Axiom® 2.0 Assay Mini 96-Array Format

Axiom® 2.0 Assay Mini 96-Array Format
Axiom® 2.0 Assay Mini 96-Array Format
Manual Protocol
UserGuide
For Research Use Only. Not for use in diagnostic procedures.
P/N 703434 Rev. 1
2
Trademarks
Affymetrix®, Axiom®, GeneChip®, Command Console®, CytoScan®, DMET™, Eureka™, Eureka Genomics®, Eureka Genotyping™, Expression
Console™, GeneAtlas®, GeneChip-compatible™, GeneTitan®, Genotyping Console™, myDesign™, MyGeneChip™, NetAffx®, OncoScan®,
PharmacoScan™, Powered by Affymetrix™, PrimeView®, and ViewRNA® are trademarks or registered trademarks of Affymetrix, Inc.
Please see www.affymetrix.com/trademarks for a complete list of Affymetrix trademarks.
All other trademarks are the property of their respective owners.
Limited License
Affymetrix hereby grants to buyer a non-exclusive, non-transferable, non-sublicensable license to Affymetrix’ Core Product IP to use the
product(s), but only in accordance with the product labels, inserts, manuals and written instructions provided by Affymetrix. “Core Product
IP” is the intellectual property owned or controlled by Affymetrix as of the shipment date of a product that covers one or more features of
the product that are applicable in all applications of the product that are in accordance with the product labels, inserts, manuals and written
instructions provided by Affymetrix. The license granted herein to buyer to the Core Product IP expressly excludes any use that: (i) is not in
accordance with the product labels, inserts, manuals and written instructions provided by Affymetrix, (ii) requires a license to intellectual
property that covers one or more features of a product that are only applicable within particular fields of use or specific applications, (iii)
involves reverse engineering, disassembly, or unauthorized analysis of the product and/or its methods of use, or (iv) involves the re-use of a
consumable product. Buyer understands and agrees that except as expressly set forth, no right or license to any patent or other intellectual
property owned or controlled by Affymetrix is granted upon purchase of any product, whether by implication, estoppel or otherwise. In
particular, no right or license is conveyed or implied to use any product provided hereunder in combination with a product or service not
provided, licensed or specifically recommended by Affymetrix for such use. Furthermore, buyer understands and agrees that buyer is solely
responsible for determining whether buyer possesses all intellectual property rights that may be necessary for buyer’s specific use of the
product, including any rights from third parties.
Patents
Axiom array plate products may be covered by one or more of the following patents: U.S. Patent Nos. 7,332,273; 7,790,389; 8,114,584;
8,273,304; 8,309,496; 8,501,122 and other U.S. and foreign patents.
The software products utilized in the Axiom Genotyping Solution may be covered by one or more of the following patents: U.S. Patent Nos.
6,090,555; 6,185,561; 6,188,783; 6,223,127; 6,229,911; 6,308,170; 6,361,937; 6,420,108; 6,484,183; 6,505,125; 6,510,391; 6,532,462; 6,567,540;
6,584,410; 6,611,767; 6,687,692; 6,826,296; 6,882,742; 6,965,704; 6,996,475; 7,068,830; 7,130,458; 7,215,804; 7,424,368; 7,634,363; 7,822,555;
7,991,564; 7,992,098; 8,190,373; 8,498,825 and other U.S. and foreign patents.
The array imaging system utilized in the GeneTitan MC Instrument may be covered by one or more of the following patents: U.S. Patent Nos.
5,981,956; 6,171,793; 6,207,960; 6,225,625; 6,490,533; 6,511,277; 6,604,902; 6,650,411; 6,643,015; 6,789,040; 6,813,567; 7,062,092; 7,108,472;
7,130,458; 7,222,025; 7,406,391; 7,689,022; 7,983,467; 7,992,098; 8,208,710; 8,233,735; 8,391,582 and other U.S. and foreign patents.
Copyright
© 2016 Affymetrix, Inc. All rights reserved.
Contents
Chapter 1
The Axiom® Mini 96 Genotyping Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
About the Axiom® Mini 96 Genotyping Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Assay Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Related Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Overview of the Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol . . . . . . . . . . . . . . . 11
Running Multiple Plate Workflows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Safety Warnings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Chapter 2
Genomic DNA Preparation and Requirements . . . . . . . . . . . . . . . . . . . . . . . . . 13
Sources of Genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
General Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Special Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Assessing the Quality of Genomic DNA Using 1% Agarose E-gels . . . . . . . . . . . . . . . . . . . .
Genomic DNA Extraction/Purification Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Genomic DNA Cleanup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Genomic DNA Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment, Consumables and Reagents Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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 Batch Registration File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chapter 3
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Preparation Before You Start . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Axiom® 2.0 Assay Mini 96 Reagent Kit, Arrays, and GeneTitan® Consumables Required . . .
Requirements and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Room Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Special Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Plate Requirements and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Thermal Cycler Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Thermal Cycler Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Oven Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Plate Centrifuge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Plate Shakers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment Care and Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Seal, Vortex, and Spin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sample Quantitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Contents
About the Reagents and Master Mix Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pipettes and Pipetting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Matrix™ 25 mL Reagent Reservoirs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Freeze-Thaw Instructions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment, Consumables, Labware, and Reagents Required . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment Required for Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol . . . . . . .
Consumables Required for Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol . . . . .
GeneTitan® MC Instrument Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Reagents for the Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol . . . . . . . . . . . . .
Chapter 4
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Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation . . . . . . . . . . 40
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Stage 1: DNA Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Input Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment, Consumables and Reagents Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1: Prepare for DNA Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2: Prepare the Denaturation Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3: Add Denaturation Master Mix to Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4: Add Neutralization Solution to Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5: Prepare the Amplification Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6: Add Amplification Master Mix to Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7: Store Remaining Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8: Freeze or Proceed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Stage 2: Fragmentation and Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Input Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment, Consumables and Reagents Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1: Prepare for Fragmentation and Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2: Incubate Samples in Preheated Ovens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3: Prepare the Fragmentation Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4: Add the Fragmentation Master Mix to Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5: Add the Stop Solution to the Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6: Prepare the Precipitation Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7: Freeze the Precipitation Plate Overnight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8: Store Remaining Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Stage 3: Centrifuge and Drying, Resuspension and Hybridization Preparation, and Sample QC
Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Input Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment, Consumables, and Reagents Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Stage 3A: Centrifuge Precipitation Plate and Dry the DNA Pellet . . . . . . . . . . . . . . . . . . . . . .
Stage 3B: Resuspension and Hybridization Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1: Prepare for Resuspension and Hybridization Preparation . . . . . . . . . . . . . . . . . . . . . . . . .
2: Prepare DNA Pellets and Warm the Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3: Thaw and Prepare the Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4: Label Tubes and Reservoirs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Contents
5: Prepare the Hybridization Cocktail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6: Add Hybridization Cocktail to DNA Pellets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7: Resuspension of DNA Pellets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8: Prepare the Hyb Ready Sample Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9: Store Remaining Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10: Freeze or Proceed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Stage 3C: Sample QC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1: Prepare for Sample QC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2: Perform QC Checks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3: Freeze or Proceed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Stage 4: Denaturation and Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Required Input from Previous Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment, Consumables, and Reagents Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1: Prepare for Denaturation and Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2: Prepare Hyb Ready Samples Stored at –20°C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3: Prepare the GeneTitan® MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4: Denature the Hyb Ready Sample Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5: Prepare Hybridization Tray and Load into GeneTitan® MC Instrument . . . . . . . . . . . . . . .
Stage 5: GeneTitan® Reagent Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment, Consumables, and Reagents Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1: Prepare for GeneTitan® Reagent Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2: Prepare the Stain, Ligation, and Stabilization Master Mixes . . . . . . . . . . . . . . . . . . . . . . .
3: Aliquot Master Mixes and Axiom Hold Buffer into Trays . . . . . . . . . . . . . . . . . . . . . . . . .
4: Store Remaining Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chapter 5
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Array Processing with the GeneTitan® Multi-Channel Instrument . . . . . . . . . 91
Before Using the GeneTitan® MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Proper Tray Alignment and Loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Stain Trays and Covers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Email and Telephone Notifications from the GeneTitan® MC Instrument . . . . . . . . . . . . . . . 96
GeneTitan® MC Instrument Lamp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Setup Options for Array Plate Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Aborting a Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Stage 1: Create and Upload Batch Registration File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Stage 2: Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Reagents Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Setup the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Procedure to Clamp a Mini 96-Array Format Plate to Hybridization Tray . . . . . . . . . . . . . . . . 106
Load Axiom® Array Plate and Hyb Tray Onto the GeneTitan® MC Instrument . . . . . . . . . . 106
Load a Second Axiom® Array Plate and Hyb Tray onto the GeneTitan® MC Instrument . . . 112
Queuing a Second Plate for Scanning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Status Window Prompts and Actions Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Stage 3: Ligate, Wash, Stain and Scan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Equipment, Consumables, and Reagents Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Proper Installation of the GeneTitan® Tray Consumables. . . . . . . . . . . . . . . . . . . . . . . . . . 119
Contents
6
Load Trays onto the GeneTitan® MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
Continuing the Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
Shutting Down the GeneTitan® MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
Chapter 6
Manual Target Preparation for Processing Three Axiom® Array Plates
per Week . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Overview of the Three-plate Workflow for Manual Target Preparation . . . . . . . . . . . . . . . . .
Timing Issues for Manual Target Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Timing Issues for GeneTitan® MC Array Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Changing Oven Temperatures for the Three Plate Workflow . . . . . . . . . . . . . . . . . . . . . . .
Thawing Frozen Plates of Amplified DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Manual Target Preparation and Array Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Day 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Day 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Day 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Day 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Day 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chapter 7
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
GeneTitan® Multi-Channel Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Miscellaneous Messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fluidic Diagnostic Messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Wash/Scan Resume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Aborting a Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Appendix A
144
144
144
144
145
145
Sample Quantitation After Resuspension . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Protocol for Sample Quantitation After Resuspension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Quantitate the Diluted Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Assess the OD Readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Suggested Protocol for OD Quantitation Using the DTX 880 . . . . . . . . . . . . . . . . . . . . . . . . .
If Performing Sample Quantitation on a Plate Reader Other than the DTX880 . . . . . . . . . . . .
Appendix C
138
139
140
143
143
Fragmentation Quality Control Gel Protocol . . . . . . . . . . . . . . . . . . . . . . . . . 144
Protocol for Running a Fragmentation Quality Control Gel . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
E-Gels and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Diluting the TrackIt™ Cyan/Orange Loading Buffer and 25 bp Ladder . . . . . . . . . . . . . . . .
Fragmentation QC Gel Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Appendix B
127
128
129
129
130
130
130
132
133
135
137
147
147
147
148
149
155
Registering Samples in Affymetrix GeneChip® Command Console® . . . . . . . 156
Creating a GeneTitan® Array Plate Registration File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
Contents
Appendix D
GeneTitan® Multi-Channel Instrument Care . . . . . . . . . . . . . . . . . . . . . . . . . . 159
Cleaning and Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Monthly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Every Six Months . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Servicing the Outer Enclosure Fan Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cleaning Schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Replacing the Bottle Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Replacing the Xenon Lamp in the GeneTitan® MC Instrument . . . . . . . . . . . . . . . . . . . . . .
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Log Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
AGCC Log Files for GeneTitan® MC Instrument Systems . . . . . . . . . . . . . . . . . . . . . . . . . .
Problems and Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Insufficient Disk Space Notice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Appendix E
7
159
159
159
159
159
161
163
168
168
169
169
170
Contact Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
Chapter 1
The Axiom® Mini 96 Genotyping Solution
About the Axiom® Mini 96 Genotyping Solution
The Axiom® Mini 96 Genotyping Solution is a genotyping technology platform that includes a manual
assay and new array configuration. This solution has applications in applied agriculture research and
human disease research. The Axiom Mini 96 solution offers the capability to genotype approximately
50,000 variants from diploid species or 32,000 variants from polyploid species in combination with a
processing throughput of greater than 3,000 samples per week. The Axiom® Mini 96-array layout retains
full compatibility with the currently existing Axiom instrumentation platform and downstream data
analysis. The Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol uses the Axiom® 2.0 Assay
Mini 96 Reagent Kit.
For agriculture applications, the Axiom Mini 96 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 human disease research applications, Affymetrix conducted an empirical screen of genomic content
from dbSNP (http://www.ncbi.nlm.nih.gov/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
Affymetrix database of validated markers that can be interrogated using the Axiom® 2.0 Assay Mini 96Array Format Manual Protocol.
The Axiom Mini 96 Solution is ideal for screening of large numbers of samples in molecular breeding
programs where turn-around time, accuracy and ease-of-use are all important. The Axiom Mini 96
Genotyping Solution enables manual preparation of DNA target preparation, DNA amplification and
enzymatic fragmentation of post-amplification products for instances when immediate processing of
samples is required. Following target preparation, arrays are processed using GeneTitan® Multi-Channel
(MC) Instrument. The Axiom Mini 96 solution also offers traceability of samples through the use of
barcoded consumables.
The Axiom 2.0 Assay interrogates biallelic SNPs and simple indels in a single, fully automated assay
protocol. Starting with genomic DNA, the samples are processed by performing a 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 hyb-ready targets are then transferred to the Affymetrix 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 Affymetrix Power Tools or Axiom™ Analysis Suite
v2.0 or later.
Chapter 1 | The Axiom® Mini 96 Genotyping Solution
9
Assay Features
The Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol User Guide provides instructions for manual
target preparation and processing of Axiom Mini 96-array format plates on the GTMC.
Target preparation for the Axiom 2.0 Mini 96 manual protocol is done in 96-array format. The hyb ready
target is then hybridized onto a partially populated 384-array format array plate, referred to as Mini 96
layout. The switch from a 96-array format to a 384-array format occurs when the user transfers the
hyb ready samples after thermal denaturation from a 96-array format PCR plate to a 384-array format
Hyb Tray.
Figure 1.1 illustrates the new Mini 96-array format plate layout. The arrays are glued only to the
quadrant 1 positions of a 384 plate. Quadrant 1 refers to the odd column well positions in rows A/C/E/
G/I/K/M/O.
Figure 1.1 Axiom Mini 96-Array Format Plate (bottom view)
Position
A1
This user guide covers the use of new GeneTitan® MC consumables and Affymetrix® GeneChip®
Command Console® v 4.3 or higher (AGCC) for the preparation of the Axiom 2.0 stain reagents. Each
tray has a unique part number and barcode that offers traceability. The new trays have the following labels
and barcodes:
Stain 1 Tray - P/N 501279
Stain 2 Tray - P/N 501394
Ligation Tray - P/N 501398
Stabilizing Tray - P/N 501396
The unique barcodes, in conjunction with the Affymetrix® GeneChip® Command Console® software,
prevents users from making errors when placing the trays in the GeneTitan MC Instrument during
Stage 3 of the array processing with the GeneTitan MC Instrument (Stage 3: Ligate, Wash, Stain and Scan
on page 118).
Chapter 1 | The Axiom® Mini 96 Genotyping Solution
10
When manually plating the GeneTitan stain reagents, it is critical that the trays are filled with the reagent
that corresponds to that particular stain tray. Stain trays filled with the incorrect reagent may lead to
failure of the Axiom assay on the GeneTitan MC Instrument.
After the trays have been prepared, the user must ensure the trays are placed in the appropriate drawer
location in the GeneTitan MC Instrument. Failure to place the proper tray in the correct location results
in an error and the GeneTitan Instrument will not proceed with the processing of the trays. Refer to Load
Trays onto the GeneTitan® MC Instrument on page 120 for detailed instruction.
Affymetrix® GeneChip® Command Console® v 4.3 or higher also offers the facility for queuing a 2nd
plate for scanning before the scan of the 1st plate is complete. The software automatically moves the 2nd
plate into the scanner when the first plate has completed scanning. Refer to Queuing a Second Plate for
Scanning on page 114 for instructions.
Related Documentation








Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol Site Preparation Guide, P/N 703435
Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol QRC, P/N 703436
Axiom® Genotyping Solution Data Analysis Guide, P/N 702961
GeneTitan® MC Protocol for Axiom® 384HT Array Plate Processing QRC, P/N 703164
GeneTitan® Multi-Channel Instrument User Guide, P/N 08-0308
GeneTitan® Multi-Channel Instrument Site Preparation Guide, P/N 08-0305
Affymetrix® GeneChip® Command Console® Software User Guide, P/N 702569
Axiom™ Analysis Suite User Guide, P/N 703307
References
1.
2.
3.
4.
5.
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
Klein RJ, Zeiss C, Chew EY, et al.: Complement factor H polymorphism in age-related macular
degeneration. Science 2005, 308:385–89
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.www.genome.gov/gwastudies. Accessed 09/28/2009.
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
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 | The Axiom® Mini 96 Genotyping Solution
11
Overview of the Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol
Running the Axiom 2.0 Assay Mini 96-Array Format Manual Protocol requires the following sets of
steps:
Genomic DNA Prep: Resulting in samples that meet requirements in Chapter 2, Genomic DNA
Preparation and Requirements on page 13
2. Target Preparation of the samples:
 See Chapter 4, Axiom ® 2.0 Assay for Mini 96-Array Manual Target Preparation on page 40
3. Array Processing, done with:
 GeneTitan® MC Instrument
 GeneTitan® Instrument Control software
 Affymetrix® GeneChip® Command Console® v 4.3 or higher software
A list of the required equipment and supplies for running the Axiom 2.0 Assay Mini 96-Array Format
Manual Protocol can be found in the Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol Site
Preparation Guide, P/N 703435.
1.
Running Multiple Plate Workflows
Affymetrix provides workflows that allow you to run a set of samples and array plates through the
protocol using a minimum of personnel and a fifty-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 hyb tray and array plate can be loaded into the GeneTitan MC Instrument.
These limitations require careful timing. For detailed information, please refer to Chapter 6, Manual
Target Preparation for Processing Three Axiom® Array Plates per Week on page 127.
Safety Warnings
CAUTION: All chemicals should be considered potentially hazardous. Therefore, we
recommend that this product should be handled only by individuals who have been trained
in laboratory techniques and used in accordance with the principles of good laboratory
practice. Wear suitable protective clothing, such as gloves, a lab coat, and safety glasses. Care
should be taken to avoid contact with skin and eyes. In case of contact with skin or eyes, wash
immediately with water. See Safety Data Sheet (SDS) for specific advice.
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.
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. Copies of the Safety Data Sheets for the kit
components are available on the Affymetrix website at www.affymetrix.com.
Chapter 1 | The Axiom® Mini 96 Genotyping Solution
12
Precautions
1.
2.
3.
4.
5.
6.
7.
GENECHIP PROBE ARRAYS AND PLATES ARE FOR RESEARCH USE ONLY; NOT
FOR DIAGNOSTIC PROCEDURES.
Avoid microbial contamination, which may cause erroneous results.
WARNING: All biological specimens and materials with which they come into contact should be
handled as if capable of transmitting infection and disposed of with proper precautions in accordance
with federal, state, and local regulations. This includes adherence to the OSHA Bloodborne
Pathogens Standard (29 CFR 1910.1030) for blood-derived and other samples governed by this act.
Never pipet by mouth. Avoid specimen contact with skin and mucous membranes.
CAUTION: Exercise standard precautions when obtaining, handling, and disposing of potentially
carcinogenic reagents.
Exercise care to avoid cross-contamination of samples during all steps of this procedure, as this may
lead to erroneous results.
Use powder-free gloves whenever possible to minimize introduction of powder particles into sample
or probe array plates.
CAUTION: Use care when handling the Scan Tray as it has protruding guiding posts that may be
sharp and can stick out of the pouch if not handled carefully.
Chapter 2
Genomic 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, you will use the Axiom® 2.0 Assay Assay Mini 96 Reagent Kit, P/N 903013 (sufficient
for two Mini 96-array format plates). These kits contain a tube labeled Genomic DNA (P/N 900421).
The use of at least one positive control DNA sample on each plate is recommended. For human samples,
the Reference Genomic DNA provided in Module 1 can serve as the control. For plant or animal samples,
use a genomic DNA sample that meets the recommended nucleic acid purity and concentration
specifications and is from the same species that is represented on the array. Ideally this control sample
has also demonstrated passing genotyping performance when used in the Axiom Genotyping Solution.
If no DNA control for your specific sample type is available, then the Axiom Reference Genomic DNA
can serve as positive control for the target preparation portion of the assay. 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. The genomic DNA requirements and preparation are described in the
following sections:
 Sources of Genomic DNA
 General Requirements on page 14
 Genomic DNA Extraction/Purification Methods on page 16
 Genomic DNA Cleanup on page 16
 Genomic DNA Preparation on page 17
Sources of Genomic DNA
The following sources of human gDNA have been successfully tested in the laboratories at Affymetrix
for DNA that meets the above requirements.
 Blood
 Saliva
 Cell line
 WGA pre-amplified DNA: Genomic DNA amplified with the REPLI-g® Kit (a whole genome
amplification kit; QIAGEN, P/N 150025) has been tested successfully with the Axiom 2.0 GenomeWide 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 Affymetrix.
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 Requirements
14
The following sources of animal gDNA have been successfully tested in the laboratories at Affymetrix
for DNA that meets the requirements below:
 Blood
 Semen
 Nasal swab
 Hair bulbs
 Ear punch tissue
The following sources of plant gDNA have been successfully tested in the laboratories at Affymetrix for
DNA that meets the requirements below:
 Seeds
 Leaves
NOTE: DNA derived from Formalin-Fixed Paraffin-Embedded (FFPE) blocks should not be used
with this assay.
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 Genomic DNA Cleanup on page 16.
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
Pre-Amplification 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 pre-amplification room or area separate from the main laboratory.
This pre-amplification 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.
Chapter 2 | Genomic DNA Preparation and Requirements
15
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 2.1 E-Gel® and Reagents Required
Item
Supplier
Mother E-Base™ Device
Daughter E-Base™ Device
(optional for running multiple gels in parallel)
E-Gel® 48 1% agarose gels
RediLoad™
E-Gel® 96 High Range DNA Marker
Part Number
EB-M03
Life Technologies
(formerly Invitrogen)
EB-D03
G8008-01
750026
12352-019
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 (RediLoad dye diluted 10-fold with nuclease free water) dye to each
sample.
 Bring each sample to a total volume of 20 μL using nuclease-free 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 spin.
To Run a 48 lane 1% agarose E-Gel:
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. Insert the 48 well 1% Agarose E-Gels into the slot.
4. Remove 2 combs.
5. Load 20 μL onto the 48 well 1% agarose E-Gel.
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 min.
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 2.1 shows gel images of intact gDNA (that is suitable for use in the Axiom 2.0 Assay Mini 96Array Format Manual Protocol) and degraded gDNA samples. Customers whose gDNA is degraded
(similar to the image in Figure 2.1) 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.
1.
Chapter 2 | Genomic DNA Preparation and Requirements
16
Figure 2.1 Gel images showing intact gDNA and degraded gDNA.
Intact Samples
Degraded Samples
10 kb —
4 kb —
2 kb —
0.8 kb —
0.4 kb —
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.
2.
3.
4.
5.
6.
7.
Add 0.5 volumes of 7.5 M NH4OAc, 2.5 volumes of absolute ethanol (stored at –20°C), to gDNA.
Vortex and incubate at –20°C for 1 hr.
Centrifuge at 12,000 xg in a microcentrifuge at room temperature for 20 min.
Remove supernatant and wash pellet with 80% ethanol.
Centrifuge at 12,000 xg at room temperature for 5 min.
Remove the 80% ethanol and repeat the 80% ethanol wash one more time.
Resuspend the pellet in reduced EDTA TE Buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA).
(See the Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol Site Preparation Guide, P/N 703435
for reagents, equipment, labware and consumables for Axiom 2.0 Assay Mini 96-Array Format
Manual Protocol).
Chapter 2 | Genomic DNA Preparation and Requirements
17
Genomic DNA Preparation
This step needs to be done before proceeding with the DNA amplification stage for manual 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. The table below shows the sample input requirements for Axiom 2.0
Assay Mini 96-Array Format Manual Protocol.
Table 2.2 Input Requirements for Axiom 2.0 Assay Mini 96-Array Format Manual Protocol
Sample Type
Volume per Well
(μL)
Input Mass per Well
(ng)
gDNA Concentration
(ng/μL)
Human
8.7
100
11.5
Diploid Plants and Animals
8.7
150
17.2
Polyploid Plants and Animals
8.7
200
23
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 Batch Registration File
Duration
Thirty minutes to an hour for reagents to thaw and half an hour for setup.
Equipment, Consumables and Reagents Required
Equipment and Consumables
The equipment and consumables listed in Table 2.3 are required for this stage.
Table 2.3 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: multi-channel P10 or P20

As required
Pipette tips
1
Plate, deep well: Eppendorf 96 Deep-well Plate, 2000 μL
1
Plate centrifuge
1
Plate spectrophotometer (required only if no OD measurements available for samples)
1
Vortexer
Chapter 2 | Genomic DNA Preparation and Requirements
18
Reagents
The reagents listed in Table 2.4 are required for this stage.
Table 2.4 Reagents Required for Genomic DNA Preparation
Reagent
Supplier
Part Number
—
—
Affymetrix
75793
From the Axiom® 2.0 Assay Mini 96 Reagent Kit

Axiom Reference Genomic DNA, P/N 900421 (use as a positive control if
genotyping Human samples). Located in Module 1, –20°C
User-supplied

Reduced EDTA TE Buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA)

Positive control gDNA (if genotyping non-Human samples)
1: Thaw Samples and Control
Thaw the components listed below to room temperature:
 gDNA samples
 gDNA positive control sample. For human studies, use Axiom Reference Genomic DNA (from the
Axiom 2.0 Assay Mini 96 Reagent Kit)
To Thaw, either:
 Place items on benchtop for one 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 Reference sample for a half-hour.
C. Remove the sample plate and/or sample tube from the water bath and wipe-dry using lab wipes.
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
To Quantitate and Dilute the gDNA:
Gently vortex (50% maximum) and spin the gDNA and control DNA.
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:
 11.5 ng/μL for Human DNA samples
 17.2 ng/μL for diploid plant and animal DNA samples
 23 ng/μL for polyploid plant and animal DNA samples
4. Seal, vortex and spin.
1.
Chapter 2 | Genomic DNA Preparation and Requirements
19
3: Aliquot the Diluted Samples and the Control
Next, the samples and control are placed in a Eppendorf 96 Deep-well Plate, 2000 μL for target
preparation.
Aliquot Diluted Samples and Reference Genomic DNA to the Deep-Well Plate as Follows:
8.7 μL of each diluted gDNA sample (this should be the equivalent of 100-200 ng of gDNA, as
required by the sample type).
2. 8.7 μL of the control DNA.
We recommend including at least one positive control on each plate.
3. Seal and spin.
1.
4: Freeze or Proceed
At this point you can:
 Store the sample plate at –20°C, or
 Proceed to DNA Amplification (see Chapter 4, Axiom® 2.0 Assay for Mini 96-Array Manual Target
Preparation on page 40).
NOTE: You can leave the gDNA sample plate at room temperature if proceeding immediately
to DNA Amplification.
5: Create a Batch Registration File
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 hyb 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 Affymetrix
GeneChip Command Console v4.3 or higher.
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.
See also Figure 2.2 for a screen shot showing an example of a batch registration file.
To Create a Batch Registration File:
1.
Open AGCC Portal  Samples, and select:
A. GeneTitan Array Plate Registration.
B. The array plate format.
C. Click Download.
Enter a unique name for each sample and any additional information.
3. Save the file.
The array plate barcode is scanned when you are ready to load the array plate and samples onto the
GeneTitan MC Instrument for processing. Please refer to Chapter 5, Stage 1: Create and Upload Batch
Registration File on page 100 for information on scanning the barcodes.
2.
Chapter 2 | Genomic DNA Preparation and Requirements
Figure 2.2 Example of a Batch Registration File
20
Chapter 3
Preparation Before You Start
Introduction
This manual assay format allows the user to run the Axiom® 2.0 Assay Mini 96-Array Format Manual
Protocol twice using one Axiom® 2.0 Assay Mini 96 Reagent Kit (P/N 903013). This section provides
information on procedures that are performed multiple times during manual target preparation and on
steps that are critical to the success of the manual target preparation. It is essential that you familiarize
yourself with the information in this section prior to running the Axiom 2.0 Assay Mini 96-Array Format
Manual Protocol.
One key item this manual assay format requires is the use of disposable reservoirs with a “trough within
a trough” design, which maximizes the amount of liquid accessible to pipette tips when using small
amounts of reagent.
A list of all equipment and resources required for the Axiom 2.0 Assay Mini 96-Array Format Manual
Protocol Manual Target Preparation is in the Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol
Site Preparation Guide, P/N 703435.
Axiom® 2.0 Assay Mini 96 Reagent Kit, Arrays, and GeneTitan® Consumables Required
The table below lists the Axiom 2.0 Assay Mini 96 reagents, manual target prep consumables, and
GeneTitan consumables required to process two Axiom Mini 96-array format plates.
Table 3.1 Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol:
Arrays, Reagents, and GeneTitan Consumables Required
Part Number
*
Description
Quantity
Available upon Design
Axiom® Mini 96-Array Format Plate
2
902629
Axiom® 384HT High Volume Consumables Kit (each kit is sufficient for
processing 5 Mini 96-Array Format Plates)
1
903014
Axiom® Mini 96 Consumables Kit for QC
(each kit is sufficient for 10 runs)
1
902986
Axiom® 2.0 Assay Mini 96 Manual Target Preparation Consumables Kit
(each kit is sufficient for preparing 4 Mini 96 target preps)
1
903013
Axiom® 2.0 Assay Mini 96 Reagent Kit*
1
Please refer to Table 3.11 on page 39 for a complete description of the kit components.
Requirements and Recommendations
This section describes requirements and recommendations for facilities and equipment needed to
perform the Axiom® 2.0 Assay for Mini 96-Array Format Manual Protocol.
Room Temperature
When referred to in the Axiom 2.0 Assay Mini 96-Array Format Manual Protocol, room temperature is
18 to 25°C.
Chapter 3 | Preparation Before You Start
22
Special Requirements
Amplification Staging Area
Precautions are required when setting up amplification reactions to avoid contamination with foreign
DNA amplified in other reactions and procedures. It is recommended that pre-amplification reaction set
up is performed in a dedicated amplification staging area separate from the main laboratory.
This amplification staging area should have a dedicated set of pipettes and plasticware. If no dedicated
amplification staging 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.
Fume Hood
At certain steps in the protocol we recommend the use of adequate local or general ventilation to keep
airborne concentrations low.
A fume hood is suggested as a way to achieve the desired concentration. Thus, a fume hood is strongly
recommended for several steps of this assay.
Control Recommendations
A negative control is not required for this assay.
We recommend including one positive control with every set of samples processed. A positive control
(Axiom Reference Genomic DNA 103) is included in the Axiom 2.0 Mini 96 Reagent Kit for human
genotyping array designs.
Plate Requirements and Recommendations
The plates listed below on Table 3.2 are required for performing manual target preparation. These plates
are available in the Axiom 2.0 Assay Mini 96 Manual Target Preparation Consumables Kit (P/N 902986)
and Axiom Mini 96 Consumables Kit for QC (P/N 903014), or purchased individually through the
manufacturer or distributor. Refer to the Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol Site
Preparation Guide, P/N 703435, for more information.
Table 3.2 Sample Plates Required for Axiom 2.0 Assay Mini 96-Array Format Manual Protocol

