Thermo Fisher Scientific Axiom 2.0 Assay 24-Array Format User guide

Brand: Thermo Fisher Scientific

Model: Axiom 2.0 Assay 24-Array Format

Type: User guide

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

Axiom™ 2.0 Assay 24-Array Format Manual

Workflow

USER GUIDE

for use with

Axiom™ 2.0 Reagent Kit 4x24 Reactions

Axiom™ 24F Array Plate

Catalog Numbers 902798

Publication Number 703335

Revision 5

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

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

DISCLAIMER

TO THE EXTENT ALLOWED BY LAW, 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. QIAGEN and REPLI-g are registered trademarks of

QIAGEN. ABgene is a trademark of Thomas Scientific. Allegra is a trademark of Beckman Coulter, Inc. BINDER is a trademark of GINDER GmbH. Bio-Rad,

Microseal, DNA Engine Tetrad, DNA Engine, and Hard-Shell are registered trademarks of Bio-Rad Laboratories, Inc. Diversified Biotech is a trademark of

Diversified Biotech. Eppendorf is a registered trademark of Eppendorf AG. Jitterbug is a trademark of Boekel Scientific. Microsoft, and Excel are either registered

trademarks or trademarks of Microsoft Corporation in the United States and/or other countries. Molecular Devices and SpectraMax are trademarks of Molecular

Devices Corporation. Sigma-Aldrich is a registered trademark of Sigma-Aldrick, Inc. VWR and Pipet-Aid are trademarks of VWR International, LLC..

Manufacturer:

Thermo Fisher Scientific Baltics UAB

V.A. Graiciuno 8, LT-02241

Vilnius, Lithuania

Products:

Axiom™ 2.0 Reagent Kit 4x24 Reactions

Manufacturer:

Affymetrix Pte Ltd

7 Gul Circle #2M-01

Keppel Logistics Building

Singapore 629563

Products:

Axiom™ 24F Array Plates

Axiom™ Microbiome Array Plates

Axiom™ myDesign™ Array Plates

Revision history of Pub. no. 703335

Revision Date Description

5 3 March 2022 • Added MicroAmp EnduraPlate™ 96 Half Skirted Plate 4483352 or 4483354,

or Bio-Rad HSS9601 as an alternative to Bio-Rad HSS9641.

• Added instructions for using the Applied Biosystems Multiskan™ Sky

Microplate Spectrophotometer for sample quantitation.

4 24 February 2021 Add the following product options:

• Bio-Rad™ HSP9631, HSP9601, HSS9601, and HSS9641 96-well PCR

plates.

• Applied Biosystems™ 25 bp Ladder.

• Applied Biosystems™ ProFlex™ PCR System thermal cycler.

• Sorvall™ Legend XTR Centrifuge (various Cat. Nos.).

• Thermo Scientific™ Digital Microplate Shaker (Cat. No. 88882005 or

88882006).

3 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™ Genomic DNA Standard (REF 103) has been

removed from the reagent kit and is available for purchase separately.

Axiom™ 2.0 Assay 24-Array Format Manual Workflow User Guide 3

Contents

CHAPTER 1 About the Axiom 2.0 Assay. . . . . . . . . . . . . . . . . . . . . . . . 8

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Overview of the Axiom 2.0 Assay 24-Array Format Manual Workflow . . . . . . . . . . . . . . . . . 10

CHAPTER 2 Genomic DNA preparation and requirements. . . . . . . . . 11

Sources of genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

General requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Special requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Assessing the quality of genomic DNA using 1% Agarose E-gels . . . . . . . . . . . . . . . . . . 13

Genomic DNA extraction/purification methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Genomic DNA cleanup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Genomic DNA preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Equipment, consumables, and reagents required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

1. Thaw samples and control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

2. Quantitate and dilute gDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

3. Aliquot the diluted samples and the control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

4. Freeze or proceed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

5. Create a GeneTitan Array Plate Registration file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

CHAPTER 3 Preparation before you start . . . . . . . . . . . . . . . . . . . . . . 21

