Stabilize Solution & Plate

Water Stabi lize Soln

[96] 10727 μL [96] 144.96 μL

(RT) (4°C)Stabi lize Solution Reservoir

Stabi lize Solution Plate

105 μL/well

[ LLLL

Stabi lize Diluent

(4°C)

[96] 1208 μL




Span-8MC



Transfer Wash A Buffer to Stain 1 & 2 Reservoirs

Stain 1 Reservoir Stain 2 Reservoir

(RT)

Wash A Buffer

[96] 22233.6 μL [96] 11596.8 μL

Continued fromGeneTitan Preparation - Page 1


Transfer Stain Buffer to Stain 1 & 2 Reservoirs


(4oC)

Stain Buffer

[96] 463.2 μL [96] 241.6 μL

Transfer Stains 1-A & 1-B to Stain 1 ReservoirStain 1-A Stain 1-B

[96] 231.6 μL [96] 231.6 μL

(4°C) (4°C)Stain 1 Reservoir




Span-8MC



Continued from GeneTitanPage 2

Transfer Stains 2-A & 2-B to Stain 2 Reservoir

Stain 2-A Stain 2-B

[96] 120.8 μL [96] 120.8 μL



Incubate Hybridization Plate in PTC200

Hyb Rxn Plate + Lid PTC200

Incubate:

1) 10 mins @ 95°C

2) 3 mins @ 48oC

Prepare Stain 1 Plates

Stain 1Plate 1 Stain 1 Plate 2

Stain 1 Reservoir

105 μL/well 105 μL/well




Span-8MC

Prepare Stain 2 PlateStain 2 Reservoir Stain 2 Plate

105 μL/well




Prepare Ligate Solution & Distribution Plate

Ligate Buffer Ligate Enzyme

[96] 7610.4 μL [96] 181.2 μL


Ligate Plate

105 μL/well

Ligate Soln 1

(4°C) [96] 1510 μL

Ligate Soln 2

[96] 362.4 μL (4°C)

[96] 1208 μL [96] 1208 μL (4°C)(4°C)

Probe Mix 1 Probe Mix 2

L





Span-8MC


Unload Hybridization Plate from PTC200

Hyb Rxn Plate + Lid Position P2

Remove Lid from Hybridization Plate

Position P2 Position P1

Process Hybridization Trayon the GeneTitan Instrument

Transfer Samples to Hybridization Tray

Hyb Rxn Plate Hyb Tray

105 μL/well




4 Target preparation on the BiomekFXP with Windows® 7

This chapter is intended for users running the Axiom 2.0 automated target preparation method using the Biomek FXP Target Prep Express software on Windows 7. If you are currently using Windows XP, see Chapter 3, ʺTarget preparation on the Biomek FXP with Windows® XPʺ on page 22

Whatʹs new for the Axiom™ 2.0 Target Preparation 96-Samples Biomek® FXP Method for Windows® 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

Axiom™ 2.0 Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

Before using the Biomek workstation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Stage 1: DNA Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

Stage 2: Fragmentation and Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

Stage 3: Centrifugation and drying pellets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169

Stage 4: Resuspension, Hybridization Preparation, and QC . . . . . . . . . . . . . . . 171

Stage 5: Preparation for the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . 184

What's new for the Axiom™ 2.0 Target Preparation 96-Samples Biomek® FXP Method for Windows® 7

Several changes have been made to the Axiom 2.0 Target Preparation 96-Samples Method for Windows® 7. Follow the new deck layouts and instructions as displayed in the user interface (while running the method), in this user guide, or in the Axiom 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP (Windows 7) Quick Reference (Pub. No. 703369).

New features • Updated user interface:

– Improved deck setup screen, featuring images of actual labware.

– Clearly designated reservoirs and labware positions.

• Countdown timers have been implemented for all on-deck incubation steps to help users track the assay run.

• New Labware: The Precipitation Plate for the new Windows 7 method uses the Eppendorf deep well plate (Cat. No. 951033481). This plate replaces the ABgene square well storage plate (Thermo Scientific AB-0932) used in the Windows® XP method.


Chapter 4 Target preparation on the Biomek FXP with Windows® 7Axiom™ 2.0 Assay 4

Method changes • Fragmentation method:

– The Fragmentation Master Mix now combines the Axiom 10X Frag Buffer, Axiom Frag Diluent, and Frag Enzyme into one master mix.

– The Isopropanol will now be added to the Precipitation Master Mix in the Precip Plate prior to the transfer of samples.

• Resuspension method:

– DNA pellet resuspension will now be performed off-deck using a microplate shaker.

– There will be 2 prompts guiding the users when to: 1) take the Sample Plate off the deck for off-deck pellet resuspension, and 2) when to return the plate back on the deck to finish the Resuspension method.

New off-deck step The user intervention steps during Resuspension now instructs the user to perform an off-deck DNA pellet resuspension step after the addition of the Axiom Resuspension Buffer. This 10-minute off-deck plate shaking protocol on a microplate shaker resuspends the pelleted DNA into the Axiom Resuspension Buffer.

Others • Updated Reservoir Stickers (Part No. 101135): The new Axiom™ 2.0 Assay method on the Biomek™ FXP Target Prep Express instrument with Windows® 7 requires new stick-on labels for the modular reagent reservoir frames for the different stages of the workflow. These stick-on labels have been updated to reflect the changes made in the Fragmentation method. They are color-coded to match the colors found on the caps of the reagent bottles in the Axiom 2.0 reagent kit.

• Labware Consumables Kit: Consumables kit for on-deck (TRobot) and off-deck (Applied Biosystems or PTC) thermal cycler users are now available to order through Thermo Fisher Scientific. See Table 34 ʺGuidance for consumable kit orderingʺ on page 124 to determine which consumables kits are applicable for your needs.

Axiom™ 2.0 Assay

The Axiom™ 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP (Windows 7) is designed for processing 96 samples at a time. The protocol is performed in 2 parts:

• Part 1: Target Preparation is performed on the Biomek FXP Target Prep Express

• Part 2: Array Processing is performed on the GeneTitan™ Multi-Channel (MC) Instrument

A list of all equipment and resources for the Axiom 2.0 Assay with Automated Target Prep is in the Axiom™ 2.0 Assay Automated Workflow for Windows® 7 Site Preparation Guide, Pub. No. 703368.

This chapter includes instructions for Part 1: Target Preparation. These instructions are presented as follows:

• ʺBefore using the Biomek workstationʺ on page 117

– ʺSeal, vortex and centrifugeʺ on page 117

– ʺBreaking the light curtainʺ on page 118

– ʺPlate centrifugeʺ on page 119

– ʺLabeling GeneTitan hybridization and reagent traysʺ on page 137

– ʺPipette tip usageʺ on page 118


Chapter 4 Target preparation on the Biomek FXP with Windows® 7Axiom™ 2.0 Assay4

– ʺSetting method preferencesʺ on page 119

– ʺEquipment, consumables, labware, and reagentsʺ on page 123

– ʺReagent block templateʺ on page 142

– ʺRelated Biomek FXP Target Prep Express documentationʺ on page 144

• ʺStage 1: DNA Amplificationʺ on page 145

• ʺStage 2: Fragmentation and Purificationʺ on page 157

• ʺStage 3: Centrifugation and drying pelletsʺ on page 169

• ʺStage 4: Resuspension, Hybridization Preparation, and QCʺ on page 171

• ʺStage 5: Preparation for the GeneTitan™ MC Instrumentʺ on page 184



Chapter 4 Target preparation on the Biomek FXP with Windows® 7Before using the Biomek workstation 4

Before using the Biomek workstation

Seal, vortex and centrifuge

Unless otherwise stated in the protocol, follow the guidelines below when instructed to seal, vortex and centrifuge plates or reagent tubes for the Biomek FXP Target Prep Express portion of this assay:

• Seal plates—we recommend using MicroAmp Clear Adhesive Films to seal your plates.

• Blot-dry: Prior to sealing plates, we recommend checking the top of the plate to ensure that there are no droplets. If droplets are present, blot-dry the top of the plate before sealing to ensure a tight seal.

– To remove droplets prior to sealing, overlay a sheet of Kimwipes laboratory tissue across the top of the plate and gently pat down to dry.

– Lift the sheet off the plate and discard. Confirm the top of the plate is dry and seal the plate as usual.

• Vortex:

– Reagents 3 times, 1 second each time at the maximum setting.

– Plates 1 second each corner, and 1 second in the center at the maximum setting.

• Centrifuge: When instructed to centrifuge plates or reagent vials, follow these guidelines unless otherwise instructed (for example, when centrifuging and drying pellets, ʺStage 3: Centrifugation and drying pelletsʺ on page 169).

– Plates:

• Centrifuge at 1,000 rpm for 1 minute at room temperature.

• Do not centrifuge for more than 1 minute.

– Reagent Vials: 3 seconds

IMPORTANT! Always ensure that your plates are tightly sealed. A tight seal will prevent sample loss and cross-well contamination, particularly when plates are being vortexed. NEVER REUSE A SEAL. ALWAYS USE A NEW SEAL.

Figure 55 Vortex plates at the corners and center


Chapter 4 Target preparation on the Biomek FXP with Windows® 7Before using the Biomek workstation4

Breaking the light curtain

For your safety, the Biomek FXP Target Prep Express is designed to immediately halt all movement when the light curtain is broken.

Light curtain broken while running the assayFor your safety, the Biomek FXP Target Prep Express is equipped with a light curtain (Figure 56). The light curtain senses when an object (such as a hand or an arm) enters the space surrounding the deck. When this curtain is broken, all movement on the deck halts until the user either clicks OK to resume the activity that was taking place, or aborts the activity. Incubation timers are not interrupted.

Pipette tip usage

Figure 56 Prompt displayed when the light curtain is broken during the process referred to as Homing All Axes.

The light curtain covers the front of the instrument. The sides are protected by Plexiglas. Both areas are shown as red in this figure. Any penetration of the light curtain from outside the deck halts all movement of the workstation. To resume activity on the deck, click OK. To abort the step or other activity, click Stop on the toolbar.

Table 29 Pipette tip usage for 1 full run of the Axiom™ 2.0 Assay on the Biomek workstation

StepMulti-Channel

P50, Pink96 tips, Cat. No. A21586

Multi-Channel AP96 P250,

Aqua96 tips,

Cat. No. 717253

Span-8 Span P250,

Green96 tips,

Cat. No. 379503

Span-8 Span P1000,

Yellow96 tips,

Cat. No. 987925

96 rxns 96 rxns 96 rxns 96 rxns

DNA Amplification — 96 tips — 33 tips

Fragmentation — 96 tips 8 tips 36 tips

Resuspension and Hybridization Preparation 96 tips 96 tips 17 tips 23 tips

Preparation for the GeneTitan™ MC Instrument• Denature samples• Transfer denatured samples to hyb tray• Prepare GeneTitan™ reagent trays

— 96 tips 26 tips 96 tips

TOTAL 96 tips 384 tips 51 tips 188 tips



Plate centrifuge One plate centrifuge is required for the Axiom™ 2.0 Assay. See the Assay 96-Array Format Automated Workflow for Biomek FXP Site Preparation Guide, Pub. No. 702984, for an appropriate plate centrifuge that can be used with the Axiom Genotyping Solution. When centrifuging and drying pellets as instructed under ̋ Stage 3: Centrifugation and drying pelletsʺ on page 169, the centrifuge must be able to spin down plates at:

• rcf: 3,200 x g (4,000 rpm for the Eppendorf 5810R with the rotor configuration described in the Axiom™ 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP Site Preparation Guide, Pub. No. 702984)

• Temperature: 4°C

In addition, the bottom of the rotor buckets should be soft rubber to ensure that the deep well plates do not crack. Do not centrifuge plates in metal or hard plastic buckets.

Setting method preferences

Typically you will set the method preferences for the Biomek software once. The settings you select will:

• Determine which process controls will be run during stages 2 and 3 (Fragmentation and Resuspension). The process controls include:

– Highly recommended: Preparation of sample dilution plates for OD and gel analysis during Resuspension and Hyb Prep. The dilution plates are taken off-deck. One is used for OD quantitation to evaluate DNA mass; the other is used to check fragment size.

– If desired: Prompt you to manually take samples for DNA quantitation prior to fragmentation.

• Select deck configuration options for the thermal cycler (choose between having an integrated Biometra TRobot 96 thermal cycler or no integrated thermal cycler).

To set the method preferences:


2. Open Project Open Project Axiom 2.0 Target Prep and click OK (Figure 57).

3. Open File Open to display the Open Method window (Figure 58).

Or click the Open Method icon


Figure 57 Opening the Axiom 2.0 Target Prep Project



.

5. In the left pane of the window, select Axiom Target Prep (Figure 59).

The method preferences selection window is displayed on the right.

Figure 58 Opening the Axiom Target Preparation method


Figure 59 Click Startup Dialog to open the Method Preferences Selection window

The selections made in this box, Device Configuration Option, are displayed each time this window appears.

The options in the Custom Run Options box must be selected prior to starting a run.

You are not prompted to select any preferences at start up.



6. Set Method Preferences:

Device Configuration Option— these settings can be changed at the start of each step

• What type of thermal cycler setup do you have?

• TRobot-select this option to perform the denaturation of the Axiom Hyb Ready Plate on the integrated Biometra TRobot 96 thermal cycler

• No integrated thermal cycler - select this option to perform the denaturation of the Axiom Hyb Ready Plate on an off-deck thermal cycler or if your Biomek does not have an integrated thermal cycler. A list of thermal cyclers that have been verified with the assay can be found below. When selecting this option, select the appropriate plate type that should be used for the Hyb Ready Plate.

– Select the Bio-Rad Skirted option when using the HSP-9631 plate for the PTC-200 or the Bio-Rad Tetrad 0240G thermal cycler.

– Select the Bio-Rad Semi-Skirted on Costar Round U-Bottom option for the Applied Biosystems 9700 or the Applied Biosystems 2720 thermal cycler. The Bio-Rad HSS-9601 plate must be stacked onto the Costar Round Bottom plate from Corning (VWR International Cat. No. 29442-392, E&K Scientific: EK 680568, Corning Mfg. Cat. No. 3795) for the Biomek FXP Target Prep Express to prepare the Hyb Ready Plate.

We have verified the performance of this assay using the Biometra TRobot 96 on the Beckman Biomek Target Prep Express liquid handler. We have also verified the performance of this assay using the following off-deck thermal cyclers, with 96-well blocks:

• Bio-Rad PTC-200G

• Applied Biosystems 9700 with a gold, silver or aluminum block

• Applied Biosystems 2720

• Bio-Rad / MJ Tetrad® 2 PTC-0240G

The performance of this assay has not been verified with other thermal cyclers.

Use of other thermal cyclers may result in assay failure and may violate the Axiom Array and Reagent replacement policy.

The thermocycler needs to be programmed with the “Axiom Denature” protocol:

a. 95°C 10 minutes

b. 48°C 3 minutes

c. 48°C hold

Use the heated lid option when setting up or running the protocol.

WARNING! Evaporation during denaturation can negatively impact assay performance. Use the recommended thermal cycler consumables and sealing film to eliminate condensation and evaporation. The arched, auto-sealing metal plate with P pads as shown in Table 35 on page 124 should be replaced as per the manufacturer’s recommendation. If using the TRobot, complete the lid setting per the manufacturerʹs instructions.



Custom Run Options— These settings are displayed in this window only. You must select/deselect here.

• QC check points

• Prompt for manual DNA quantitation before fragmentation—the workstation will pause following inactivation of the DNA amplification reaction to allow you to manually remove an aliquot of each sample for off-line (manual) DNA quantitation. This extra quality control step is available for troubleshooting the DNA amplification reaction.

• Prepare plates for gel QC and OD after hyb rxn transfer— the workstation will prepare 2 plates of resuspended samples properly diluted for the fragmentation gel QC and OD quantitation process control checks. See Appendix B, ̋ Fragmentation quality control gel protocolʺ on page 292 and Appendix C, ʺSample quantitation after resuspensionʺ on page 295 for instructions and result assessment guidelines.

• Run method in test mode—select this option to skip all of the incubation timers in a step. If selected, a prompt is displayed asking you to confirm that you want to run a step in test mode (Figure 60). Use this option to perform runs using deionized water only, not actual reagents or samples.

7. Click Start at the top of the left pane to close the default settings window.

IMPORTANT! For troubleshooting and support purposes, we strongly recommend that you perform the gel QC and OD quantitation process controls after resuspension.

CAUTION! Never process samples in test mode. The assay will fail; all of your samples and reagents will be lost.

Figure 60 Prompt displayed when the custom run option, Run method in test mode, is selected.

IMPORTANT! The method preferences (Figure 59 on page 120) should be chosen prior to starting the method. These settings are not prompted for at runtime.


Chapter 4 Target preparation on the Biomek FXP with Windows® 7Equipment, consumables, labware, and reagents 4

Equipment, consumables, labware, and reagents

Thermo Fisher Scientific now offers labware consumables kits for target preparation:

• Axiom 96 Consumable Kit for Biomek FXP (Cat. No. 902800)

• Axiom 96 Consumable Kit for QC (Cat. No. 902801)

• Axiom 96 Consumable Kit for Applied Biosystems TC (Cat. No. 902803)

• Axiom 96 TC Plate Sealing Kit (Cat. No. 902802)

Note: Beckman plates, reservoirs, and tips are not included in the kits prepared by Thermo Fisher Scientific. You must order all Beckman supplies directly from Beckman.

Table 30 Axiom 96 Consumable Kit for Biomek FXP (Cat. No. 902800)

Labware item Part No. Quantity

Bio-Rad Hard-Shell 96-well Plate

203015 40

Eppendorf Deep Well Plate 203079 4

Table 31 Axiom 96 Consumable Kit for QC (Cat. No. 902801)


OD Plate, UV 202609 40

Table 32 Axiom 96 Consumable Kit for off-deck (Applied Biosystems) TC (Cat. No. 902803)


Bio-Rad Hard-Shell 96-well semi-skirted PCR Plate 203055 5

Costar Clear Polystyrene 96-Well Plates 202165 5

Table 33 Axiom 96 TC Plate Sealing Kit (Cat. No. 902802)


Bio-Rad Metal Lid 202519 1

Bio-Rad Microseal P Pad 202958 1


Chapter 4 Target preparation on the Biomek FXP with Windows® 7Equipment, consumables, labware, and reagents4

Labware and materials used on the Biomek workstation deck

Table 34 Guidance for consumable kit ordering

Consumable kit

Thermal cycler option

On-deck,

TRobot

Off-deck PTC-200

Off-deck Applied

Biosystems

Axiom™ 96 Consumable Kit for Biomek FXP (Cat. No. 902800)

Axiom™ 96 Consumable Kit for QC (Cat. No. 902801)

Axiom™ 96 Consumable Kit for Off-Deck (Applied Biosystems) TC (Cat. No. 902803)

Axiom™ 96 TC Plate Sealing Kit (Cat. No. 902802)

Table 35 Labware and materials used on the Biomek workstation deck

Labware Supplier and Cat. No. Image

Biomek AP96 – P250 Pipette Tips (aqua box; pre-sterile, barrier)


Biomek Span P250 Pipette Tips (green box; pre-sterile, barrier)




Biomek Span P1000 Pipette Tips (yellow box; pre-sterile, barrier, conductive)


Biomek Span P50 Pipette Tips (pink box; pre-sterile, barrier)


Bio-Rad Hard-Shell 96-well Plate(available in multiple colors)

Part of the Axiom™ 96 Consumable Kit for Biomek FXP Cat. No. 902800

Plate Part No. 203015

Alternate Vendor:Bio-RadCat. No. HSP-9631

Eppendorf 96 Deep-well Plate, 2,000 μL(available in multiple colors)

Part of the Axiom™ 96 Consumable Kit for Biomek FXP Cat. No. 902800


Alternate Vendor:Eppendorf951033481

Table 35 Labware and materials used on the Biomek workstation deck (Continued)




OD Plate, UV Part of the Axiom™ 96 Consumable Kit for QCCat. No. 902801


Alternate Vendor:E & K ScientificEK-25801

Lid, metal(arched, auto-sealing with P pads)

Part of the Axiom™ 96 TC Plate Sealing Kit Cat. No. 902802

Lid Part No. 202519P Pad Part No. 202958

Alternate Vendor:Bio-RadCat. No. MSL-2032 andP Pad Cat. No. MSP-1003

Hard-Shell Full-Height 96-Well Semi-Skirted PCR Plate • This consumable is required only if

using off-deck Applied Biosystems 2720 or Applied Biosystems 9700 Thermal cyclers

Part of the Axiom™ 96 Consumable Kit for Off-deck (Applied Biosystems) TCCat. No. 902803


Alternate Vendor:Bio-RadCat. No. HSS-9601



Front view

Side view



Plate, Costar Brand Serocluster round Bottom Plate from Corning• Note: this consumable is required

only if using an off-deck Applied Biosystems 9700 or Applied Biosystems 2720 thermal cycle

Part of the Axiom™ 96 Consumable Kit for Off-deck (Applied Biosystems) TCCat. No. 902803


Alternate Vendors:VWR International Cat. No. 29442-392E&K Scientific Cat. No. EK 680568Corning Mfg Cat. No. 3795

Beckman Deep Well Titer Plate(polypropylene)


Frame for reservoirs Beckman CoulterCat. No. 372795



Frame



Half reservoirHalf module, 75 mL capacity


Quarter Reservoirs• Quarter module, 40 mL capacity• Quarter module divided by width,

19 mL capacity each receptacle

Beckman Coulter• Cat. No. 372790 (40 mL)• Cat. No. 372792 (19 mL)

Reagent block, chilled to 4°C Beckman CoulterCat. No. A83054

24-Position Tube Rackwith one 11 mm tube insert in position A6.

Beckman CoulterCat. No. 373661 (rack)Cat. No. 373696 (insert)



Divided by width19 mL capacity

Undivided40 mL capacity

Metal posts on block circled in red.A1

Tube inserA6



Adaptor, Deep Well Plate (installed on the Shaking Peltier)

This adaptor is typically installed by a Beckman Coulter field service technician during new system installation or a system upgrade. Ensure that you have one of these adaptors on the deck prior to running this assay.


The Applied Biosystems 9700 and the Applied Biosystems 2720 use the semi-skirted 96-well plates (Cat. No. HSS-9601). For use on the Biomek deck, the semi-skirted PCR plate must be stacked on a Costar brand Serocluster 96-well Round Bottom Microtitration plate as shown in the figure to the right.

Reagent Block Template(designed specifically for use with the Axiom 2.0 Reagent Kit)

Contact Thermo Fisher Scientific

Zerostat Anti-static Gunand Ion-Indicator Cap

Milty Zerostat,

Thermo Fisher Scientific Cat. No. 74-0014



The metal block is the adaptor.

Template on reagent block. Metal posts on block circled in red.



GeneTitan MC Instrument ConsumablesAll consumables for the GeneTitan MC Instrument are provided by Thermo Fisher Scientific. The following table provides guidance on the consumables that are shipped with the array plate.

IMPORTANT! All covers must have barcodes. Discard any cover without a barcode.

Table 36 Axiom™ GeneTitan™ MC Instrument consumables (Axiom 96-array plate)

Item Part number Labware image Information

Axiom 96-array plate, various designs

Varies, depending on array design.

Axiom 96-Array plate:• Comprised of 3 parts:

clear plastic cover, array plate, and blue array plate protective base.

• The clear plastic cover for the array plate protects the array plate during transport. Discard after opening pouch.

• The array plate must always be kept in the blue array plate protective base at all times.

• The blue array plate protective base in the package holds the array and protects it from damage or exposure to dust.

Note: Array plate is not included in the Axiom GeneTitan Consumables Kit.


1

2

3

1

2

3



Table 37 Axiom™ GeneTitan™ MC Instrument Consumables (from the Axiom™ Array GeneTitan™ Consumables Kit, Cat. No. 901606)


Scan Tray 900746 Box501006 Pouch

96-Plate Scan Tray:• Comprised of 3

parts: scan tray, black protective base, and a scan tray cover.

• The black scan tray protective base in the package protects the glass bottom of the scan tray from damage before it is loaded into the GeneTitan MC Instrument.

• The scan tray cover protects the contents in the scan tray and must be deionized before used. See Appendix E, "Deionization procedure for GeneTitan™ trays and covers" on page 307.

• Remove the black scan tray protective base before loading the scan tray with the scan tray cover into the GeneTitan MC Instrument.

• The Scan Tray must be loaded into the GeneTitan Instrument with the Scan Tray Cover only.

• Do not load the Scan Tray with the protective base still on.



Black Scan Tray Protective Base, shown without the Scan Tray with cover

• The black scan tray protective base in the package is used to protect the bottom of the scan tray glass from damage. The black scan tray is distinct from the blue array plate protective base and must not be used with the array plate.

• Remove and set aside the protective base from the scan tray before loading.

Scan Tray with cover, shown without the black protective base

• The GeneTitan scan tray must be loaded with the scan tray cover into the GeneTitan MC Instrument.

• Do not load the scan tray with the protective base.





GeneTitan 5 Stain Trays Kit

4249910 Kit501025 Tray

• The GeneTitan Stain Tray Kit comes with 5 stain trays packaged in zip-top bags to keep them free of dust.