Plate Description

Bio-Rad Hard-Shell® High-Profile 96-Well Semi-Skirted PCR Plate

Eppendorf 96 Deep-well Plate, 2000 μL

Bio-Rad Hard-Shell® Low-Profile 96-Well Skirted PCR Plate

Greiner Bio-One 96 Well UV-Star® Microplate
Manufacturer/
Distributor
Part Number
Bio-Rad
HSS-9641
Eppendorf
951033481
Bio-Rad
HSP-9631
E&K Scientific
25801
Chapter 3 | Preparation Before You Start
23
Thermal Cycler Recommendations
The following thermal cyclers are recommended:
 Bio-Rad PTC-200, or
 Bio-Rad DNA Engine Tetrad 2 #PTC-0240, or
 ABI 9700 (with gold, sliver, or aluminum block), or
 ABI 2720
IMPORTANT: Always use the heated lid option when programming protocols.
We have verified the performance of this assay using the following thermal cyclers: Bio-Rad PTC-200,
ABI 9700 (with a gold, silver or aluminum block), ABI 2720 and the Bio-Rad PTC-0240. 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 thermal
cycler needs to be programmed with the Axiom 2.0 Denature protocol:
95°C 10 min
2. 48°C 3 min
3. 48°C hold
Use the heated lid option when setting up or running the protocol.
1.
WARNING: Evaporation during denaturation can negatively impact assay performance. Use
the recommended thermal cycler consumables and sealing film to eliminate condensation
and evaporation. For thermal cyclers with variable lid tension (such as the Bio-Rad PTC-200 or
Tetrad 0240) please follow the manufacturer’s instructions for adjusting lid tension.
Thermal Cycler Consumables
Table 3.3 provides details into the consumables to be used with each thermal cycler.
Table 3.3 Thermal Cycler Consumables for the Axiom 2.0 Assay Mini 96-Array Format Manual Protocol
Thermal Cycler
Model
PCR Plate Type
Bio-Rad PTC-200


Seal*
Bio-Rad Hard-Shell® Low-Profile 96-Well Skirted PCR
Plate (Bio-Rad P/N HSP-9631)
Bio-Rad Hard-Shell® High-Profile 96-Well Semi-Skirted
PCR Plate (Bio-Rad P/N HSS-9641)†
MicroAmp Clear Adhesive Film from
Applied Biosystems (P/N 4306311)
ABI 9700

Bio-Rad Hard-Shell® High-Profile 96-Well Semi-Skirted
PCR Plate (Bio-Rad P/N HSS-9641)†
MicroAmp Clear Adhesive Film from
Applied Biosystems (P/N 4306311)
ABI 2720

Bio-Rad Hard-Shell® High-Profile 96-Well Semi-Skirted
PCR Plate (Bio-Rad P/N HSS-9641)†
MicroAmp Clear Adhesive Film from
Applied Biosystems (P/N 4306311)
Bio-Rad Tetrad® 2
PTC-0240

Bio-Rad Hard-Shell® Low-Profile 96-Well Skirted PCR
Plate (Bio-Rad P/N HSP-9631)
Bio-Rad Hard-Shell® High-Profile 96-Well Semi-Skirted
PCR Plate (Bio-Rad P/N HSS-9641)†
MicroAmp Clear Adhesive Film from
Applied Biosystems (P/N 4306311)

*Microseal
“B” film from Bio-Rad (P/N MSB-1001) may be used in place of MicroAmp Clear Adhesive Film for the Bio-Rad
and ABI thermal cyclers.
† Included in the Axiom® 2.0 Assay Mini 96 Manual Target Preparation Consumables Kit (P/N 902986)
Chapter 3 | Preparation Before You Start
24
Oven Recommendations
The following ovens are recommended:
 BINDER ED 56 Drying and Heating Chamber. Refer to the Axiom® 2.0 Assay Mini 96-Array Format
Manual Protocol Site Preparation Guide, P/N 703435, for ordering information.
 Affymetrix GeneChip Hyb Oven 645
NOTE: The GeneChip® Hybridization Oven 640 is currently not supported with the Axiom 2.0
Assay; however, if you want to utilize it in the workflow please contact your Field Service
Engineer (FSE) or Affymetrix Technical Support regarding the compatibility of this oven with
the Axiom 2.0 Assay.


If using an Affymetrix GeneChip Hyb Oven, set the rotation speed to 15 RPM to aid in even heat
distribution.
For either Affymetrix GeneChip Hyb Oven, plates are placed in the bottom of the oven. To avoid
interfering with the rotation apparatus, do not stack plates in the oven.
Plate Centrifuge
One plate centrifuge is required for the Axiom 2.0 Assay Mini 96-Array Format Manual Protocol. Refer
to the Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol Site Preparation Guide, P/N 703435, for
an appropriate plate centrifuge that can be used with the Axiom Genotyping Solution. When
centrifuging and drying pellets as instructed under Stage 3A: Centrifuge Precipitation Plate and Dry the
DNA Pellet on page 59, the centrifuge must be able to spin down plates at:
 Rcf: 3200 xg (4000 RPM for the Eppendorf 5810R with the rotor configuration described in the
Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol Site Preparation Guide, P/N 703435).
 Temperature: 4°C and room temperature.
In addition, the bottom of the rotor buckets should be soft rubber 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.
Plate Shakers
We recommend using one of the following shakers listed in Table 3.4.
Table 3.4 Plate Shakers
Shaker
Supplier
Part Number
Thermo Scientific™ Compact Digital
Microplate Shaker
Thermo Scientific
88880023
Jitterbug™
Boekel Scientific
Model 130 000
Equipment Care and Calibration
Lab instrumentation plays an important role in the successful completion of this assay. To aid in
maintaining consistency across samples and operators, all equipment must be regularly calibrated and well
maintained, including:
 All pipettes, thermal cyclers, and ovens
 Plate spectrophotometer
Chapter 3 | Preparation Before You Start
25
Procedures
This section covers procedures you may need to do repeatedly during the workflow, or which are critical
to the performance of the assay.
Seal, Vortex, and Spin
Unless otherwise noted, when the protocol instructs you to seal, vortex and spin:
 Seal plates: We recommend using MicroAmp Clear Adhesive Films to seal your plates.
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.

Blot-dry: Prior to sealing plates, we recommend checking the top of the plate to make sure 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 Kimwipe 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
 Plates:
 For deep well plates (such as Eppendorf 96 Deep-well Plate, 2000 μL plate), vortex at max speed
for 5 seconds in each sector for a total of 5 sectors (Figure 3.1).
 For PCR plates (such as Bio-Rad Hard Shell or semi-skirted plates, vortex at max speed for
2 seconds in each sector for a total of 5 sectors (Figure 3.1).
 Reagent Vials: 3 times at max speed for, 1 sec each time.
Figure 3.1 Vortexing Plates
NOTE: In the procedures, “vortex twice” means to repeat the vortexing step.

Spin: When instructed to spin plates or reagent vials, follow these guidelines unless otherwise
instructed (for example, when centrifuging and drying pellets, see Step 2 in the section Stage 3A:
Centrifuge Precipitation Plate and Dry the DNA Pellet on page 59).
 Plates:
 Spin at 1000 rpm for 30 sec at room temperature.
 Do not spin for more than 1 min.
 Reagent Vials: 3 sec
Chapter 3 | Preparation Before You Start
26
Sample Quantitation
This protocol has been optimized using a PicoGreen assay to determine genomic DNA concentrations.
Other quantitation methods such as UV Absorbance may give different readings. Therefore, you should
correlate readings from other methods to the equivalent PicoGreen-determined concentration.
Please refer to Chapter 2, Genomic DNA Preparation and Requirements on page 13 for more information.
About the Reagents and Master Mix Preparation
Axiom 2.0 Assay Mini 96 Reagent Kit Components
Each Axiom 2.0 Assay Mini 96 Reagent Kit (P/N 903013) is sufficient to run two Mini 96-array format
plates. Refer to Table 3.11 for a full description of the kit.
 Caps on the vials are color-coded by assay stage.
 Properly store all enzyme reagents, especially enzyme-containing vials. Improper storage methods can
profoundly impact activity.
IMPORTANT: The Axiom 2.0 Assay for Mini 96 is compatible only with reagents from an
Axiom Reagent Kit. These reagents are not interchangeable with reagents from other
Affymetrix reagent kits, such as SNP 6.0, DMET Plus, etc.
Reagents from Other Suppliers
 Use only fresh reagents from the recommended suppliers to help eliminate changes in pH or the salt
concentration of buffers.
 Consult the appropriate SDS for reagent storage and handling requirements.
Master Mix Preparation
 Carefully follow each master mix recipe. Use pipettes that have been calibrated to ± 5%.
 If you run out of master mix during any of these procedures, a volume error has been made or the
pipettes are not accurate. We recommend that you stop and repeat the experiment.
NOTE: The volumes of Master Mixes prepared are designed to provide consistent handling of
reagents and consistent assay results. The percent overage of different master mixes may
differ, depending upon the reagent volumes involved.
When Using Reagents at the Lab Bench
 Properly chill essential equipment such as reagent coolers before use.
 Ensure that enzymes are kept at –20°C until needed. When removed from the freezer, immediately
place in a cooler that has been chilled to –20°C.
Chapter 3 | Preparation Before You Start
27
Pipettes and Pipetting
To efficiently process samples:
 Use a pipette of appropriate size for the volume of liquid being transferred (Table 3.5).
Table 3.5 Recommended Pipette Sizes
Pipette Size
Recommended Volume Range
Single channel and multi-channel P20
1-20 μL
Single channel and multi-channel P200
20-200 μL
Single channel and multi-channel P1200
200-1000 μL



We recommend the use of Rainin pipettes and tips. Affymetrix has only verified the use of Rainin
multi-channel pipettes in this assay. The use of other pipettes may impact the timing of the protocol
and may adversely impact the assay. Pipette substitution may violate the terms of the Axiom 2.0 Assay
Mini 96-Array Manual Protocol and array replacement policy.
Always use pipettes that have been calibrated.
It is essential that you be proficient with the use of single- and multi-channel pipettes. To familiarize
yourself with the use of multi-channel pipettes, we strongly recommend practicing several times before
processing actual samples. Use water and reagent reservoirs to get a feel for aspirating and dispensing
solutions to multiple wells simultaneously.
Single-channel Pipettes and Serological Pipettes
Use single-channel pipettes for preparing Master Mixes and for puncturing bubbles in GeneTitan trays.
The single-channel pipettes will not be used for working with the plates or trays otherwise.
 Use single channel pipettes for volumes less than or equal to 2 mL. For volumes between 1 and 2 mL,
add the reagent in two portions with a fresh tip for each portion.
 Use serological pipette for volumes  2 mL.
Multi-Channel Pipettes
Use 8 or 12-channel pipettes when working to add Master Mix or to transfer samples to plates and
GeneTitan trays.
 Use a pipette of appropriate size for the volume of liquid being transferred.
 Change pipette tips after each transfer or addition.
Matrix™ 25 mL Reagent Reservoirs
The Axiom 2.0 Assay Mini 96-Array Format Manual Protocol requires the use of disposable reservoirs
with a “trough within a trough” design. This special design maximizes the amount of liquid accessible to
pipette tips when using small amounts of reagent.
Figure 3.2 Dispense Reagents from Matrix™ 25 mL Reagent Reservoirs
NOTE: During the precipitation step, the Precipitation Master Mix working volume exceeds
the reservoir capacity. The reservoir must be filled twice.
Chapter 3 | Preparation Before You Start
28
Freeze-Thaw Instructions
The Axiom 2.0 Assay Mini 96 Reagent Kit is sufficient to run two Mini 96-array format plates. Excess
volume of the Axiom 2.0 Assay Mini 96 Reagent Kit after the first use may be stored in a freezer at
–25°C to –15°C or a refrigerator at 2°C to 8°C to be used for a second experiment for up to 60 days after
initial use (Table 3.6). Affymetrix recommends that reagents not exceed three freeze-thaw cycles. Please
monitor the freeze-thaw cycles of the reagents by following the guidelines below.
Mark Reagent Pouches, Tubes and Bottles to Track Use
To keep track of usage, we recommend that users mark the pouch while the reagents are thawing.
 Using a permanent marker, label the module pouch with “Thaw #1: XX/XX/XX” and any other useful
information (i.e., experiment name, user name, etc.).
Figure 3.3 Example of Labeling a Reagent Pouch

Using a permanent marker, make a tally mark on each reagent tube or bottle to indicate how many
times the reagent has been thawed.
Figure 3.4 Example of a properly marked reagent bottle that has been thawed once.
Thaw tally mark

After the experiment, place all tubes and bottles back in the appropriate pouch and place at proper
storage temperature. See Table 3.6.
Table 3.6 Reagent Storage Temperature
Storage
Temperature
Module 1
Module 2-1
Module 4-1

2°C to 8°C
–25°C to –15°C
Module 2-2


Module 4-2


Chapter 3 | Preparation Before You Start
Equipment, Consumables, Labware, and Reagents Required
Equipment Required for Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol
Thermal Cycler
Refer to Thermal Cycler Recommendations on page 23.
Oven
Refer to Oven Recommendations on page 24.
Plate Centrifuge
Refer to Plate Centrifuge on page 24.
Plate Shaker
Refer to Plate Shakers on page 24.
Consumables Required for Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol
Table 3.7 Consumables Required for Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol
Labware
Eppendorf 96 Deep-well Plate,
2000 μL
Eppendorf Deep-well Plate 96/2000 μL,
wells clear, PCR clean, border blue
Supplier and Part
Number
Part of the Axiom® 2.0
Assay Mini 96 Manual
Target Preparation
Consumables Kit
(Kit P/N 902986,
Plate P/N 203079).
Alternate Supplier:
Eppendorf,
P/N 951033481
P/N 0030501349
Matrix™ Reagent Reservoirs, 25mL
Thermo Scientific™ Matrix™ Reagent
Reservoirs, 25 mL
Part of the Axiom® 2.0
Assay Mini 96 Manual
Target Preparation
Consumables Kit
(Kit P/N 902986,
P/N 203077).
Alternate Supplier:
Thermo Scientific
P/N 8093-11
Image
29
Chapter 3 | Preparation Before You Start
Table 3.7 Consumables Required for Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol (Continued)
Labware
Bio-Rad Hard Shell 96-well plate
Bio-Rad Hard-Shell® Low-Profile 96-Well
Skirted PCR Plates
Supplier and Part
Number
Part of the Axiom® 2.0
Assay Mini 96 Manual
Target Preparation
Consumables Kit
(Kit P/N 902986,
Plate P/N 203015).
Alternate Supplier:
Bio-Rad,
P/N HSP-9631
96 Half-Skirt Plate
Bio-Rad Hard-Shell® High-Profile 96-Well
Semi-Skirted PCR Plates
Part of the Axiom® 2.0
Assay Mini 96 Manual
Target Preparation
Consumables Kit
(Kit P/N 902986,
Plate P/N 203009).
Alternate Supplier:
Bio-Rad, P/N HSS-9641
96 Well UV Plate
Greiner UV-Star® 96 well plates
Part of the Axiom® Mini
96 Consumables Kit for
QC
(Kit P/N 903014,
Plate P/N 202609).
Alternate Suppliers:
Fisher Scientific,
E&K Scientific,
Greiner Bio-One
P/N 655801
50 mL Conical-bottom Centrifuge
Tubes, Polypropylene
Various
15 mL Conical-bottom Centrifuge
Tubes, Polypropylene
Various
Image
30
Chapter 3 | Preparation Before You Start
31
Table 3.7 Consumables Required for Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol (Continued)
Labware
Supplier and Part
Number
96-well Block
Diversified Biotech
Cooling Chamber for 0.2 mL tubes,
96 holes (4 for 1.5 mL & 6 for 0.5 mL
tubes), Dim.: 6 1/8”L x 3 1/8”W x 1” H
P/N CHAM-1000
96-Well PCR Racks
Various
Image
GeneTitan® MC Instrument Consumables
All consumables for the GeneTitan MC Instrument are provided by Affymetrix. The following table
provides guidance on the consumables that are shipped with the array plate. Consult Chapter 5, Before
Using the GeneTitan® MC Instrument on page 91 for information on aligning and loading trays into the
GeneTitan MC Instrument.
IMPORTANT: All covers must have barcodes. Discard any cover without a barcode.
Table 3.8 Axiom® 384HT High Volume Consumables Kit, P/N 902629*
Qty
10
5
5
5
5
5
25
*
Item
384 Layout GeneTitan® Stain Tray (Stain 1)
384 Layout Axiom® Stain2 Tray
384 Layout Axiom® Stab. Tray
384 Layout Axiom® Ligation Tray
384 Layout GeneTitan® Hyb Tray
384 Layout GeneTitan® Scan Tray
384 Layout GeneTitan® Scan and Stain Tray Cover
Each Axiom® 384HT High Volume Consumable Kit is sufficient to process 5 Axiom Mini 96-array format plates. These trays are required for
processing Axiom Mini 96-array format plates on the GeneTitan® Multi-Channel Instrument. Please use the new Axiom 384HT High Volume
Consumable Kit when running the Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol.
Chapter 3 | Preparation Before You Start
32
Table 3.9 Axiom® Mini 96-Array Format Plate
Item
Part Number
Axiom
Mini
96-Array
Format
Plate,
various
designs
Varies, depending
on array design.
Labware Image
Information
NOTE: Array plate is
not included in the
Axiom 384HT High
Volume Consumables
Kit.
(All array plates
have the P/N
202091 etched
on the plastic)
Shipping Cover
(to be discarded)
Array Plate
protective
base
Array Plate
The Axiom array plate
shipping package includes
the following:
 The function of the
white plastic cover for
the array plate is to
protect the array plate
during transport. You
can discard this after
removing the array
plate.
 The array plate must be
protected at all times
from damage or
exposure to dust. The
array plate must be in
the blue array plate
protective base at all
times.
 The blue array plate
protective base in the
package must be used
to protect the array
plate from damage.
Chapter 3 | Preparation Before You Start
33
Table 3.10 Axiom® GeneTitan® MC Instrument Consumables Axiom® 384HT High Volume Consumables Kit, P/N 902629
Item
Part Number
Labware Image
902279/501280
384HT
GeneTitan
Scan Tray
and Cover*
Information
Barcoded Scan
Tray Cover
GeneTitan® Scan Tray
Scan Tray
protective base
Blue Scan
Tray
Protective
Base
P/N 202096
The Axiom scan tray
shipping package
includes the following:
 The GeneTitan scan tray
includes a scan tray
cover. The tray cover
should be used to cover
the scan tray before
placing the tray in the
GeneTitan MC
Instrument.
 The scan tray must be
protected at all times
from damage or
exposure to dust. The
scan tray must be in the
blue plate cover at all
times except when
loaded into the
GeneTitan MC
Instrument.
 The blue scan tray
protective base in the
package is used to
protect the bottom of
the scan tray glass from
damage. Remove the
protective base from the
scan tray before loading
the scan tray with the
scan tray cover in the
GeneTitan MC
Instrument.