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Equipment required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Consumables required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

GeneTitan MC Instrument consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Axiom 2.0 Reagent Kit, arrays, and GeneTitan consumables required . . . . . . . . . . . . . . . 31

Requirements and recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Room temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Special requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Plate requirements for target preparation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Contents

4Axiom™ 2.0 Assay 24-Array Format Manual Workflow User Guide

Thermal cycler consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Equipment care and calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Guidelines for handling plates and reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Sample quantitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

About the reagents and master mix preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Pipettes and pipetting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Using the divided reservoir . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Freeze-thaw instructions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Label GeneTitan hybridization and reagent trays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Reagent kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

CHAPTER 4 24-array format manual target preparation. . . . . . . . . . . 42

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Stage 1: DNA amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Input required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Equipment, consumables, and reagents required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

1: Prepare for DNA Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

2: Prepare the Denaturation Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

3: Add Denaturation Master Mix to samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

4: Add Neutralization Solution to samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

5: Prepare the Amplification Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

6: Add the Amplification Master Mix to samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

7: Freeze the reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

8: Freeze or proceed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

Stage 2: Fragmentation and precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

Input required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

Equipment, consumables, and reagents required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

1: Prepare for fragmentation and precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

2: Incubate samples in preheated ovens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

3: Prepare the Fragmentation Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

4: Add the Fragmentation Master Mix to samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

5: Add the Stop Solution to the samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

6: Prepare the Precipitation Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

7: Prepare and add isopropanol to Precipitation Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

8: Freeze the Precipitation Plate overnight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

9: Freeze reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

Stage 3: Centrifuge and dry, resuspend and hybridize preparation, and sample QC . . . . . . 60

Contents

Axiom™ 2.0 Assay 24-Array Format Manual Workflow User Guide 5

Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Input required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Equipment, consumables, and reagents required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

Stage 3A: Centrifuge the Precipitation Plate and dry the DNA pellet . . . . . . . . . . . . . . . . . . 63

Stage 3B: Resuspension and hybridization preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

1: Prepare for resuspension and hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

2: Prepare DNA pellets and warm the resuspension buffer . . . . . . . . . . . . . . . . . . . . . . . . 64

3: Thaw and prepare the reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

4: Label tubes and reservoirs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

5: Add resuspension buffer to DNA pellets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

6: Resuspension of DNA pellets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

7: Prepare the Hybridization Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

8: Prepare the Hyb-Ready Sample Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

9: Freeze or proceed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

Stage 3C: (recommended) Perform quantitation and fragmentation QC checks . . . . . . . . . 67

1: Prepare for sample QC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

2: Perform QC Checks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

3: Freeze or proceed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

Stage 4: Denaturation and hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

Required input from previous stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

Equipment, consumables, and reagents required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

1: Prepare for denaturation and hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

2: Prepare hybridization-ready samples that are stored at –20°C . . . . . . . . . . . . . . . . . . . 74

3: Prepare the GeneTitan MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

4: Denature the Hyb-Ready Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

5: Prepare hybridization tray and load into GeneTitan MC Instrument. . . . . . . . . . . . . . . . 76

Stage 5: GeneTitan reagent preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

Equipment, consumables, and reagents required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

1: Prepare for GeneTitan reagent preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

2: Prepare the Stain, Ligation, and Stabilization Master Mixes . . . . . . . . . . . . . . . . . . . . . 86

3: Aliquot master mixes and Axiom Hold Buffer into trays . . . . . . . . . . . . . . . . . . . . . . . . . 89

4: Store remaining reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

CHAPTER 5 Array processing with the GeneTitan MC Instrument . . . 97

Before using the GeneTitan MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

Proper tray alignment and loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

Stain trays and covers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

Email and telephone notifications from the GeneTitan MC Instrument . . . . . . . . . . . . . . 101

GeneTitan MC instrument lamp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

Contents

6Axiom™ 2.0 Assay 24-Array Format Manual Workflow User Guide

Setup options for array plate processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