• The GeneTitan stain trays are barcoded and the trays have separator walls that are flush with the frame of the stain tray, as shown by the yellow line and the yellow oval in the lower photo.

GeneTitan™ Stain and Scan Tray Cover

202757 • The GeneTitan stain and scan tray covers prevent evaporation of the stains in stain trays and the array holding buffer in the scan tray.

• All stain and scan trays must be placed in the GeneTitan MC Instrument with the GeneTitan stain tray cover.

• All tray covers must be deionized to remove static electricity prior to placing the cover on the tray.

• See the section "Deionization procedure for GeneTitan™ trays and covers" on page 307 for the anti-static procedure.






Proper alignment and loading of plates, covers and trays is critical when using the GeneTitan MC Instrument. Each plate, cover and tray has 1 notched corner. The notched corner of plates, trays, covers and bases must be in vertical alignment with each other, and placed in position A1 per the Tray Alignment guide inside each GeneTitan MC Instrument drawer (Figure 61 and Figure 62 on page 136).


Note: The instrument control software will display a warning if it detects a problem during the fluid dispense operations. The filters in the GeneTitan Wash A, Wash B and Rinse bottles should be replaced if the software displays such a warning. See Appendix F, ʺGeneTitan™ Multi-Channel Instrument Careʺ on page 313 for the

GeneTitan stain tray shown with the stain tray cover

Tray 501025Cover 202757

Hybridization Tray

900747 • After aliquoting the denatured hybridization ready samples into the hybridization tray, the tray should be immediately loaded into the GeneTitan MC Instrument with the barcode facing away from the operator, i.e., Barcode should be on the back side.







message displayed to the user and the procedure for replacing the filters.







2

3


1

2

344

5

5

6

7

7

6



Figure 62 Array plate with protective blue base and the hyb tray aligned and properly loaded into drawer 6

Array Plate with Protective Blue Base

Hyb Tray

1

2

1 2

IMPORTANT! When you install the consumables, ensure that the fingers are retracted. Do not lay the consumables on top of the drawer fingers - this indicates that the instrument is not functioning correctly. Notify your field service engineer if the fingers do not retract automatically. You should place the trays into the instrument drawers when a drawer is fully extended by the instrument. The fingers are retracted when the drawer is open and are extended when the drawer is closed in order to restrain the consumable. See Figure 63 for an image showing the location of the tabs.


2

1



1

2





Proper labeling for hyb trays and reagent trays is described in:

• ʺLabeling for hyb traysʺ, below


Labeling for hyb trays

You may label the hyb tray on the front part of the short side of the tray, next to the notch at the left, as shown in Figure 65. The proper section for labeling is closest to the notched corner, corresponding to the A1 and B1 wells.




IMPORTANT! It is critical that you write only on the proper locations of the proper sides of hyb and stain trays. Do NOT write in any other location, as this can interfere with sensors inside the GeneTitan MC Instrument and result in experiment failure. To ensure proper placement of lids onto stain trays, and trays onto the GeneTitan MC Instrument, you can also mark the notched corner of the trays and lids.



Figure 65 Labeling GeneTitan hyb trays

CAUTION! Writing on the wrong side of the hyb tray may interfere with the operation of sensors in the GeneTitan MC Instrument.


Notched corner of the hyb tray should face the front.

Label the hyb tray in this area.

1

2

3

1 2 3





IMPORTANT! Do not confuse hyb trays with stain trays.





1 2 3

1

2

3



Loading tray consumables onto the GeneTitan™ MC Instrument

Loading, or installing, the trays and plates onto the GeneTitan MC Instrument is a simple procedure, but there are certain precautions that you must take in order to ensure an error-free procedure. The following figures show you how to do it. See Figure 67 through Figure 70.

Figure 67 Scan tray loaded in drawer 2

IMPORTANT! When you load the plates, or trays, insert them under the tabs, or fingers, that may protrude into the stage. Confirm that the tray is not resting on these fingers. See Figure 108 on page 211.




Figure 68 Stain 1 tray and Ligation tray loaded in drawer 3

Figure 69 Stain 2 tray and Stabilization tray loaded in drawer 4



Reagent block template

The Axiom™ 2.0 Reagent Kit template fits precisely onto the top of the chilled reagent block, and is held in place by the metal posts on the block (Figure 71). Using this template will help ensure the proper placement of reagent tubes onto the block for each method.

Reservoir stickers

The reservoir labels are stick-on labels for the modular reagent reservoir frames for the different stages of the automated workflow. These stick-on labels are color-coded to match the colors found on the caps of the reagent tubes in the Axiom 2.0 Reagent Kit. Using these labels helps ensure the proper placement of reservoirs and reagents for each method.

Figure 70 Stain 1 tray loaded in drawer 5

Figure 71 Axiom 2.0 Reagent Template for the reagent block

CAUTION! The reservoir stickers for Windows® 7 (Part No. 101135) are different than the XP stickers. Ensure you are using the correct version.



There are 4 reservoir holders used in the Axiom 2.0 method. Three of these will have templates on 2 sides and the remaining reservoir holder will have a template on 1 side for a total of 7 templates.

Remove the protective surface from the back of the label and place on the reservoir frames as directed in the Figure 72 through Figure 75.

Reservoir frame 1

Reservoir frame 2

Figure 72 Reservoir frame 1 for Windows® 7 users


Side A

Side B

Side A

Side B



Reservoir frame 3

Reservoir frame 4

Related Biomek FXP Target Prep Express documentation

The following user manuals are installed at the same time as the Biomek FXP Target Prep Express software (Start All Programs Beckman Coulter Manuals). See these for troubleshooting the Biomek workstation.

• Biomek® Liquid Handler User’s Manual, Beckman Coulter Pub. No. 987834

• Biomek® Software User’s Manual, Beckman Coulter Pub. No. B30026AA



Side A

Side B

Side A


Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 1: DNA Amplification 4


Note: For this protocol, the term samples includes the positive control.

Duration

Note: A 22–24 hours incubation is required at the end of this stage.

Equipment, consumables, labware and reagents required



Table 38 Time required for "Stage 1: DNA Amplification"

Activity Time



Incubation 23 hours

Total time ~24 hours

Table 39 Equipment and labware required

Quantity Item






1 Oven (must maintain a constant temperature of 37°C for at least 23 hours with a temperature accuracy of 1°C)• >3 array plates per week—we recommend using the BINDER ED 56• 3 array plates per week—OK to use the GeneChip Hybridization Oven

or the BINDER ED 56

1 Plate centrifuge

1 Vortex


1 box of each Barrier pipette tips:• Biomek Span P1000 (yellow)• Biomek AP96, P250 (aqua)



Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 1: DNA Amplification4

Reagents required

2 Plate, deep well titer


3 Reservoir, quarter module (40 mL)

2 Reservoir, half module (75 mL)

Table 40 Reagents required for DNA Amplification

Axiom 2.0 Reagent Kit Module

Axiom 2.0 Denat Soln 10X

Module 1, –20°C

Axiom 2.0 Neutral Soln

Axiom 2.0 Amp Soln

Axiom 2.0 Amp Enzyme

Axiom Water

IMPORTANT! The Axiom 2.0 Assay is compatible with only reagents from an Axiom reagent kit. These reagents are not interchangeable with reagents from other Applied Biosystems reagent kits, such as CytoScan, SNP 6.0, DMET Plus, etc.

Table 39 Equipment and labware required (Continued)

Quantity Item




Check the water and waste containers

To check the system water and waste containers:

1. If necessary, fill the system water container using deionized water (no need for ultra-pure water).

2. If necessary, empty the system waste bottle.

Turn on the Biomek FXP Target Prep Express

To turn on the workstation:

1. Power on the workstation.

2. Ensure that all of the peripherals are powered on.

• Two Inheco Single Tec controllers

Each 1 controls the Static Peltier (Pelt_1) or the Shaking Peltier (SPelt_96); no additional power supply.

• Thermal cycler:

Biometra TRobot 96 if present on the Biomek workstation deck. Otherwise, a stand-alone thermal cycler can be used.

3. Launch the Biomek Software by double-clicking the Biomek Software icon on the desktop .

You can also open Start All Programs Beckman Coulter Biomek Software.

Home All AxesThis procedure will home all axes and prime the fluidics lines.

To Home All Axes:

1. Open Instrument Home All Axes.

2. Ensure all conditions in the Warning prompt Figure 76-A are met, then click OK.

An icon instructing you to Stop and wait while the instrument homes is displayed (Figure 76-B).

Figure 76 Homing All Axes

A B



3. When:

a. The Warning prompt in Figure 77-A is displayed, confirm that no tips are loaded in the Span-8 Pod, and click OK.

The lines for the Span-8 tips are primed and the next prompt shown in Figure 77-B is displayed.

b. When the intake (syringes and tubing) for the Span-8 tips is clear of bubbles, click OK.

2. Thaw and prepare the reagents and Sample Plate

Thaw and prepare the reagents and Sample Plate


1. Thaw the Sample Plate (containing 100 ng or 5 ng/µL of gDNA for human, 150 ng or 7.5 ng/µL of gDNA for diploid plants and animals, or 200 ng or 10 ng/µL of gDNA for polypoid plants and animals) on the benchtop at room temperature and centrifuge.

Note: Do not place a frozen Sample Plate directly on the workstation deck.


• Axiom 2.0 Denat Soln 10X

• Axiom 2.0 Neutral Soln

• Axiom 2.0 Amp Soln

• Axiom Water

Leave the Axiom 2.0 Amp Enzyme in the freezer until ready to use.

Note: Allow ~1 hour for Axiom 2.0 Amp Soln to thaw on the benchtop at room temperature. If the solution is not completely thawed after 1 hour, vortex briefly and return to the benchtop to complete thawing. The bottles can also be thawed in a dish with Millipore water. The Axiom 2.0 Amp Soln must be thoroughly mixed before use.

3. Vortex all reagents (except Axiom 2.0 Amp Enzyme), then place at room temperature.

• Vortex the Axiom 2.0 Amp Soln and Axiom 2.0 Neutral Soln for 30 seconds to thoroughly mix.

• Vortex and centrifuge the Axiom 2.0 Denat Soln 10X before placing on the deck.

• For the Axiom 2.0 Amp Enzyme, just before placing on the deck gently flick the tube 3 times to mix and centrifuge.

4. Preheat the Oven to 37°C.

We recommend using one of these ovens:

• BINDER ED 56

• GeneChip Hybridization Oven 645 (turn rotation on to 15 rpm)

Figure 77 Prompts displayed for priming the Span-8 pod fluidic lines

A B



3. Run the DNA Amplification step

To run the DNA Amplification step:


2. Open File Open to display the Open Method window.

Or click the Open Method icon .

3. Select Axiom 2.0 Target Prep and click OK

4. At the top of the main window (Figure 78-A), click the Run button to open the Axiom™ Target Prep window.

5. In the Axiom Target Preparation window (Figure 78-B):

a. Select DNA Amplification.

b. Click OK.

The Deck Layout for DNA Amplification is displayed (Figure 79 on page 151).


1

1







• Figure 81 on page 153—reagent block

Figure 78 Opening the Axiom Target Preparation methods window

A B

IMPORTANT! Axiom 2.0 Amp Enzyme—Immediately prior to placing on the reagent block, gently flick the tube with your finger 2 to 3 times to mix; then centrifuge. Do NOT vortex.



Figure 79 Deck layout window for the DNA Amplification method




Table 41 Labware and Reagent Locations on the Deck for the DNA Amplification Method

Position on Deck

Labware Reagent or Samples


P1 Beckman Deep Well Titer Plate gDNA samples

P4 Bio-Rad Hard-Shell 96-well plate (any color) —


Pour Axiom 2.0 Neutral Solution into Reservoir 2

P11 Reservoirs in frame:• Half module (1)• Half module (2)

• Pour Axiom Water into reservoir 1• Pour Axiom 2.0 Amp Soln into reservoir 2

Pelt_1 Reagent block, chilled to 4°C See Figure 81.


P15 Bio-Rad Hard-Shell 96-well plate (any color)

SPelt96_1 Beckman Deep Well Titer Plate —

1Empty

Reservoir

2Axiom

2.0 Neutral

Soln

3Empty

Reservoir

1

Axiom Water

2

Axiom 2.0 Amp Soln



7. Check the deck layout to ensure that all labware and reagents are in the proper locations.


8. Click OK.

The system flushes the Span-8 fluidics system. Observe the lines and syringes for air bubbles.

Figure 81 Placement of reagents on chilled reagent block for the DNA Amplification step.

Important:

• Position all plates and the chilled reagent block with A1 in the upper left corner of the frame.• centrifuge all reagent tubes prior to placing in block to avoid loss of solution volume to the cap and sides of the tube.• Press reagent tubes into the block to ensure that they are fully seated.

Axiom 2.0Amp

Enzyme

Axiom 2.0 Denat Soln

10X



Flick and centrifuge the Amp Enzyme before placing in block.


A1

A1



9. At the prompt to repeat the Span-8 fluidics system flush (Figure 82):



The DNA Amplification step runs until the Amplification Master Mix has been added to the Sample Plate. Once complete, the instructions and prompt shown in Figure 83 are displayed.

• Follow the instructions for the Sample Plate (see also ʺUser interventionʺ below).

• Follow the instructions for clearing the workstation deck.

User intervention

1. Remove the Sample Plate.

2. Blot the top of the plate with a Kimwipes laboratory tissue to remove any droplets that may be present.

3. Tightly seal the plate.

4. Vortex and centrifuge the plate.

5. Place in a preheated oven and incubate at 37°C for 23 1 hour.


6. After 22–24 hours of incubation, do one of the following:

• Proceed directly to ʺStage 2: Fragmentation and Purificationʺ on page 157

• Tightly seal and store the amplified samples at –20°C.

Figure 82 Flushing the Span-8 fluidics system to purge air bubbles

Figure 83 Instructions for clearing the workstation deck

IMPORTANT! • Seal the Sample Plate before placing in the oven.





Summary of DNA Amplification

Incubate on Deck10 mins @ RT


Stage 1: DNA Amplification Page 1

Transfer Denat Soln 1X to Sample Plate

Prepare Denat Soln 1X & Distribution Plate

Span-8

Denat Soln 10X Axiom Water

488 μL 4392 μL

(4°C) (RT)D1 Reservoir

D1 Plate

30 μL/well

D1 Plate Sample Plate

20 μL/well

MC

Continued on AmplificationPage 2

Fill Neutral Soln Plate

N1 Reservoir N1 Plate

140 μL/well




Span-8MC

Continued from AmplificationPage 1

Prepare Amplification Master Mix & Distribution Plate

Amp Soln Amp Enzyme

26374 μL 586 μL


Amp Mastermix Plate

255 μL/well

Stage 1: DNA Amplification Page 2


Transfer Neutral Soln to Sample Plate

N1 Plate Sample Plate

130 μL/well

Incubate Sample PlateOffline for 23 (±1) hr. @ 37°C

Transfer Amplification Master Mix to Sample Plate

Amp Mastermix

PlateSample Plate

230 μL/well


Final Volume = 400 mL


Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 2: Fragmentation and Purification 4

Stage 2: Fragmentation and Purification

Note: Purification is done by precipitation (See Step ʺ4. Precipitationʺ on page 165).

Duration



Table 42 Time required for "Stage 2: Fragmentation and Purification"

Activity Time

Hands-on time ~25 minutes~50 minutes if frozen DNA from Step 1

Biomek FXP Target Prep Express– Deactivation incubation—20 minutes to

deactivate the amplification reaction and 20 minutes to equilibrate to the fragmentation temperature

– Fragmentation incubation—30 minutes

1 hour 20 minutes

Total time 1 hour 45 minutes to 2 hours 20 minutes

Table 43 Equipment, consumables and labware required

Quantity Item



1 Freezer, –20°C






1 Plate centrifuge

1 Vortex (for plates and microtubes)


1 box of each Barrier pipette tips:• Biomek AP96, P250 (aqua)• Biomek Span P250 (green)• Biomek Span P1000 (yellow)



Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 2: Fragmentation and Purification4

Reagents required








1 Eppendorf 96 Deep-well Plate, 2,000 μL


3 Reservoir, quarter module

1 Reservoir, quarter module, divided by half

1 Reservoir, half module

Table 44 Reagents required for the Fragmentation Method

Reagent Module


Axiom Frag Enzyme (leave at –20°C until ready to use)Module 2-1, –20°C

Axiom 10X Frag Buffer

Axiom Precip Soln 2

Axiom Frag DiluentModule 2-2, 2–8°C

Axiom Frag Rxn Stop

Axiom Precip Soln 1

User-supplied

Isopropanol, 99.5% 96 samples: 65 mL

Table 43 Equipment, consumables and labware required (Continued)

Quantity Item



2. Thaw and prepare the amplified DNA samples and reagents

Thaw and prepare the Amplified DNA Sample Plate

If the plate of amplified samples is frozen:













• Axiom 10X Frag Buffer

• Axiom Precip Soln 2

2. Vortex all reagents (except Axiom Frag Enzyme), then place on ice.

• Vortex and centrifuge Axiom Precip Soln 2 before placing onto deck.

• For the Axiom Frag Enzyme: Leave at –20°C until ready to use. Just before placing on the deck gently flick the tube 3 times to mix and centrifuge.

3. Run the Fragmentation step

To open the Target Preparation methods window:



3. Click the Run button.

4. Select Fragmentation, then click OK (Figure 84).



The deck layout for Fragmentation is displayed (Figure 85).






Figure 84 Selecting the Fragmentation method

IMPORTANT! Remove the seal from the Sample Plate before placing on the deck.



Figure 85 Deck layout for the Fragmentation method




Table 45 Labware and reagent locations on the deck for the Fragmentation step

Position on deck





P7 Beckman Deep Well Titer Plate Amplified gDNA

P8 Eppendorf 96 Deep-well Plate, 2,000 μL —

P10 Reservoirs in frame:• Quarter module (1)• Quarter module (2)• Quarter module, divided (3 and 4)

• Pour Axiom Frag Rxn Stop into reservoir 1• Pour Axiom 10X Frag Buffer into reservoir 2

P11 Reservoirs in frame:• Half module (1)• Quarter module (2)

• Pour Isopropanol into reservoir 1• Pour Axiom Precip Soln 1 into reservoir 2


P13 Biomek Span P250 Pipette Tips (green)


3Empty

4Empty

1AxiomFragRxnStop

2Axiom10X Frag

Buffer

1

Isopropanol

2AxiomPrecipSol 1





7. Click OK to continue.





Figure 87 Placement of reagents on chilled reagent block for the Fragmentation step

IMPORTANT:

• Always position the chilled reagent block with A1 in the upper left corner of the frame.• centrifuge all reagent tubes prior to placing in block to avoid loss of solution volume to the cap and sides of the tube.• Press reagent tubes into the block to ensure that they are fully seated.

Axiom Precip Soln 2

Axiom Frag

Diluent

Axiom Frag

Enzyme




Axiom Frag Enzyme:

Flick to mix 3 times and centrifuge immediately prior to placing on the chilled reagent block.

A1

A1



The Fragmentation step begins. The Sample Plate is incubated at 65°C to inactivate amplification.

9. If you selected Prompt for manual DNA quantitation in the default software settings, you will then be prompted to remove an aliquot of each sample for a optional quantitation process control (Figure 84). The plate will remain at 36.5°C until the aliquots have been collected. Remove 4 µL aliquots from each well into a 96 well PCR plate and set aside for later quantitation (e.g., using the Quant-iT™ PicoGreen® dsDNA Kit from Life Technologies). Immediately continue sample processing by placing the amplification reactions back onto the shaking peltier and clicking OK at the prompt shown in Figure 88.

If Prompt for manual DNA quantitation was not selected, the box in Figure 88 will not appear.

Note: Remain near the Biomek FXP Target Prep Express if you are going to remove aliquots for quantitation. Avoid leaving the samples at 36.5°C for a long period of time.

10. Once the Fragmentation Complete message box appears (Figure 89), follow the instructions in the message box discarding used labware and tips, storing unused tips and storing the cold block at 4°C. Click OK when ready to continue and proceed to Step ʺ4. Precipitationʺ on page 165.

Figure 88 Prompt for manual DNA quantitation

Figure 89 Fragmentation complete message



4. Precipitation To precipitate the DNA samples:

1. Remove the Precipitation Plate from the deck.

2. Blot the top of the plate with a Kimwipes laboratory tissue and seal tightly.

3. Place the plate in a –20°C freezer overnight to precipitate.

4. Return to the Biomek workstation and clear the deck.



Summary of Fragmentation


Span-8MC


Inactivate DNA Amplification Reaction

Sample Plate

Incubate:

1) 20 min @ 65.0°C2) 20 min @ 36.5°C

SPelt



Prepare Fragmentation Master Mix & Distribution Plate

Frag Di luent Frag Enzyme

1551.2 μL 150.6 μL

(4°C) (4°C)Frag MM Reservoir

Frag MM Distr ibution Plate

67 μL/well

FragB Reservoir

(RT)

6882.4 μL



Prepare Precipitation Soln & Distribution Plate

Span-8MC


Incubate on Deck

30 mins @ 36.5°C

Transfer Fragmentation Master Mix to Sample Plate

Sample Plate

57 μL/well

Fill Stop Solution Plate

Stop Reservoir Stop Plate

29 μL/well

Precip Soln 2 Precip Soln 1

222 μL

(4°C)

Precip Plate

240 μL/well





FragMM Distribution Plate

Transfer Isopropanol to Precipitation Plate

Iso Reservoir Precip Plate

600 μL/well




Span-8MC



Transfer Stop Solution to Sample Plate

Stop Plate Sample Plate

19 μL/well

Offline PrecipitationSee user guide

Transfer Sample to Precip Plate

Sample Plate Precip Plate

Plate Contents


Final Volume = 1316 μL


Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 3: Centrifugation and drying pellets 4

Stage 3: Centrifugation and drying pellets

Duration


To centrifuge and dry the pellets:

1. Turn the oven on and preheat to 37°C.

Use an oven that can sustain a constant temperature of 37°C and has a temperature accuracy of 1°C (we recommend the BINDER ED 56). If processing 3 or fewer array plates, you can use a GeneChip Hybridization Oven.

2. Transfer the Precipitation Plate from the –20°C freezer to a pre-chilled centrifuge. Centrifuge the plate for 40 minutes at 4°C at 3,200 x g (4,000 rpm for the Eppendorf 5810R centrifuge with the rotor configuration described in the Axiom™ 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP Site Preparation Guide, Pub. No. 702984).

Table 46 Time required for Centrifugation and drying pellets

Activity Time


Centrifugation 40 minutes

Drying 25 minutes

Total time 75 minutes

Table 47 Equipment and consumables required

Quantity Item



1 Plate centrifuge, precooled to 4°C


Sample Plate One plate of precipitated samples from Stage 2 in an Eppendorf 96 Deep-well Plate

CAUTION! During this step, handle the plate gently to avoid disturbing the pellets. Do not bump or bang the plate.

WARNING! Use rotor buckets with a soft rubber bottom to ensure that the deep well plates do not crack. Do not use buckets where the plates sit directly on a metal or hard plastic bottom, such as the A-4-62 rotor with a WO-15 plate carrier (hard bottom) for the Eppendorf 5810R centrifuge. Use of hard bottom plate carriers may result in cracked plates, loss of sample, unbalanced centrifugation, damage to the instrument and possible physical injury.


Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 3: Centrifugation and drying pellets4

3. Immediately after the 40 minutes centrifugation period, empty the liquid from the plate as follows:

a. Remove the seal.

b. Invert the plate over a waste container and allow the liquid to drain.

c. While still inverted, gently press the plate on a pile of Kimwipes laboratory tissues on a bench and leave it for 5 minutes.

4. Turn the plate right side up and place in an oven for 20 minutes at 37°C to dry.



• Proceed directly to ʺStage 4: Resuspension, Hybridization Preparation, and QCʺ on page 171, even if some droplets of liquid remain. Leave the Sample Plate at room temperature. It is helpful to begin preparing reagents for Stage 4 while centrifuging and drying pellets.

• Store the plates for resuspension later in the same day:

• Tightly seal the plates.

• If resuspension will be carried within 4 hours, keep the plates at room temperature.

• If resuspension will be carried out in more than 4 hours, store the plates in a refrigerator (2–8°C).

• Store the plates for resuspension on another day:

• Tightly seal the plate and store at –20°C.


Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 4: Resuspension, Hybridization Preparation, and QC 4

Stage 4: Resuspension, Hybridization Preparation, and QC

Note: In this stage, resuspension buffer is delivered to the plate of pelleted DNA by the Biomek FXP Target Prep Express. The samples are then resuspended by shaking off deck. The user must pay careful attention to pop-up prompts that inform the user when to remove the plate from the deck to perform an off-deck pellet resuspension step, and when the user must replace the plate with resuspended DNA back on the deck to complete the method.