The blue scan tray
protective base in the
package is used to
protect the bottom of
the scan tray glass from
damage. The blue scan
tray is distinct from the
blue array plate
protective base and
must not be used with
the array plate.
Remove the protective
base from the scan tray
before loading in the
GeneTitan MC
Instrument.
Chapter 3 | Preparation Before You Start
34
Table 3.10 Axiom® GeneTitan® MC Instrument Consumables Axiom® 384HT High Volume Consumables Kit, P/N 902629 (Continued)
Item
Part Number
Scan Tray
with
cover*
Scan Tray 501280
Cover 501315
Labware Image
Information


GeneTitan
Stain
Trays*
501279 - Stain 1
501394 - Stain 2
501398 - Ligation
501396 - Stab

Stain 1 Tray
Stain 2 Tray
Ligation Tray
Stabilizing Tray
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.
The GeneTitan stain
trays are packaged in
zip-top bags to keep
them free of dust. Each
GeneTitan stain tray is
uniquely barcoded.
IMPORTANT: Each
GeneTitan stain tray is
labeled with a name
and an individual
barcode. Ensure that
you always use the
appropriate tray with
the corresponding
reagent. Failure to do
so may result in assay
failure. When
transferring the trays
to the GeneTitan
Instrument ensure that
the trays are placed in
the proper location in
the drawer. Failure to
do so results in an error
and the GeneTitan
Instrument will not
proceed with the
processing of the trays.
Chapter 3 | Preparation Before You Start
35
Table 3.10 Axiom® GeneTitan® MC Instrument Consumables Axiom® 384HT High Volume Consumables Kit, P/N 902629 (Continued)
Item
Part Number
Labware Image
Information
GeneTitan
Scan and
Stain Tray
cover*
501315

The GeneTitan scan and
stain tray covers are
provided to prevent any
evaporation of the
stains in stain trays and
the array holding buffer
in the scan tray. The
GeneTitan scan and
stain tray covers are
barcoded.
GeneTitan
stain tray
cover,
shown on
top of the
stain tray*
Cover 501315

The GeneTitan stain
trays must be placed in
the GeneTitan MC
Instrument with the
GeneTitan stain tray
cover.
384 Hyb
Tray
501278

The GeneTitan
hybridization trays are
packaged in white
pouches with the label
“384 Layout
GeneTitan® Hyb Tray”
ref# 501278 (pouch)/
902278 (box)
The hybridization trays
are packaged with a
protective cover which
should be discarded
prior to use. 384 Hyb
Tray Cover, P/N 203006

Discard Hyb Tray Cover
*
NOTE: After aliquoting the appropriate solution to each tray type, the tray should be loaded into the GeneTitan® MC Instrument with the
barcode facing away from the operator, i.e., barcode should be on the back side.
Chapter 3 | Preparation Before You Start
36
Proper Tray Alignment and Loading
Proper alignment and loading of trays and covers is critical when using the GeneTitan MC Instrument.
Each tray and cover has one notched corner. The notched corner of the tray and its corresponding cover
or protective base must be in vertical alignment with each other. Consult Chapter 5, Before Using the
GeneTitan® MC Instrument on page 91 for important information on aligning trays and loading them into
the GeneTitan MC Instrument.
TIP: Mark the notched corner of each tray and cover with permanent marker to help ensure
proper alignment and loading onto the GeneTitan MC Instrument.
CAUTION: Take care not to damage the consumables or bend the blue base posts or scan tray
posts.
IMPORTANT: Always place the flat side of the cover against the stain tray.
Figure 3.5 Placement of Covers on Trays
Correct placement of cover on stain tray.
Incorrect placement of cover on stain tray.
Chapter 3 | Preparation Before You Start
37
Labeling GeneTitan® Hybridization and Reagent Trays
When 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.
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.
Proper labeling for hyb trays and reagent trays is described in:
 Labeling for Hyb Trays, below
 Labeling for Stain Trays on page 38
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 3.6. The proper section for labeling is closest to the notched corner, corresponding to
the A1 and F1 wells.
Figure 3.6 Labeling GeneTitan® Hyb Trays
Do NOT label trays on
the long side of the tray
Notched corner of the hyb tray
should face the front
Label the hyb tray here
CAUTION: Writing on the wrong side of the Hyb tray may interfere with the operation of
sensors in the GeneTitan MC Instrument.
Chapter 3 | Preparation Before You Start
38
Labeling for Stain Trays
You may label the stain trays on the left side of the front of the tray as shown in Figure 3.7. The correct
side is closest to the notched corner, corresponding to the A1 through F1 wells.
IMPORTANT: Do not confuse hyb trays with stain trays.
Figure 3.7 Labeling GeneTitan® Stain Tray (Stain Tray shown with Lid)
Do NOT label trays on the
long side of the tray
Notched corner of the stain
tray should face the front
Label the stain tray here
Chapter 3 | Preparation Before You Start
39
Reagents for the Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol
The Axiom® 2.0 Assay for Mini 96-Array Format Manual Protocol uses the Axiom 2.0 Assay Mini 96
Reagent Kit (P/N 903013). Kits consist of 4 modules for different stages of the assay with some modules
having both 4°C and –20°C pouches. There are specific instructions for which reagents are needed and
how to treat them within each stage.
Table 3.11 Axiom® 2.0 Assay Mini 96 Reagent Kit, P/N 903013 (sufficient for processing two Axiom Mini 96-array format plates)*
Component
Module 1: P/N 901711
Storage



Module 2—Pouch 1 of 2: P/N 901528



Module 2—Pouch 2 of 2: P/N 901529



Module 3



Module 4—Pouch 1 of 2: P/N 901278

Axiom Frag Diluent
Axiom Frag Rxn Stop
Axiom Precip Soln 1







Axiom Hold Buffer (1 bottle)


Axiom Reference gDNA 103
Axiom 2.0 Amp Soln
Axiom 2.0 Amp Enzyme
–25°C to –15°C
Axiom Hyb Buffer
Axiom Hyb Soln 1
–25°C to –15°C
Axiom Resusp Buffer
Axiom Hyb Soln 2
Axiom Wash Buffer A: P/N 901446 (4 bottles per kit)
Axiom Wash Buffer B: P/N 901447 (2 bottles per kit)
Axiom Water: P/N 901578 (2 bottles per kit)
Axiom Ligate Soln 2
Axiom Probe Mix 2
Axiom Wash A
Axiom Stain 1-A
Axiom Stain 1-B


*
Axiom Frag Enzyme
Axiom 10X Frag Buffer
Axiom Precip Soln 2



Axiom Hold Buffer: P/N 903012

Axiom Ligate Buffer
Axiom Ligate Enzyme
Axiom Ligate Soln 1


Module 4—Pouch 2 of 2: P/N 901276
Axiom 2.0 Denat Soln 10X
Axiom 2.0 Neutral Soln
Axiom Water






Axiom Probe Mix 1
Axiom Stain Buffer
Axiom Stabilize Soln
Axiom Stain 2-A
Axiom Stain 2-B
Axiom Stabilize Diluent
Axiom Water
Axiom Hold Buffer
2°C to 8°C
room temperature
15°C to 30°C
–25°C to –15°C
2°C to 8°C
2°C to 8°C
Axiom® 2.0 Assay Mini 96 Reagent Kit only states Axiom 2.0 on Mod 1. Do not use reagents from DMET™ Plus Solution, CytoScan® Reagent
Kit or any Expression reagent kits.
Chapter 4
Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
Introduction
Manual target preparation for the Axiom Mini 96-array enables you to process 96 samples at a time
without the use of automation equipment. The protocol is performed in two parts:
 Part 1: Manual Target Preparation, as described in this chapter
 Part 2: Array Processing is performed on the GeneTitan® Multi-Channel (MC) Instrument
Array handling and processing protocols require the use of a GeneTitan MC Instrument, as described
in Chapter 5, Array Processing with the GeneTitan® Multi-Channel Instrument on page 91.
IMPORTANT: Read all the instructions in Chapter 3, Preparation Before You Start on page 21,
before performing manual target preparation.
A list of all equipment and resources required for the Axiom 2.0 Assay for Mini 96-Array Format Manual
Protocol is in the Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol Site Preparation Guide,
P/N 703435.
The protocol for manual target preparation is presented in the following sections:
 Stage 1: DNA Amplification on page 41
 Stage 2: Fragmentation and Precipitation on page 48
 Stage 3: Centrifuge and Drying, Resuspension and Hybridization Preparation, and Sample QC on page 56
 Stage 3A: Centrifuge Precipitation Plate and Dry the DNA Pellet on page 59
 Stage 3B: Resuspension and Hybridization Preparation on page 60
 Stage 3C: Sample QC on page 63
 Stage 4: Denaturation and Hybridization on page 67
 Stage 5: GeneTitan ® Reagent Preparation on page 74
Using the manual target preparation protocol, a single operator can process three gDNA and array plates
a week during a forty-hour work week for a total of 288 arrays. See Chapter 6, Manual Target Preparation
for Processing Three Axiom® Array Plates per Week on page 127 for further information.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
41
Stage 1: DNA Amplification
IMPORTANT: Before proceeding to DNA Amplification, perform the gDNA preparation
described in Chapter 2, Genomic DNA Preparation and Requirements on page 13.
NOTE: For this protocol, the term samples includes the positive control.
The following sets of steps are necessary to perform DNA amplification:
1: Prepare for DNA Amplification on page 43
2: Prepare the Denaturation Master Mix on page 44
3: Add Denaturation Master Mix to Samples on page 44
4: Add Neutralization Solution to Samples on page 45
5: Prepare the Amplification Master Mix on page 45
6: Add Amplification Master Mix to Samples on page 46
7: Store Remaining Reagents on page 46
8: Freeze or Proceed on page 46
IMPORTANT: Amplification preparation should take place in an a dedicated area such as a
biosafety hood with dedicated pipettes, tips, vortex, etc. See Amplification Staging Area on
page 22 for more information.
Duration
For 96 samples:
 Time to thaw materials: 1 hr
 Hands-on time: approximately 0.5 hr
 Incubation at 37°C: 23 ±1 hr
 Total time required: approximately 24.5 hr
Input Required
The gDNA Sample Plate: an Eppendorf 96 Deep-well Plate, 2000 μL with 8.7 μL of each gDNA diluted to
a concentration of 11.5 ng/μL, 17.2 ng/μL, or 23 ng/μL as required according to the sample type.
See Genomic DNA Preparation on page 17 for more information.
Equipment, Consumables and Reagents Required
Equipment and Consumables
The equipment and consumables listed in Table 4.1 are required for this stage.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
Table 4.1 Equipment and Consumables Required for Stage 1: DNA Amplification
Quantity
As required
Item
Adhesive seals for 96-well plate - Applied Biosystems MicroAmp Clear adhesive film
1
Cooler, chilled to –20°C
1
Microcentrifuge tube holder
1
15 mL tube holder
1
Marker, fine point, permanent
1
Mini microcentrifuge (microfuge with microtube rotor)
1 each
Rainin Pipettes:
Single-channel P200
 Single-channel P1000
 Multi-channel P20
 Multi-channel P200
 Multi-channel P1200

As needed
As needed
Pipette tips
Pipette, serological
5 x 1/10 mL
 10 x 1/10 mL

1
Pipet aid
1
Plate centrifuge, at room temperature
1
Oven, set at 37°C
2
15 mL conical tube
1
Vortexer
1
Timer
3
Matrix™ 25 mL Reagent Reservoir P/N 8093-11
42
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
43
Reagents Required
Table 4.2 Reagents Required for Stage 1: DNA Amplification
From the Axiom® 2.0 Assay Mini 96 Reagent Kit
Module
Axiom 2.0 Denat Soln 10X
Axiom 2.0 Neutral Soln
Axiom 2.0 Amp Soln
Module 1, –20°C
P/N 901711
Axiom Water
Axiom 2.0 Amp Enzyme
1: Prepare for DNA Amplification
To Prepare for DNA Amplification
Set an incubator/oven temperature at 37°C.
We recommend using one of these ovens:
 BINDER ED 56
 Affymetrix GeneChip® 645 Hybridization Oven (turn rotation on to 15 rpm)
2. Set the centrifuge temp to room temperature.
3. Thaw and prepare the reagents and sample plate.
1.
To Thaw and Prepare the Reagents:
1.
Thaw the gDNA sample plate on the benchtop at room temperature and pulse-spin to get all the
droplets down.
IMPORTANT:

gDNA samples must be brought to room temperature before proceeding with
denaturation.

gDNA samples must be 8.7 μL volume of each gDNA at a concentration of 11.5 ng/μL,
17.2 ng/μL, or 23 ng/μL, depending on the sample type, in an Eppendorf 96 Deep-well
Plate, 2000 μL (see Genomic DNA Preparation on page 17).
Thaw the following reagents in a small water bath on the benchtop at room temperature (small water
bath: small tray or container, such as a pipet tip box, filled with fresh filtered water):
 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 cooler in the freezer until ready to use.
3. Vortex all reagents (except Axiom 2.0 Amp Enzyme), then place at room temperature.
 Axiom 2.0 Amp Soln: Vortex for 30 sec to thoroughly mix.
 Axiom 2.0 Neutral Soln: Vortex for 30 sec to thoroughly mix.
 Axiom 2.0 Denat Soln 10X: Vortex and pulse-spin before use.
 Axiom 2.0 Amp Enzyme: Gently invert and flick the tube 3 times to mix and pulse-spin just before use.
2.
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.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
4.
44
Label the 15 mL conical tubes as indicated in the table below:
Table 4.3 Labeling Tubes
Label
Tube Size
Temperature
Contents