Aborting a process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

Stage 1: Create and upload GeneTitan Array Plate Registration file . . . . . . . . . . . . . . . . . . 106

Stage 2: Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

Reagents required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

Set up the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

Load an array plate and hybridization tray . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

Load a second array plate and hybridization tray . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

Queue a second plate for scanning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

Status window prompts and actions required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

Stage 3: Ligate, Wash, Stain, and Scan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

Equipment, consumables, and reagents required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

Proper installation of the GeneTitan tray consumables . . . . . . . . . . . . . . . . . . . . . . . . . . 126

Load trays onto the GeneTitan MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

Continuing the workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

Shutting down the GeneTitan MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

CHAPTER 6 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

GeneTitan MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

Miscellaneous messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

Fluidics diagnostic messages. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

Fluidics diagnostic messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

Wash/Scan Resume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

Aborting a run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

APPENDIX A Fragmentation quality control gel protocol . . . . . . . . . 144

Protocol for running a fragmentation quality control gel . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

Equipment required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

E-Gels and reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

Prepare the gel diluent and 25 bp DNA ladder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

Run the fragmentation QC gel protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

APPENDIX B Sample quantitation after resuspension . . . . . . . . . . . 147

Equipment required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

Quantify the diluted samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

Install Axiom™ OD methods on the Multiskan™ Sky Microplate Spectrophotometer . . . . . 148

Use a Multiskan™ Sky session . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

Contents

Axiom™ 2.0 Assay 24-Array Format Manual Workflow User Guide 7

OD yield evaluation guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

Plate reader guidelines for sample quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

APPENDIX C Register samples in GeneChip Command Console . . 155

Creating a GeneTitan Array Plate Registration file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

APPENDIX D Deionize GeneTitan trays and covers . . . . . . . . . . . . . 158

Deionization procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159

Ion-indicator cap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

APPENDIX E GeneTitan MC Instrument care . . . . . . . . . . . . . . . . . . 162

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

Cleaning and maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

Monthly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

Every 6 months . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

Servicing the outer enclosure fan filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

Replacing the bottle filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164

Replacing the xenon lamp in the GeneTitan MC Instrument . . . . . . . . . . . . . . . . . . . . . . 166

Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170

Log files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170

GCC log files for GeneTitan MC systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171

Problems and solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171

Insufficient disk space notice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171

APPENDIX F Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174

Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175

Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176

Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176

Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

8Axiom™ 2.0 Assay 24-Array Format Manual Workflow User Guide

1About the Axiom 2.0 Assay

Introduction

The Axiom™ Solution consists of a technology platform that includes assay

biochemistry, automated and manual target preparation options, multiple array plate

formats, and array processing on the GeneTitan™ MC Instrument. This solution has

applications in human genetics research, basic and applied agriculture research, and

microbiome research.

For

human genotyping 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 database of

validated markers that can be interrogated using the Axiom™ Human Genotyping

Solution. There are multiple arrays available for use with the Axiom Human

Genotyping Solution which leverage the content of this proprietary database.

For agriculture applications, the Axiom™ Agrigenomics Genotyping Solution is

capable of genotyping samples using DNA extracted from leaves and seeds. The use

of DNA microarrays for easy, cost-effective genotyping of single nucleotide

polymorphisms (SNPs) and insertion/deletion polymorphisms (indels) plays an

important role in genotype-trait association studies and marker-assisted selection in

both plant and animal breeding programs.

For microbiome research, Axiom™ Microbiome Solution enables researchers to detect

all known microorganisms in a sample with a single assay. Using Axiom™ assay

biochemistry, Axiom™ Microbiome Solution interrogates non-polymorphic sequences

in both family-conserved and target-specific regions from NCBI database sequences.

Axiom™ Microbiome Array detects over 12,000 species including archaea, bacteria,

fungi, protozoa, and viruses. The array content is sample type agnostic, suitable for

applications in nutrigenomics, agrigenomics, and animal research and modeling.