Duration Resuspension and hybridization preparation



Table 48 Time required for Resuspension

Activity Time


Frozen pellet equilibration to room temperature 1.5 hours


Table 49 Equipment, consumables and labware required

Quantity Item






1 Plate centrifuge, at Room Temp

1 Vortex


1 box of each Barrier pipette tips:• Biomek Span P50 (pink)• Biomek AP96, P250 (aqua)• Biomek Span P250 (green)• Biomek Span P1000 (yellow)


Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 4: Resuspension, Hybridization Preparation, and QC4

Reagents required

5 platesOR:

Bio-Rad Hard-Shell PCR 96-well (HSP-9631)For on-deck thermal cycling or when using the PTC-0240/PTC-200 off-deck thermal cycler

6 plates for off deck

cycling with Applied

Biosystems thermal cyclers

4 Bio-Rad (HSP-9631) Hard-Shell PCR 96-well Plate and 1 HSS-9601 Hard-Shell Full-Height 96-Well Semi- Skirted PCR Plate1 Plate, Costar Brand Serocluster round bottom plate

For off-deck thermal cycling using the Applied Biosystems 9700 or Applied Biosystems 2720 thermal cycler– Note: The HSS-9601 plate stacked on the Costar brand serocluster

round-bottom plate should only be used on position P2 on the Biomek FXP deck. See Figure 97 on page 187.

1 Plate, OD


1 Reservoir, 19 mL (quarter module, divided)

3 Reservoir, 40 mL (quarter module)

Table 50 Reagents required for Resuspension and Hybridization

Reagent Module


Axiom Hyb Buffer Module 2-1, –20°C

Axiom Hyb Soln 1

Axiom Resusp Buffer Module 2-2, 2–8°C

Axiom Hyb Soln 2

Other reagents required

Nuclease-Free Water, ultrapure MB grade(Thermo Fisher Scientific, Cat. No. 71786; for OD and gel plate preparation)

To fill line of divided reservoir

TrackIt Gel Loading Buffer, diluted 1,000-fold(see Appendix B, "Fragmentation quality control gel protocol" on page 292 for dilution instructions.)

13 mL

Table 49 Equipment, consumables and labware required (Continued)

Quantity Item



1. Preparing frozen pellets and Axiom Resusp Buffer

The equilibration of resuspension buffer and pellets to room temperature (18°C to 25°C) is very critical for the success of the Axiom 2.0 assay. When either is cooler than room temperature, pellets may not resuspend completely. This may result in compromised assay performance. Note following guidelines on how to work with plates with fresh, cold or frozen pellets:

Pellets:

• Plates with fresh pellets can be kept at room temperature for up to 1 hour if proceeding with the resuspension and hybridization protocol within an hour.

• Plates with fresh pellets that are not processed within an hour can be transferred to a refrigerator (2-8°C) for few hours only if processed within a day. However, it is critical to equilibrate the plate to room temperature for at least 30 minutes before proceeding with the resuspension and hybridization protocol.

• Plates with frozen pellets (e.g., on Day-5 of 8-plate workflow) must be pre-equilibrated at room temperature for at least 1.5 hour before proceeding with the resuspension and hybridization protocol.

Resuspension Buffer:

• Resuspension buffer, when pulled out from Module 2-2 at 2-8°C, needs at least 1 hour to equilibrate to room temperature.






3. Launch the Biomek software.


3. Thaw and prepare the reagents



1. Thaw Axiom Hyb Soln 1 and warm Axiom Resusp Buffer on the benchtop at room temperature.

2. Vortex the Axiom Resusp Buffer and the Axiom Hyb Buffer.

3. Vortex and centrifuge Axiom Hyb Soln 1 and Axiom Hyb Soln 2.

4. Run the Resuspension, Hybridization Preparation, and QC step

Note: We strongly recommend that you run 2 quality process controls during this step:

• A gel to verify successful fragmentation

• An OD quantitation of each resuspended sample

The Biomek FXP Target Prep Express can be set to prepare fragmentation and OD plates that are ready for processing. These process controls must be selected as a run preference prior to starting a run. See ʺSetting method preferencesʺ on page 119 for instructions.



To run the Resuspension, Hybridization Preparation, and QC step:



3. Click the Run button.The Axiom 2.0 Method selection dialog box appears.

4. Select Resuspension, Hybridization Preparation, and QC and click OK.The deck layout for this method is displayed (Figure 90 on page 175).






6. Label the Bio-Rad plates placed on the deck in positions P2, P9 and P11. For example:

• P2—Hyb Ready + <sample description>

• P9—Dil QC

• P11—Gel QC

Note: Verify the appropriate plastic consumables are being used on the deck for the Hyb Reaction Plate when using the Applied Biosystems 9700 or Applied Biosystems 2720 thermal cycle.

IMPORTANT! The pellets and the resuspension buffer must be at room temperature before proceeding with this step.



Figure 90 Deck layout for the Resuspension, Hybridization Preparation, and QC

If Prepare plates for gel QC and OD after hyb rxn transfer is selected in method preferences, the following labware is required on the deck:

• Multichannel P50 pipette tips

• Dilution QC plate• Gel QC plate• OD QC plate (do NOT touch

bottom of plate)If not selected, these labware are not needed and will not appear in the deck layout.

See "Setting method preferences" on page 119 for more information.




Table 51 Labware and reagent locations on the deck for Resuspension, Hybridization Preparation, and QC

Positionon deck


TL1 Biomek AP96 – P250 pipette tips (aqua) —

P11 Biomek Span P50 Pipette Tip (pink) —

P2 Hyb Reaction Plate:• On-deck TRobot 96 or off-deck PTC-200 or PTC 0240 users:

Bio-Rad Hard-Shell 96-well plate (HSP-9631) or

• Off-deck Applied Biosystems 9700 or 2720 users: Bio-Rad Hard-Shell Full-Height 96-Well Semi-Skirted PCR Plate (HSS-9601) stacked on a Costar Round Bottom plate

WARNING: When using the Applied Biosystems 9700 or the Applied Biosystems 2720 off-deck thermal cycler for denaturing the Hyb Ready Plate, the Bio-Rad HSS-9601 Hard-Shell Full-Height 96-Well Semi-Skirted PCR Plate must be stacked on a CoStar Brand Serocluster Round Bottom Plate (Axiom 96 Consumables kit for off-deck Applied Biosystems users (Part No. 902803)) as shown in Table 35 on page 124.The HSS-9601 and HSP-9631 PCR plates are not interchangeable on the Biomek FXP deck.

P3 Eppendorf 96 Deep-well Plate, 2,000 μL Pelleted samples



P91 Bio-Rad Hard-Shell 96-well plate Dilution QC Plate

P10 Reservoirs in frame:• Quarter module (1)• Quarter module (2)• Quarter module (3)• Quarter module, divided (4 and 5)

• Pour Axiom Resus Buffer into reservoir 1• Pour Axiom Hyb Buffer into reservoir 2• Leave reservoir 3 empty• Pour Gel Diluent (diluted gel loading buffer) into reservoir 4:

13 mL• Pour nuclease-free water into reservoir 5 to fill line

1Axiom

ResuspBuffer

2AxiomHyb

Buffer

3Empty

Reservoir

4Gel

Diluent

5Water



P111 Bio-Rad Hard-Shell 96-well plate (for Gel QC) Gel QC Plate

P121 OD plate, 96-well UV OD QC Plate


P13 Biomek Span P250 pipette tips (green) —

P14 Biomek Span P1000 pipette tips (yellow) —

1 If QC is not selected, leave deck positions P1, P9, P11, and P12 empty.

Table 51 Labware and reagent locations on the deck for Resuspension, Hybridization Preparation, and QC

Positionon deck


Figure 92 Placement of reagents on chilled reagent block for Resuspension, Hybridization Preparation, and QC

IMPORTANT:


Axiom Hyb

Soln 2

Axiom Hyb

Soln 1




A1

A1





8. Click OK to continue the method.





10. There are 2 pop-up prompts that inform the user: 1) when to remove the plate from the deck to perform an off-deck pellet resuspension step, and 2) when the user must return the plate with resuspended DNA back on the deck to complete the method.

Note: Seal the resuspension plate tightly before placing it on the off-deck microplate shaker.

a. The first prompt appears following the addition of the Resuspension Buffer to the DNA pellets (see Figure 93).

• Remove the Sample Plate from Position 3. This plate contains the DNA pellets in Axiom Resuspension Buffer.

b. Follow the instructions below to carry out the DNA pellet resuspension by off-deck shaking. Upon completion of the on-deck method that aliquots the Axiom Resuspension Buffer to the Sample Plate containing the pelleted samples, resuspension is carried out by shaking off-deck using the following steps:

• Blot the top of the Sample Plate using a Kimwipes laboratory tissue to remove any droplets that may be present.

• Seal the plate tightly; blue pellets should be visible at the bottom of the wells.

• Place the Sample Plate onto one of the following shakers for 10 minutes:

– Thermo Scientific Compact Digital Microplate Shaker: set at 900 rpm

– Jitterbug: set at speed of 7

– Inspect the plate from the bottom. If the pellets are not dissolved, repeat the shaking step.

– Centrifuge the plate in a room temperature centrifuge.

c. Ensure that the instructions in the pop-up message box were followed.

d. When ready, click OK to proceed with the method.

The method continues by preparing the Hybridization Master Mix and, if selected, the Sample QC Plates.

Figure 93 Instructions to perform the off-deck DNA pellet resuspension.



e. A second prompt appears which guides the user to return the Sample Plate back on the deck to prepare the final Hyb Reaction Plate.

f. Centrifuge the Precipitation Plate with resupended DNA samples to get all the droplets down, minimizing sample loss.

g. Unseal the Precipitation Plate before placing it back on the deck at Position 3.

h. Ensure that the instructions in the pop-up message box were followed.

i. When ready, click OK to proceed with the method.

The method continues by preparing the Hyb Reaction Plate (the final hybridization ready samples) and, if selected, the Sample QC Plates.

11. Run the fragmentation gel and OD quantitation process controls.

For instructions and guidelines on assessing gel and OD results, see:

• Appendix B, ʺFragmentation quality control gel protocolʺ on page 292

• Appendix C, ʺSample quantitation after resuspensionʺ on page 295


• If the GeneTitan MC Instrument is free, and if the gel and OD quantitation results are good:

• Discard used labware and reagents

• Discard used tips

• Store unused Span-8 tips

• Click OK in the Resuspension and Hyb Prep complete dialog box and proceed to ʺStage 5: Preparation for the GeneTitan™ MC Instrumentʺ on page 184.

• If the GeneTitan MC Instrument is not free, then follow the instructions shown in Figure 94:

• Tightly seal the Hyb Rxn Plate and store at –20°C.

• Discard used labware and reagents

• Discard used tips

• Store unused Span-8 tips

• Store cold block at 4°C.

• Click OK when complete.

IMPORTANT! Only proceed with the method once the DNA pellets have been fully resuspended into Axiom Resuspension Buffer using an off-deck microplate shaker.

Figure 94 Instructions to follow if the GeneTitan MC Instrument is not free.



Summary of Resuspension and Hybridization Preparation


Span-8MC


Prepare Resuspension Buffer Plate

ResB Reservoir ResB Plate

45 μL/well

Transfer Resuspension Buffer to Precipitation Plate

ResB Plate Precip Plate

35 μL/well

Prepare Hybridization Master Mix & Distribution Plate

Hyb Soln 2 Hyb Soln 1

1197 μL 66.5 μL

(4°C) (4°C)MM Reservoir

Hyb MM Plate

90 μL/well

Hyb Buffer

(RT)

9376.5 μL

Continued on ResuspensionPage 2


Follow the user interface instructions to perform the

Off-line Pellet Resuspension(See user guide for details)

You must click OKK to continue with method.

A second message will appear later when it is time to place the Sample Plate with resuspended DNA back

on deck.





Span-8MC


Continued from ResuspensionPage 1

Transfer Hybridization Master Mix to Precipitation Plate

Hyb MM Plate Precip Plate

80 μL/well

See QC flowchart if performing analysis after Resuspension

Transfer Hybridization Mix to Hyb Reaction Plate

Precip Plate Hyb Rxn Plate

115 μL/well

Store HybridizationReaction Plate



Follow user interface instructionsto return the sample plate back on the deck

after completing the Off-line Pellet Resuspension(See user guide for details)

Click OKK to continue with method



Stage 4: Resuspension & Hybridization Prep QC

Span-8MC

Stage 4: Resuspension & Hybridization Prep QC, Page 1

Fill Dilution QC Plate with Water

Water Reservoir Dilution QC Plate

55 μL/well

Fill OD QC Plate with Water

Water Reservoir OD QC Plate

90 μL/well

Fill Gel QC Plate with Dye

Dye Reservoir Gel QC Plate

120 μL/well

Continued on QCPage 2

Transfer Hybridization Mix to Dilution Plate

Hyb Rxn Plate Dilution QC Plate

5 μL/well



Wait for the message prompt on whento return the sample plate back on the deck

after completing the Off-line Pellet Resuspension(See user guide for details)

Follow the instructions on the pop-up message and Click OKK to continue with method



Perform Offline Analysis

Stage 4: Resuspension & Hybridization Prep QC

Span-8MC

Stage 4: Resuspension & Hybridization Prep QC, Page 2

Transfer Diluted Sample to OD QC Plate

Dilution QC Plate OD QC Plate

10 μL/well

Continued from QCPage 1

Transfer Diluted Sample to Gel QC Plate

Dilution QC Plate Gel QC Plate

3 μL/well





Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument4

Stage 5: Preparation for the GeneTitan™ MC Instrument

About Stage 5 You will proceed to Stage 5 in 1 of 2 ways:

• Directly from Stage 4 without interruption.

• With samples that were stored at –20°C after Stage 4.

This stage, Preparation for GeneTitan, can include any combination of the options shown in Figure 95. The first 2 options complete target preparation on the Biomek FXP Target Prep Express.

The options are:

Option 1

• Denature samples - the Hyb Rxn plate is placed on the thermal cycler and the samples are denatured. At this step, you must also select the “Transfer denatured samples to hyb tray” option. After the denature program has completed, the block will hold temperature until the user has dismissed the prompt, indicating that they are ready to continue the method to transfer the samples to the hyb tray and then carry it to the GeneTitan MC Instrument. Do not leave samples on the thermal cycler for a long period of time.

Option 2

• Transfer denatured samples to hyb tray - the denatured samples are transferred from the Hyb Rxn plate to the hyb tray, and are ready to load onto the GeneTitan MC Instrument.

Note: When using an Applied Biosystems 9700 or the Applied Biosystems 2720 thermal cycler for off-deck denaturation, the Bio-Rad Hard-Shell Full-Height 96-Well Semi-Skirted PCR Plate (Hyb Reaction Plate) must be stacked on a Costar brand Serocluster Round Bottom plate from Corning (Corning Mfg Cat. No. 3795)


You can run one step, or a combination of steps.


Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument 4

Option 3

• Prepare GeneTitan™ reagent trays - the solutions required for the fluidics stage of array processing on the GeneTitan MC Instrument are prepared and aliquoted to the appropriate trays (3 stain trays, 1 ligation tray, 1 stabilization tray and 1 scan tray with Holding Buffer).

When performing a 1 plate workflow, select 2 options (option 1 and option 2).

• Denature samples and Transfer denatured samples to hyb tray when you are preparing the samples to begin hybridization in the GeneTitan MC Instrument.

- or select one option (option 3)

• Prepare GeneTitan™ reagent trays to prepare reagents for loading onto the GeneTitan MC Instrument the following day when the plate is finished with the hybridization period and about to begin GeneTitan fluidics processing.

Options for performing a multi-plate workflow

When performing a multi-plate workflow (see Chapter 6, ʺProcessing 8 Axiom™ array plates per weekʺ on page 246 for a description of the 8 plate workflow), the need to carry out preparation of reagents for 1 plate while simultaneously hybridizing a second plate will arise. In this case, all 3 options in the Biomek FXP method can be carried out concurrently. Select all 3 options when using an on-deck thermal cycler.

• Denature samples and Transfer denatured samples to hyb tray will prepare samples for the new plate going into the GeneTitan MC Instrument to begin hybridization.

-and-

• Prepare GeneTitan™ reagent trays will prepare reagents for the plate that is already in the GeneTitan MC Instrument hybridization oven and is ready to go into the Wash, Ligation, Stain, and Scan phases of GeneTitan array processing.

Note: In the 8 plate workflow, all 3 options will only be selected for middle days of the workflow. On the first day of the workflow, only the Denature samples and Transfer denatured samples to hyb tray options will be used. On the last day of the workflow, only the Prepare GeneTitan™ reagent trays option will be used.

Note: When using an off-deck thermal cycler, avoid letting the samples sit at room temperature for an extended period of time after denaturation. Do not begin denaturation at the same time as the GeneTitan reagent preparation.

Off-deck thermal cycler option

The steps for performing a simultaneous preparation of GeneTitan reagent trays and hybridization of a second plate required in the course of a multi-plate workflow are modified slightly when the off-deck thermal cycler option is selected. The reagent trays to be loaded into the GeneTitan MC instrument are prepared in an initial run of the method.

Denaturation of the hybridization ready samples in the off-deck thermal cycler begins mid way through the GeneTitan reagent prep on the Biomek deck.

After loading the reagent trays into the GeneTitan MC instrument, you must perform a second run of the Biomek method to transfer the denatured samples to the hyb tray for loading into the GeneTitan MC instrument. You will need a timer for this modified step.



The modified steps are:

1. Select Preparation for GeneTitan step with only the Prepare GeneTitan™ reagent trays box checked, click OK.

2. Prepare deck as shown in Figure 96.

3. Click OK.

4. Start timer. Wait 25–30 minutes, then begin denaturation of the hybridization ready samples using the off-deck thermal cycler.

5. Upon completion of the GeneTitan reagent tray preparation, load reagent trays and scan tray into the GeneTitan MC instrument

6. Once the reagents are loaded into the GeneTitan MC instrument and the denaturation method on the thermal cycler is complete, retrieve the denatured hybridization ready samples from the thermal cycler

7. Return to Biomek FXP and begin a new method, select Preparation for GeneTitan step with only the Transfer denatured samples to hyb tray box checked, click OK.

8. Prepare deck as shown in Figure 97 (note that a spacer must be used with HSS-9601 plates for Applied Biosystems thermal cyclers).

Figure 96 Deck layout for “Prepare GeneTitan™ Reagent Trays” only (selected for Off-deck Thermal Cycler option)



9. Click OK.

10. After the denatured samples have been transferred to the GeneTitan hyb tray, load hyb tray into the GeneTitan MC Instrument.

Figure 97 Deck layout for “Transfer Denatured Samples to Hyb Tray”: Off-deck Thermal Cycler option and position P2

-

Bio-Rad Hard-Shell Full-Height 96-Well Semi-Skirted

PCR Plate (HSS-9601) stacked on a Costar Round

Bottom plate for off-deck thermal cycling with the

Applied Biosystems 9700 or 2720 thermal cycler

The Hyb Reaction Plate in deck position P2 must be

one of the following:

Bio-Rad Hard-Shell 96-well plate (HSP-9631) for

on-deck or off-deck thermal cycling using the

TRobot 96, PTC-200 or PTC 0240

OR

--



If a plate is already in the hybridization ovenFor the plate that is already in the GeneTitan hybridization oven and that is ready to go into Ligation, Wash-Stain and Scan, select option 3, Prepare GeneTitan™ reagent trays. The solutions required for the fluidics stage of array processing on the GeneTitan MC Instrument are prepared and aliquoted to the appropriate trays (3 stain trays, 1 ligation tray, 1 stabilization tray and 1 scan tray with Holding Buffer).

Duration of GeneTitan™ MC Instrument reagent preparation and sample preparation

Sample denaturation

Denatured sample transfer to hyb tray

IMPORTANT! The reagent trays prepared in the third sub-step, Prepare GeneTitan™ reagent trays:

• Are NOT for use with the hyb tray currently being prepared on the Biomek workstation.

• Are for the continued processing of an Axiom array plate that is:

– already on the GeneTitan MC Instrument.

– has completed the hybridization stage.

– is ready for transfer to the fluidics area.

The reagent trays for the fluidics stage on the GeneTitan MC Instrument CANNOT be prepared in advance. Do not prepare these plates if there is no array plate ready for the fluidics stage. Once prepared, these plates must be loaded onto the instrument as soon as possible and cannot be stored.

Table 52 Time required to denature samples

Activity Time

Hands-on time: ~3 minutes



Table 53 Time required to transfer denatured samples to the hyb tray

Activity Time






Preparation of GeneTitan Reagent Trays

Note: When you select Denaturation and Preparation of the GeneTitan Reagent Trays, the on-deck processes run concurrently.


Sample Denaturation

Transfer to hyb tray

Table 54 Time Required to Prepare Reagent Trays for the GeneTitan MC Instrument

Activity Time

Prepare reagents (thaw and organize reagents)

~30 minutes




Table 55 Equipment required for denaturing sample

If denaturing samples: Item Quantity

• On the Biomek workstation Lid, arched metal 1

Table 56 Consumables required for transferring denatured samples to a hyb tray

Item Quantity

Item required from the GeneTitan™ Consumable Kit, Cat. No. 901606:• hyb tray 1


If using off deck Applied Biosystems 9700 or Applied Biosystems 2720 thermal cyclers: Costar brand Serocluster Round Bottom plate from Corning Mfg. Cat. No. 3795) to be used for stacking under the HSS-9601 semi-skirted plate (Hyb Reaction Plate).

1



Prepare GeneTitan™ reagent trays

Note: See Table 36 on page 130 for GeneTitan MC Instrument Consumable catalog numbers.








Table 57 Consumables and other equipment required

Item Quantity

Items required from the GeneTitan™ Consumable Kit, Cat. No. 901606: • Scan tray with cover and protective base• Stain trays• Covers for stain trays

155


Pipette tips, Biomek Span P1000 (yellow) 1 full box

Pipette tips, Biomek Span P250 (green) 1 full box

Reagent block, chilled to 4°C 1

Reservoirs, quarter module divided 3

Reservoirs, quarter module 3

Tube rack, 24 position with insert (room temperature rack) 1

Zerostat Anti-Static Gun 1



2. Prepare the reagents for GeneTitan Reagent Tray Preparation

Note: Ligation Buffer and Ligation Solution 2 require approximately 30 to 40 minutes to thaw on the benchtop at room temperature.

Reagents required

Preparing Axiom Wash A and Axiom Stabilize DiluentDuring storage of the Axiom Wash A and Axiom Stabilize Diluent (in Module 4-2 stored at 4°C), precipitation in the form of clear crystals can sometimes occur. Therefore, follow the procedure below to ensure that any precipitate is returned to solution prior to use.

Note: The presence of some precipitate is okay and will not adversely impact assay performance. Follow the instructions below to resuspend any precipitate before use.

Table 58 Reagents required for GeneTitan MC Instrument Reagent Tray Preparation

Module Reagent Thaw on benchtop, then place on ice

Place on ice Place on benchtop at room temperature

Module 3Room

Temperature

Axiom Wash Buffer A

Axiom Wash Buffer B

Axiom Water

Module 4-1–20°C

Axiom Ligate Buffer - for 30 minutes

Axiom Ligate Enzyme Keep at –20°C until ready to use

Axiom Ligate Soln 1

Axiom Probe Mix 1

Axiom Stain Buffer

Axiom Stabilize Soln

Module 4-22 to 8°C

Axiom Ligate Soln 2 - for 30 to 40 minutes

Axiom Probe Mix 21

Axiom Wash A - for 30 minutes

Axiom Stain 1-A1

Axiom Stain 1-B1

Axiom Stain 2-A1

Axiom Stain 2-B1


Axiom Water

Axiom Hold Buffer1

1 These solutions are light sensitive. Keep tubes out of direct light for a prolonged period of time.



To prepare the Axiom Wash A:

1. Vortex the bottle for 30 seconds.

2. Place on the benchtop at room temperature for 30 minutes.

3. Examine the reagent for precipitate (look into the top of the bottle).

4. If precipitate is still present, vortex again for 30 seconds.

5. Pour Axiom Wash A into the appropriate reagent reservoir and leave on the benchtop until ready to use.

To prepare the Stabilize Diluent:

If crystals are observed in the Axiom Stabilize Diluent:

1. Vortex and centrifuge.

2. Look for precipitate. If any, warm tube to room temperature and vortex again.

Preparing Axiom Ligate BufferWhite precipitate is sometimes observed when the Axiom Ligate Buffer is thawed.

Note: The presence of some precipitate is okay and will not adversely impact assay performance. Follow the instructions below to attempt to resuspend a majority of precipitate before use.

To prepare the Axiom Ligate Buffer:

1. Place on the benchtop at room temperature for 30 minutes. This bottle can also be thawed in a dish with room temperature Millipore water.

2. Examine the buffer for precipitate (look into the top of the bottle).

3. If precipitate is present, vortex the bottle for 30 seconds.

4. Re-examine the buffer for precipitate.

5. If precipitate is still present, warm the bottle with your hands and vortex again for 30 seconds.

6. If precipitate is still present after hand warming proceed with the protocol below.

7. Pour the Axiom Ligate Buffer into the appropriate reagent reservoir and leave on the benchtop until ready to use.

Prepare the remaining reagents

To prepare the remaining reagents for GeneTitan MC Instrument Plate Preparation:

1. Leave the Axiom Ligate Enzyme at –20°C until ready to use.

2. Thaw the following reagents from Module 4-1 at –20°C on the benchtop at room temperature, then vortex, centrifuge, and place on ice:

• Axiom Ligate Soln 1

• Axiom Probe Mix 1

• Axiom Stabilize Soln

• Axiom Stain Buffer

3. Prepare the remaining reagents from Module 4-2 as follows:

a. Gently flick each tube 2 to 3 times to mix, then centrifuge.

b. Place reagents on ice, except for the Axiom Hold Buffer, Axiom Ligate Soln 2 and Axiom Water— leave these reagents on the benchtop at room temperature until ready to use.