D MM
15 mL
leave tube at room temperature
Denaturation Master Mix

Amp MM
15 mL
leave tube at room temperature
Amplification Master Mix
5.
Label three Matrix 25 mL Reagent Reservoirs (P/N 8093-11) as indicated in the table below:
Table 4.4 Labeling Reagent Reservoirs for DNA Amplification
Label
Temperature
Contents

D MM
Leave reservoir at room temperature
Denaturation Master Mix

N Soln
Leave reservoir at room temperature
Neutralization Solution

Amp MM
Leave reservoir at room temperature
Amplification Master Mix
2: Prepare the Denaturation Master Mix
To Prepare the Denaturation Master Mix (carry out the following steps at room temperature):
1.
Per Table 4.5, dilute the appropriate volume of Axiom 2.0 Denat Soln 10X using the Axiom Water.
Table 4.5 Preparing Denaturation Master Mix (D MM)
Reagent
per Sample
Master Mix 96+
Axiom Water
7.8 μL
1.652 mL
Axiom 2.0 Denat Soln 10X
0.9 μL
183.6 μL
Total Volume
8.7 μL
1.836 mL
To the 15 mL tube marked D MM, add:
2.
Vortex and leave at room temperature.
3: Add Denaturation Master Mix to Samples
To Add the Denaturation Master Mix to Your Samples (carry out the following steps at room
temperature):
Ensure the samples in the Sample Plate are fully thawed. Pulse-spin the plate to get all the
droplets down.
Remember: Samples must be at room temperature for this step.
2. Using a P1000, gently pipet or pour the Denaturation Master Mix into the reagent reservoir marked
D MM.
3. Carefully remove the seal from the Sample Plate and discard the seal.
4. Using a P20 12-channel pipette, add 8.7 μL of Denaturation Master Mix to each sample
(Total Volume: 17.4 μL/well).
 Pipet directly into the liquid of each well. Do not mix by pipetting up and down.
 Change tips between each addition.
 This plate is now known as the Denaturation Plate.
1.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
5.
Seal and vortex the Denaturation Plate. Start the timer for a 10 minute incubation after vortexing.
6.
Pulse-spin the Denaturation Plate to 1000 rpm at room temperature.
45
NOTE: The quick spin time is included in the 10 minute incubation.
7.
Visually examine the volume in each well.
A. Keep a record of any wells that visually appear to have a particularly low or high volume; these
samples may need to be repeated.
B. Do NOT stop to measure volumes; proceed without delay.
8. Complete the 10 minute incubation on the benchtop at room temperature.
While completing the incubation at room temperature, prepare the Neutralization Soln as described
in Step 1 on page 45.
9. After incubation immediately add the Neutralization Soln as described in 4: Add Neutralization
Solution to Samples on page 45.
4: Add Neutralization Solution to Samples
To Add the Neutralization Master Mix to Your Samples (carry out the following steps at room
temperature):
1.
2.
3.
4.
5.
Measure 6.4 mL of Axiom 2.0 Neutral Soln and slowly pipet the reagent into the reagent reservoir
marked N Soln.
Carefully remove the seal from the Denaturation Plate and discard the seal.
Using a P200 12-channel pipette, add 56.6 μL of Axiom 2.0 Neutral Soln to each sample
(total volume: 74 μL/well).
 Pipet down the wall of each well. Change tips between each addition.
 The plate is now known as the Neutralization Plate.
Seal, vortex, and pulse-spin the Neutralization Plate.
Visually examine the volume in each well (should be ~74 μL/well) and:
A. Keep a record of any wells that visually appear to have a particularly low or high volume; these
samples may need to be repeated.
B. Do NOT stop to measure volumes.
6.
Proceed immediately to 5: Prepare the Amplification Master Mix on page 45.
5: Prepare the Amplification Master Mix
To Prepare and Add the Amplification Master Mix (carry out the following steps at room temperature):
1.
Per Table 4.6, pipet the appropriate amount of Axiom 2.0 Amp Soln into the tube labeled Amp MM
at room temperature.
TIP:
The Amp Soln is a viscous solution. To ensure the Amp Soln reagent transfer is accurate:

Pipet slowly.

Allow bubbles generated from mixing to settle at the top before pipetting.

Use a 10 mL serological pipette and a P1000 to transfer the Amp Soln into the Amp MM tube.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
46
Table 4.6 Amplification Master Mix (Amp MM)
Reagent
Per Sample
Master Mix 96+
To the 15 mL tube marked Amp MM, add:
Axiom 2.0 Amp Soln
97.9 μL
10.9 mL
Axiom 2.0 Amp Enzyme
2.2 μL
241 μL
100.1 μL
11.1 mL
Total Volume
2.
Remove the Axiom 2.0 Amp Enzyme from the freezer and place in a portable cooler at –20°C.
A. Invert and flick the Axiom 2.0 Amp Enzyme tube three times, then pulse-spin.
B. Per Table 4.6 on page 46, add the appropriate amount of Axiom 2.0 Amp Enzyme to the tube
labeled Amp MM.
C. Vortex the Amplification Master Mix well, invert the tube 2 times, and then vortex again.
6: Add Amplification Master Mix to Samples
Slowly pour the Amplification Master Mix to the reagent reservoir labeled Amp MM.
2. Carefully remove the seal from the Neutralization Plate and discard the seal.
3. Using a P200 12-channel pipette, slowly add 100.1 μL of Amplification Master Mix to each sample of
the Neutralization Plate.
 Pipet down the wall of the well (Total Volume: 174.1 μL/well). Do not mix by pipetting up and
down.
 Change tips between each addition.
1.
NOTE: After adding the Amplification Master Mix, the plate is now known as the
Amplification Plate.
Seal tightly, vortex twice, and spin the Amplification Plate for one minute at 1000 rpm (as described
in Seal, Vortex, and Spin on page 25).
5. Place the sealed Amplification Plate in an oven set at 37°C and leave undisturbed for 23 ±1 hr.
4.
NOTE: If using a GeneChip® Hybridization Oven, place the plate on the bottom of the
oven. Plates do not rotate. Set the rotor for 15 rpm speed. See Oven Recommendations
on page 24 for more information.
7: Store Remaining Reagents
Store remaining Module 1 reagents for future use. Follow guidelines presented in the section FreezeThaw Instructions on page 28.
8: Freeze or Proceed
After the incubation finishes, you can either:
 Proceed to Stage 2: Fragmentation and Precipitation on page 48.
 Store the Amplification Plate at –20°C.
NOTE: If freezing, do not perform the stop amplification reaction step before you store the
Amplification Plate at –20°C. The Stop Amplification Reaction step will be performed after
thawing the frozen plate, as described in 1: Prepare for Fragmentation and Precipitation on
page 49.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
Figure 4.1 Stage 1: Amplification Workflow Diagram
DNA Amplification
Denaturation Step
Labware and Reagents Needed
Axiom Water
Denat Soln 10X
1652 μL
183.6 μL
QTY 3
QTY 1
(with gDNA)
QTY 2
Axiom® 2.0 Module 1
Vortex
Prepare for DNA Amplification
Pour into reservoir
Amp Soln
Amp Enzyme
8.7 μL/well
Denaturation Plate
Vortex, Pulse-spin
Incubate at RT for 10 min.
10.9 mL
241 μL
Vortex
Pour into reservoir
Neutralization Step
100.1 μL/well
Neutral Soln
Amplification Plate
Vortex for 30 sec
Pulse-spin
6.4 mL
Carefully pipet into
reservoir
Final Volume: 174.1 μL/well
56.6 μL/well
Neutralization Plate
Vortex, Pulse-spin
Incubate Sample Plate
@ 37°C for 23 ±1 hours
DNA Amplification – Page 1 of 1
47
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
Stage 2: Fragmentation and Precipitation
The following sets of steps are necessary to perform fragmentation and precipitation:
1: Prepare for Fragmentation and Precipitation on page 49
2: Incubate Samples in Preheated Ovens on page 51
3: Prepare the Fragmentation Master Mix on page 52
4: Add the Fragmentation Master Mix to Samples on page 52
5: Add the Stop Solution to the Samples on page 53
6: Prepare the Precipitation Master Mix on page 53
7: Freeze the Precipitation Plate Overnight on page 54
8: Store Remaining Reagents on page 54
Duration
Total time: approximately 2 hours.
Input Required
Amplification Plate from Stage 1: DNA Amplification on page 41.
Equipment, Consumables and Reagents Required
Equipment and Consumables
The equipment and consumables listed in Table 4.7 are required for this stage.
Table 4.7 Equipment and Consumables Required for Stage 2: Fragmentation and Precipitation
Quantity
As required
Item
Adhesive seals for 96-well plates
1
Freezer set to –20°C (Designate a shelf where the precipitation plates can be left undisturbed)
1
Cooler, chilled to –20°C
1
Ice bucket, filled with ice
1
Marker, fine point, permanent
1 each
Rainin Pipettes:





As needed
1
As needed
Single channel P1000
Single channel P200
Multi-channel P20
Multi-channel P200
Multi-channel P1200
Pipette tips for pipettes listed above
Pipet-aid
Pipette, serological


5 x 1/10 mL
10 x 1/10 mL
1
Plate centrifuge set at room temp
1
Mini microcentrifuge (microfuge with microtube rotor)
2-3
Ovens (see Oven Recommendations on page 24):


One oven set at 37°C
One oven set to 65°C
48
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
49
Table 4.7 Equipment and Consumables Required for Stage 2: Fragmentation and Precipitation
Quantity
Item
1
15 mL conical tube
1
50 mL conical tube
1
50 mL conical tube holder
3
Matrix™ 25 mL Reagent Reservoir P/N 8093-11
1
Vortexer
Reagents Required
Table 4.8 Reagents Required for Stage 2: Fragmentation and Precipitation
Reagent
Module
From the Axiom® 2.0 Assay Mini 96 Reagent Kit
Axiom Frag Enzyme (leave at –20°C until ready to use)
Axiom 10X Frag Buffer
Module 2-1, –20°C
P/N 901528
Axiom Precip Soln 2
Axiom Frag Diluent
Axiom Frag Rxn Stop
Module 2-2, 2–8°C
P/N 901529
Axiom Precip Soln 1
User-supplied - Refer to the Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol Site Preparation Guide,
P/N 703435
Isopropanol (2-Propanol), 99.5%
1: Prepare for Fragmentation and Precipitation
Set Ovens and Centrifuge
1.
Set the incubators/ovens.
A. If you are running one plate per week, you will need to set two incubators/ovens as follows,
preferably the night before:
 One oven set at 37°C. Use an oven that can sustain a constant temperature of 37°C and has a
temperature accuracy of 1°C.
 One oven set at 65°C.
B. If you are running the three plate per week manual target preparation workflow, three ovens are
recommended. See Chapter 6, Manual Target Preparation for Processing Three Axiom® Array Plates
per Week on page 127 for further information.
2. Set the centrifuge temp to room temperature.
TIP: Keep a set of balance plates ready to minimize any time delays before spinning the
Fragmentation plate in-between steps.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
50
Thaw and Prepare the Amplified DNA Samples and Reagents
Thaw and Prepare the Amplified DNA Sample Plate
If the Plate of Amplified DNA Samples is Frozen (skip this step if the Amplified Sample Plate was not
frozen at the end of the previous stage):
Place the deep-well plate in a small water bath.
For example, pour fresh filtered 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 min until all wells have thawed.
3. Spin down the plate at 1000 rpm for 30 sec.
4. To avoid cross-contamination of wells during vortexing:
1.
A. Remove the seal and blot the top of the plate with a Kimwipe.
B. Tightly re-seal the plate using a fresh seal.
Vortex the plate for 30 sec to thoroughly mix.
6. Spin at 1000 rpm for 30 sec.
5.
Thaw and Prepare the Reagents
Prepare reagents as shown below at the start of the 65°C incubation of the Amplification Plate.
To Thaw and Prepare the Fragmentation Reagents:
Axiom 10X Frag Buffer:
 Thaw on the bench top at room temperature then place on ice.
 Vortex before use.
2. Axiom Frag Diluent:
 Place on ice.
 Vortex and pulse-spin before use.
3. Axiom Frag Rxn Stop:
 Place on bench top to warm to room temperature.
 Vortex before use.
4. Axiom Frag Enzyme: Leave at –20°C until ready to use. Just before use, gently flick the tube 3 times
to mix and pulse-spin.
1.
To Thaw and Prepare the Precipitation Reagents:
Axiom Precip Soln 1
 Place on bench top to warm to room temperature.
 Vortex before use
2. Axiom Precip Soln 2:
 Thaw on the bench top at room temperature then place on ice.
 Vortex and pulse-spin before use
3. Isopropanol (user-supplied)
 Keep in room temperature
1.
Label Tubes And Reagent Reservoirs
1.
Label the 15 mL conical tubes as indicated in the table below:
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
51
i
Table 4.9 Label Conical Tubes
Label
Tube Size
Temperature
Contents

Frg MM
15 mL
Place tube on ice
Fragmentation Master Mix

Precip MM
50 mL
Place tube at room temperature
Precipitation Master Mix
2.
Label three Matrix 25 mL Reagent Reservoirs (P/N 8093-11) as indicated in the table below.
i
Table 4.10 Label Reagent Reservoirs for Fragmentation and Precipitation
Label
Temperature
Contents

Frg MM
Leave reservoir at room temperature
Fragmentation Master Mix

Stop
Leave reservoir at room temperature
Frag Rxn Stop

Precip MM
Leave reservoir at room temperature
Precipitation Master Mix
2: Incubate Samples in Preheated Ovens
OPTIONAL: Remove samples for quantifying amplification yield by the PicoGreen Assay.
A. Carefully remove the seal from the Amplification Plate and discard the seal.
B. Transfer 4 μL of samples from each well to a 96 well PCR plate such as a Bio-Rad Hard-Shell
96-well plate, HSP-9631 and set aside for later quantitation (e.g., using the Quant-iT™
PicoGreen® dsDNA Kit from Thermo Fisher Scientific (formerly Life Technologies/
Invitrogen)).
C. Reseal the Amplification Plate and proceed to Stop the DNA Amplification Reaction:.
Stop the DNA Amplification Reaction:
Place the Amplification Plate in the 65°C oven:
 If proceeding directly from the end of Stage 1: DNA Amplification on page 46, transfer the
Amplification Plate from the 37°C oven to the 65°C oven. Ensure the seal is still securely attached
to the plate to minimize evaporation.
 If working with a thawed plate, change the seal, vortex, and pulse-spin the Amplification Plate as
instructed in Thaw and Prepare the Amplified DNA Sample Plate on page 50 before placing it in the
65°C oven.
2. Incubate for 20 minutes.
1.
Prepare for Fragmentation:
Transfer the Amplification Plate from the 65°C oven to the 37°C oven.
 Press on the seal, if needed.
2. Incubate for 45 minutes.
1.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
52
3: Prepare the Fragmentation Master Mix
To Prepare the Fragmentation Master Mix:
1.
Start making the Fragmentation Master Mix when there is still five minutes to the finish of the 37°C
incubation, using the values in the table below.
Transfer the Axiom Frag Enzyme to a –20°C portable cooler. Keep in cooler until ready to use.
Table 4.11 Axiom Fragmentation Master Mix
Reagent
per Sample
Master Mix 96+
Axiom 10X Frag Buffer
19.9 μL
2.7 mL
Axiom Frag Diluent
4.5 μL
610.7 μL
Axiom Frag Enzyme
0.4 μL
59.3 μL
24.8 μL
3.4 mL
To the 15 mL tube marked Frg MM, add:
Total Volume