The Axiom™ 24-Format Solution utilizes the Axiom™ assay biochemistry, array

configuration and processing, and manual target preparation. This solution has

applications in human disease research and basic and applied agriculture research.

The Axiom™ 24-array layout, 96-array layout, and the 384HT-array layout retains full

compatibility with the currently existing Axiom™ instrumentation platform and

downstream data analysis. The Axiom™ 2.0 Assay 24-Array Format Manual

Workflow uses Axiom™ 2.0 Assay 24 Sample Reagents (Table 9 on page 31).

The Axiom™ Solution offers a choice of innovative, predesigned or custom Axiom™

myDesign™ arrays. “Off-the-shelf” human genotyping arrays cover more populations

than any other technology and are available for discovery of disease-associated

markers and drug response variants or for population genetics. These arrays have

optimized designs enabling combined GWAS, replication and fine-mapping in 1

study. For applied agriculture research, predesigned arrays are available for numerous

species. Axiom™ Microbiome Array is available as a predesigned array for the

detection of microorganisms. Predesigned Axiom™ arrays deliver cost-effective

Axiom™ 2.0 Assay 24-Array Format Manual Workflow User Guide 9

Chapter 1 About the Axiom 2.0 Assay

Introduction 1

discovery power with the most recent genomic content. For a complete list of

supporting human genetics research, agrigenomics genotyping, and microbiome

research products, visit our website.

Axiom™ myDesign™ Genotyping Arrays allow you to select your own content,

enabling you to include markers relevant for your specific study. Markers for Axiom™

myDesign™ arrays can be chosen from many sources including, but not limited to,

SNPs from the Axiom™ Genomic Database; sequencing initiatives; or your own

personally selected variants. From whole-genome to targeted variant studies, Axiom™

myDesign™ Arrays enable rapid advances in genetics research.

In summary, the Axiom™ Solution provides:

• Optimized arrays for high coverage, cost-effective genetics and microbiome

studies.

• Automated and manual target preparation, which includes methods for DNA

amplification, fragmentation, purification, and resuspension of the target in

hybridization cocktail.

• Hands-free processing of array plates on the GeneTitan™ Multi-Channel (MC)

Instrument.

• Automated software packages for stream-lined analysis of all Axiom™ arrays.

References 1. Klein RJ, Zeiss C, Chew EY, et al.: Complement factor H polymorphism in age-

related macular degeneration. Science 2005, 308:385–89

2. 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.

3. Paux, E., et al., Sequence-based marker development in wheat: Advances and

applications to breeding. Biotechnology Advances, Corrected Proof, online 1

October 2011. doi:10.1016/ j.biotechadv.2011.09.015

4. Rincon, G., et al.: Hot topic: Performance of bovine high-density genotyping

platforms. Holsteins and Jerseys Journal of Dairy Science 2011, 94: 6116-6121

Chapter 1 About the Axiom 2.0 Assay

Overview of the Axiom 2.0 Assay 24-Array Format Manual Workflow

10 Axiom™ 2.0 Assay 24-Array Format Manual Workflow User Guide

Overview of the Axiom 2.0 Assay 24-Array Format Manual

Workflow

Running the Axiom 2.0 Assay 24-Array Format Manual Workflow requires the

following sets of steps:

1. Genomic DNA preparation, resulting in samples that meet requirements spelled

out in Chapter 2, "Genomic DNA preparation and requirements" on page 11.

2. Manual target preparation of the samples.

See Chapter 4, "24-array format manual target preparation" on page 42.

3. Array processing, done with:

• GeneTitan MC Instrument

• GeneTitan Instrument Control software

• GCC Portal software

See Chapter 5, "Array processing with the GeneTitan MC Instrument" on page 97.

A list of the required equipment and supplies for running the Axiom 2.0 Assay 24-

Array Format Manual Workflow can be found in the Axiom™ 2.0 Assay 24-Array Format

Manual Workflow Site Preparation Guide, Pub. No. MAN0018141.