3. Prepare the Sample Plate (if stored at –20°C) and the array plate

To prepare samples that were stored at –20°C:

1. Warm the Hyb Ready Plate at room temperature for 5 minutes. It is not necessary to equilibrate the plate for longer duration.

2. Ensure that the Hyb Ready Plate is sealed well. If the plate is not sealed well:

a. centrifuge the plate and carefully remove the old seal.

b. If there is condensation on the top of the plate, blot dry gently with a Kimwipes laboratory tissue.

c. Use a fresh seal and tightly reseal the plate.

3. Vortex the Hyb Ready Plate briefly, then centrifuge to 1,000 rpm.

4. Place the Hyb Ready Plate at room temperature.

To prepare the array plate:

1. Warm up the array plate on the benchtop before setting up hybridization on the GeneTitan MC Instrument.

a. Leave the array plate in the pouch at room temperature, for a minimum of 25 minutes, before opening and loading on the GeneTitan MC Instrument to allow the plate to come to room temperature.

b. At the end of the array warm up time, open the pouch and scan the array plate barcode into the Batch Registration file (see ̋ Stage 1: Create and upload Batch Registration fileʺ on page 219).

4. Prepare the GeneTitan™ MC Instrument

Before your samples are being denatured and/or the Biomek FXP Target Prep Express is preparing reagent trays, ensure that the GeneTitan MC Instrument is ready for use.

One or more of the following steps may need to be performed:

1. Launch GCC and select GCC GeneTitan Control.

2. Upload your sample registration file now, if you have not done so (see ʺStage 1: Create and upload Batch Registration fileʺ on page 219).

If you do not upload your samples before scanning the array plate barcode, the software will assign names to your samples.

3. On the GCC GeneTitan Instrument Control, select the System Setup tab (Figure 98).


a. Setup Option: Hyb-Wash-Scan


b. Click Next.

c. Plate Information:

• Barcode: Scan or manually enter the Axiom array plate barcode and click Next.


5. Fill the Wash A, Wash B and Rinse bottles.




6. Empty the Waste bottle.

• After preparing the GeneTitan MC Instrument, perform steps 6a, 6b or 6c as appropriate for your workflow:

– Go to Step 6a on page 201: if you are denaturing the samples and transferring them to the hyb tray.

– Go to Step 6b on page 202: if you are preparing reagents for the Ligation-Wash-Stain.

– Go To Step 6c on page 202: if you are performing a 2-8 plate workflow, and you are denaturing samples for the 2nd plate and are preparing ligation reagents for the 1st plate. The samples for the 2nd Axiom array plate will be denatured and transferred into the Hyb tray. The Hyb tray will be placed in the GeneTitan MC Instrument with the array plate in preparation for hybridization. The Biomek FXP Target Prep Express will also prepare reagents for Ligation-Wash-Stain for the 1st array plate that was placed in the GeneTitan MC Instrument hybridization oven 24 hours ago.

Figure 98 Setup options for processing array plates.

System Setup tab

Select Hyb-Wash-Scan option



5. Run the Preparation for GeneTitan™ step

To run the Preparation for GeneTitan step:



3. Click the Run button.The Axiom 2.0 Target Prep run options window appears (Figure 99).

4. Select Preparation for GeneTitan™ and the sub-steps that you wish to run; then click OK.

Based upon your choices, the appropriate deck layout is displayed (Figure 100).

5. Deionize the GeneTitan stain trays. See Appendix E, section entitled ʺDeionization procedure for GeneTitan™ trays and coversʺ on page 307 for the deionization procedure.






• Figure 103 on page 200—tube block

Note: Prior to placing the arched metal lid on the deck, be sure to clean the attached Microseal® P pad with 70% ethanol and dry it. See the package insert for this product for further information on cleaning and replacement.


You can run each step individually, or any combination of steps. If you do not select Prepare GeneTitan™ reagent trays, the deck layout will be different than the one shown in Figure 100.

• Select Denature Samples and Transfer denatured samples to hyb tray– if you are preparing to load the array plate into the GeneTitan

Instrument for Hybridization.• Select Prepare GeneTitan™ Reagent Trays

– if you are preparing to load the GeneTitan Instrument for Wash-Stain and Imaging.

• Select Denature Samples and Transfer denatured samples to hyb tray and Prepare GeneTitan™ Reagent trays

– if you are running an 8 plate workflow then you will need to select all 3 check boxes in some instances in preparation for the plate that will go into the GeneTitan Instrument for Hybridization.

• Select Prepare GeneTitan™ Reagent trays for the array plate that has completed hybridization and is ready to be processed in the GeneTitan Instrument for Ligation-Wash-Stain and Imaging.

IMPORTANT! It is important to deionize the GeneTitan MC Instrument trays to remove any static electricity on the trays. Static attraction by the trays may prevent the tray cover from being lifted up by the instrument.



Figure 100 Deck layout for Preparation for GeneTitan—denature, transfer to hyb tray, and GeneTitan plate preparation

The deck layout displayed is based upon the selections you make for this step.

IMPORTANT: Destatic the stain trays prior to placing them on the deck. See the section "Deionization procedure for GeneTitan™ trays and covers" on page 307.

Included in deck layout if the following options are selected:

• Denature samples• Transfer denatured samples to

Hyb Tray

Included in deck layout if Prepare GeneTitan™ reagent trays is selected.




Table 59 Labware and Reagent Locations on the Deck for GeneTitan Reagent Preparation

Position on deck


If Denature samples and Transfer denatured samples to Hyb Tray are selected, (no reagent tray preparation), only the labware listed below is required:

TL1 Biomek AP96 – P250 pipette tips (aqua) —

P1 Lid, arched metalClean before use (70% ethanol).

—

P2 Hyb Reaction Plate1 Hyb-ready samples

P3 Hyb Tray2 —

If Prepare GeneTitan™ reagent trays is selectd the labware listed below is required:

P4 Stain 12, 3 (second of 2 trays with Stain 1) —

P5 Stain 22, 3 —

P6 Lig2, 3 —

P7 Scan Tray on protective base* —

P8 Stbl2, 3 —

P9 Stain 12, 3 (first of 2 trays with Stain 1) —

P10 Tube block with 1 insert, room temperature See Figure 103 on page 200.

P11 Reservoirs in frame:• Quarter module, divided (1 and 2)• Quarter module, divided (3 and 4)• Quarter module, divided (5 and 6)

• Pour Axiom Water into reservoir 1• Pour Axiom Ligation Buffer into reservoir 5

1Axiom Water

3Empty

5Axiom LigateBuffer

2Empty

4Empty

6Empty





• Pour Axiom Hold Buffer into reservoir 1• Pour Axiom Wash A into reservoir 2• Leave reservoir 3 empty

Pelt_1 Reagent block, chilled to 4°C See Figure 102 on page 199

P13 Biomek Span P250 pipette tips (green) —

P14 Biomek Span P1000 pipette tips (yellow) —

1 For on-deck or off-deck denaturation using the TRobot 96, PTC-200, or PTC 0240, use the Bio-Rad Hard-Shell 96-well plate (Cat. No.HSP-9631). For off-deck denaturation using the Applied Biosystems 9700 or 2720 thermal cycler, use the Bio-Rad Hard-Shell Full-Height96-Well Semi-Skirted PCR Plate (Cat. No. HSS-9601) and place the semi-skirted PCR plate on top of a Costar Round Bottom plate (Cat.No. 3795) for “Transfer Denatured Samples to Hyb Tray”.

2 These trays are included in Axiom Array GeneTitan Consumables Kit (Cat. No. 901606).3 Label each of these stain trays as described above as described in "Labeling GeneTitan hybridization and reagent trays". For example,

label the Stain tray to be placed in P6 with the word “Lig”. This tray will contain the Ligation master mix.

Table 59 Labware and Reagent Locations on the Deck for GeneTitan Reagent Preparation (Continued)

Position on deck


1Axiom Hold

Buffer

2Axiom

Wash A

3Empty

Reservoir

IMPORTANT! It is critical that you write only on the proper location of the hyb tray (on the edge in front of wells A1 and B1) as illustrated in Figure 65 on page 138 or on the proper location of the stain/reagent trays (on the edge in front of wells A1 to C1) as illustrated in Figure 66 on page 139. Do NOT write on any other side, as this can interfere with sensors inside of the GeneTitan MC Instrument and result in experiment failure. To ensure proper placement of lids onto stain trays, and trays onto the GeneTitan MC Instrument, you can also mark the notched corner of the trays and lids.

IMPORTANT! Do not confuse hyb trays with stain trays.



Figure 102 Placement of reagents on chilled reagent block for GeneTitan Reagent Tray Preparation

IMPORTANT:


Axiom Ligate

Enzyme

Axiom Probe Mix 1

Axiom Probe Mix 2

Axiom Ligate Soln 1

Axiom Stain 2-B

Axiom Stain Buffer

Axiom Stabilize

Soln


Axiom Stain 2-A

Axiom Stain 1-B

Axiom Stain 1-A




A1

A1





8. Click OK to continue the step.




Click Yes if air bubbles are present. Repeat the flush until no air bubbles are present.

If the Denature Samples option is selected, the Biomek FXP Target Prep Express places the samples onto the thermal cycler and runs the denaturation program. If Prepare GeneTitan™ reagent trays is also selected, reagent tray preparation for the GeneTitan MC Instrument starts while the samples are being denatured.

Figure 103 Placement of reagents on room temperature tube block

AxiomLigationSoln 2

A1



6a. Complete Stage 4: Preparation for GeneTitan™ - Hybridization Trays

If you selected Denature samples and Transfer denatured samples to Hyb tray in Step 4 (See Step ʺ5. Run the Preparation for GeneTitan™ stepʺ on page 195 and Figure 99 on page 195), use the following instructions:

1. Once the GeneTitan MC Instrument is ready, return to the Biomek FXP Target Prep Express click OK (prompt shown in Figure 104 above).

The denatured samples are then taken off the thermal cycler and are transferred to the hyb tray.

• Ensure that there are no air bubbles present in the hyb tray. Puncture any air bubbles that you see using a pipette tip.

2. Transfer the hyb tray to the GeneTitan MC Instrument and load. See the section ʺArray processing with the GeneTitan™ MC Instrumentʺ on page 209 for the proper way of loading the array plate and the hyb tray.

3. Return to the Biomek FXP Target Prep Express and clear the deck (Figure 105).





Figure 104 Prompt indicating denaturation is complete.

IMPORTANT! Immediately load the array plate and hyb tray into the GeneTitan MC Instrument.

Figure 105 Instructions displayed at the end of Step 4.



6b. Complete Stage 4: Preparation for GeneTitan™ - Reagent Trays

If you selected Prepare GeneTitan™ Reagent trays in Step 4 (See Step ʺ5. Run the Preparation for GeneTitan™ stepʺ on page 195 and Figure 99 on page 195), use the following instructions:

1. Once the prompt shown in Figure 105 appears, remove the reagent trays and scan tray with Hold Buffer from the deck and cover with the appropriate lids.




3. Transfer the reagent trays, scan tray to the GeneTitan MC Instrument and load. See the section ʺStage 3: Ligate, Wash, Stain, and Scanʺ on page 235 to continue the process on the GeneTitan MC instrument.

4. Return to the Biomek FXP Target Prep Express and clear the deck (see Figure 98 on page 194).





6c. Complete Stage 4: Preparation for GeneTitan™ - Multiple Plate Workflow

If you selected all 3 options in Step 4 (See Step ʺ5. Run the Preparation for GeneTitan™ stepʺ on page 195 and Figure 99 on page 195) and are preparing hyb tray and reagent trays in a 2+ plate workflow, use the following instructions:

The Hyb tray is prepared for a new Axiom array plate that will be loaded into the GeneTitan MC Instrument. The reagent trays are prepared for the Axiom array plate that is in the hybridization oven in the GeneTitan MC Instrument and is ready to move to the next stage of the process - Ligation, Wash-Stain and Scan.

Note: When using an off-deck thermal cycler, see the instructions for the ʺOff-deck thermal cycler optionʺ on page 185.

1. Once the reagent trays are prepared and sample denaturation is complete, the prompt in Figure 104 on page 201 is displayed (do not click OK yet). Remove the reagent trays and scan tray with Hold Buffer from the deck and cover with the appropriate lids.




IMPORTANT! Immediately load the reagent trays onto the GeneTitan MC Instrument. Do not leave denatured samples or reagent trays at room temperature for any length of time.



3. Transfer the reagent trays, scan tray and array plate to the GeneTitan MC Instrument and load.

4. Return to the Biomek FXP Target Prep Express and click OK at the prompt shown in Figure 104 on page 201.

The denatured samples are then taken off the thermal cycler and are transferred to the hyb tray.

5. Transfer the hyb tray to the GeneTitan MC Instrument and load. See the section ʺArray processing with the GeneTitan™ MC Instrumentʺ on page 209 for the proper way of loading.

6. Transfer the reagent trays, scan tray to the GeneTitan MC Instrument and load. See the section ʺArray processing with the GeneTitan™ MC Instrumentʺ on page 209 to continue the process on the GeneTitan MC Instrument.

7. Return to the Biomek FXP Target Prep Express and clear the deck (Figure 105 on page 201).





IMPORTANT! Immediately load the reagent trays and the hyb tray onto the GeneTitan MC Instrument. Then load the array plate and hyb tray. Do not leave denatured samples or reagent trays at room temperature for any length of time.



Summary of Preparation for GeneTitan MC™ Instrument


Span-8MC


Move Hyb Lid onto Hyb Rxn Plate

Hyb Lid Hyb Rxn Plate

Fill Scan Tray with Hold Buffer

Hold Buffer Reservoir Hold Buffer Plate

150 μL/well

Continued on GeneTitan Preparation - Page 2


Prepare Stabilize Solution & Plate

Water Stabi lize Soln

10727 μL 144.96 μL

(RT) (4°C)Stabi lize Solution Reservoir

Stabi lize Solution Plate

105 μL/well

Stabi lize Diluent

(4°C)

1208 μL






Span-8MC


Continued on GeneTitan Preparation - Page 3

Transfer Wash A Buffer to Stain 1 & 2 Reservoirs

Stain 1 ReservoirStain 2

Reservoir

(RT)

Wash A Buffer

22233.6 μL 11596.8 μL

Continued from GeneTitan Preparation - Page 1


Transfer Stain Buffer to Stain 1 & 2 Reservoirs


(4°C)

Stain Buffer

463.2 μL 241.6 μL

Transfer Stains 1-A & 1-B to Stain 1 ReservoirStain 1-A Stain 1-B

231.6 μL 231.6 μL





Span-8MC




Transfer Stains 2-A & 2-B to Stain 2 Reservoir

Stain 2-A Stain 2-B

120.8 μL 120.8 μL



Prepare Stain 1 Plates

Stain 1 Plate 1 Stain 1 Plate 2

Stain 1 Reservoir

105 μL/well 105 μL/well





Span-8MC

Prepare Stain 2 PlateStain 2 Reservoir Stain 2 Plate

105 μL/well




Prepare Ligate Solution & Distribution Plate

Ligate Buffer Ligate Enzyme

7610.4 μL 181.2 μL


Ligate Plate

105 μL/well

Ligate Soln 1

(4°C) 1510 μL

Ligate Soln 2

362.4 μL (RT)

1208 μL 1208 μL(4°C)(4°C)

Probe Mix 1 Probe Mix 2




Incubate Hybridization Plate in TRobotHyb Rxn Plate + Lid TRobot

Incubate:

1) 10 min. @ 95°C 2) 3 min. @ 48°C




Span-8MC


Unload Hybridization Plate from TRobotHyb Rxn Plate + Lid

Position P2

Remove Lid from Hybridization Plate

Position P2 Position P1

Process Hybridization Trayon the GeneTitan Instrument

Transfer Samples to Hybridization Tray

Hyb Rxn Plate Hyb Tray

105 μL/well





5 Array processing with theGeneTitan™ MC Instrument

The Axiom™ 2.0 Assay is designed for processing 96 samples at a time on Axiom™ Genome-Wide and Custom myDesign™ array plates. The protocol is performed in 2 sets of steps:

• Target Preparation: Automated target prep, performed with the Biomek FXP Target Prep Express.

• Array Processing: performed on the GeneTitan Multi-Channel (MC) Instrument.

This chapter includes instructions for Part 2: Array Processing.

Before using the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

Stage 1: Create and upload Batch Registration file . . . . . . . . . . . . . . . . . . . . . . . 219

Stage 2: Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220

Status window prompts and actions required . . . . . . . . . . . . . . . . . . . . . . . . . . . 233

Stage 3: Ligate, Wash, Stain, and Scan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

Continuing the workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244

Shutting down the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . 245

Before using the GeneTitan™ MC Instrument

Note: For optimal GeneTitan MC Instrument performance, the maximum relative humidity must be 80% for temperatures up to 75.2°F (24°C) and a minimum humidity of 30 ±7% relative humidity. Operating outside the working environment specifications leads to higher static levels and results in the evaporation of reagents from stain trays.


Proper alignment and loading of plates, covers and trays is critical when using the GeneTitan MC Instrument. Each plate, cover and tray has one notched corner. The notched corner of plates, trays, covers and bases must be in vertical alignment with each other, and placed in position A1 per the Tray Alignment guide inside each GeneTitan MC Instrument drawer (Figure 106 and Figure 107 on page 211).





Chapter 5 Array processing with the GeneTitan™ MC InstrumentBefore using the GeneTitan™ MC Instrument5

Note: The instrument control software will display a warning if it detects a problem during the fluid dispense operations. The filters in the GeneTitan Wash A, Wash B and Rinse bottles should be replaced if the software displays such a warning. See Appendix F, ʺGeneTitan™ Multi-Channel Instrument Careʺ on page 313 for the message displayed to the user and the procedure for replacing the filters.







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Chapter 5 Array processing with the GeneTitan™ MC InstrumentBefore using the GeneTitan™ MC Instrument 5

Figure 107 Array plate with protective blue base and the hybridization tray aligned and properly loaded into drawer 6

Array plate with protective blue base

Hybridization tray

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IMPORTANT! When you install the consumables, ensure that the fingers are retracted. Do not lay the consumables on top of the drawer fingers - this indicates that the instrument is not functioning correctly. Notify your field service engineer if the fingers do not retract automatically. You should place the trays into the instrument drawers when a drawer is fully extended by the instrument. The fingers are retracted when the drawer is open and are extended when the drawer is closed in order to restrain the consumable. See Figure 108 for an image showing the location of the tabs.


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Proper labeling for hybridization trays and reagent trays is described in:

• ʺLabeling for hybridization traysʺ, below





IMPORTANT! It is critical that you write only on the proper locations of the proper sides of hyb and stain trays. Do NOT write in any other location, as this can interfere with sensors inside the GeneTitan MC Instrument and result in experiment failure. To ensure proper placement of lids onto stain trays, and trays onto the GeneTitan MC Instrument, you can also mark the notched corner of the trays and lids.




Labeling for hybridization trays

You may label the hybridization tray on the front part of the short side of the tray, next to the notch at the left, as shown in Figure 110. The proper section for labeling is closest to the notched corner, corresponding to the A1 and B1 wells.

Figure 110 Labeling GeneTitan hybridization trays

CAUTION! Writing on the wrong side of the hybridization tray, or on the wrong part of the long side, may interfere with the operation of sensors in the GeneTitan MC Instrument.


Notched corner of the hybridization tray should face the front.

Label the hybridization tray in this area.

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Email and telephone notifications from the GeneTitan™ MC Instrument

We strongly recommend that you configure the Applied Biosystems™ GeneChip™ Command Console (GCC) software to send you GeneTitan MC Instrument notifications. It is critical that you know when the instrument requires your attention—either for sample handling or troubleshooting. Rapid notification can lessen the risk of sample loss.

Notifications can be sent to email addresses and telephones. See the Applied Biosystems™ GeneChip™ Command Console User Guide for instructions.

The types of notifications available will let you know when a process:

• Starts

• Completes

• Aborts

• Encounters an error





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GeneTitan™ MC Instrument lamp

The GeneTitan MC Instrument uses a xenon arc lamp system that is warranted to provide 500 hours of illumination for imaging the array at 2 wavelengths. The xenon lamp has a limited lifetime and needs to be replaced at regular intervals.

The GeneTitan Instrument Control software provides a timer that indicates the remaining useful life of the bulb and notifies you when it requires replacement. It is important to adhere to the warnings specified in the GeneTitan MC Instrument user guide.

See the GeneTitan MC Instrument User Guide, Pub. No. 08-0308, or Appendix F, ʺGeneTitan™ Multi-Channel Instrument Careʺ on page 315 of this user guide for details on replacing the lamp.

See the GeneTitan MC Instrument User Guide, Pub. No. 08-0308, for the Lambda LS and Smart controller system. The Lamp and the controller should NEVER be switched ON or OFF manually. The GeneTitan MC Instrument control software manages the lamp activity and will switch the lamp ON and OFF as required. It takes 10 minutes to warm-up the lamp. In idle mode the lamp will remain ON for 2 hours before it is automatically switched OFF and if there are no more plates being transferred from the fluidics to the imaging station. This is by design and is intended behavior. Do not try to save the lamp life by turning OFF the switch on the lamp.

Note: The power switch on the shutter box should be ON at all times. The OPEN/CLOSE switch on the shutter box should be at AUTO position at all times.

Setup options for array plate processing

The processes (setup options) available for processing array plates are shown in Figure 112. A brief description of each option is given below.

Figure 112 Setup Options for Processing Array Plates

System Setup tab

Setup options

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Hyb-Wash-ScanThis setup option enables you to hybridize, wash-ligate-stain-stabilize, and scan an array plate on the GeneTitan MC Instrument.

• Hyb: the array plate is moved to the hybridization oven inside the instrument. Each denatured sample in the hybridization tray is hybridized to an array on the array plate.

– Duration for 96 samples = 23.5 hours

• Wash: samples on arrays are ligated, washed, stained and stabilized.

– Duration for 96 samples = ~5 hours


• Scan: The array plate is moved to the imaging device in the GeneTitan MC Instrument and each array is scanned.

– Duration for 96 samples = ~7.5 hours

Hyb-WashIf this setup option is selected, array plate processing will stop after the array has gone through fluidics processing. Use this option if an array plate cannot be scanned on the same GeneTitan MC Instrument as the one used for hybridization and fluidics processing.

If the array plate cannot be scanned immediately after the Hyb-Wash process is complete:

1. Wrap the array plate (in the scan tray with black protective base) in aluminum foil to protect from light.

No lid is required. Do not invert the plate stack. If inverted, the Hold Buffer will spill out of the tray. To prevent liquid spillage, try to keep the plate level when handling the plates. Do not touch the bottom optical surface of the scan tray.

2. Store at 4°C.

3. Scan the array plate within 3 days or less.

When ready to scan the array plate:

1. Keeping the plate protected from light, bring the plate to room temperature for ~20 minutes.

2. Remove the aluminum foil and load onto the GeneTitan MC Instrument.

Wash-ScanUse this option if:

• You wish to bypass the Hybridization step and perform only the Wash/Stain and Scan steps.




Note: It usually takes 25-30 minutes to warm up Wash B if this option is selected.

Wash-Scan-ResumeUse this option if:

• It was necessary to hybridize the array plate in an oven separate from the GeneTitan MC Instrument.

• Fluidics processing has been interrupted (e.g., a power failure occurs at your facility).

ScanUse this option:

• To rescan an entire array plate or specific arrays on a plate that failed to scan for reasons such as bubbles or gridding failure.

• If you have hybridized and performed the fluidics processes on a different GeneTitan MC Instrument than the one you will currently use for the scan, or at a different time.

Unload Plates Use this option to unload plates and trays from the instrument when:

• Array plate processing is complete.

• Array plate processing has been aborted.



Aborting a process If necessary, you can abort the processing of one or more array plates. Instructions and an example are shown below in Figure 113.

If the instrument aborts a process, you can retrieve the array plate and related consumables as described in Figure 113. An instrument-initiated abort may occur:

• Due to improper placement of plates.

• If the uninterrupted power supply (UPS) detects a long power interruption, draining the UPS to 75% power.

Figure 113 Manually aborting an array plate

To abort array plate processing:

1. Click the Stop button.2. Select the array plate that you want to abort.3. Click Abort.4. Click Yes.5. Wait until the status of the array plate in the

WorkFlow window changes from AbortRequest… to Aborted.

6. Once aborted, retrieve the array plate and other related consumables by:• Using Setup Option: Unload Plates• Loading a new array plate.

Exception: If reagents are loading, abort the plate using the Cancel button displayed in the reagent load step.

Note: If the gripper is required to complete the Abort process, the plate will remain in the “AbortRequest” state until the gripper becomes available.

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Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 1: Create and upload Batch Registration file 5

Stage 1: Create and upload Batch Registration file

You must create and upload a Batch Registration file in the Applied Biosystems™ GeneChip™ Command Console software before you begin ̋ Stage 2: Hybridizationʺ on page 220 (example shown in Figure 114). This file contains information critical for:

• Data file generation during scanning

• Tracking the experimental results for each sample loaded onto an array plate

1. If you have not already created a batch registration file, create one now. (See Appendix D, ʺRegistering samples in GeneChip™ Command Console™ʺ on page 304 for detailed instructions.)