Add the reagents from Table 4.11 to the Frg MM tube in the order shown, using appropriate single
channel pipettes.
Just before the end of the 45 minute 37°C incubation, flick the Axiom Frag Enzyme tube 2 to
3 times, and spin.
Add the Axiom Frag Enzyme to the Frg MM tube at the end of the 45 minute 37°C incubation.
NOTE: Leave the Axiom Frag Enzyme at –20°C until ready to use.
Vortex twice and place on ice.
3. Slowly pour the Fragmentation Master Mix in the reagent reservoir labeled Frg MM placed at room
temperature.
2.
4: Add the Fragmentation Master Mix to Samples
IMPORTANT: Work quickly to perform this set of steps to minimize the time that the
Fragmentation Plate is out of the 37°C oven.
1.
2.
3.
4.
5.
Carefully remove the Amplification Plate from the 37°C oven and place on the bench top at room
temperature.
Do not place the Amplification Plate on ice.
Carefully remove the seal from the Amplification Plate and discard the seal.
Pipetting directly into the liquid of each well, use a P200 12-channel pipette to add 24.8 μL of
Fragmentation Master Mix to each reaction. Do not mix by pipetting up and down.
 Change tips after each addition.
 After adding the Fragmentation Master Mix to the plate, the plate is now known as the
Fragmentation Plate.
Seal the Fragmentation Plate and vortex twice.
Start the timer for 30 min.
IMPORTANT: Keep your timer in a safe place. It is helpful to note down the actual time when
the incubation began in case the timer stops accidentally.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
53
Pulse-spin the Fragmentation Plate to 1000 rpm in the plate centrifuge at room temperature.
7. Quickly transfer plate to 37°C oven and incubate for 30 min.
6.
CAUTION: Be watchful for the end of the thirty minute incubation period.
Fragmentation is an exact 30 minute incubation step. Longer and shorter incubation
times may lead to poor performance of the assay.
Prepare the Stop solution a few minutes before the end of the 30 minute incubation period, as
described in 5: Add the Stop Solution to the Samples, below.
5: Add the Stop Solution to the Samples
To Add the Stop Solution (carry out the following steps at room temperature):
1.
2.
3.
4.
5.
6.
A few minutes before the end of the 30 minute incubation period, measure 1.8 mL of the Axiom Frag
Rxn Stop solution and transfer into the reagent reservoir labeled Stop.
Remove the Fragmentation Plate from the oven and place on the bench top at room temperature.
At the end of the 30 minute fragmentation incubation period, carefully remove the seal from the
Fragmentation Plate and discard the seal.
Using a P20 12-channel pipette, end the fragmentation reaction by adding 8.3 μL of Stop Solution to
each reaction. Do not mix by pipetting up and down.
 Pipet directly into the liquid of each well.
 Change tips after each addition.
 Proceed immediately to the next step.
Seal and vortex and do a quick spin at 1000 rpm.
Leave the Fragmentation Plate on the benchtop while you prepare the Precipitation Master Mix.
6: Prepare the Precipitation Master Mix
To Prepare and Add Precipitation Master Mix (carry out the following steps at room temperature):
1.
Prepare Precipitation Master Mix in the 50 mL conical tube labeled Precip MM.
Table 4.12 Precipitation Master Mix
Reagent
per Sample
Master Mix 96+
To the 50 mL tube marked Precip MM, add:
Axiom Precip Soln 1
103.5 μL
10.9 mL
Axiom Precip Soln 2
0.9 μL
91.9 μL
Isopropanol
261 μL
27.6 mL
365.4 μL
38.6 mL
Total Volume
NOTE: Use a 5 or 10 mL serological pipette to pipet Axiom Precip Soln 1.
2.
Vortex the Precip MM tube and place on benchtop at room temperature.
3.
Pour approximately half of the Precipitation Master Mix into the reagent reservoir labeled
Precip MM.
NOTE: The total volume of the Precipitation Master Mix exceeds the reservoir capacity
(25 mL). Pour approximately half of the Precipitation Master Mix and re-fill the reservoir
with the rest of the Precipitation Master Mix after the first half has been exhausted.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
54
Carefully remove the seal from the Fragmentation Plate and discard the seal.
5. Using a P1200 12-channel pipette, add 365.4 μL of Precipitation Master Mix to each sample. Mix well
by pipetting up and down 6-7 times within the solution. Observe the solution while it is within the
tips—it should look homogeneous after pipetting 5-7 times. If not, repeat mixing a few more times
until the solution looks homogeneous.
 Do not vortex the plate after isopropanol addition to avoid cross-contamination of the samples.
 Change tips after each addition.
4.
NOTE: After adding the Precipitation Master Mix, the plate is now known as the
Precipitation Plate.
6.
Blot the top of the plate with a Kimwipe and seal tightly with a Microamp seal.
7: Freeze the Precipitation Plate Overnight
Carefully transfer the Precipitation Plate into the –20°C freezer and incubate overnight (16-24 hrs).
TIP: It is recommended to designate a shelf in a –20°C freezer where the plates can be left
undisturbed.
8: Store Remaining Reagents
Store remaining Module 2-1 and Module 2-2 reagents for future use. Follow guidelines presented in the
section Freeze-Thaw Instructions on page 28.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
Figure 4.2 Stage 2: Fragmentation & Precipitation
Fragmentation
Labware and Reagents Needed
Incubate Samples in Pre-Heated Ovens
1) 20 min. @ 65°C
2) 45 min. @ 37°C
28 mL
2-Propanol
QTY 3
QTY 1
(Samples)
Axiom® 2.0:
Module 2-1
Module 2-2
QTY 1 QTY 1
Fragmentation Step
10X Frag
Buffer
2.7 mL
Frag
Diluent
610.7 μL
Frag
Enzyme
Precipitation Step
Precip
Soln 1
Precip
Soln 2
2-Propanol
(User-supplied)
10.9 mL
91.9 μL
27.6 mL
59.3 μL
Invert tube
and vortex to mix
Pour into reservoir
Vortex to mix well
24.8 μL/well
Fragmentation Plate
Vortex, Pulse-spin
Incubate at 37qC
for 30 min.
Pour half the volume of the
Master Mix, two times.
Transfer to samples and refill reservoir
365.4 μL/well
Stop Fragmentation Reaction
Precipitation Plate
Pipet up and down to mix
Do not vortex.
Stop Soln
Final Volume: 572.6 μL/well
1.8 mL
Carefully pipet into
reservoir
Incubate Precipitation Plate
@ –20°C overnight
8.3 μL/well
Fragmentation Plate
Vortex, Pulse-spin
Fragmentation – Page 1 of 1
55
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
56
Stage 3: Centrifuge and Drying, Resuspension and Hybridization Preparation,
and Sample QC
This stage requires the following sets of steps:
Stage 3A: Centrifuge Precipitation Plate and Dry the DNA Pellet on page 59
Stage 3B: Resuspension and Hybridization Preparation on page 60
1: Prepare for Resuspension and Hybridization Preparation on page 60
2: Prepare DNA Pellets and Warm the Reagents on page 60
3: Thaw and Prepare the Reagents on page 60
4: Label Tubes and Reservoirs on page 61
6: Add Hybridization Cocktail to DNA Pellets on page 62
7: Resuspension of DNA Pellets on page 62
5: Prepare the Hybridization Cocktail on page 61
8: Prepare the Hyb Ready Sample Plate on page 62
10: Freeze or Proceed on page 62
Stage 3C: Sample QC on page 63
1: Prepare for Sample QC on page 63
2: Perform QC Checks on page 63
3: Freeze or Proceed on page 64
CAUTION: Some of the steps in this stage should be performed under a fume hood.
IMPORTANT: For troubleshooting and support purposes, we strongly recommend that you
perform the gel QC and OD quantitation process controls after Resuspension.
Duration
Centrifuge and dry plates: 1 hour 20 minutes
Resuspension and hyb mix preparation: 25 min
 Gel QC and OD: 45 min
Total: 2.5 hr


Input Required
Precipitation Plate from Stage 2: Fragmentation and Precipitation on page 48.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
Equipment, Consumables, and Reagents Required
The equipment and consumables listed in Table 4.13 are required for this stage.
Table 4.13 Equipment and Consumables Required for Stage 3: Drying, Resuspension and Sample QC
Quantity
As required
1
1 each
As needed
Item
Adhesive seals for 96-well plates
Marker, fine point, permanent
Rainin Pipettes:
 Single channel P20
 Single channel P-100
 Multi-channel P20
 Multi-channel P-200
Pipette tips for pipettes listed above
2
Bio-Rad Hard-Shell 96-well plate, Bio-Rad P/N HSP-9631 or any 96-well PCR plate for making the
dilutions:
 Dilution QC Plate
 Gel QC Plate
1
Bio-Rad Hard-Shell 96-well plate, Bio-Rad P/N HSP-9631
or
96 Half-Skirt Plate, Bio-Rad P/N HSS-9641
 Hyb Ready Plate
1
96-Well PCR Racks, if needed (to hold the 96 Half-Skirt Plate)
1
96 Well UV Plate
OD plate

1
Oven set at 37°C
1
Mini microcentrifuge (microfuge with microtube rotor)
1
Fume Hood
1
Plate centrifuge set at 4°C
1
15 mL conical tube
1
5 mL Serological Pipette
1
Pipet aid
1
Shaker, either:
™ Compact Digital Microplate Shaker
 Thermo Scientific
 Jitterbug
1
Vortexer
4
Matrix™ 25 mL Reagent Reservoir P/N 8093-11
57
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
58
Reagents Required
Table 4.14 Reagents Required for Stage 3: Drying, Resuspension and QC
Reagent
Module
From the Axiom® 2.0 Assay Mini 96 Reagent Kit
Axiom Hyb Buffer
Axiom Hyb Soln 1
Axiom Resusp Buffer
Axiom Hyb Soln 2
Module 2-1, –20°C
P/N 901528
Module 2-2, 2–8°C
P/N 901529
Other Reagents Required for QC Steps (optional)
Gel Diluent, 15 mL of 1000-fold dilution of TrackIt™ Cyan/Orange Loading
Buffer (see Appendix A, Fragmentation Quality Control Gel Protocol on
page 144 for dilution instructions.)
15-fold dilution of TrackIt™ 25 bp DNA Ladder (P/N 10488-022)
Nuclease-free water, ultrapure MB Grade (P/N 71786; for OD and Dilution
QC Plate preparation)
Gels and Related Materials Required
At the end of this stage, verifying the fragmentation reaction is highly recommended. See Appendix A,
Fragmentation Quality Control Gel Protocol on page 144 for the required gel and related materials.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
59
Stage 3A: Centrifuge Precipitation Plate and Dry the DNA Pellet
CAUTION: During this step, handle the Precipitation Plate gently to avoid disturbing the
pellets. Do not bump or bang the plate against another object.
To Centrifuge and Dry the DNA Pellets:
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 using an Affymetrix GeneChip Hyb Oven, set the rotation
speed to 15 rpm to distribute heat.
2. Transfer the Precipitation Plate from the –20°C freezer to a pre-chilled centrifuge. Centrifuge the
plate for 40 min at 4°C at 3200 xg (4000 RPM for the Eppendorf 5810R centrifuge with the rotor
configuration described in the Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol Site
Preparation Guide, P/N 703435).
1.
NOTE: If you are processing two plates at the same time, as in the three plate/week
manual preparation workflow, you can centrifuge both plates at the same time.
.
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.
3.
Immediately after the 40 min centrifugation period, empty the liquid from each plate as follows:
A. Carefully remove the seal from the Precipitation Plate and discard the seal.
B. Invert plate over a clean waste container to allow the liquid to drain. Collect liquid and dispose of
liquid according to local, state, and federal regulations.
C. While still inverted, gently press the plate on a pile of Kimwipes on a bench and allow them to
4.
drain for 5 min. Transfer the plate to a new pile of Kimwipes twice during the 5 min period.
Turn the plate right side up and place in an oven for 20 min at 37°C to dry.
 Tightly seal the plate upon completion
NOTE: If using an Affymetrix 645 oven:
5.

Place the plate on the bottom of the oven. Plates do not rotate.

Turn off the rotor during the 20 min drying time.
Do one of the following:
 Proceed directly to Stage 3B: Resuspension and Hybridization Preparation on page 60, even if some
droplets of liquid remain. Leave the sample plate at room temperature. It is helpful to begin
preparing reagents for Stage 3B while centrifuging and drying pellets.
 Store the plate for resuspension later in the same day:
 Tightly seal the plate.
 If resuspension will be carried within 4 hours, keep the plate at room temperature.
 If resuspension will be carried out in more than 4 hours, store the plate in a refrigerator (2-8°C).
 Store the plate for resuspension on another day:
 Tightly seal the plate.
 Store the plate at –20°C.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
60
Stage 3B: Resuspension and Hybridization Preparation
1: Prepare for Resuspension and Hybridization Preparation
To Prepare for Resuspension and Hybridization
1.
Set the centrifuge to room temperature.
2: Prepare DNA Pellets and Warm the Reagents
IMPORTANT: The plate of pelleted DNA and resuspension reagent must be at room
temperature before proceeding with this step.
The equilibration of the plate of pelleted DNA, resuspension buffer, and hybridization buffer to room
temperature (18-25°C) is very critical for the success of the Axiom 2.0 Mini 96 Assay. When any of these
are cooler than room temperature, pellets may not resuspend completely. This may result in compromised
assay performance. Please note following guidelines on how to work with plates with fresh, cold, or frozen
pellets:
Pellets:



Fresh Pellets: A plate with fresh pellets can be kept at room temperature if proceeding with the
Resuspension and Hybridization Preparation protocol within 4 hours.
Cold Pellets: A plate with fresh pellets that are not processed within 4 hours can be transferred to a
refrigerator (2-8°C) if processed during the same day. However, it is critical to equilibrate the plate to
room temperature for at least 30 minutes before proceeding with the Resuspension and Hybridization
Preparation protocol.
Frozen Pellets: A plate with frozen pellets must be pre-equilibrated at room temperature for at least
1.5 hour before proceeding with the Resuspension and Hybridization Preparation protocol.
Resuspension and Hybridization Reagents:

Resuspension buffer, hybridization buffer, Hyb Soln 1, and Hyb Soln 2 need at least 1 hour to
equilibrate to room temperature.
3: Thaw and Prepare the Reagents
To Thaw and Prepare the Reagents:
Thaw Axiom Hyb Soln 1 on the benchtop at room temperature.
Warm Axiom Resusp Buffer, Axiom Hyb Buffer, and Axiom Hyb Soln 2 on the benchtop at room
temperature for at least one hour.
3. Vortex the Axiom Resusp Buffer and the Axiom Hyb Buffer. Keep at room temperature.
4. Vortex and pulse-spin Axiom Hyb Soln 1 and Axiom Hyb Soln 2 before use.
1.
2.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
61
4: Label Tubes and Reservoirs
1.
Label the 15 mL tube as indicated in the table below:
i
Table 4.15 Label Tube
Label

2.
Hyb C
Tube Size
Temperature
Contents
15 mL
Room Temperature in Fume Hood
Hybridization Cocktail
Label one Matrix 25 mL Reagent Reservoirs (P/N 8093-11) as indicated in the table below.
i
Table 4.16 Label Reagent Reservoirs for Resuspension and Hybridization Preparation
Label