IMPORTANT! Note for Axiom™ Microbiome Array users: For guidance on DNA

sample preparation and requirements for the Axiom Microbiome Array, see

Section 1, Chapter 2 “DNA Preparation and Requirements” of the Axiom™

Microbiome Solution User Guide (Pub. No. 703408).

Axiom™ 2.0 Assay 24-Array Format Manual Workflow User Guide 11

2Genomic DNA preparation and

requirements

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, use the Axiom™ 2.0 Reagent Kit 4x24 Reactions (Cat. No. 902678) for

Axiom™ genotyping assays, or Axiom Microbiome Reagent Kit 4x24 Reactions (Cat.

No. 902910) for Axiom™ microbiome assays. (Table 9 on page 31).

Axiom™ Genomic DNA Standard (Ref. 103) (Part No. 951957) is available for purchase

separately. This DNA meets the requirements outlined in this chapter, and is included

for use 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 here. However, the reliability of any given result should be

assessed in the context of overall experimental design and goals.

The genomic DNA requirements and preparation are described in the following

sections:

•"Sources of genomic DNA"

•"General requirements" on page 12

•"Genomic DNA extraction/purification methods" on page 14

•"Genomic DNA cleanup" on page 14

•"Genomic DNA preparation" on page 15

Sources of genomic DNA

The following sources of human gDNA have been successfully tested in the

laboratories at Thermo Fisher Scientific for DNA that meets requirements.

• Blood

• Saliva

• Cell line

• WGA preamplified 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 Assay. The REPLI-g™ Kit was used to amplify

IMPORTANT! Note for Axiom™ Microbiome Array users: For guidance on DNA

sample preparation and requirements for the Axiom Microbiome Array, see Section 1,

Chapter 2 “DNA Preparation and Requirements” of the Axiom™ Microbiome Solution

User Guide (Pub. No. 703408).

Chapter 2 Genomic DNA preparation and requirements

General requirements

12 Axiom™ 2.0 Assay 24-Array Format Manual Workflow User Guide

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.

Success with other types of samples depends on quality (degree of degradation, level

of purity, and so on) and quantity of gDNA extracted.

The following sources of animal gDNA have been successfully tested in the

laboratories at Thermo Fisher Scientific and meet the requirements outlined under

"General requirements" on page 12.

• Blood

• Semen

• Nasal swab

• Hair bulbs

• Ear punch tissue

The following sources of plant gDNA have been successfully tested and meet the

requirements:

• Seeds

• Leaves

The following sources of microbial gDNA and cDNA from RNA viruses have been

successfully tested and meet the requirements:

• Stool

Note: DNA derived from formalin-fixed paraffin-embedded (FFPE) blocks should not

be used with this assay.

Success with other types of samples depends on quality (degree of degradation, level

of purity, and so on) and quantity of gDNA extracted.

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 (that is, 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

"Genomic DNA cleanup" on page 14.

• 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.

Axiom™ 2.0 Assay 24-Array Format Manual Workflow User Guide 13

Chapter 2 Genomic DNA preparation and requirements

General requirements 2

Special

requirements

Preamplification area

Precautions 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.

Ideally, this preamplification area would be separate from the amplification staging

area described in Chapter 3, on page 32, however these areas may be combined due to

space and equipment limitations.

Assessing the

quality of genomic

DNA using 1%

Agarose E-gels

We recommend this quality control step to asses the quality of the gDNA prior to

starting the assay.

Equipment and reagents recommended

Unless otherwise indicated, all materials are available through thermofisher.com.

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.

To run a 48-lane 1% agarose E-Gel:

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

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

3. Adjust the run time to ~27 minutes.

4. Insert the 48-well 1% Agarose E-Gels into the slot.

5. Remove the combs.

6. Load 20 µL from the above plate onto two 48-well 1% agarose E-Gels.

Table 1 E-Gel® and reagents required.

Item Source

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 requirements

Genomic DNA extraction/purification methods

14 Axiom™ 2.0 Assay 24-Array Format Manual Workflow User Guide

7. Load 15 µL of diluted High Range DNA Marker (1:3 dilution or ~0.34 X from

stock) into all marker wells (as needed).