2. In GCC, select the array plate format (96 samples) and open a batch registration file template.

3. Scan the array plate barcode into the yellow barcode field.

4. Enter a unique name for each sample and any additional information.

5. Save the file.

6. Upload the file.

IMPORTANT! It is very important to create and upload a batch registration file with your sample information prior to starting ʺStage 2: Hybridizationʺ on page 220.

Figure 114 Example of a Batch Registration file for an array plate


Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 2: Hybridization5

Stage 2: Hybridization

Reagents required Reagents required

An Axiom Genome-Wide Human or non-Human array plate or an Axiom myDesign™ Genotyping 96-Array Plate is required for this step. Prior to inserting this plate into the GeneTitan MC Instrument for hybridization, the array plate should be brought to room temperature as described below:

Warm up the array plate on the benchtop before setting up hybridization on the GeneTitan MC Instrument.

1. Remove the array plate box from the 4°C refrigerator where it is stored.

2. Open the box and remove the pouch containing the array plate and protective base.

3. Leave the array plate in the pouch, unopened but placed on the bench for a minimum of 25 minutes before opening and loading on the GeneTitan MC Instrument to allow the plate to come to room temperature.

4. At the end of the array warm up time, open the pouch and scan the array plate barcode into the Batch Registration file (see ʺStage 1: Create and upload Batch Registration fileʺ on page 219).

A hybridization tray containing denatured samples (from Step 2 on page 106 of Chapter 3) is also required for this step. The samples should be denatured and transferred to the hybridization tray only after the GeneTitan MC Instrument is ready for loading the hybridization tray in the ʺLoad Axiom™ array plate and hybridization tray onto the GeneTitan™ MC Instrumentʺ section on page 225.

Table 60 Reagents required from the Axiom 2.0 Reagent Kit

Reagent Module

Axiom Wash Buffer A(both bottles; 1L) Module 3,

Room TemperaturePart No. 901472

Axiom Wash Buffer B

Axiom Water



Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 2: Hybridization 5

Setup the instrument

To setup the instrument:

1. Launch GCC Launcher and select GCC GeneTitan Control (Figure 115).

The system initializes. After initialization, the System Status tab is selected and the status of the Hybridization Oven is displayed at the bottom of the Log window. The status should read: <Time of day> System Ready


IMPORTANT! Do not close the scanner application by right-clicking on it and choosing the “Close” option. This will cause the scanner application to exit abnormally and cause undue delay in processing the next plate. The correct way to close the application is described in ʺShutting down the GeneTitan™ MC Instrumentʺ on page 245.

Figure 115 Launching GCC and initializing the GeneTitan MC Instrument

System ready



2. Select the System Setup tab (Figure 116).


d. Setup Option: Hyb-Wash-Scan


e. Click Next.

Figure 116 System Setup tab and the information displayed in this pane

Status: This field displays the actions that must be performed to prepare or unload the GeneTitan MC Instrument for the setup option that has been selected.

After each action you are instructed to click the Next button or to press the blinking blue Confirmation button located on the GeneTitan MC Instrument.

System Setup tab

Setup Option: The various options you can choose for processing Axiom array plates.

Workflow Steps: This field displays an overview of the user actions required to process an array plate based on the setup option selected.

Barcode: The array plate barcode. Can be scanned or entered manually.

Protocol Name: The protocol that GeneTitan MC Instrument will run. The list of protocols displayed is based on the first 6 digits of the array plate barcode. Only the protocols that are valid for the type of array plate loaded are displayed.

NOTE: If there is not enough disk space, a message is displayed.

• Delete or move .dat files to another location to free up enough disk space for the data that will be generated by 8 Axiom array plates.

• One 96 Axiom array plate requires ~80 GB



f. Plate Information:

• Barcode: Scan or manually enter the Axiom array plate barcode and click Next.

The first 6 characters of the barcode identify the type of plate being loaded, the protocol GeneTitan MC Instrument will use to process the plate, and the imaging device parameters required for this type of plate.


The system reads the first 6 digits of the array plate barcode to determine which protocols can be run for the type of array plate that has been loaded. Only valid protocols are displayed.

4. Complete the remaining workflow steps as follows:

a. Refill bottles with buffer (Figure 118 on page 224).Fill these bottles:

• Wash A: fill with Axiom Wash Buffer A—keep at 2L full

• Wash B: fill with Axiom Wash Buffer B—Use all 600 mL of Wash B from the reagent kit per Axiom plate. Fill to 1L mark when processing 2 plates on the same day.

• Rinse: fill with Axiom Water—keep at 1L full f

Figure 117 Barcode error message

IMPORTANT! • Always ensure that the GeneTitan bottles containing Wash A and Rinse are above

the 50% mark when setting up the system to process an Axiom array plate. All 600 mL of the Wash buffer B from the Axiom 2.0 reagent Kit should be emptied into the GeneTitan Wash B bottle when setting up the system to process a plate. This ensures that the GeneTitan Wash B bottle is filled to more than the requisite 35% of Wash B bottle volume. Also, do not overfill the bottles. Fill Wash Buffer B and Rinse bottles to the 1L mark only. Wash A keep at 2L. We strongly recommend refilling these bottles every time you are prompted to do so.

If the volume in any of these bottles becomes too low during a run, a message is displayed (see Chapter 8, ʺTroubleshootingʺ on page 281). However, even if you fill the bottle at this time, the instrument may not be able to successfully complete the step that was in progress.

• Wash B—if you intend to load 2 array plates on the same day, fill the Wash B bottle to the 1L mark (use both bottles from the Axiom 2.0 Reagent Kit).

If this error message is displayed:

• Ensure that the library files for the type of array plate you are using are correctly installed.

• Try manually entering the array plate barcode.• Library files must be installed prior to launching the GeneTitan MC Instrument. If a

library file must be installed, exit the GeneTitan MC Instrument, install libraries and relaunch the GeneTitan MC Instrument.



b. Empty the waste bottle.

c. Press the Confirmation button on GeneTitan MC Instrument to continue. A fluidics check is run (~1 minute).

d. Empty trash bin

• Open the trash bin and empty.

If already empty, the trash bin remains locked and the Status pane reads “Trash bin is empty.”

• Press the Confirmation button to continue.

e. Remove consumable trays and plates

• Remove used trays and plates when drawers open.

• If no consumables to remove, the Status window reads “Drawers are empty.”


f. Continue to ʺLoad Axiom™ array plate and hybridization tray onto the GeneTitan™ MC Instrumentʺ on page 225.

Figure 118 Example of the remaining workflow steps

Workflow step

Specific instructions for each workflow step



Load Axiom™ array plate and hybridization tray onto the GeneTitan™ MC Instrument

The System Layout pane indicates the position of the various trays in each drawer during a GeneTitan MC Instrument run at maximum throughput. This pane does not change as plates are loaded or removed (Figure 119).

To load an Axiom array plate and hybridization tray onto GeneTitan MC Instrument:

1. When drawer 6 opens, load the array plate and hybridization tray as follows:

a. Examine the wells of the hybridization tray for bubbles; puncture any bubbles with a pipette tip.

b. Load the hybridization tray on the right side of the drawer (Figure 121 on page 226).

c. Remove the array plate and protective blue base from its package.

To avoid dust or other damage, leave the array plate packaged until ready to load onto the GeneTitan MC Instrument (Figure 120).

The array plate must be loaded on its protective blue base, as shown in Figure 121 on page 226. The clear plastic cover on top of the array plate SHOULD NOT be loaded in the GeneTitan MC Instrument. See Figure 106 on page 210 for more details on the correct way of loading the array plate.

Figure 119 System layout—location of plates inside the GeneTitan MC Instrument

IMPORTANT! Removing bubbles at this step greatly reduces the chance of bubbles under the arrays when the hybridization tray and the Axiom array plate are clamped. Bubbles under an array can result in black spots on the array image.

Drawers showing contents.

Each line corresponds to a specific drawer number. In this example “Used Hyb Tray” is in the right side of Drawer 1, and “Hyb Tray” is the right side of Drawer 6.Note: Earlier versions of the software may show

as “Fix Tray” rather than “Stabilizing Tray”.

Right side of drawerLeft side of drawer



d. Load the array plate with the protective blue base on the left side of the drawer (Figure 121).

Figure 120 Array plate packaging

Figure 121 Array plate with protective blue base and the hybridization tray properly loaded into drawer 6.

CAUTION! The notched corner of each plate, cover and tray must be aligned. When loading onto the GeneTitan MC Instrument, the notched edge plates, covers and trays must be aligned as indicated by the Tray Alignment guide in the drawer (Figure 121 on page 226).

The error message shown in may be displayed. Plate barcodes must face the internal barcode reader (back of the drawer). Improper tray positioning can cause the GeneTitan MC Instrument to crash, and can result in substantial damage to the instrument and loss of samples.

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Array plate with protective blue base Hybridization tray



e. Press the Confirmation button.

When you load the array plate on the left side of the drawer, the internal bar code reader reads the barcode of the array plate and compares it with the barcode and the plate type specified in the Barcode and Plate Type fields on the Setup page. If the information is correct, the application allows you to proceed to the next step. If the instrument is unable to read the barcode, it will push the tray out and will prompt (Figure 122) you to load the correct plate with the proper orientation into the instrument (Figure 121).

– Click OK to retry and check the loading of the array plate; or

– Click Skip if the instrument has problems reading the barcode and after verifying that the trays have been placed in the proper orientation.

Figure 122 Barcode error message

IMPORTANT! Do not install a 3 plate stack of trays (Figure 123). Confirm that you have removed the clear plastic shipping cover as shown in Figure 106 on page 210.

Figure 123 Do Not install a 3 plate stack of trays



f. Select the arrays to scan (instructions in Figure 124).

By default, all arrays are selected.

2. Click Next, then click OK to begin processing the samples.

The array plate is placed on top of the hybridization tray and clamped (now referred to as the plate stack).

The software starts the process for clamping the array plate to the hybridization tray. Press OK on the dialog shown in Figure 125 and wait for the drawer to open before retrieving the array plate and hybridization tray combo for inspection. The sandwich of the array plate and hybridization tray needs to be manually inspected before the array processing can begin. Once clamping is complete the dialog shown in Figure 126 on page 229 will be displayed. If you do not press OK in Figure 125 the dialog box will go away without intervention and Figure 126 on page 229 will be displayed.

Figure 124 Selecting which arrays to scan on an array plate

Figure 125 Clamping in progress notification

Default – all arrays are selected

Single array - click one box

Multiple arrays:

– Click one box– Hold down the Ctrl key– Click another box in the same column

Group of arrays:

– Click one box– Hold down the Shift key– Left-click and drag the mouse

Message in Status window.



3. When drawer 6 opens and the prompt in Figure 126 is displayed:

a. Remove the plate stack and gently press the 2 plates together at each clamping point.

Listen for a clicking sound which indicates that the plates are now clamped. No clicking sound indicates the plates are already clamped. (The figure below shows an example of an array plate and hybridization tray stack.)

Figure 126 Location of camping points on the array plate and hybridization tray

Clamping points on an Axiom array plate and hybridization tray

Array Plate

Hybridization tray

Notched corners

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b. Inspect the bottom of the plate stack for bubbles under the arrays—do NOT invert the plates.

c. If bubbles are present, gently tap the plate until the bubbles move out from under the arrays—do NOT unclamp the plate stack.

d. Return the plate stack to the drawer, and press the Confirmation button to proceed.

The message below may be displayed again if plate orientation is incorrect or if the hybridization tray barcode cannot be read. Click OK to proceed.



Load a second Axiom™ array plate and hybridization tray onto the GeneTitan™ MC Instrument

When you can load a second array plate and hybridization trayOnce processing begins, you have a specific period of time during which you can load another Axiom array plate and hybridization tray. This period of time is displayed above the Hyb Oven Status pane (Figure 127). You cannot load another hybridization tray before or after this period of time.

While the first plate is in the oven, you can load another plate if the time spacing requirement is met. This is to ensure that the second plate does not have to wait for system resources in its workflow. The time spacing is roughly equal to the longer of the wash-stain or scan time of the first plate.

IMPORTANT! You must load the next array plate and hybridization tray during the period of time displayed above the Hyb Oven Status. You cannot load another hybridization tray before or after this period of time. You will have to wait until the current process is finished which will result in disruption of the 8 plate workflow and fewer than 8 plates processed per week.

Figure 127 Loading a second hybridization tray based on hybridization oven status information

This pane displays the period of time during which another array plate and hybridization tray can be loaded.

Additional plates cannot be loaded before or after this period of time while the instrument is operating.

In this example the system is currently available.

Position of plate stack in the hybridization oven.

Position 1 - left side of oven

Position 2 - right side of oven

Oven Temperature.

• Green indicates the current oven temperature is within the target temperature range.

• Yellow indicates oven temperature outside of target temperature range.

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2

3



1. Select the System Setup tab.

2. Load an Axiom array plate and hybridization tray in the same manner that you loaded the previous plate and tray.

a. Scan or manually enter the Axiom array plate barcode, then click Next.

b. Load the Axiom array plate with the blue base (without the cover) and the hybridization tray (as shown in Figure 121 on page 226), then press the Confirmation button.

c. Select the arrays to scan, then click Next.

d. Ensure that the plates are clamped securely when prompted, then press the Confirmation button.

e. Click OK when prompted to resume plate processing.

Select the System Status tab to view Axiom array plate status in the WorkFlow window (Figure 128).

Figure 128 Example of the workflow window when 2 plates are loaded and are in the hybridization oven

Location: Left and Right positions = the position of the scan tray in drawer 2 (left or right side of the drawer).

1

1


Chapter 5 Array processing with the GeneTitan™ MC InstrumentStatus window prompts and actions required 5

Status window prompts and actions required

As a part of normal GeneTitan MC Instrument operations you may see the following status prompts. Table 61 explains the necessary actions required.

Table 61 Refilling buffer bottles and emptying the waste bottle

Status window prompt Action required Receptacle – Reagent

Buffer bottles have been depressurized. Refill buffer into the bottles. Empty the waste bottle.

• Replenish the fluid in Wash Bottles A and B, and the Rinse bottle.1

• Empty the Waste Bottle.• Press the Confirmation button to

continue.

• Wash Bottle A – fill with Axiom Wash Buffer A up to 2L.

• Wash Bottle B – fill with Axiom Wash Buffer B to the 1L mark.

• Rinse – fill with Axiom Water to the 1L mark.

Do not overfill these bottles.

1 Every time you are prompted to refill the buffer bottles, the system runs a fluidics check (duration ~1 minute).

Table 62 Emptying the trash bin

Status window prompt Action required Receptacle – Reagent

Empty trash bin • Open and empty the trash bin.• Press the Confirmation button to

continue.

Note: If the trash bin is empty, you will not be able to open it. Continue the process by pressing the blue confirmation button

—


Chapter 5 Array processing with the GeneTitan™ MC InstrumentStatus window prompts and actions required5

Table 63 Loading the array plate and hybridization tray; barcode error messages

Status window prompt Action required Reagent – Receptacle

Load array plate tray on [Left/Right] side of drawer. Load hybridization tray without cover on [Left/Right] side of drawer.

Load the array plate with the blue base and the hybridization tray in drawer 6.• IMPORTANT! The blue base must remain in “left side

HTA in” even when empty.• IMPORTANT! The trays must be positioned correctly. If

the trays are placed incorrectly, the software will display an error dialog box indicating the barcode could not be read.


• Hybridization tray loaded with denatured samples.

These messages are displayed if:• A plate has been loaded

improperly.• The bar code is missing

or obscured

Text version of the error message

WARNING! The system was not able to verify the array plate barcode.

Verify that the tray on the left side of the drawer has a blue protective base and if applicable, an array plate, in the correct ORIENTATION. The right side of the drawer should contain a hybridization tray, if applicable, in the correct ORIENTATION.

Details:

The consumable is either not the correct consumable, not loaded correctly, or its barcode is not readable. Proceeding with an incorrect or incorrectly loaded consumable can result in a loss of consumables, loss of samples and may require a Field Service Engineer to service the instrument.

See the System Setup tab or the user guide provided with the Assay or GCC for instructions on proper consumable placement.

Press the flashing blue confirmation button or...

Press OK, the GeneTitan MC Instrument will verify the barcode and orientation.

Press Skip, the GeneTitan MC Instrument will NOT verify the barcode and orientation. The barcode entered at registration will be used.

Table 64 Selecting which arrays to scan

Status window prompt Action required Reagent – Receptacle

Select arrays to scan • Accept the default (all arrays selected) if appropriate. Otherwise, select the arrays to be scanned.

• Click Next, then click OK to start processing.

—


Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 3: Ligate, Wash, Stain, and Scan 5

Stage 3: Ligate, Wash, Stain, and Scan

Equipment, consumables, and reagents required

Scan tray with Axiom Hold Buffer• Cover the tray by orienting the notched corner of the cover over the notched edge

of the tray and leave on the benchtop (no need to protect from light).

CAUTION! Do not remove the scan tray from its protective black base. Leave the scan tray in the base until loaded onto the GeneTitan MC Instrument. When handling the scan tray, the bottom glass surface of the tray should not be touched.

Notched corner of the cover is aligned with the notched corner of the scan tray.

Always leave the scan tray in its protective black base.

1

1

2

2


Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 3: Ligate, Wash, Stain, and Scan5

Proper installation of the GeneTitan™ tray consumables

It is very important that you load the GeneTitan tray consumables in the proper orientation. The barcode faces into the instrument (see Figure 129 and Figure 130).


Figure 129 You must rotate and load the trays so that the barcode faces into the instrument.

Figure 130 The proper loading of the GeneTitan Tray consumables is shown(the image shows the Stain Tray and the Stain Tray cover as an example).

Notch(This faces out and left)

Barcode(This faces BACK TO THE REAR of the instrument)

Turn the tray and cover combo so that the barcodes face BACK AND INTO the instrument and the notch faces OUT AND TO THE LEFT.

FRONT(OF INSTRUMENTFACING YOU)

Notch faces out and left.“For Research Use Only” faces out.

Barcode faces in and back.

FOR RESEARCHUSE ONLY

FOR RESEARCHUSE ONLY



Load trays onto the GeneTitan™ MC Instrument

To load trays onto the GeneTitan MC Instrument:

When hybridization of an Axiom array plate has finished, a message alerts you to resume the workflow setup. Press OK and the software takes you directly back to the System Setup tab.

This prompt to continue into reagent load occurs when the hyb is complete. “Estimated Time Remaining” displayed under “Hybridization Oven Status” may display a time remaining of 0 to 30 minutes when the prompt occurs.

The GeneTitan MC Instrument will allow reagent load to take place after either:

• the estimated time counts down to zero, or

• the actual real world hyb time (as indicated by the computer clock) indicates the hyb is complete.

Note: The time estimate displayed on some systems may lag due to high CPU utilization. The GeneTitan MC Instrument allows the workflow to synchronize with the system clock to compensate for this situation during the final half hour of the hyb time estimate. When this prompt to resume reagent loading is displayed to the user there is no need to wait for the estimated time to count down to zero.

Follow the prompts displayed to continue with staining, ligation, stabilizing and scanning.

1. Follow the prompts in the Status window.

a. Wash Bottles A and B, and the Rinse Bottle—refill as necessary (the system will prime itself again); Waste bottle—empty if necessary.

• Wash bottle A—2L

• Wash Bottle B and Rinse Bottle—fill to 1L mark only.

b. Empty the trash bin.

c. Remove consumable trays and plates as instructed, except for the blue base.Leave the blue array plate base in drawer 6 even though the base is empty.

2. Load consumable trays and plates as follows:

a. Follow the prompts in the Status window (load sequence and prompts in Table 65).

b. Once loaded, examine each cover for droplets of liquid.

c. If any liquid is present, remove the tray, clean the cover and top of the tray with Kimwipes laboratory tissues, and reload the tray.

CAUTION! • Orient trays as indicated by the guide inside the drawer. Improper

orientation may cause the run to fail.

• Remove the protective black base from the scan tray immediately prior to loading Figure 131 on page 239).

• Examine each cover for droplets of liquid after loading. Liquid on the cover can result in capillary phenomenon. As a result, the tray may stick to the cover and be lifted out of place inside the instrument.



Table 65 Sequence for loading the trays with reagents

Loading sequence by

drawer number

Left Right

Note: If the software is unable to verify the barcode on the scan tray and the scan tray cover, the software will display the following error message.

2 Scan Tray with cover—do not load the protective black base(left side of drawer as indicated in Status window)

Figure 131 on page 239

3 Stain Tray with Stain 1 Ligation Tray


4 Stain Tray with Stain 2 Stabilization Tray with Stabilization Reagent




5 Stain Tray with Stain 1 Empty

Table 65 Sequence for loading the trays with reagents (Continued)

Loading sequence by

drawer number

Left Right

Figure 131 Scan tray loaded in drawer 2




IMPORTANT! When you load the plates, or trays, insert them under the tabs, or fingers, that may protrude into the stage. Confirm that the tray is not resting on these fingers.

1

1

1 Tab or “finger” in GeneTitan drawer.

Figure 132 Stain 1 tray (left) and Ligation tray (right) loaded in drawer 3



3. At the prompt, click Yes to load another Axiom array plate and hybridization tray. Right or left position is determined by the position of Axiom array plates already in the GeneTitan MC Instrument.

4. Follow the prompts and:

a. Setup Option: select Setup Another Run, then click Next.

b. Scan or manually enter the Axiom array plate barcode, then click Next.

c. Select a protocol, then click Next.

Figure 133 Stain 2 tray (left) and Stabilization tray (right) loaded in drawer 4.

Figure 134 Stain 1 Tray loaded in drawer 5



d. When drawer 6 opens:

• Remove the blue base from the previous Axiom array plate.

• Load a new Axiom array plate and new blue base on the left; load a new hybridization tray on the right.

• Press the Confirmation button.

e. Click OK when prompted in the Confirm Resume Processing message.

f. When drawer 6 opens, confirm that the plate stack is securely clamped, then press the Confirmation button.

The following is a description of array plate movements in the GeneTitan MC Instrument as users execute a multi-plate workflow.

1. The plate stack which has finished hybridization is moved from the hybridization oven to drawer 1 (temporarily).

2. The new plate stack in drawer 6 is moved to the hybridization oven.

3. The plate stack currently in drawer 1 (see Step 1) is moved to the unclamping station where it is unclamped and moved into the fluidics section of the GeneTitan MC Instrument.

Note: At the end of a Hyb-Wash-Scan run, all plate and tray covers should be in the trash.

Figure 135 is an example of how the System Status Workflow window appears when 3 Axiom array plates are being processed.



Figure 135 Example of the System Status window—3 Axiom array plates are being processed

Workflow indicates the number of plates being processed and where they are in the instrument. In this example, 3 Axiom array plates are being processed: 2 in the hybridization oven and 1 in fluidics.Estimated Completion Time is for the current process.

Status area: Current status indicates that another (4th) plate cannot be added to the GeneTitan hybridization oven because both oven slots are currently in use.

Estimated Time Remaining for fluidics is adjusted as necessary. Adjustments can be due to process interruptions such as a drawer being opened.

Step currently executing in fluidics.

1

1

2

2

3

4

3

4


Chapter 5 Array processing with the GeneTitan™ MC InstrumentContinuing the workflow5

Continuing the workflow

Once a plate has gone through the fluidics stage of the process, it is moved to the imaging device.

When the scanning process begins, the window shown in Figure 136 is displayed. This window must remain open while Axiom array plates are being scanned.

CAUTION! • The Scan Control window must remain open while Axiom array plates are

being scanned. Closing this window will halt the scanning process. You can minimize this window if necessary without creating any interference to the imaging.

• Do not manually, or through the GCC transfer utility, move any data associated with the current plate that is being processed/scanned. Transferring data will dramatically slow scanning and may cause the computer to freeze.

Figure 136 Scan Control window


Chapter 5 Array processing with the GeneTitan™ MC InstrumentShutting down the GeneTitan™ MC Instrument 5

Shutting down the GeneTitan™ MC Instrument

This procedure assumes that all of the Axiom array plates loaded onto the GeneTitan MC Instrument have been processed.

To Shutdown the GeneTitan MC Instrument:

1. On the System Setup page, open the Setup Options drop-down menu and select Unload Plates.

2. Unload all of the consumables as prompted.

3. Power off the GeneTitan MC Instrument by opening Tools Shutdown from the menu.

4. Exit the Applied Biosystems™ GeneChip™ Command Console software if it does not close automatically.

Note: If the instrument is processing an array plate, the software will not allow you to shut down the system.

WARNING! Do not attempt to shut down the GeneTitan MC Instrument while array plates are being processed.


6 Processing 8 Axiom™ array platesper week

Using 1 Biomek FXP Target Prep Express (Biomek workstation) and 1 GeneTitan MC Instrument, the Axiom™ 2.0 Genotyping Assay (using the 96-array plate) can be run at a throughput of 8 Axiom™ array plates per 5-day work week. This chapter includes tables that present the timing of the steps required to perform this workflow per 5-day work week, 8 hours per day.