Hyb C
Temperature
Contents
Room Temperature in Fume Hood
Hybridization Cocktail
5: Prepare the Hybridization Cocktail
NOTE: If a plate was stored at –20°C after drying the pellets, it must be allowed to sit at room
temperature for 1.5 hour before carrying out resuspension.
NOTE: Make sure all reagents have equilibrated to room temperature before preparing the
Hybridization Cocktail.
CAUTION: It is recommended that the remainder of the steps in this stage be performed
under a fume hood.
1.
Prepare the Hybridization Cocktail in the Hyb C 15 mL tube.
A. Add the reagents in Table 4.17 to the Hyb C tube in the order shown.
Table 4.17 Hybridization Cocktail
Reagent
per Sample
Master Mix 96+
Axiom Resuspension Buffer
15.2 μL
1.8 mL
Axiom Hyb Buffer
30.7 μL
3.6 mL
Axiom Hyb Soln 1
0.22 μL
25.2 μL
Axiom Hyb Soln 2
3.9 μL
454.1 μL
Total Volume
50 μL
5.8 mL
To the 15 mL tube labeled Hyb C, add:
B. Vortex twice to mix.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
62
6: Add Hybridization Cocktail to DNA Pellets
To Resuspend the Pellets (carry out the following steps at room temperature):
1.
2.
3.
4.
5.
Pour the Hyb Cocktail into the reagent reservoir labeled Hyb C.
Carefully remove the seal from the Precipitation Plate and discard the seal.
Using a P200 12-channel pipette, transfer 50 μL of Hyb Cocktail to each well of the Precipitation
Plate. Avoid touching the pellets with the pipette tips.
 Change pipette tips after each addition.
 After adding Hybridization Cocktail, the plate is known as the Resuspension Plate.
Seal the Resuspension Plate.
Briefly spin down the plate in a room temperature centrifuge for 30 seconds.
7: Resuspension of DNA Pellets
Place the sealed Resuspension Plate on one of the following shakers:
 Thermo Scientific ™ Compact Digital Microplate Shaker: at speed 900 rpm for 15 min
 Jitterbug: at speed 7 for 15 min
2. Inspect the Resuspension Plate from the bottom. If the pellets are not dissolved, repeat Step 1.
3. Pulse-spin plate to 1000 rpm.
1.
8: Prepare the Hyb Ready Sample Plate
To Prepare the Hyb Ready Sample Plate
1.
Choose a 96-well plate that will be compatible with the thermo cycler model that will be used for
sample denaturation. See Table 3.3 on page 23, Thermal Cycler Consumables for the Axiom 2.0 Assay
Mini 96-Array Format Manual Protocol.
NOTE: The Axiom® Mini 96 Consumables Kit includes the 96 Half-Skirt Plate as the Hyb
Ready Sample Plate.
Label the 96-well PCR plate as Hyb Ready [Sample ID].
3. Set a P200 12-channel pipette to 55 μL (this is slightly higher than the volume of the sample in each
well of the Resuspension Plate).
4. Using the P200 pipette, transfer the entire contents of each well in the Resuspension Plate to the
labeled Hyb Ready Plate.
 Change pipette tips after each transfer.
5. Seal and pulse-spin.
2.
9: Store Remaining Reagents
Store remaining Module 2-1 and Module 2-2 reagents for future use. Follow the guidelines presented in
the section Freeze-Thaw Instructions on page 28.
10: Freeze or Proceed
At this point you can:
 Proceed to Stage 3C: Sample QC (highly recommended), below; or
 Proceed to Stage 4: Denaturation and Hybridization; or
 Store the Hyb Ready samples at –20°C.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
63
Stage 3C: Sample QC
Before proceeding to Stage 4: Denaturation and Hybridization, we highly recommend that you perform
quantitation and fragmentation quality control checks.
1: Prepare for Sample QC
To Prepare for Sample QC:
Prepare the Reagents
Obtain the Reagents for Sample QC:
15 mL of nuclease-free water.
2. 15 mL of Gel Diluent.
The Gel Diluent is a 1000-fold dilution of the TrackIt Cyan/Orange Loading Buffer as described in
Diluting the TrackIt™ Cyan/Orange Loading Buffer and 25 bp Ladder on page 145.
3. 90 μL of a 15-fold dilution of TrackIt™ 25 bp DNA Ladder as described in Diluting the TrackIt™
Cyan/Orange Loading Buffer and 25 bp Ladder on page 145.
4. One E-Gel 48 Agarose Gel, 4% Agarose
1.
Label Reservoirs
Label two Matrix 25 mL Reagent Reservoirs (P/N 8093-11) as indicated below:
Table 4.18 Label Reagent Reservoirs for QC
Label
Temperature
Contents

H2O
Leave reservoir at room temperature
Nuclease-free Water

Gel Dil
Leave reservoir at room temperature
Gel Diluent
Prepare Sample QC Plates
Label two Bio-Rad Hard Shell 96-well plates, or any 96-well PCR plate for making the dilutions:
 Label one plate as Dil QC
 Label the second plate as Gel QC
2. Obtain one 96-well UV Star, 370 μL/well plate
1.
NOTE: Change tips while transferring samples from the Hyb Ready Plate and the QC Dilution
Plate to avoid cross-contamination.
2: Perform QC Checks
To Perform the QC Checks (carry out the following steps at room temperature):
1.
Prepare Dilution QC Plate and OD Plate.
Pour 15 mL nuclease-free water into the reagent reservoir labeled H20.
The water will be used to make the Dilution QC Plate and the OD Plate.
A. Add 33 μL nuclease-free water to each well of the Dil QC Plate.
B. Add 90 μL nuclease-free water to each well of the OD Plate.
2.
Prepare the Dilution QC Plate:
A. Transfer 3 μL of the Hyb Ready sample from each well of the Hyb Ready Plate to the corresponding
well of the Dil QC Plate. Change pipette tips after each transfer.
B. Seal, vortex, and pulse-spin.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
3.
Prepare OD Plate:
A. Carefully remove the seal from the QC Dilution Plate and discard the seal.
B. Transfer 10 μL of each Dil QC sample to the to the corresponding wells of the 96-well UV Star
plate. Change pipette tips after each transfer.
C. Mix by pipetting up and down.
 Change pipette tips after each addition.
 Final sample mass dilution is 120-fold.
See Appendix B, Sample Quantitation After Resuspension on page 147 for more information on
performing the Sample Quantitation.
4. Prepare Gel QC Plate:
A. Pour 15 mL of Gel Diluent into the reagent reservoir labeled Gel Dil.
B. Add 120 μL of Gel Diluent to each well of the Gel QC Plate.
C. Transfer 3 μL of each Dil QC sample to the corresponding wells of the Gel QC Plate.
Change pipette tips after each transfer.
D. Seal, vortex, and pulse-spin the plate.
5. Run gel as described in Appendix A, Fragmentation Quality Control Gel Protocol on page 144.
After the QC checks, the Dilution QC Plate, OD plate, and remaining Gel QC samples can be
discarded once satisfactory results from the gel and OD 260 readings have been obtained.
3: Freeze or Proceed
At this point you can:
 Proceed to Stage 4: Denaturation and Hybridization, below; or
 Store the Hyb Ready samples at –20°C.
64
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
Figure 4.3 Stage 3B: Resuspension and Hybridization Preparation
Resuspension and Hybridization Preparation
Centrifuge Precipitation Plate
to Pellet DNA
Labware and Reagents Needed
Speed: 3200 xg
Duration: 40 minutes
Temperature: 4°C
Axiom® 2.0:
Module 2-1
Module 2-2
QTY 1
QTY 1
Precip Plate
(Samples)
QTY 1
QTY 1
96 Half-Skirt Plate
Dry DNA Pellets
x Decant liquid by inverting plate
x Blot-Dry inverted plate for 5 minutes
x Incubate @ 37qC for 20 minutes rightside up
Add Hybridization Cocktail
Resusp
Buffer
Hyb
Buffer
Hyb
Soln 1
Hyb
Soln 2
Resuspension of DNA Pellets
Duration: 15 minutes
Jitterbug: Speed 7
Thermo Sci Titer Plate Shaker: 900 rpm
Prepare Hyb Ready Sample Plate
Resuspension
Plate
With Resuspended
DNA
1.8 mL
3.6 mL
25.2 μL
454.1 μL
Entire well contents
(50 μL/well)
Hyb Cocktail
Vortex to mix
Hyb Ready
Sample Plate
Vortex well,
Pulse-spin.
Pour into reservoir
50 μL/well
96 Half-Skirt Plate
Final Volume: 50 μL/well
Resuspension Plate
Resuspend pellets on plate
shaker for 15 min.
Pulse-spin.
Proceed to Sample QC
Resuspension and Hybridization Preparation – Page 1 of 1
65
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
Figure 4.4 Stage 3C: QC
Sample QC
Aliquot Water to
Dilution QC and OD Plates
Labware and Reagents Needed
15 mL Water
15 mL Water
Pour into reservoir
QTY 1
(Hyb Ready
Sample Plate)
15 mL
Gel Diluent
QTY 2
QTY 1
33 μL/well
90 μL/well
QTY 2
Prepare OD Plate
Dilution QC Plate
OD QC Plate
Dilution QC Plate
Aliquot Gel Diluent to Gel QC Plate
10 μL/well
Read Abs260, 280,
and 320 nm
15 mL Gel
Diluent
OD QC Plate
Final Volume: 100 μL/well
Pour into reservoir
120 μL/well
Prepare Gel QC Plate
Dilution QC Plate
Gel QC Plate
3 μL/well
Vortex, Pulse-spin.
Prepare Dilution QC Plate
Gel QC Plate
Hyb Ready Plate:
Vortex, Pulse-spin
Load 20 µL into a
4% agarose E-Gel for
analysis
Final Volume: 123 μL/well
3 μL/well
Vortex, Pulse-spin
Proceed to Stage 4 or
Store Hyb Ready Samples at –20qC
Dilution QC Plate
Final Volume: 36 μL/well
Sample QC – Page 1 of 1
66
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
67
Stage 4: Denaturation and Hybridization
You will proceed to Stage 4 in one of two ways:
 Directly from Stage 3 without interruption.
 With Hyb Ready samples that were stored at –20°C after Stage 3.
This stage requires the following sets of steps:
1: Prepare for Denaturation and Hybridization on page 69
2: Prepare Hyb Ready Samples Stored at –20°C on page 69
3: Prepare the GeneTitan® MC Instrument on page 69
4: Denature the Hyb Ready Sample Plate on page 70
5: Prepare Hybridization Tray and Load into GeneTitan® MC Instrument on page 71
To Perform Stage 4:
 If the Hyb Ready Plate was stored at –20°C, go to 2: Prepare Hyb Ready Samples Stored at –20°C on
page 69.
 If you are proceeding directly from the end of Stage 3 on page 64, go to 4: Denature the Hyb Ready
Sample Plate on page 70.
CAUTION: Parts of this stage should be performed under a fume hood.
Duration


Hands-on: 45 minutes including denaturation time
GeneTitan MC Instrument: 23.5 to 24 hours Hyb Time
Required Input from Previous Stage

Hyb Ready Sample Plate
Equipment, Consumables, and Reagents Required
The following thermal cyclers are recommended:
 Bio-Rad PTC-200, or
 Bio-Rad DNA Engine Tetrad 2 #PTC-0240, or
 ABI 9700, or
 ABI 2720
IMPORTANT: Always use the heated lid option when programming protocols.
The thermal cycler needs to be programmed with the Axiom 2.0 Denature protocol (see Thermal Cycler
Recommendations on page 23).
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
Table 4.19 Equipment Required for Stage 4: Denaturation and Hybridization
Quantity
Equipment
1
GeneTitan MC Instrument
1
Rainin P200 12-channel Pipette
As needed
*The
Pipette tips
1
Thermal Cycler
1
96 well metal chamber warmed in a
48°C oven*
Appropriate thermal cycler, programmed with the
Axiom 2.0 Denature protocol (see Thermal Cycler
Recommendations on page 23).
metal chamber coming out of a 48°C oven is warm to the touch. Gloves and mitts can be used if it feels too hot.
Table 4.20 Consumables Required for Stage 4: Denaturation and Hybridization
Quantity
Consumable
Supplier and
Part Number
1

One Axiom myDesign Genotyping Mini 96-Array Format Plate in a protective base
Affymetrix
Various P/Ns
1

384 Hyb Tray*
P/N 501278
*The
Consumables for the GeneTitan MC Instrument are packaged separately from the Axiom array plates. The 384 Hyb
Tray, along with other GeneTitan MC consumables, are included in the Axiom® 384HT High Volume Consumables Kit
(P/N 902629).
Table 4.21 Reagents Required from the Axiom® 2.0 Assay Mini 96 Reagent Kit
Reagent
Module
Axiom Wash Buffer A (both bottles; 1L), P/N 901446
Axiom Wash Buffer B (P/N 901447)
Axiom Water (P/N 901578)
Module 3,
Room Temperature
68
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
69
1: Prepare for Denaturation and Hybridization
To Prepare for Denaturation and Hybridization
Preheat the 96-well metal chamber in a 48°C oven.
2. Allow array plate to equilibrate to room temperature for a minimum of 25 minutes.
1.
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
C. Batch Registration file as described in Appendix C, Registering Samples in Affymetrix GeneChip ®
Command Console® on page 156.
WARNING: Do not remove the array plate from the protective base or touch the
surface of any arrays.
3.
Power up the thermal cycler and prepare for the Axiom 2.0 Denature protocol to run with the heated
lid option selected.
2: Prepare Hyb Ready Samples Stored at –20°C
To Prepare Hyb Ready Samples That Were Stored at –20°C:
Warm up the Hyb Ready Plate at room temperature for 5 minutes. It is not necessary to equilibrate
the plate for longer duration.
2. Make sure the Hyb Ready Plate is sealed well.
If the plate is not sealed well:
1.
A. Spin the plate and carefully remove the old seal.
B. If there is condensation on the top of the plate, blot dry gently with a Kimwipe.
C. Use a fresh seal and tightly reseal the plate.
Vortex the Hyb Ready Plate briefly, then spin at 1000 rpm for 30 seconds.
4. Place the Hyb Ready Plate at room temperature.
3.
3: Prepare the GeneTitan® MC Instrument
Before you denature your Hyb Ready samples, ensure that the GeneTitan MC Instrument is ready for
use by following the instructions given in Chapter 5, Stage 2: Hybridization on page 101 and Appendix C,
Registering Samples in Affymetrix GeneChip® Command Console® on page 156.
A brief summary of the steps which need to be performed is:
1.
Prepare the reagents from Module 3 as described in Table 4.22:
Table 4.22 Reagents from Module 3
Reagent
Temperature
Treatment
Axiom Wash Buffer A (P/N 901446)
Room Temp
Invert 2-3X for mixing before filling GT bottle
Axiom Wash Buffer B (P/N 901447)
Room Temp
Invert 2-3X for mixing before filling GT bottle
Axiom Water (P/N 901578)
Room Temp
None
2.
Launch AGCC and select AGCC GeneTitan Control.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
3.
70
Upload your sample registration file now.
If you do not upload your samples before scanning the array plate barcode, the software will assign
names to your sample.
NOTE: When creating the sample registration file, you have the ability to add the barcode
of the hybridization tray as a sample file attribute, to enable traceability in the system.
Refer to the AGCC User Guide, Chapter 4, for details about adding attributes to your
sample files.
4.
Select the System Setup tab (Figure 4.5).
Figure 4.5 Setup Options for Processing Array Plates
5.
Configure the software as follows:
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.
 Protocol Name: Select the protocol name and click Next.
6. Fill the Wash A, Wash B and Rinse bottles.
7. Empty the Waste bottle.