8. Fill all empty wells with water.

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

When run time is reached (the ladder band reaches the end of the lane), the system

automatically shuts 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 assay prior to beginning any large scale projects.

Genomic DNA extraction/purification methods

Genomic DNA extraction and purification methods that meet the general

requirements 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 the supernatant and wash the pellet with 80% ethanol.

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

6. Remove the 80% ethanol, then 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).

Figure 1 Gel images showing intact gDNA and degraded gDNA.

Axiom™ 2.0 Assay 24-Array Format Manual Workflow User Guide 15

Chapter 2 Genomic DNA preparation and requirements

Genomic DNA preparation 2

Genomic DNA preparation

This step needs to be done before proceeding with the DNA amplification stages.

The genomic DNA (gDNA) that you 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 is 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. For Axiom Microbiome Arrays, a total of 50 ng of gDNA is

required per array.

To Prepare gDNA:

"1. Thaw samples and control"

"2. Quantitate and dilute gDNA".

"3. Aliquot the diluted samples and the control"

"4. Freeze or proceed"

"5. Create a GeneTitan Array Plate Registration file"

Note: For detection of RNA viruses, RNA must be reverse transcribed to yield input

amenable to Axiom target preparation using the protocol outlined in Section 1,

Chapter 3, of the Axiom™ Microbiome Solution User Guide (Pub. No. 703408).

Duration Allow 30-60 minutes for reagents to thaw and 30 minutes for setup.

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

Stool 20 µL 50 ng 2.5 ng/µL

Chapter 2 Genomic DNA preparation and requirements

Genomic DNA preparation

16 Axiom™ 2.0 Assay 24-Array Format Manual Workflow User Guide

Equipment,

consumables, and

reagents required

Equipment and consumables

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

Reagents

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

Unless otherwise indicated, all materials are available through thermofisher.com.

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 Deepwell plate:

•For Axiom Genotyping: ABgene 96 Square Well Storage; Cat. No.

AB-0932 or Eppendorf 96 Deepwell Plate, 2,000 µL; Eppendorf, Cat.

No. 951033481.

•For Axiom Microbiome: Eppendorf 96 Deepwell Plate, 2,000 µL;

Eppendorf, Cat. No. 951033481.1

1This is the only plate supported for Axiom microbiome use. Using a different plate type

may result in assay failure.

1 Plate centrifuge

1 Plate spectrophotometer (required only if no OD measurements

available for samples)

1 Vortexer

Table 4 Reagents required for Genomic DNA preparation.

Reagent Source

Genomic DNA Standard (includes Axiom™ Genomic DNA Standard (Ref.

103) for use as a positive control), –20°C

951957

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

Positive control gDNA (if genotyping non-Human samples)

No template control (if assaying microbial samples)

Axiom™ 2.0 Assay 24-Array Format Manual Workflow User Guide 17

Chapter 2 Genomic DNA preparation and requirements

Genomic DNA preparation 2

1. Thaw samples

and control

Thaw the following components to room temperature:

• gDNA samples.

• gDNA positive control sample. For human and microbiome studies, use 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 immerse the sample plate

or tube when placing it in the bath.

b. Thaw the sealed sample plate and control sample for a half-hour.

c. Remove the sample plate and/or sample tube from the water bath, then wipe-

dry using laboratory wipes. Ensure that 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

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

concentration.

1. Gently vortex (50% maximum) and centrifuge the gDNA and gDNA positive

control sample (if not using Axiom Reference Genomic DNA 103).

2. Recommendation: quantitate each sample (for example, 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

• 2.5 ng/µL for stool samples

4. Seal, vortex and centrifuge.

3. Aliquot the

diluted samples and

the control

Next, place the samples and control in a deepwell plate for target preparation.

Axiom genotyping

• For Axiom genotyping arrays, use the ABgene 96 Square Well Storage Plate, Cat.

No. AB-0932, or Eppendorf 96 Deepwell Plate, 2,000 µL; Eppendorf, Cat. No.