Overview of the 8-plate workflow

During the initial week of startup, all work is done on the Biomek FXP Target Prep Express. You will process 8 plates of genomic DNA samples. At the end of this week, you will have what are now referred to as 8 plates of hyb-ready target (target).

Subsequent weeks of the workflow involve the simultaneous processing of plates on the Biomek FXP Target Prep Express and on the GeneTitan MC Instrument. Each week:

• Eight plates of target from the previous week are denatured and transferred to the GeneTitan MC Instrument for hybridization and array plate processing.

• Eight new plates of target are prepared on the Biomek FXP Target Prep Express.

Plate numbering scheme

• Target preparation on the Biomek FXP Target Prep Express: Sample Plates are numbered 1 through 8.

• Target prepared the previous week is processed on the GeneTitan MC Instrument. The plates denatured and loaded onto the GeneTitan™ MC Instrument are now referred to as plates A through H. Target prepared on the Biomek FXP Target Prep Express can be run on the GeneTitan MC Instrument in random order.

IMPORTANT! Experienced users and careful timing are critical for the successful execution of this workflow.


Chapter 6 Processing 8 Axiom™ array plates per weekOverview of the 8-plate workflow 6

Week 1

Table 66 Overview of week 1: Target preparation on the Biomek FXP Target Prep Express

Day Activities Plates

1 • Amplify 8 plates of genomic DNA. 1 through 8

2 • Fragment and precipitate 3 plates amplified on Day 1.• Freeze 5 plates of amplified gDNA for fragmentation later in the week.

• 1, 5, 8• 2, 3, 4, 6, 7

3 • Fragment and precipitate 2 more amplified plates.• Centrifuge, dry, resuspend and QC the 3 plates precipitated on Day 2.

• 2, 3• 1, 5, 8

4 • Fragment and precipitate the 3 remaining amplified plates.• Centrifuge and dry the 2 plates precipitated on Day 3.

• 4, 6, 7• 2, 3

5 • Resuspend and QC the 2 plates dried on Day 4.• Centrifuge, dry, resuspend and QC the 3 plates precipitated on Day 4.

• 2, 3• 4, 6, 7

Table 67 Hands-on time required for target preparation on the Biomek FXP Target Prep Express per 96-array plate

Steps on the Biomek FXP Target Prep Express Time required

Amplification 30 minutes

Fragmentation 2 hours

Resuspension 45 minutes

Off-deck centrifugation and drying 75 minutes

Off-deck QC gel and OD 45 minutes

Denaturation only 30 minutes

Denaturation and GeneTitan reagent tray preparation 45 minutes

Transfer denatured samples to the GeneTitan MC Instrument 15 minutes

Transfer reagent trays to the GeneTitan MC Instrument 15 minutes

Total time required (includes setup) 7 hours total


Chapter 6 Processing 8 Axiom™ array plates per weekOverview of the 8-plate workflow6

Week 2

The hybridization time for the Axiom 2.0 Assay on the GeneTitan MC Instrument is 23.5–24 hours (Table 69). This provides a 30 minutes window during which you are prompted by the instrument control software to load the reagents required for washing and staining. We recommend that you begin loading the reagent trays onto the GeneTitan MC Instrument at the mid-point of this 30 minutes window. As such, the wash procedures will begin 24 hours after the start of hybridization. If catch-up is required in the framework of the 8-plate workflow, begin loading reagents at the beginning of this 30 minutes window (i.e., immediately after prompted by the software).

IMPORTANT! Maintaining consistent timing during the set up of the GeneTitan MC Instrument is critical to containing the user interventions of the 8 plate workflow within an 8 hour work day. Once a process begins late, there is little opportunity to catch up until the end of the workflow.

Table 68 Overview of week 2: Array processing in the GeneTitan™ MC Instrument

Day Activities Plates

1 • Hybridize 2 plates of denatured target • A and B

2 • Hybridize 2 plates of denatured target• Load reagent trays for fluidics and imaging of plates loaded on day 1

• C and D• A and B


• E and F• C and D


• G and H• E and F

5 • Load reagent trays for fluidics and imaging of plates loaded on day 4 • G and H

Table 69 Time required for Axiom array plate processing on the GeneTitan MC Instrument

Steps on the GeneTitan MC Instrument Time required

Hybridization of 2 plates• First plate loaded at 9:30 a.m.• Second plate loaded at 5:00 p.m.

23.5 hours each plate

Loading reagent trays that were prepared on the Biomek FXP Target Prep Express

15 minutes

Fluidics 5 hours each plate

Imaging 96 arrays: 7.5 hours


Chapter 6 Processing 8 Axiom™ array plates per weekThawing frozen plates of amplified DNA 6

Thawing frozen plates of amplified DNA

To thaw frozen plates of amplified DNA:



2. Leave the plate in the water bath for ~ 50 minutes until all wells have thawed.







Automated target preparation for processing 8 Axiom™ array plates per week

Using 1 Biomek FXP Target Prep Express (Biomek workstation) and 1 GeneTitan MC Instrument, the Axiom™ 2.0 Genotyping Assay (using the 96-array plate) can be run at a throughput of 8 Axiom™ array plates per 5-day work week. This chapter includes tables that present the timing of the steps required to perform this workflow per 5-day work week, 8 hours per day.

During the initial week of startup, all work is done on the Biomek FXP Target Prep Express. You will process 8 plates of genomic DNA samples. At the end of this week, you will have what are now referred to as 8 plates of hyb-ready target (target). Subsequent weeks of the workflow involve the simultaneous processing of plates on the Biomek FXP Target Prep Express and on the GeneTitan MC Instrument. Each week:

• Eight plates of target from the previous week are denatured and transferred to the GeneTitan MC Instrument for hybridization and array plate processing.

• Eight new plates of target are prepared on the Biomek FXP Target Prep Express.



Chapter 6 Processing 8 Axiom™ array plates per weekInitial target prep week—Biomek FXP Target Prep Express6

Initial target prep week—Biomek FXP Target Prep Express

Initial target prep week—Day 1

• Amplify 8 plates of gDNA samples.

IMPORTANT! • All amplifications should be set up on Day 1 to allow for a 23 1 hour

amplification incubation for each plate.

• Begin thawing the amplification reagents, particularly the Axiom 2.0 Amp Soln, 60 minutes prior to the start of each reaction.

Monday a.m. Monday p.m.

8 9 10 11 12 1 2 3 4 5

1

2

3

4

5

6

7

8

Figure 137 Initial target prep week—Day 1 activities

Table 70 Initial target prep week—Day 1 activities

Activity Plate number

Instrument Approximate start times

DNA Amplification 1

Biomek FXP Target Prep Express

10:00 a.m.

DNA Amplification 2 10:30 a.m.



DNA Amplification 5 12:00 p.m.





Chapter 6 Processing 8 Axiom™ array plates per weekInitial target prep week—Biomek FXP Target Prep Express 6


• Fragment and precipitate plates 1, 5 and 8.

• Freeze amplified plates 2, 3, 4, 6, and 7 after each plate has incubated for 23 hours.

IMPORTANT! • Plates 1, 5 and 8 are fragmented and precipitated on Day 2 without freezing to

preserve a 23 hours amplification incubation.

• Store all plates not fragmented and precipitated on Day 2 at –20°C following 23 hours of amplification reaction incubation.

• Precipitation is carried out at –20°C overnight.

Tuesday a.m. Tuesday p.m.

8 9 10 11 12 1 2 3 4 5

1—Fragment/Precipitate

s2

s3

s4


s6

s7


s = Seal tightly and store at –20°C.





• Thaw plates 2 and 3 (see ʺThawing frozen plates of amplified DNAʺ on page 249).

• Fragment and precipitate plates 2 and 3.

• Centrifuge, dry, resuspend and QC plates 1, 5 and 8.




Fragment and precipitate 1 Biomek FXP Target Prep Express 9:30 a.m.

Freeze (–20°C) 2 — 10:00 a.m.

Freeze (–20°C) 3 — 10:30 a.m.

Freeze (–20°C) 4 — 11:00 a.m.


Freeze (–20°C) 6 — 12:30 p.m.

Freeze (–20°C) 7 — 1:00 p.m.

Fragment and precipitate 8 Biomek FXP Target Prep Express 1:30 p.m.

IMPORTANT! • Amplified plates that are frozen must be thawed and thoroughly mixed by

following the procedure under ʺThawing frozen plates of amplified DNAʺ on page 249.

• After being centrifuged and dried, plates 1, 5 and 8 are sealed and placed in a 4°C refrigerator until further processing later the same day.


• Prior to resuspension and QC, plates 1, 5 and 8 must be brought to room temperature (place on benchtop for 30 minutes).



Wednesday a.m. Wednesday p.m.

8 9 10 11 12 1 2 3 4 5

2 - RT


1, 5, 8Centrif/Dry

3 - RT


1-RT 1-Resus

1-QC

5-RT 5-Resus

5-QC

8-RT 8-Resus

8-QC

RT = Bring plate to room temperatureCentrif = centrifuge offlineResus = resuspendQC = fragmentation QC gel and OD quantitation





Bring plate to room temperature (RT) 2 — 8:30 a.m.


Centrifuge and dry 1, 5, 8 Plate centrifuge and oven 9:45 a.m.



Resuspension 1 Biomek FXP Target Prep Express, 1:30 p.m.

Off-deck QC 1 Plate spectrophotometer, e-gel system 2:15 p.m.

Resuspension 5 Biomek FXP Target Prep Express 2:15 p.m.








• Centrifuge and dry plates 2 and 3.




• After being centrifuged and dried, plates 2 and 3 are sealed and stored at –20°C.

Thursday a.m. Thursday p.m.

8 9 10 11 12 1 2 3 4 5

4 - RT


2, 3Centrif/Dry

6 - RT


7 - RT


RT = Bring plate to room temperatureCentrif = Centrifuge



Activity Plate number Instrument Approximate start times

Thaw 4 — 8:30 a.m.


Centrifuge and dry 2, 3 Plate centrifuge and oven 9:45 a.m.

Thaw 6 — 10:30 a.m.


Thaw 7 — 12:30 p.m.





• Centrifuge and dry plates 4, 6 and 7.

• Resuspend and QC plates 2, 3, 4, 6 and 7.

IMPORTANT! • Plates 2 and 3 must be brought to room temperature for 90 minutes prior to

resuspension.

• After being centrifuged and dried, plates 4, 6 and 7 are sealed. Place plates 6 and 7 in a 4°C refrigerator until further processing later the same day. Plate 4 can be left on the benchtop.

• Prior to resuspension and QC, plates 6 and 7 must be brought to room temperature (place on benchtop for 30 minutes).

Friday a.m. Friday p.m.

8 9 10 11 12 1 2 3 4 5

2 - RT

3 - RT

2-Resus

2-QC

4, 6, 7Centrif/Dry

3-Resus

3-QC

4-Resus

4-QC

6-RT 6-Resus

6-QC

7-RT 7-Resus

7-QC

RT = Bring plate to room temperature.Resus = ResuspensionQC = fragmentation QC gel and OD quantitation




Table 74 Initial target prep week—day 5 activities


Bring to room temperature 2 — 8:00 a.m.


Resuspension and QC 2 Biomek FXP Target Prep Express 9:30 a.m.


Resuspension and QC 3


10:15 a.m.

Resuspension and QC 4 11:00 a.m.

Resuspension and QC 6 1:15 p.m.



Chapter 6 Processing 8 Axiom™ array plates per weekSimultaneous 8-plate workflow 6

Simultaneous 8-plate workflow

The tables on the following pages provide a breakdown of the timing involved to simultaneously:

• GeneTitan MC Instrument: Process 8 array plates per week with target prepared the previous week on the Biomek FXP Target Prep Express.

• Biomek FXP Target Prep Express: Prepare 8 new plates of target for processing the following week on the GeneTitan MC Instrument.

Eight-plate workflow—Day 1

Biomek FXP Target Prep Express activities• Denature 2 plates of target prepared the previous week (Plates A and B).

• Amplify 8 new plates of genomic DNA (1 through 8)

GeneTitan MC Instrument activities• Transfer denatured plates to the GeneTitan MC Instrument and begin

hybridization (Plates A and B).

IMPORTANT! Timing is critical.

• The Biomek FXP Target Prep Express will be in operation for a large percentage of each work day.

• Plates for target preparation on the Biomek FXP Target Prep Express are numbered 1 through 8.

• Plates with target that are now ready for denaturation and transfer to the GeneTitan MC Instrument for hybridization, fluidics processing, and imaging are lettered A through H.

• Plates of target from the previous week (1–8) can be processed on the GeneTitan MC Instrument in any order.

IMPORTANT! • Plates 1 through 8 are referred to as A through H when denatured and loaded onto

the GeneTitan MC Instrument. Plates 1 through 8 can go onto the GeneTitan MC Instrument in any order.

• All amplifications are set up on Day 1 to maintain a 23 1 hour amplification incubation for each plate.

• Begin thawing the amplification reagents, particularly the Axiom 2.0 Amp Soln, 60 minutes prior to the start of each reaction.


Chapter 6 Processing 8 Axiom™ array plates per weekSimultaneous 8-plate workflow6

Monday a.m. Monday p.m.

8 9 10 11 12 1 2 3 4 5

Den A Hyb A

1

2

3

4

5

6

7

8

Den B Hyb B

• Den = denature• Den time period includes 15 minutes to transfer the denatured Sample Plate from the Biomek workstation to the

GeneTitan MC Instrument.• Begin thawing the amplification reagents, particularly the Axiom 2.0 Amp Soln, 60 minutes prior to the start of each

reaction.

Figure 142 Eight-plate workflow—Day 1 activities

Table 75 Eight-plate workflow—Day 1 activities


Denature on the FX A Biomek FXP Target Prep Express 8:45 a.m.

Load onto the GT and hybridize A GeneTitan MC Instrument 9:30 a.m.

DNA Amplification 1


10:00 a.m.




DNA Amplification 5


12:00 p.m.




Denature on the FX B Biomek FXP Target Prep Express 4:15 p.m.

Load onto the GT and hybridize B GeneTitan MC Instrument 5:00 p.m.




Biomek FXP Target Prep Express activities• Denature 2 plates of target from the previous week (plates C and D).

• Prepare reagent trays for the GeneTitan MC Instrument (for Plates A and B already on the GeneTitan MC Instrument).

• Transfer the denatured samples and reagent trays to the GeneTitan MC Instrument.


• Freeze amplified plates 2, 3, 4, 6, and 7 after each plate has incubated for 23 hours

GeneTitan MC Instrument activities• Load reagent trays for A and B. These plates are moved from the hybridization

oven to the fluidics area. After fluidics, the plates move to the imaging area of the instrument. Load Plates C and D for hybridization.

IMPORTANT! • Plates 1, 5 and 8 are fragmented and precipitated without freezing to preserve a

23 hours amplification incubation.

• All plates not fragmented and precipitated on Day 2 should be stored at –20°C following 23 hr of amplification reaction incubation.




Tuesday a.m. Tuesday p.m.

8 9 10 11 12 1 2 3 4 5

Thaw GT

rgntsDen C Hyb C

End Hyb

A

GT rgnt plates for A Fluidics and Scan A


s2

s3

s4


s6

s7


Thaw GT

rgntsDen D

Hyb D

Hyb B23.5–24 hours

GT rgnt plates for B

**B

** B = Fluidics and Scan B

Den = denature

Denature and GT rgnt plate time periods include 15 minutes to transfer the denatured plate and reagent trays from the Biomek to the GeneTitan MC Instrument.

s = Seal tightly and store at –20°C.

GT rgnt plates = reagent trays for GeneTitan array plate processing.





Activity Plate designation


Thaw reagent for GT rgnt plate preparation

A — 8:00 a.m.

Concurrently:• Denature C• Prepare GT rgnt plates for A• Transfer to the GeneTitan MC

Instrument

Denature CReagents for A

Biomek FXP Target Prep Express 8:30 a.m.

• Load GT rgnt plates and begin fluidics processing for A

• Load C and begin hybridization

A to fluidicsC to hybridization

oven

GeneTitan MC Instrument 9:30 a.m.


Freeze (–20°C) 2 — 10:00 a.m.

Freeze (–20°C) 3 — 10:30 a.m.

Freeze (–20°C) 4 — 11:00 a.m.


Freeze (–20°C) 6 — 12:30 p.m.

Freeze (–20°C) 7 — 1:00 p.m.


Thaw reagent for GT rgnt plate preparation

B — 3:30 p.m.

Concurrently:• Denature D• Prepare GT rgnt plates for B• Transfer to the GeneTitan MC

Instrument

Denature DReagents for B

Biomek FXP Target Prep Express 4:00 p.m.

• Load GT rgnt plates and begin fluidics processing for B

• Load D and begin hybridization

B to fluidicsD to hybridization

oven

GeneTitan MC Instrument 5:00 p.m.

Denature and GT rgnt plate preparation time periods include 15 minutes to transfer the denatured plate and reagent trays from the Biomek to the GeneTitan MC Instrument.




Off-deck activities• Thaw plates 2 and 3.

• QC—OD quantitation and fragmentation gel for Plates 1, 5, 8.

Biomek FXP Target Prep Express activities• Denature 2 plates of target from the previous week (Plates E and F).

• Prepare reagent trays for the GeneTitan MC Instrument (for Plates C and D already on the GeneTitan MC Instrument).


• Fragment and precipitate Plates 2 and 3.

• Centrifuge, dry, resuspend and QC Plates 1, 5, 8.

GeneTitan MC Instrument activities• Load reagent trays for Plates C and D. These plates are moved from the

hybridization oven to the fluidics area. After fluidics, the plates will move to the imaging area of the instrument. Also load Plates E and F for hybridization.



Wednesday a.m. Wednesday p.m.

8 9 10 11 12 1 2 3 4 5

Thaw GT

rgntsDen E Hyb E

End Hyb C

GT rgnt plates for C

Fluidics and Scan C

2 – RT


1, 5, 8Centrif/Dry

3 - RT


1-RT 1-Resus 1-QC

5-RT 5-Resus 5-QC

8-RT 8-Resus 8-QC

Thaw GT

rgntsDen F Hyb F

Hyb DGT rgnt

plates for DFluidics

and Scan D

RT = Bring plate to room temperatureCentrif = centrifugeResus = resuspendQC = fragmentation QC gel and OD quantitationGT rgnt plates = reagent trays for GeneTitan array plate processing• Den time period includes 15 minutes to transfer the denatured Sample Plate and reagent trays from the Biomek to the

GeneTitan MC Instrument






Thaw reagents for GT rgnt plate preparation

C — 8:00 a.m.


Concurrently:• Denature E• Prepare GT rgnt plates for C• Transfer to the GeneTitan MC Instrument

• Denature E• Reagents for C


• Load GT rgnt plates and begin fluidics processing for C

• Load E and begin hybridization

• C to fluidics

• E to hyb oven












F — 3:30 p.m.


Concurrently:• Denature F• Prepare GT rgnt plates for D• Transfer to the GeneTitan MC Instrument

• Denature F• Reagents for D


• Load GT rgnt plates and begin fluidics processing for D

• Load F and begin hybridization

• D to fluidics

• F to hyb oven





Off-Deck activitiesBring Plates 4, 6 and 7 to room temperature.

Biomek FXP Target Prep Express activities• Denature 2 plates of target from the previous week (Plates G and H).

• Prepare reagent trays for the GeneTitan MC Instrument (for Plates E and F already on the GeneTitan MC Instrument).


• Fragment and precipitate Plates 4, 6, 7.

• Centrifuge and dry Plates 2 and 3.

GeneTitan MC Instrument activities• Load reagent trays for E and F. These plates are moved from the hybridization

oven to the fluidics area. After fluidics, the plates move to the imaging area of the instrument. Load Plates G and H for hybridization.




• After being centrifuged and dried, Plates 2 and 3 are sealed and stored at –20°C.



Thursday a.m. Thursday p.m.

8 9 10 11 12 1 2 3 4 5

Thaw GT

rgntsDen G Hyb G

End Hyb E

GT rgnt plates for E

Fluidics and Scan E

4 - RT


2, 3 Centrif/Dry

6 - RT


7 - RT


Thaw GT

rgntsDen H Hyb H

Hyb FGT rgnt

plates for FFluidics

and Scan F

RT = Bring plate to room temperatureCentrif = centrifugeDenature and GT rgnt plate preparation time periods include 15 minutes to transfer the denatured plate and reagent trays from the Biomek to the GeneTitan MC Instrument.

Figure 145 Eight-plate workflow— Day 4 Activities



Table 78 Eight-plate workflow— Day 4 activities

Activity Plate designation



E — 8:00 a.m.


Concurrently:• Denature G• Prepare GT rgnt plates for E• Transfer to the GeneTitan MC Instrument

• Denature D• Reagent for E


• Load GT rgnt plates and begin fluidics processing for E

• Load G and begin hybridization

• E to fluidics

• G to hyb oven



Centrifuge and dry 2, 3 Plate centrifuge and oven 9:45 a.m.






F — 3:30 p.m.

Concurrently:• Denature H• Prepare GT rgnt plates for F• Transfer to the GeneTitan MC Instrument

• Denature H • Reagents for F


• Load GT rgnt plates and begin fluidics processing for F

• Load H and begin hybridization

• F to fluidics

• H to hyb oven





Off-Deck activitiesBring Plates 2 and 3 to room temperature.

Biomek FXP Target Prep Express activities• Centrifuge and dry Plates 4, 6, 7.

• Resuspend and QC Plates 2, 3, 4, 6, 7.

GeneTitan MC Instrument activitiesLoad reagent trays for plates E and F. These plates are moved from the hybridization oven to the fluidics area. After fluidics, the plates will move to the imaging area of the instrument. Also load Plates G and H for hybridization.

IMPORTANT! • Plates 2 and 3 must be brought to room temperature for 90 minutes prior to

resuspension.

• After being centrifuged and dried, plates 4, 6 and 7 are sealed. Place Plates 6 and 7 in a 4°C refrigerator until further processing later the same day. Plate 4 can be left on the benchtop.

• Prior to resuspension and QC, Plates 6 and 7 must be brought to room temperature (place on benchtop for 30 minutes).



Friday a.m. Friday p.m.

8 9 10 11 12 1 2 3 4 5

End Hyb G

GT rgnt plates for G

Fluidics and Scan G

2 - RT

3 - RT

2-Resus 2-QC

4, 6, 7Centrif/Dry

3-Resus 3-QC

4-Resus 4-QC

6-RT 6-Resus 6-QC

7-RT 7-Resus 7-QC

Hyb HRgnt Plate

Prep HFluidics

and Scan H

RT = Bring plate to room temperature.Resus = ResuspensionQC = fragmentation QC gel and OD quantitationGT rgnt plate preparation time periods include 15 minutes to transfer the denatured plate and reagent trays from the Biomek to the GeneTitan MC Instrument.







Prepare GeneTitan reagent trays

G Biomek FXP Target Prep Express 8:30 a.m.

Load reagent trays and begin fluidics processing for G

G GeneTitan MC Instrument 9:30 a.m.


Resuspension and QC 2 Biomek FXP Target Prep Express 9:30 a.m.


Resuspension and QC 3


10:15 a.m.

Resuspension and QC 4 11:00 a.m.



Prepare GeneTitan reagent trays

H Biomek FXP Target Prep Express 4:00 p.m.

Load reagent trays and begin fluidics processing for H

H GeneTitan MC Instrument 5:00 p.m.


7 Processing 3 Axiom™ array platesper week

The 3 array plate/week automated target prep workflow enables you to do the target preparation and array processing for 3 Axiom array plates in the same week.

This workflow is performed using 1 Biomek FXP Target Prep Express (Biomek workstation) (see Chapter 3, ʺTarget preparation on the Biomek FXP with Windows® XPʺ on page 22) and 1 GeneTitan MC Instrument (see Chapter 5, ʺArray processing with the GeneTitan™ MC Instrumentʺ on page 209). This chapter includes tables that present the timing of the steps required to perform this workflow per 5-day work week, 8 hours per day.

The 3 plate per week automated workflow is described in the following sections:

• ʺOverview of the 3-plate workflow for automated target preparationʺ

• ʺThawing frozen plates of amplified DNAʺ on page 274

• ʺTarget prep and array processingʺ on page 275



Chapter 7 Processing 3 Axiom™ array plates per weekOverview of the 3-plate workflow for automated target preparation7

Overview of the 3-plate workflow for automated target preparation

The table below displays the timing and duration of the hands-on processing necessary for performing the 3-plate workflow by 1 person.

The 3 plates are referred to as Plates A, B and C in the target prep and in the GeneTitan Array Processing.

In order to process 3 plates during a 40-hour week, the steps should be performed in the order and with the timing described in this chapter.