951033481.

Aliquot diluted samples and control gDNA to columns 5, 7, and 9 of the ABgene 96

Square Well Storage Deepwell Plate or Eppendorf 96 Deepwell Plate:

1. Aliquot 20 µL of each diluted gDNA sample to columns 5, 7, and 9 (this should

be the equivalent of 100 to 200 ng of gDNA, as required by the sample type)

reserving at least 1 empty well if planning to include a positive control.

Note: Thermo Fisher Scientific recommends including at least 1 positive control

on each plate.

2. If including a positive control, aliquot 20 µL of gDNA control to the empty well

reserved in Step 1.

Chapter 2 Genomic DNA preparation and requirements

Genomic DNA preparation

18 Axiom™ 2.0 Assay 24-Array Format Manual Workflow User Guide

3. Seal and centrifuge.

Axiom Microbiome

• For Axiom™ Microbiome Arrays, use the Eppendorf™ 96 Deepwell Plate,

2,000 µL; Cat. No. 951033481. This plate is the only plate supported for Axiom

microbiome use. Using a different plate type may result in assay failure.

Aliquot diluted samples and controls to columns 5, 7, and 9 of the Eppendorf Deepwell

Plate for 24-format manual target preparation

1. Aliquot 20 µL of each diluted gDNA to columns 5, 7, and 9. This should be the

equivalent of 50 ng of gDNA, as required.

2. Aliquot the controls:

• Positive control: Aliquot 20 µL of the Reference Genomic DNA 103 control into

well H09.

• Negative control: Aliquot 20 µL of no template control (elution buffer or

reduced EDTA TE buffer) into well G09.

3. When including cDNA templates (see Section 1, Chapter 3, “cDNA Synthesis for

RNA Samples” in the Axiom™ Microbiome Solution User Guide, Pub. No. 703408)

first transfer 2.5 µL of Reduced EDTA TE Buffer to the sample plate. Then add

17.5 µL of cDNA template generated.

4. Seal and centrifuge.

Note: Thermo Fisher Scientific requires including Axiom Reference Genomic

DNA 103 as a positive control and the use of a no template control on each plate.

Figure 2 Aliquot diluted gDNA samples to columns 5, 7, and 9 only.

123456789101112

Axiom™ 2.0 Assay 24-Array Format Manual Workflow User Guide 19

Chapter 2 Genomic DNA preparation and requirements

Genomic DNA preparation 2

4. Freeze or

proceed

At this point you can:

• Store the gDNA sample plate at –20°C, or

• Proceed to DNA Amplification for manual target preparation. See Chapter 4, "24-

array format manual target preparation" on page 42.

Note: You can leave the sample plate at room temperature if proceeding

immediately to DNA Amplification.

5. Create a

GeneTitan Array

Plate 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 C, "Register samples

in GeneChip Command Console" on page 155. Figure 4 shows an example of a batch

registration file.

1. Open GCC Portal  Samples, then:

a. Select GeneTitan Array Plate Registration.

b. Select 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 is not scanned until you are ready to load the array plate and

samples onto the GeneTitan MC Instrument for processing.

Figure 3 Aliquot diluted samples to columns 5, 7, and 9 only.

123456789101112

REF

103

NTC

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 in the GeneTitan 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 scan the array plate barcode and upload

the file to GeneChip Command Console (GCC).

Chapter 2 Genomic DNA preparation and requirements

Genomic DNA preparation

20 Axiom™ 2.0 Assay 24-Array Format Manual Workflow User Guide

Figure 4 Example of a GeneTitan Array Plate Registration file.

Your specific information is populated here.

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Thermo Fisher Scientific Axiom 2.0 Assay 24-Array Format User guide

Brand: Thermo Fisher Scientific

Model: Axiom 2.0 Assay 24-Array Format

Type: User guide

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Thermo Fisher Scientific Axiom 2.0 Assay 24-Array Format User guide

Thermo Fisher Scientific Axiom 2.0 Assay 24-Array Format User guide

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