Figure 147 Three plate per week automated target prep workflow

PlateDay 1 Day 2 Day 3 Day 4 Day 5

AM PM AM PM AM PM AM PM AM PM8 9 10 11 12 1 2 3 4 5 6 8 9 10 11 12 1 2 3 4 5 6 8 9 10 11 12 1 2 3 4 5 6 8 9 10 11 12 1 2 3 4 5 6 8 9 10 11 12 1 2 3 4 5 6

A

B

C

NotesBegin thawing required reagents for the process.Begin warming Axiom array plate to room temperature

x Day 2 11 AM: Freeze Plate Cy Day 3 Noon: start plate C thawingz Day 4 5:00 PM: Coupled operations on GeneTitan MC Instrument: Load reagents for Plate A and hybridization tray

and array plate for Plate CColor Code

Amplification Fragmentation and Precipitation Centrifugation and Drying Resuspension and Hybridization Mix Prep QC Sample Denature/load array plate and hybridization tray into the GeneTitan MC Instrument GeneTitan MC reagent trays prep and loading

x y z

z


Chapter 7 Processing 3 Axiom™ array plates per weekOverview of the 3-plate workflow for automated target preparation 7

Table 80 Daily steps for automated target prep workflow for 3 plates/week

Day Activities

1 Amplify 3 plates of genomic DNA

2 1. Fragment and precipitate 2 plates amplified on Day 12. Freeze Plate C at –20°C

3 1. Centrifuge and dry Plate A and Plate B2. Resuspend and QC Plate A3. Resuspend and QC Plate B4. Denature Plate A and load samples and array plate into the GeneTitan MC

Instrument5. Store Plate B at –20°C6. Thaw Plate C frozen on Day 27. Fragment and precipitate Plate C amplified on Day 1

4 1. Denature Plate B and load samples and array plate into the GeneTitan MC Instrument

2. Centrifuge, dry, resuspend, and QC Plate C3. Prepare GeneTitan reagent trays for Plate A and load into the GeneTitan

MC Instrument and denature Plate C

5 1. Prepare GeneTitan reagent trays for Plate B and load into the GeneTitan MC Instrument

2. Prepare GeneTitan reagent trays for Plate C and load into the GeneTitan MC Instrument

Table 81 Time Required for Target Preparation on the Biomek FXP Target Prep Express per 96-Array Plate

Steps on the Biomek FXP Target Prep Express Time required

Amplification 30 minutes

Fragmentation 2 hours

Resuspension 45 minutes

Off-deck centrifugation and drying 75 minutes

Off-deck QC gel and OD 45 minutes

Denaturation only 30 minutes

Transfer denatured samples to the GeneTitan MC Instrument 15 minutes

Prepare and Load reagent trays to the GeneTitan MC Instrument 60 minutes

Total Time Required includes deck setup. 7 hours


Chapter 7 Processing 3 Axiom™ array plates per weekThawing frozen plates of amplified DNA7

Thawing frozen plates of amplified DNA

The automated 3 plate workflow described in this chapter requires freezing Plate C of DNA following amplification on Day 2. You should thaw Plate C prior to the fragmentation step on Day 2.

To thaw frozen plates of amplified DNA:











Chapter 7 Processing 3 Axiom™ array plates per weekTarget prep and array processing 7

Target prep and array processing

The tables in the sections below show the steps that need to be performed on each day of the workflow.

Three plate/week workflow—Day 1

Note: Genomic DNA sample for amplification should be prepared as described in ʺGenomic DNA preparation and requirementsʺ on page 13.

See ʺStage 1: DNA Amplificationʺ on page 51 for more information on the protocol.

Figure 148 Three plate/week automated target prep workflow—Day 1 activities

Plate

Day 1 a.m. Day 1 p.m.

8 9 10 11 12 1 2 3 4 5

A Amp

B Amp

C Amp

Notes

Begin thawing reagents and materials for the process

Color Code

Amp Amplification (see "Stage 1: DNA Amplification" on page 51)

Table 82 Three plate/week auto target prep workflow—Day 1

Time Topic

9:30 Prepare DNA amplification reagents for Plate A

10:30 Prepare DNA amplification reagents for Plate C

10:30 Start DNA amplification Plate A

11:30 Start DNA amplification Plate C

1:30 Prepare DNA amplification reagents for Plate B

2:30 Start DNA amplification Plate B


Chapter 7 Processing 3 Axiom™ array plates per weekTarget prep and array processing7


Figure 149 Three plate/week workflow—Day 2 activities

Plate


8 9 10 11 12 1 2 3 4 5

A Frag

B Frag

C

Notes

Begin thawing required reagents

X Freeze Plate C

Color Code

Frag Fragmentation and precipitation (see "Stage 2: Fragmentation and Purification" on page 65)

X


Time Topic

9:30 Prepare Frag reagents for Plate A

10:00 Start Fragmentation Plate A

11:00 Transfer Amplified Plate C to –20°C

1:30 Prepare Frag reagents for Plate B

2:00 Start Fragmentation Plate B




Figure 150 Three plate/week automated target prep workflow—Day 3 activities

Plate


8 9 10 11 12 1 2 3 4 5

A Cent/Dry R/HP QC Denat/ hyb

B Cent/Dry R/HP QC

C Frag

Notes


Begin warming Axiom array plate to room temperature

X Store Hyb Ready Plate A and Plate B at –20°C

Y Begin thawing Amp Plate C

Color Codes

Frag Fragmentation and Precipitation (see "Stage 2: Fragmentation and Purification" on page 65)

Cent/Dry Centrifugation and Drying (see "5. Centrifuge and dry pellets" on page 75)

R/HP Resuspension and Hyb Mix Prep (see "Stage 3: Resuspension and hybridization preparation" on page 76)

QC Quality control checks

Denat/hyb

Sample Denature/load array plate and hybridization tray into the GeneTitan MC Instrument (see "Stage 4: Preparation for the GeneTitan™ MC Instrument" on page 89)

X

Y

X

Table 84 Three plate/week automated target prep workflow—Day 3

Time Topic

9:00 Start Centrifugation of Plate A and Plate B.

9:40 Start Drying of Plate A and Plate B.

10:05 Start Resuspension and Hybridization Preparation Plate A, keep Plate B sealed at room temperature.

10:45 Start Resuspension and Hybridization Preparation Plate B.

11:30 Run QC on Plate A and Plate B.

12:00 Transfer Hyb-ready Plate A and Plate B to freezer (–20°C).Thaw Amplified Plate C.

12:30 Prepare fragmentation reagents for Plate C.

1:00 Start fragmentation of Plate C.

4:30 Start denature Sample Plate A

5:00 Start Hyb-Wash-Scan run Plate A.



Three plate/week workflow—Day 4 IMPORTANT! The GeneTitan reagent trays for array processing cannot be loaded

until the array plate has finished hybridization, and they should not be prepared more than 1.5 hours before hybridization will finish. The GeneTitan reagent trays cannot be prepared ahead of time and stored.

Figure 151 Three plate/week automated target prep workflow—Day 4

Plate


8 9 10 11 12 1 2 3 4 5

A

GT Reagent

Prep/Load

B Denat/hyb

C Cent/Dry R/HP QCDenat/hyb

Notes


Begin warming Axiom array plate to room temperature

X Store Hyb Ready Plate C at –20°C

Z Coupled GeneTitan Instrument operations: Load reagent trays for Plate A and hyb tray/array plate for Plate C.

Color Codes

Cent/Dry Centrifugation and Drying (See "5. Centrifuge and dry pellets" on page 75)

R/HP Resuspension and Hyb Mix Prep (see "Stage 3: Resuspension and hybridization preparation" on page 76)

QC Quality Control Checks

Denat/Hyb Sample Denature/load array plate and hybridization tray into the GeneTitan MC Instrument (see "Stage 4: Preparation for the GeneTitan™ MC Instrument" on page 89)

GT Reagent prep/load

GeneTitan reagent trays prep and load (see "Stage 4: Preparation for the GeneTitan™ MC Instrument" on page 89)

ZX

Z



Table 85 Three Plate/Week Auto Target Prep Workflow—Day 4

Time Topic

9:00 Start Denature Sample for Plate B (see "Stage 4: Preparation for the GeneTitan™ MC Instrument" on page 89)

9:30 Start Hyb-Wash-Scan run for Plate B

9:30 Start centrifugation of Plate C

10:10 Start drying of Plate C

10:35 Start resuspension and hybridization preparation Plate C

11:10 Run QC on Plate C

11:40 Transfer Plate C to freezer (–20°C)

3:30 Prepare wash and stain reagents for Plate A

4:00 Prepare GeneTitan reagent trays for Plate A (see "Stage 4: Preparation for the GeneTitan™ MC Instrument" on page 89)

4:30 Start denature samples for Plate C

5:00 Load GeneTitan reagent trays for Plate A

5:00 Start Hyb-Wash-Scan run for Plate C



Three plate/week workflow—Day 5 IMPORTANT! The GeneTitan reagent trays for array processing cannot be loaded

until the array plate has finished hybridization, and they should not be prepared more than 1.5 hours before hybridization will finish. The GeneTitan reagent trays cannot be prepared ahead of time and stored.

Figure 152 Three plate/week automated target prep workflow—Day 5

Plate


8 9 10 11 12 1 2 3 4 5

A

B

GT Reagent

Prep/Load

C

GT Reagent

Prep/Load

Notes


Color Codes

GT Reagent

Prep/Load

GeneTitan reagent trays prep and load (see "Stage 4: Preparation for the GeneTitan™ MC Instrument" on page 89)


Time Topic

8:00 Prepare Wash-Stain reagents for Plate B

8:30 Prepare GeneTitan reagent trays for Plate B (see "Stage 4: Preparation for the GeneTitan™ MC Instrument" on page 89)

9:30 Load GeneTitan reagent trays for Plate B

3:30 Prepare wash and stain reagents for Plate C

4:00 Prepare GeneTitan reagent trays for Plate C (see "Stage 4: Preparation for the GeneTitan™ MC Instrument" on page 89)

5:00 Load GeneTitan reagent trays for Plate C


8 Troubleshooting


If a hardware problem is encountered while running the Axiom target preparation methods on the Biomek FXP Target Prep Express, you can do the following:

• See these documents:

– Biomek® Liquid Handler User’s Manual, Beckman Coulter Pub. No. 987834

– Biomek® Software User’s Manual v4.1, Beckman Coulter Pub. No. B30026AA

• For information on recovering a run, contact your Thermo Fisher Scientific Field Application Scientist.

• For additional information on Biomek FXP Target Prep Express hardware, error messages, or to request service, contact Beckman Coulter. Be sure to have the serial number of your workstation available.

GeneTitan™ Multi-Channel Instrument

See the GeneTitan™ Multi-Channel Instrument User Guide, Pub. No. 08-0306 for further troubleshooting information.

Table 87 GeneTitan Multi-Channel Instrument troubleshooting guidelines for the Axiom 2.0 Assay

Problem Possible causes Possible actions

Plate trapped in GeneTitan Multi-Channel Instrument.

• Plate (or plate with lid) not properly loaded in drawer.

• Notched edge of lid and plate not aligned.

• Gripper failed to retrieve plate.• System requires adjustment.

1. Restart the GeneTitan Multi-Channel Instrument.

2. Run the setup option Unload Plates.3. If the plate remains trapped in the

instrument, call Thermo Fisher Scientific support.

Computer frozen. • Too many processes running.• Attempting to transfer data while an

array plate is being scanned (imaged).

Restart the computer and unload all of the plates.• Plates in Hyb station: finish hybridization off-

line.• Plate in Scanner: rescan using Scan Only

function• Plate in Fluidics: use Wash/Scan Resume to

resume the fluidics process.Do not manually, or through the GCC transfer utility, move any data associated with the current plate that is being processed/scanned.


Chapter 8 TroubleshootingGeneTitan™ Multi-Channel Instrument8

Miscellaneous messages

Hybridization aborted:• System-initiated abort• User-initiated abort

System-initiated abort:• Power lossUser-initiated abort:• User error• Other

Array plate and hybridization tray are still clamped:• Contact your local Field Service Engineer

with information on the workstation model.• The plate stack is moved to drawer 1.• Remove the plate stack and finish

hybridization offline.• Return the hybridized array plate stack to the

GeneTitan Multi-Channel Instrument and finish processing using the Wash/Scan process.

FAILED messages See "GeneTitan MC Instrument messages that appear when the instrument has a fluidics problem" on page 284

FLUIDIC DIAGNOSTIC messages

See "Fluidic diagnostic messages" on page 284.

Fluidics aborted:• System-initiated abort• User-initiated abort

System-initiated abort:• Power lossUser-initiated abort:• Incorrect protocol selected

Follow the recommendations and instructions under "Wash/Scan Resume" on page 288.

Table 87 GeneTitan Multi-Channel Instrument troubleshooting guidelines for the Axiom 2.0 Assay (Continued)

Problem Possible causes Possible actions

Table 88 Miscellaneous messages and recommended actions

Message and recommended action

Indicates that an item is in the gripper, and normal startup of the GeneTitan Multi-Channel Instrument is not possible. The item must be removed from the instrument before you can begin processing array plates.

Recommendation: click Yes.If you click No, nothing will occur. Homing will not complete and you will not be able to use the system.The item held by the gripper will be moved to either:• Drawer 2—plates and trays• Trash Bin—coversThe drawer names will reflect the location (left or right) and the drawer number (1 through 6).Examples: Drawer2L_Hta_DOWN = Scan tray on left side of drawer 2HtaHyb = Clamped hybridization tray and array plateDrawer(n)L/R_Hta_DOWN where n is the drawer number and L or R to indicate the left or right side.The _Hta_ (second term) indicates the item held. An example is drawer1R_HtaHyb_DOWN indicating it is an array plate with a hybridization tray or Drawer2L_ScanHta_Pk_DOWN indicating it is an array plate with a scan tray


Chapter 8 TroubleshootingGeneTitan™ Multi-Channel Instrument 8

The drawer listed in the message is not fully closed. Manually push the drawer back into the instrument until it is fully closed. There are 2 stop positions with audible clicks; push until you hear the second click and the drawer is fully seated.

• Check that the array plate barcode has been entered correctly.

• Ensure that the library files required for the type of array plate you are using have been installed, and are installed in the correct directory.

• Restart the GeneTitan Instrument control software after library files have been installed.

Table 88 Miscellaneous messages and recommended actions (Continued)

Message and recommended action



Fluidic diagnostic messages

Table 89 GeneTitan MC Instrument messages that appear when the instrument has a fluidics problem

Problem and possible causes

Rinse bottle—fluid level too low or bottle empty.

If this message is displayed:• during a water wash step, array processing has

been compromised.• during cleanup, array processing is okay, but

cleanup will not be complete.Always ensure that the GeneTitan bottles containing Wash A and Rinse are above the 50% mark when setting up the system to process an Axiom array plate.All 600 mL of the Wash Buffer B from the Axiom 2.0 reagent Kit should be emptied into the GeneTitan Wash B bottle when setting up the system to process a plate. This ensures that the GeneTitan Wash B bottle is filled to more than the requisite 35% of Wash B bottle volume.

About this message:• BUFFERX = Buffer bottle A, B or Rinse• WASHX = Wash A or B reservoir in the fluidics

station.Recommended actions:• Replenish fluid level in the Rinse or Wash Bottle B

to the 1L mark. Do not overfill.– Only replenish bottles when prompted by the UI.

Replenishing during fluidic processing may cause system malfunction including overflowing inside the system and more problems. The only thing to do while a plate is running is to ensure that bottle caps are secure.

• Replenish fluid level in Wash Bottle A to 2L.• Secure the bottle cap.• Replace the filterInstructions for filter replacement in the GeneTitan™ Multi-Channel Instrument User Guide, Pub. No. 08-0308.If the problem persists, call Thermo Fisher Scientific Support.



The typical cause is an unsecure bottle cap.

If the failure is detected during priming, the instrument will pause and wait for the problem to be corrected.

If the failure is detected during another process, and if the cause is a clogged filter, wait until the end of the run to replace the filter.Instructions for filter replacement in the GeneTitan™ Multi-Channel Instrument User Guide, Pub. No. 08-0306.

When the instrument experiences a loss in Clean Dry Air (CDA) pressure, the software will display the warning message.

When the pressure is detected again, a dialog message confirming the availability of CDA pressure is displayed.

Possible causesVerify that the facility CDA or the portable CDA compressor is in working condition. See the GeneTitan MC Instrument Site Preparation Guide for the portable compressor model that has been validated with the GeneTitan MC instrument.Contact your local Field Service Engineer and notify them about the error message.





Leak DetectedLeak checks are performed at application startup and any time a fluidic process (priming filling draining etc.) is performed. The leak detection is a hard-wired sensor which will shut off fluid flow without software control. Leaks are normally confined to the drip pan located inside the system.

Causes:• System malfunction• User killing the application using task manager

during a fill operation resulting in application exit without stopping flow.

Solution: Contact Support. The system cannot be used for any fluidic processing until this is resolved.

Leak Resolved This message is displayed when the leak is resolved (meaning the sensor LED is again lit up). If the original leak detected message was not acknowledged it will be automatically removed from the GUI and replaced by the following message. It will remain displayed until another leak is detected or the user acknowledges it by pressing OK. To resolve this issue complete the following tasks• Verify all internal and external tubing is connected

and clean• Verify wash reservoirs are clean• Verify all bottle caps are secure and that no bottle

cap is crimping a supply line.• Verify vacuum is working properly• Do not refill bottles or empty waste except when

prompted to by the GeneTitan application.• Contact your facility group to ensure CDA is

supplied to your GeneTitan system.Contact your Thermo Fisher Scientific Field Service Engineer to have the sensor adjusted or replaced if the problem persists even after correcting for the usual causes outlined above.





Filter Error Message: Dispense related check

Filter Error Message: Fill related check

The filters in the GeneTitan fluidics bottles (Wash A, Wash B and Rinse) need to be replaced when the filters are worn out. The software displays warning message boxes for the filter in each reagent bottle when it detects a problem or shows a trend of increased fill times during fluid fill operations. If an error is detected as described above, then a message box titled “Filter Change Required” is displayed along with the information on the specific dispense operation. You should change all 3 filters when a warning is displayed for any 1 of the 3 filters.See the section "Replacing the filter" on page 314 in Appendix F.





Wash/Scan Resume

If a run is aborted during fluidics processing, the instrument will place the aborted array plate into the scan tray. To restart this process, remove the Axiom array plate from the scan tray and place the array in its protective blue base.

The step at which the run was aborted can be identified by:

• Viewing the System Status window if you are aborting the last plate through the fluidics system.

• Initiating the resume process.

1. System Setup tab: Select Wash/Scan Resume

2. Follow the prompts to unload and reload all drawers.

The trays will be loaded. It is up to you to determine whether or not to load fresh reagents or reuse the trays already in the GeneTitan Multi-Channel Instrument. Base your decision upon the step where the problem occurred.

To help ensure that the samples are processed correctly, we recommend that you:

1. Load new stain trays with fresh reagents.

2. Load a new scan tray.

We do not recommend the use of trays without reagents or holding buffers for steps that appear to have already executed.

Resume stepYou must select the step at which you wish to resume plate processing. You can select any step that has not yet been started.

For certain steps, you can enter a duration in seconds (even if the step requires >1 hour to run, you must enter the duration in seconds). You can set a step for less time than normal, but not for longer than the normal duration.

Aborting a run • Abort can take up to 3 minutes if a plate is in the fluidics station. Status window Abort Requested changes to Abort Completed.

• Clamped Array-Plate-Hybridization tray stack that is aborted from the oven or from drawerIN (drawer 6) is moved to drawer 1.

• Proceed as follows:

– Use the Unload Plates option to remove the aborted plate(s).

– Start another run which will force an unload of the aborted plate(s)

System-initiated• Power interruption

• Plate loaded incorrectly

• Equipment malfunction

The system will abort the processing. Follow the instructions displayed in the user interface.

User-initiatedCan abort processing of individual array plates.

If multiple plates are being processed, the gripper may continue to process the remaining array plates.


A Safety

General safety

For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals.

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, etc). To obtain SDSs, see the ʺDocumentation and supportʺ section in this document.

WARNING! The following components contain harmful or toxic ingredients:

• Axiom Stabilize Soln: 8% Gluteraldehyde

• Axiom HybSoln 2: 100% Formamide

• Axiom Hyb Buffer: <55% Tetramethylammonium Chloride

In all cases customers should use adequate local and general ventilation in order to minimize airborne concentrations.


Appendix A SafetyChemical safetyA

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, and 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 ʺDocumentation and supportʺ 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 adequate ventilation (for example, fume hood).

• Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturerʹs 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 necessary) 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.


Appendix A SafetyBiological hazard safety A

Biological hazard safety

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. All work should be conducted in properly equipped facilities using the appropriate safety equipment (for example, physical containment devices). Safety equipment also may 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:www.cdc.gov/biosafety/publications/bmbl5/BMBL.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


http://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf

http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

B Fragmentation quality control gelprotocol

Protocol for running a fragmentation quality control gel

Equipment required

E-Gels and reagents

Consumables

Table 90 Equipment required

Item Supplier Part number

Gel imager Your choice —

Pipette, multichannel or single channel P20 Your choice —

Plate centrifuge Your choice —

Vortex Your choice —

Table 91 E-Gel and reagents required


Mother E-Base Device

Life Technologies(formerly

Invitrogen)

EB-M03

Daughter E-Base Device EB-D03

E-Gel® 48 4% agarose gels G8008-04

TrackIt 25 bp DNA Ladder 10488-022

TrackIt Cyan/Orange Loading Buffer 10482-028

Nuclease-free Water Your choice —

Table 92 Gel and reagents required


Adhesive film – use 1 of the following:• MicroAmp Clear Adhesive Film• Microseal 'B' Film

Thermo Fisher ScientificBio-Rad

4306311MSB1001

Pipette Tips Same brand as pipette —


Appendix B Fragmentation quality control gel protocolProtocol for running a fragmentation quality control gel B

Diluting the TrackIt™ Cyan/Orange Loading Buffer

The following recipe is for preparing a 1000-fold dilution of the TrackIt Cyan-Orange Loading Buffer.

To dilute the TrackIt Cyan/Orange Loading Buffer:

1. Add 50 µL of TrackIt Cyan/Orange Loading Buffer to 49.95 mL nuclease-free water.

Total volume 50 mL.

2. Vortex tube to mix well.

3. Store at room temperature.

To dilute the TrackIt 25bp Ladder:

The following recipe is for preparing a 15-fold dilution of the Invitrogen TrackIt 25 bp DNA Ladder.

1. In a 1.5 mL microcentrifuge tube, add 6 µL of TrackIt 25 bp Ladder to 84 µL nuclease-free water. Total volume: 90 µL.

2. Vortex tube to mix well. Centrifuge to get droplets down.

Note: The recipe has enough volume to fill 4 marker wells of 1 E-Gel® 48 4% agarose gel. Scale up as needed if running multiple gels.

Fragmentation QC gel protocol

This protocol is based on running QC gels for 96 samples.

To run a fragmentation QC gel:

1. Tightly seal the gel QC plate prepared during automated target preparation.

2. Vortex the center of the plate for 3 sec. Centrifuge to 1,000 rpm to get droplets down.

3. Connect an E-Base™ device(s) to an electrical outlet.

4. Push the Power/Prg button on each to ensure the program is in EG mode (not EP mode).

5. Take the gel out of the pouch and remove the combs.

6. Place the E-Gel® 48 gel into an E-Base unit.

7. Load 20 µL from each well of the Gel QC plate onto the gels.

8. Load 15 µL of 15-fold diluted TrackIt 25 bp ladder into the marker wells (M).

9. Load 20 µL nuclease-free water into any unused wells.

10. Run the gels for 22 minutes.

11. Image the gel.

Fragmentation QC gel images should look similar to the gel shown in Figure 153.


Appendix B Fragmentation quality control gel protocolProtocol for running a fragmentation quality control gelB

Figure 153 Example of a typical fragmentation QC E-gel

125 bp

25 bp

25 bp

125 bp

Fragments should fall between 125 bp and 25 bp.

25 bp ladder 25 bp ladder


C Sample quantitation afterresuspension

Protocol for sample quantitation after resuspension

Equipment required The following equipment is required for this protocol.

Quantitate the diluted samples

During target prep, 2 plates of diluted samples are prepared: 1 for OD quantitation and 1 for a QC gel to check the fragmentation reaction.

For OD quantitation, readings should be taken at wavelengths of 260, 280, and 320 nm. See ʺSuggested protocol for OD quantitation using the DTX 880ʺ on page 297 for more information.

To quantitate the diluted samples prepared for OD quantitation:

1. Launch the Multimode Analysis Software.

2. When the Protocol Selection List is displayed, select the appropriate protocol.

3. Right-click the protocol and select Run the selected protocol.

4. In the Result Name field, enter your experiment name.

5. Click the Eject Plate Carrier icon.

6. Load the OD plate onto the DTX 880.

7. Click the Close Plate Carrier icon.

8. Click the Run the Selected Protocol icon at the bottom of the window.

When the protocol is finished running, a list of results is displayed. If you used the formula provided in this appendix, 2 XML files are generated (Figure 154). Open the ResultData file with Microsoft® Excel® to view and assess the OD readings. RawData file information is included in the ResultData file.

Table 93 Equipment required for sample quantitation after resuspension

Quantity Item

1 DTX 880 Multimode Detector with genomic filter slide

Figure 154 List of files that are generated post DTX-880 scan.


Appendix C Sample quantitation after resuspensionProtocol for sample quantitation after resuspensionC

Assess the OD readings

If using the formula provided in this appendix, the raw data is included in the final Result Data file. Figure 155 is an example of a Result Data file. Your OD readings should be similar to those displayed below.

OD yield assessment guidelinesThe measurement of the yield of DNA after resuspension of the pellets is an important QC checkpoint in the Axiom 2.0 Assay. If the median yield for the plate is < 1200 µg DNA per sample:

• Pause the protocol.

• Assess each of the steps performed to that point to determine the possible source of the low yields.

This DNA yield corresponds to an A260 value of approximately 0.59 and an A260-A320 value of approximately 0.50.

Figure 155 Example of Result data file with acceptable OD readings


Appendix C Sample quantitation after resuspensionSuggested protocol for OD quantitation using the DTX 880 C

Suggested protocol for OD quantitation using the DTX 880

The formula suggested below requires 6 passes. The settings and formula are shown below.

Protocol Type—Analysis

General Settings—Enter a name for the protocol


Appendix C Sample quantitation after resuspensionSuggested protocol for OD quantitation using the DTX 880C

Technique Type—Select Absorbance

Labware—x_Abs_Greiner 96 UV clear std (96 Microplate Format)



Layout Settings—as appropriate for 96-array format plates

Method Selection—add (+) the 3 formulas created on the Data Reduction Page to the Group 1 box.



Data Reduction Page—create the formulas required for scans at 260, 280 and 320This protocol consists of 6 passes. Click Add new Pass to create passes 2 through 6, shown in these figures below.



1

k




Appendix C Sample quantitation after resuspensionIf performing sample quantitation on a plate reader other than the DTX880 C

Output Settings—select Export to Microsoft® Excel® and Show Result Viewer

Save the protocol.

If performing sample quantitation on a plate reader other than the DTX880

Your plate reader should be calibrated to ensure accurate readings.

The total yield in µg per well can be calculated as:

(A - C)*D*V*E/P

Where:

A = the observed OD260

C = the observed OD320 (an estimate of a blank reading)

D = 120 (the net dilution factor when preparing the OD Sample plate as described in the Automated Target Preparation Protocol)

V = 115 (the volume of the sample in µL after the resuspension step)

E = 0.05 (the extinction coefficient of duplex DNA at 260 nm)

P = the optical path length for the plate type and plate reader used.

If your plate reader does not record the OD320, the OD260 of a blank solution of water only should be used for the parameter “C” above.

The optical path length is dependent on the type of plate and spectrophotometer used. Check your manufacturerʹs recommendations for the path length for your instrument and plate type or for recommendations on how to measure this quantity. The SpectraMax Plus384, described as an alternative spectrophotometer in the Assay 96-Array Format Automated Workflow for Beckman Biomek FXP Site Preparation Guide, Pub. No. 702984, can employ an automated path length detection system. Consult this instrument’s manual for more information.

The resulting yield calculations can be compared against the typical yields shown in column H of Figure 155 on page 296 and against ʺOD yield assessment guidelinesʺ on page 296.


D Registering samples in GeneChip™

Command Console™

Creating a GeneTitan™ Array Plate Registration file

A GeneTitan Array Plate Registration file is a Microsoft® Excel® spreadsheet that includes information on the samples you are processing on a single array plate. This information includes the array plate format, the array plate barcode, and sample file names so that you can track the samples that are loaded onto a particular array plate.

The version of Microsoft Excel must be 1997-2000 (file extension is .xls; not .xlsx).

To create a GeneTitan Array Plate Registration file:

1. In GCC Portal, open the Samples menu and select GeneTitan Array Plate Registration (Figure 156).

Figure 156 Selecting GeneTitan Array Plate Registration


Appendix D Registering samples in GeneChip™ Command Console™

Creating a GeneTitan™ Array Plate Registration file D

2. Select the array plate to be processed on the GeneTitan MC Instrument (Figure 157 on page 305).

a. Select the array plate type.

b. Click Download.

3. Complete the registration file as follows:

a. Click the Microsoft Excel box on the bottom bar of the monitor to open the Excel spreadsheet.

b. Enter a unique name for each sample (Sample File Name) and any additional information you would like to include (Figure 158).

c. Do one of the following:

• If you are ready to load the array plate onto the GeneTitan MC Instrument, scan the array plate barcode and proceed to the next step.

• If you are not ready to load the array plate onto the GeneTitan MC Instrument, proceed directly to the next step.

4. Save the file as follows:

a. Open File Save As.

b. Enter a name for the array plate registration file.

c. Click Save.

By default, the file is saved in the Affymetrix_Downloads folder.

Figure 157 Selecting the type of array plate to be processed

Figure 158 Entering sample information into an Array Plate Registration file


Appendix D Registering samples in GeneChip™ Command Console™

Creating a GeneTitan™ Array Plate Registration fileD

5. When ready to load the array plate onto the GeneTitan MC Instrument:

a. Click the Browse button, navigate to the file, and click Open.

b. Scan the array plate barcode if not already scanned.

c. Click the Upload button (Figure 159), wait for the information to load, then click the Save button located at the bottom of the next page that is displayed.

If the samples are successfully registered, the message in Figure 160 is displayed.

Figure 159 Uploading the Array Plate Registration file to GCC

Figure 160 Array plate samples successfully registered


E Deionization procedure forGeneTitan™ trays and covers

We recommend the use of the Zerostat 3 Anti-Static Gun (Cat. No. 74-0014) to deionize GeneTitan™ MC Instrument stain tray trays and lids.

Deionize the inner surface of each tray and cover:

• The surface of the tray with the wells that will hold reagents.

• The surface of the cover that will face the reagents.

IMPORTANT! Except for the Axiom™ array plates, scan tray and the hybridization tray, you must deionize all GeneTitan stain trays, stain tray covers and scan tray cover using an anti-static gun. You must do this before you fill the trays with reagents and before you place the covers on the trays. Deionization removes the static electricity. The presence of static electricity on the underside of the cover can cause the gripper to lift the tray along with the tray cover and can result in an aborted run. See Figure 161, Figure 162 and Figure 163.

CAUTION! Do not deionize the scan tray or hybridization tray.

Figure 161 Scan tray with cover. Deionize only the cover.


Appendix E Deionization procedure for GeneTitan™ trays and coversDeionization procedureE

Deionization procedure

The following process provides guidance on how to use the anti-static gun on the stain and scan tray covers only. See Figure 163.

1. Treat the plate or lid as if it were divided into 6 sections (see Figure 163), and deionize as follows.

2. Place a Kimwipes laboratory tissue on the benchtop.

3. Place the stain tray on a table top. Use the anti-static gun to aim at the center of each of the 6 sections on a 96-well tray and pull the trigger. Ensure that a stream of ionized particles settles on all wells of the stain tray to dissipate the static electricity. Squeeze and release the trigger slowly 3 times over each section (Squeeze for approximately 2 seconds and release for approximately 2 seconds).

4. Place the stain tray cover with the flat surface facing upward on the Kimwipes laboratory tissue.

5. Aim the anti-static gun (Cat. No. 74-0014) approximately one-half inch away from the flat surface and pull the trigger. As you pull the trigger move the gun across the cover so that the stream of ionized particles settles on all areas of the cover and dissipates the static electricity. Squeeze and release the trigger slowly 3 times over each section (squeeze for approximately 2 seconds and release for approximately 2 seconds).

Figure 162 Stain tray with cover. Deionize the cover and the tray.

WARNING! The deionization steps 4 and 5 will damage the Axiom arrays on the plate. Before using the anti-static gun, ensure that the Axiom array plates remain in their protective pouch and placed away from the deionization area. You must place the scan tray and hybridization tray away from the area where you are performing deionization.


Appendix E Deionization procedure for GeneTitan™ trays and coversDeionization procedure E

6. Place the treated cover or tray on the tissue and lift it up (see Figure 163).


• If the tissue does not cling to the plastic, proceed with the protocol.

• If the tissue still clings to the plastic, then perform steps 3 and 4 again. If it continues to cling to the plastic, test the device using the ion-indicator cap to confirm that the unit is still releasing ions. Otherwise, it may be time to replace the unit.

Figure 163 Removing the static charge from stain trays and lids.

Treat the inside surface of stain trays (right) and cover (left).

• If a tissue clings to treated surface, try the deionization procedure again. • If the tissue still clings, it may be time to replace the anti-static gun.

1 2

3 4

5 6


Appendix E Deionization procedure for GeneTitan™ trays and coversIon-indicator capE

Ion-indicator cap

The ion-indicator cap is a testing device used to verify the release of ions when the anti-static gun is in use (Cat. No. 74-0014, Figure 163).

Testing the Zerostat 3 with the ion-indicator cap

1. Insert the ion-indicator cap into the nose of the Zerostat and then slowly squeeze the release trigger (see Figure 164).

2. Observe the discharge through the viewing slot on the ion-indicator cap of the anti-static gun. A visible light is observed in the viewing window on the cap when charged ions are discharged.

3. If you cannot see the light, the gun may be unusable and you should replace it.

4. Each Zerostat anti-static gun is capable of 50,000 trigger operations, which is sufficient for approximately 200-250 runs on the GeneTitan MC Instrument.

Figure 164 Zerostat 3 anti-static gun (Cat. No. 74-0014) with ion-indicator cap to test functionality

IMPORTANT! Ensure to remove the cap from the gun before deionizing a tray or cover.

Ion-indicator cap

The Ion-Indicator Cap is attached to the Zerostat to test the functionality of the anti-static gun.

IMPORTANT: Do not leave the Ion-Indicator Cap on the Zerostat gun when deionizing trays and lids.


F GeneTitan™ Multi-ChannelInstrument Care

Cleaning and maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311

Servicing the outer enclosure fan filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312

Replacing the bottle filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313

Replacing the xenon lamp in the GeneTitan™ MC Instrument . . . . . . . . . . . . . 315

Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320

This chapter provides instructions on caring for and maintaining the instrument and on troubleshooting if problems arise.

• Always run a Shutdown protocol when the instrument will be off or unused overnight or longer. This will prevent salt crystals from forming within the Fluidics system.

• Always use deionized water to prevent contamination of the lines. Swap out old buffers with freshly prepared buffer at each system startup.

The GeneTitan™ Instrument should be positioned on a sturdy level bench away from extremes in temperature and away from moving air.

Cleaning and maintenance

The GeneTitan family of instruments require little in the way of customer maintenance. The instruments must be kept clean and free of dust. Dust buildup can degrade performance. Wipe the exterior surfaces clean using a mild dish detergent solution in water. Do not use ammonia based cleaners or organic solvents such as alcohol or acetone to clean the system because they may damage the exterior surfaces.

The following tasks should be performed regularly to ensure the Imaging Device remains in working order.

Monthly Wipe down the outer surface of the Imaging Device with a dry cloth.

Every 6 months Replace the cooling fan air filters at the rear of the instrument.

Replace the Micropore filters in the Wash A, Wash B, and Rinse bottles. If you run 4-8 plates/week then the micro-pore filters need to be replaced more frequently.

IMPORTANT! Before performing maintenance turn off power to the instrument to avoid injury in case of an electrical malfunction.


Appendix F GeneTitan™ Multi-Channel Instrument CareServicing the outer enclosure fan filtersF

Servicing the outer enclosure fan filters

Cleaning schedule The GeneTitan fan filter cartridge (Figure 165) should be cleaned at least every 90 days of service. Note that in some service locations, the presence of excessive dust or particulate matter may necessitate cleaning the cartridge more often than 90 days.

A plugged filter cartridge can cause excessive temperatures within the machine that can cause unwanted evaporation of GeneTitan reagents.

Part details for GeneTitan fan filter:Thermo Fisher Scientific Cat. No. 01-0669

Number of filters required per GeneTitan instrument: 3

Cleaning procedure 1. Slide the filter cartridge from the fan filter cartridge at the rear of the GeneTitan MC Instrument.

2. Submerse in clean DI water. Rinse and agitate gently to dislodge material.

3. Remove from water and dry with clean compressed air or towels.

4. When the filter cartridge is completely dry to the touch, re-install the cartridge.

Figure 165 The GeneTitan filter cartridge


Appendix F GeneTitan™ Multi-Channel Instrument CareReplacing the bottle filters F

Replacing the bottle filters

The bottles used in GeneTitan MC Instrument contain a filter to remove particulates that may exist in the buffers and DI water. The filters in the GeneTitan fluidics bottles (Wash A, Wash B and Rinse) need to be replaced when the filters are clogged.

The message boxes displayed in Figure 166 will provide information on fluid dispense errors that were detected by the instrument for any of the bottles or when the instrument detects an increase in the amount of time that is required to perform the fill operations.

If an error is detected as described above, then a message box titled “Filter Change Required” is displayed (Figure 166) along with the information on the specific dispense operation. You should change all 3 filters when a warning is displayed for any 1 of the 3 filters.

Note: The reagent bottles are depressurized when this warning message is displayed. It is safe to change the filters in all 3 fluidic bottles when this message is displayed.

After changing the filters in all 3 bottles using the procedure described below, press the Yes button to continue. If you choose to ignore the error message, press the No button. This warning message will be displayed each time GCC instrument control software is launched. You may also experience data quality issues if particulate matter cannot be trapped by the filters because they are clogged.

Figure 166 Filter Change Required messages


Appendix F GeneTitan™ Multi-Channel Instrument CareReplacing the bottle filtersF

We recommend that your site keep 3 spare filters on hand in the event the filters need to be replaced. The procedure for replacing the filters is simple.

GeneTitan reagent bottle filters part details:Thermo Fisher Scientific Cat. No. 01-0671

Removing and inspecting the filter

1. Loosen and remove the cap on the bottle.

2. Carefully remove the filter from the end of the filter body.

3. Visually inspect the filter. If one of the filters appears to have a concentration of dirt or contaminate in it, discard it and replace the filter with a new one.

Replacing the filter 1. Insert the filter into the end of the filter body.

2. Replace the cap onto the bottle and tighten it.

3. Repeat for each bottle.

Figure 167 Replacing the filter

Buffer supply line

Filter holder

Filter

1

2

3

1 2 3

IMPORTANT! Replace one filter at a time to ensure the correct connection of the buffer supply tube to its respective bottle. The color of the buffer supply tubing matches the bottle color code.


Appendix F GeneTitan™ Multi-Channel Instrument CareReplacing the xenon lamp in the GeneTitan™ MC Instrument F

Replacing the xenon lamp in the GeneTitan™ MC Instrument

This section applies to your site only if you have the GeneTitan Multi-Channel (MC) instrument. After the normal life expectancy of the lamp has expired, the software application will alert you to the requirement to replace the lamp. This procedure is simple but you must follow good health and safety precautions.

GeneTitan xenon lamp catalog number: Thermo Fisher Scientific 01-0740

Lamp life/imaging device status notices

The Imaging Status pane displays lamp life and Imaging Device status notices for the GeneTitan MC Instrument.

In normal operation, the pane displays the hours of life left in the lamp (Figure 168):

It displays a red or yellow notice when the lamp life is getting short (Figure 169):

It also displays a red notice when the Imaging Device is offline (Figure 170):

Note: The 300 watt xenon lamp in the GeneTitan MC Instrument is warranted for 500 hours. The instructions to replace the lamp are available on the following page. After changing the lamp, it is necessary to reset the lamp life clock manually.

IMPORTANT! Do not try to replace the lamp when a plate is being processed either in the fluidics or scanner system.

Figure 168 Lamp Life above tolerance

Figure 169 Lamp Life above tolerance

Figure 170 Imaging Device offline

WARNING! You must turn off the lamp using the power switch in the rear of the unit and remove the power cord. Allow the lamp to cool before attempting to replace the lamp.


Appendix F GeneTitan™ Multi-Channel Instrument CareReplacing the xenon lamp in the GeneTitan™ MC InstrumentF

Removing the xenon lamp

1. Unscrew the 4 retaining bolts. They should be finger tight (Figure 171).

2. Remove and set aside the warning cover to reveal the xeon lamp contained within.

3. Place each hand on each side of the blue plastic flange and lift out the lamp in a vertical motion (Figure 172). You must use both hands to remove the lamp successfully. Apply equal pressure on each side of the lamp and gently lift.

Figure 171 Unscrewing the Bolts

Figure 172 Lifting out the lamp

Unscrew these 4 bolts.



Replacing the lamp

1. Hold the lamp by the blue plastic flanges. Ensure that the lamp bulb faces inward toward the reflecting mirror (Figure 173) and vertically insert the lamp (Figure 174).

2. Replace the warning cover and hand tighten the bolts (Figure 171).

CAUTION! Ensure that you install the lamp in the correct orientation.

Figure 173 The reflecting mirror

Reflecting mirror


Appendix F GeneTitan™ Multi-Channel Instrument CareReplacing the xenon lamp in the GeneTitan™ MC InstrumentF

Figure 174 Inserting the lamp

IMPORTANT: The lamp bulb faces away from the fan and toward the reflecting mirror.



Resetting the lamp counter

You must alert the software application that you have replaced the lamp so that the hours of the lamp counter are reset to zero. This menu option is only available when the system is not processing any plates.

1. On the software application click Tools Reset Counter for Life Remaining (Figure 175).

2. The software will display a message that asks you to confirm the lamp life counter is being reset as a result of lamp replacement (Figure 176).

Figure 175 Inserting the lamp


Appendix F GeneTitan™ Multi-Channel Instrument CareTroubleshootingF

3. Click Yes if you want to reset the counter. The software will display a message that confirms that the software has reset the counter (Figure 177).

Troubleshooting

This section provides instructions on how to identify and solve simple problems with the GeneTitan MC Instrument. If a problem or error occurs that is not listed in this chapter contact Thermo Fisher Scientific Technical Support for assistance.

For software errors that do not involve hardware crashes the most common solution is to shut down the application and then restart it. If the same error occurs shut down both the application and the computer and then restart. If it still occurs shut down the GeneTitan MC Instrument and then restart.

Log files The log files are produced by different GCC components. The logs provide a record of the tasks performed by different components, such as the migration tools and installer. These log files provide useful information for troubleshooting problems. These files may be requested by your Field Application Scientist (FAS), Field Service Engineer (FSE), or the call center.

Figure 176 Are you sure?

Figure 177 The counter is reset.


Appendix F GeneTitan™ Multi-Channel Instrument CareTroubleshooting F

GCC log filesThe following files are generated by the GeneTitan Instruments. All the GCC log files are from the following path: C:\Command_Console\Logs. The different log files include:

Other GCC filesYour FAS and/or FSE may request you to send the following files for troubleshooting:

1. Library files (*.PARAMS, *.MASTER, *.WORKFLOW, *.SMD, *.MEDIA) located in C:\Command_Console\Library, excluding the large analysis library files (CDF, PSI, GRC).

2. Provide a list of all sub folders and their contents under the library files folder located in C:\Command_Console\Library. Ensure there are no duplicate library files, as these can cause problems.

3. GCC system configuration file located at C:\Command_Console\Configuration\Calvin.System.config

4. Pending job order files located in C:\Command_Console\Jobs

5. Other GCC related information, such as:

a. The number of files under C:\Command_Console\Data, including sub directory.

b. If the system is a networked system or a standalone system.

c. Other applications installed on the system, such as antivirus application, MS Office, and Internet Explorer versions.

GCC log files for GeneTitan™ MC Instrument Systems

Log files for the GeneTitan MC Instrument control processes are placed in subdirectories of the C:\Command_Console\Logs\ folder. Thermo Fisher Scientific may need the following files for troubleshooting:

GeneTitan MC Instrument fluidics

1. C:\Command_Console\Logs\96F\

a. Subdirectories named by date (e.g., Log7-29-2009)

• Collect all dated directories and contents since the GeneTitan application was started, not just the date of the event (some logging goes into files from the date the application started so this can be critical for us).

• Absolutely required are all the log directories from the date the run was started to the date of the event.

2. C:\Command_Console\Logs\96F\FluidicErrorLog - all files in this directory.

Systemlog.XML XML file with system information.

DEC.log Text file with information on the use of the Data Exchange Console (DEC).

DECError.log Text file with information on errors created while using DEC.

AGCC_LibFileImporter. log (with date and time code)

Text file with info on use of the Library File Importer.


Appendix F GeneTitan™ Multi-Channel Instrument CareTroubleshootingF

GeneTitan MC Instrument imaging device

1. C:\Affymetrix\GeneChipHTScanControlMC\Log - collect all dated directories and contents since the GeneTitan application was started.

2. C:\Affymetrix\GeneChipHTScanControlMC\RunLog - collect all dated directories and contents since the GeneTitan application was started.

Insufficient disk space notice

If there is not enough memory on the computer’s drives to save the data from an array plate, a notice appears (Figure 178) when:

• you first initialize the software and instrument.

• you select arrays for imaging.

If you see this notice, you will need to free up sufficient disk space before imaging starts.

Figure 178 Insufficient disk space notice


Documentation and support

Related documentation

Table 94 Documents related to the Axiom™ 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP

Document Publication number Description

Axiom™ 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP Site Preparation Guide

702984 Provides guidance on reagents, instruments, and supplies required to run the Axiom 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP.

Axiom™ 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP (Windows XP) QRC

702962 An abbreviated reference for the target preparation step of the Axiom 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP running on the Windows XP operating system. This quick reference document is intended for experienced users.

Axiom™ 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP (Windows® 7) QRC

703369 An abbreviated reference for the target preparation step of the Axiom 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP running on the Windows 7 operating system. This quick reference document is intended for experienced users.

Axiom™ 2.0 gDNA Sample Prep Protocol QR

702987 An abbreviated reference on the genomic DNA sample preparation protocol.

Axiom™ gDNA Sample Prep for Genome-Wide BOS 1 Array Plate QR

702975 An abbreviated reference on the genomic DNA sample preparation protocol for the Genome-Wide BOS 1 Array Plate.

GeneTitan™ MC Protocol for Axiom™ 2.0 Array Plate Processing QR

702988 An abbreviated reference for processing Axiom 2.0 array plates with the GeneTitan Multi-Channel Instrument.

GeneTitan™ Multi-Channel Instrument User Guide

08-0308 The GeneTitan Multi-Channel (MC) Instrument automates array processing from target hybridization to data generation by combining a hybridization oven, fluidics processing, and state-of-the art imaging device into a single bench-top instrument. This document detailing the use, care, and maintenance for the GeneTitan MC Instrument.

GeneTitan™ Multi-Channel Instrument Site Preparation Guide

08-0305 Provides guidance on creating and maintaining the proper environment required for the GeneTitan Multi-Channel Instrument.


Documentation and supportRelated documentation

Analysis and Software

Axiom™ Genotyping Solution Data Analysis Guide

702961 This guide provides information and instructions for analyzing Axiom genotyping array data. It includes the use of Axiom™ Analysis Suite, Applied Biosystems Microarray Power Tools (formerly APT) and SNPolisher R package to perform quality control analysis (QC) for samples and plates, SNP filtering prior to downstream analysis, and advanced genotyping methods.

Applied Biosystems ™ GeneChip™ Command Console™ Software User Guide

702569 This user guide provides instructions on using Applied Biosystems GeneChip Command Console Software (GCC) used to control GeneChip instrument systems. Command Console Software provides an intuitive set of tools for instrument control and data management used in the processing of GeneChip Arrays.

Axiom™ Analysis Suite User Guide 703307 This user guide provides instructions on using Axiom™ Analysis Suite—a single-source software package to enable complete genotyping analysis of all Axiom arrays.

Axiom 2.0 Assay Manual Protocol

Axiom™ 2.0 Assay 96-Array Format Manual Protocol User Guide

702990 This user guide provides comprehensive instructions on running the Axiom 2.0 Assay 96-Array Format Manual Protocol.

Axiom™ 2.0 Assay 96-Array Format Manual Protocol Site Preparation Guide

702991 Provides guidance on reagents, instruments, and supplies required to run the Axiom 2.0 Assay 96-Array Format Manual Protocol.

Axiom™ 2.0 Assay 96-Array Format Manual Protocol QRC

702989 An abbreviated reference for the target preparation step of the Axiom 2.0 Assay 96-Array Format Manual Protocol. This quick reference document is intended for experienced users.

Beckman Coulter documents

Biomek® Liquid Handler User’s Manual 987834 This document is installed at the same time as the Biomek FXP Target Prep Express software. To access, click Start All Programs Beckman Coulter Manuals.

Biomek® Software User’s Manual B30026AA This document is installed at the same time as the Biomek FXP Target Prep Express software. To access, click Start All Programs Beckman Coulter Manuals.

Table 94 Documents related to the Axiom™ 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP

Document Publication number Description


Documentation and supportCustomer and technical support

Customer and technical support

Visit thermofisher.com/support for the latest in services and support, including:

• Worldwide contact telephone numbers

• Product support, including:

– Product FAQs

– Software, patches, and updates

• Order and web support

• Product documentation, including:

– 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 found on Life Technologies’ website at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, please contact Life Technologies at thermofisher.com/support.


http://thermofisher.com/support

http://www.thermofisher.com/us/en/home/global/terms-and-conditions.html

http://www.thermofisher.com/us/en/home/global/terms-and-conditions.html


References

Hindorff LA, Junkins HA, Mehta JP, and Manolio TA. A Catalog of Published Genome-Wide Association Studies. Available at: www.genome.gov/gwastudies. Accessed 09/28/2009.

Klein RJ, Zeiss C, Chew EY, et al. Complement factor H polymorphism in age-related macular degeneration. Science 2005, 308:385–89

Manolio T.A. and Collins F.S. The HapMap and Genome-Wide Association Studies in Diagnosis and Therapy. Annu Rev Medicine 2009, 60:443–56


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04 September 2018


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