4: Denature the Hyb Ready Sample Plate
Make sure the thermal cycler is powered on and the Axiom 2.0 Denature protocol with the heated lid
option has been selected.
2. Open the lid of the thermal cycler and place the sealed Hyb Ready Plate on the thermal cycler. Check
the integrity of the seal as evaporation during denaturation can negatively impact assay performance.
3. Close the lid. For thermal cyclers with variable lid tension (such as the Bio-Rad PTC-200 or the
Bio-Rad DNA Engine Tetrad 2 #PTC-0240) follow manufacturer’s instructions for adjusting lid
tension.
4. Start the Axiom 2.0 Denature protocol, described on Thermal Cycler Recommendations on page 23).
1.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
71
5: Prepare Hybridization Tray and Load into GeneTitan® MC Instrument
Plate Format Switching during Hyb Transfer Step: During this step, you will be switching plate formats.
The denatured Hyb Ready samples will be transferred from a 96-format PCR plate to a 384-format Hyb
Tray. The samples must only be transferred to the Quadrant 1 wells of the 384-format Hyb Tray.
Figure 4.6 illustrates the hyb transfer step when the switch from 96-format to 384-format occurs.
Figure 4.6 Plate Format Switching during Hyb Transfer
1
A
2
3
4
5
6
7
A1
A2
A3
A4
B1
B2
B3
B4
C1
C2
C3
C4
8Quadrants
9 10 11 Explained:
12 13 14 15 16 17 18 19 20 21 22 23 2
C
D
E
D1
D2
D3
D4
E1
E2
E3
E4
A6
A7
A8
A9
A10
A11
A12
C5
C6
C7
C8
C9
C10
C11
C12
The red well numbers in the 384 images represent the
corresponding well transferred from the 96 Hyb Ready Plate
D6
D8 left quadrant
D9
D10
D11
D12
(all D5
in Quadrant
1, D7
the upper
of a section).
F
G
A5
A 384 plate consists of 96 4-well sections (96 x 4 = 384).
Each of the 4 wells in a section is a “quadrant”.
In the
to the
wells A1,
C3 of B12
B5 example
B6
B7 left, B8
B9 A3, C1,
B10 and B11
the 384 plate are the designated Quadrant 1 (Q1) wells
within their respective 4-well section.
B
H
I
1
2
3
4
5
6
7
8
9
10
11
A
12
1
A A1
2
K4
3
A2
C B1
D
C
E
5
F1
6
7
8
F2
9
A3
A4
M
B3
G1
B4
G2 B5
N
C3
C4
C5
O
H1
L
B
B
E5
E6
E7
B2
A5
10 F3
11 12 13 F4
14 15 16 F5
17 18 19 F6
20 21 22F723 24
A6
A7
B6
G3
B7
C6
C7
G4
A8
A9
B8
B9
G5
C8
C9
A10
A11
A12
B10G6 B11
B12
G7
C1
C2
G D1
D2
D3
D4
D5
D6
D7
D8
D9
D10
D11
D12
E1
E2
E3
E4
E5
E6
E7
E8
E9
E10
E11
E12
F
K
F1
F2
F3
F4
F5
F6
F7
F8
F9
F10
F11
F12
G
M G1
G2
G3
G4
G5
G6
G7
G8
G9
G10
G11
G12
H
O H1
H2
H3
H4
H5
H6
H7
H8
H9
H10
H11
H12
F
D
P
H
E
E8
E9
E10
E11
E12
F8
F9
F10
F11
F12
G8
G9
G10
G11
G12
H8
H9
H10
H11
H12
J
I
H2
H3
H4
C10
H5
C11
H6
C12
H7
J
L
N
P
96 Hyb Ready Plate
384 Hyb Tray
CAUTION: It is recommended to perform the next set of steps under a fume hood.
1.
2.
3.
4.
5.
After the Axiom 2.0 Denature protocol has completed, remove the Hyb Ready Plate from the thermal
cycler and place into a 96-well block that has been pre-warmed in an oven at 48°C.
Move the warm 96-well block containing the denatured Hyb Ready Plate to a fume hood.
Remove seal from Hyb Ready Plate and discard.
Remove the 384 Hyb Tray from packaging.
Label the hyb tray. See the note below and Figure 3.6 on page 37 for more information.
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 F1) as illustrated in Figure 3.6 on page 37. Do NOT write on any
other side, as this can interfere with sensors inside the GeneTitan MC Instrument and
result in experiment failure.

Be sure to remove the hyb tray cover before transferring the denatured samples.
IMPORTANT: Do not confuse hyb trays with stain trays.
6.
Place the hyb tray under the fume hood. Remove the hyb tray cover.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
7.
72
Obtain a P200 12-channel pipette and set it at 35 μL. Slowly transfer the denatured samples from the
96-well Hyb Ready Plate into the corresponding quadrant 1 wells of the 384 Hyb Tray as instructed
below in Table 4.23 Plate Format Switching Guidance.
Table 4.23 Plate Format Switching Guidance: Transfer denatured samples from
a 96-array format PCR plate to Wells in Quadrant 1 of a 384-array format Hyb Tray.
96-array format Hyb Ready PCR Plate
384-array format Hyb Tray
Row A
Row A, odd wells
Row B
Row C, odd wells
Row C
Row E, odd wells
Row D
Row G, odd wells
Row E
Row I, odd wells
Row F
Row K, odd wells
Row G
Row M, odd wells
Row H
Row O, odd wells




8.
Dispense to the first stop to avoid creating bubbles.
Change pipette tips after each transfer; discard the tip even if it shows some volume left.
Ensure that there are no air bubbles present in the hyb tray. Puncture any air bubbles that you see
using a clean pipette tip.
There is no need to spread the sample around the bottom of the hyb tray wells. Sample distribution
across the well will occur when the array plate is stacked together with the hyb tray by the
GeneTitan MC Instrument.
Load the array plate and hyb tray into the GeneTitan MC Instrument (see Load Axiom® Array Plate
and Hyb Tray Onto the GeneTitan® MC Instrument on page 106).
IMPORTANT: The sandwich of the array plate and hybridization tray needs to be
manually clamped and inspected before the array processing can begin. Carefully review
and execute the array plate/hyb tray clamping procedure steps as detailed in Figure 5.20,
Array Plate/Hyb Tray Clamping Procedure on page 110.
Hybridization will continue on the GeneTitan MC Instrument for 23.5-24 hours before you can load the
Ligation/Staining/Stabilization reagent trays into the GeneTitan MC Instrument. Near the end of the
23.5 to 24 hour hybridization period in the GeneTitan MC, proceed to Stage 5: GeneTitan® Reagent
Preparation.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
Figure 4.7 Stage 4: Denaturation and Hybridization
Denaturation and Hybridization
Labware and Reagents Needed
Denature Hyb Ready Samples
in Thermal Cycler
1) 10 min. @ 95°C
2) 3 min. @ 48°C
QTY 1
(Hyb Ready
Sample Plate)
QTY 1
Transfer Denatured Hyb Ready Samples
to Hyb Tray
Denatured Hyb
Ready Samples
Keep samples on a
48°C heat block
35 μL/well into Quadrant 1 only
Hyb Tray with
Samples in Q1
Process Hybridization Tray on
the GeneTitan® MC Instrument
73
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
74
Stage 5: GeneTitan® Reagent Preparation
This stage needs to be done when hybridization in the GeneTitan MC Instrument is near completion
(1.5 hours before completion), so the reagent trays can be loaded for the GeneTitan MC array processing
steps.
Total time for this step: 1.5 hours, including reagent preparation, hands-on time and GeneTitan MC
Instrument loading.
IMPORTANT: The reagent trays prepared in this step, Stage 5: GeneTitan® Reagent
Preparation are for the continued processing of an Axiom array plate that:

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 should not 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 should not be stored.
The following instructions are for manually preparing the reagents and trays required to process Axiom
array plates on the GeneTitan MC Instrument:
1: Prepare for GeneTitan® Reagent Preparation on page 77
2: Prepare the Stain, Ligation, and Stabilization Master Mixes on page 80
3: Aliquot Master Mixes and Axiom Hold Buffer into Trays on page 83
4: Store Remaining Reagents on page 87
The reagents and trays required are as follows:
Table 4.24 Reagent Trays Required for the Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol on the GeneTitan®
MC Instrument
Type of Tray
Quantity
Tray Designation
Master Mix/Reagent
384 Layout GeneTitan Stain Tray
2
S1
Stain 1 Master Mix
384 Layout Axiom® Stain2 Tray
1
S2
Stain 2 Master Mix
384 Layout Axiom® Stab Tray
1
Stbl
Stabilization Master Mix
384 Layout Axiom® Ligation Tray
1
Lig
Ligation Master Mix
Scan Tray
1
Scan Tray
Hold Buffer
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
75
Equipment, Consumables, and Reagents Required
Table 4.25 Equipment Required for Stage 5: Manually Preparing Ligation, Staining, and Stabilization Reagent Trays for
the GeneTitan® MC Instrument
Quantity
Equipment
1
GeneTitan MC Instrument
1
Ice bucket with ice
As Needed
Kimwipes
As Needed
Markers
1
Cooler for enzyme
1
Microcentrifuge
1
Pipetaid
1 each
Rainin Pipettes: single channel
P200
 P1000
Rainin Pipettes: 12-channel:
 P200

1
Vortexer
Table 4.26 Consumables Required for Stage 5: Manually Preparing Ligation, Staining, and Stabilization Reagent Trays for the
GeneTitan® MC Instrument
Quantity
As required
1 kit* includes:
10
5
5
5
5
5
25
1
As required for pipettes
listed in Table 4.25
*
Consumable
Part Number
Aluminum foil (optional)
Axiom® 384HT High Volume Consumables Kit (Sufficient for 5 x Mini 96-Array
Format Plates)
®
 384 Layout GeneTitan Stain Tray (Stain 1)
®
 384 Layout Axiom Stain2 Tray
®
 384 Layout Axiom Stab. Tray
®
 384 Layout Axiom Ligation Tray
®
 384 Layout GeneTitan Hyb Tray
®
 384 Layout GeneTitan Scan Tray
®
 384 Layout GeneTitan Scan and Stain Tray Cover
Affymetrix P/N 902629
Pipette, serological
5 x 1/10 mL

Pipette tips
5
Matrix™ 25 mL Reagent Reservoir
4
15 mL conical tube
P/N 8093-11
Each Axiom® 384HT High Volume Consumable Kit is sufficient to process 5 Axiom Mini 96- or 384-array format plates. These trays are
required for processing Axiom 384 array plates on the GeneTitan® Multi-Channel Instrument.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
Reagents Required
Table 4.27 Axiom 2.0 Reagents Required for Stain and Ligation Stage (for processing 2 Mini 96-Array Format Plates)
Reagent
Module
Axiom Ligate Buffer
Axiom Ligate Enzyme
Axiom Ligate Soln 1
Module 4-1, –20°C
P/N 901278
Axiom Probe Mix 1
Axiom Stain Buffer
Axiom Stabilize Soln
Axiom Ligate Soln 2
Axiom Probe Mix 2*
Axiom Wash A
Axiom Stain 1-A*
Axiom Stain 1-B*
Axiom Stain 2-A*
Module 4-2, 2-8°C
P/N 901276
Axiom Stain 2-B*
Axiom Stabilize Diluent
Axiom Water
Axiom Hold Buffer*
Additional Axiom Hold Buffer (1 bottle)
(For processing the second Axiom Mini 96-array format plate)
*
These solutions are light sensitive. Keep tubes out of direct light for a prolonged period of time.
2°C to 8°C
P/N 903012
76
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
77
1: Prepare for GeneTitan® Reagent Preparation
Thaw and Prepare the Reagents
NOTE: Ligation Buffer and Ligation Solution 2 require approximately 30 to 40 min to thaw on
the benchtop at room temperature.
Table 4.28 Reagents Required for GeneTitan® MC Instrument Reagent Tray Preparation
Module
Module 4-1
–20°C
Reagent
Thaw on Benchtop,
then Place on Ice
Axiom Ligate Buffer*
 for 30 min
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

Place on Ice
 for 30 to 40 min
Axiom Ligate Soln 2
Axiom Probe Mix 2†

 for 30 min
Axiom Wash A
Module 4-2
2 to 8°C
*
Axiom Stain 1-A†

Axiom Stain 1-B†

Axiom Stain 2-A†

Axiom Stain 2-B†

Axiom Stabilize Diluent

Axiom Water

Axiom Hold Buffer†
(1 bottle required)‡

This bottle can also be thawed in a dish with room temperature Millipore water.
These solutions are light sensitive. Keep tubes out of direct light for a prolonged period of time.
‡ Axiom Hold Buffer for preparing the Scan Tray for the second plate are provided in P/N 903012.
†
Place on Benchtop at
Room Temperature
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
78
Preparing Axiom Wash A and Axiom Stabilize Diluent
During 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.
To Prepare the Axiom Wash A:
1.
2.
3.
4.
5.
Vortex the bottle for 30 sec.
Place on the benchtop at room temperature for 30 min.
Examine the reagent for precipitate (look into the top of the bottle).
If precipitate is still present, vortex again for 30 sec.
Leave on the benchtop until ready to use.
To Prepare the Stabilize Diluent:
If crystals are observed in the Axiom Stabilize Diluent:
Vortex and spin.
2. Look for precipitate.
If any:
 Warm tube to room temperature and vortex again.
1.
Preparing Axiom Ligate Buffer
White precipitate is sometimes observed when the Axiom Ligate Buffer is thawed.
NOTE: The presence of some precipitate is OK 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.
2.
3.
4.
5.
6.
7.
Place on the benchtop at room temperature for 30 min. This bottle can also be thawed in a dish with
room temperature Millipore water.
Examine the buffer for precipitate (look into the top of the bottle).
If precipitate is present, vortex the bottle for 30 sec.
Re-examine the buffer for precipitate.
If precipitate is still present, warm the bottle with your hands and vortex again for 30 sec.
If precipitate is still present after hand warming proceed with the protocol below.
Leave the Axiom Ligate Buffer on the benchtop until ready to use.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
79
Prepare the Remaining Reagents
To Prepare the Remaining Reagents for GeneTitan MC Instrument Plate Preparation:
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, spin 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:
1.
A. Gently flick each tube 2 to 3 times to mix, then spin.
B. Place reagents on ice, except for the Axiom Hold Buffer, Axiom Ligate Soln 2 and Axiom Water—
leave these reagents at room temperature.
Label the Master Mix Tubes
1.
Mark the side of each tube with one of designations shown in Table 4.29.
Table 4.29 Labeling Master Mix Tubes for Stain, Ligation, and Stabilization Reagents
Conical Tube
Number of Tubes
Tube Designation
Contents
Place Tube:
15 mL
1
S1

Stain 1 Master Mix
On ice
15 mL
1
S2

Stain 2 Master Mix
On ice
15 mL
1
Stbl

Stabilization Master Mix
On ice
15 mL
1
Lig

Ligation Master Mix
On ice
Label the Reagent Reservoirs
1.
Label five Matrix 25 mL Reagent Reservoirs (P/N 8093-110) as indicated in the table below.
Table 4.30 Labeling Reagent Reservoirs
Reservoir Designation
Contents
S1

Stain 1 Master Mix
S2

Stain 2 Master Mix
Stbl

Stabilization Master Mix
Lig

Ligation Master Mix
Hold

Axiom Hold Buffer
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
80
2: Prepare the Stain, Ligation, and Stabilization Master Mixes
Prepare Stain 1 Master Mix
To Prepare the Stain 1 Master Mix:
1.
Use appropriate serological and single-channel pipettes to add reagents to the 15 mL tube labeled S1
in the order shown in Table 4.31. This recipe will provide enough for both S1 reagent trays.
Table 4.31 Stain 1 Master Mix
Reagent
per Array
Master Mix 96+
To the tube marked S1, add:

Axiom Wash A
67.2 μL
7.392 mL

Axiom Stain Buffer
1.4 μL
154 μL

Axiom Stain 1-A
0.7 μL
77 μL

Axiom Stain 1-B
0.7 μL
77 μL
70 μL
(35 μL x 2)
7.7 mL
Total
Gently invert the tube 10 times to mix. Do not vortex.
3. Place on ice and protect from direct light (e.g., cover with aluminum foil or ice bucket lid).
2.
Prepare Stain 2 Master Mix
To Prepare the Stain 2 Master Mix:
1.
Use appropriate single-channel pipettes to add reagents to the 15 mL tube labeled S2 in the order
shown in Table 4.32.
Table 4.32 Stain 2 Master Mix
Reagent
per Array
Master Mix 96+
To the tube marked S2, add:

Axiom Wash A
33.6 μL
4.2 mL

Axiom Stain Buffer
0.70 μL
87.5 μL

Axiom Stain 2-A
0.35 μL
43.8 μL

Axiom Stain 2-B
0.35 μL
43.8 μL
35 μL
4.4 mL
Total
Gently invert the S2 tube 10 times to mix. Do not vortex.
3. Place on ice and protect from direct light (e.g., cover with aluminum foil or ice bucket lid).
2.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
81
Prepare Stabilization Master Mix
To Prepare the Stabilization Master Mix:
1.
Use appropriate single-channel pipettes to add reagents to the 15 mL tube labeled Stbl in the order
shown in Table 4.33.
Table 4.33 Stabilization Master Mix
Reagent
per Array
Master Mix 96+
To the tube marked Stbl, add:

Axiom Water
31.1 μL
3914 μL

Axiom Stabilize Diluent
3.5 μL
441 μL

Axiom Stabilize Soln
0.4 μL
55 μL
35 μL
4.4 mL
Total
Vortex the master mix at high speed for 3 sec.
3. Place on ice.
2.
Prepare Ligation Master Mix
The Ligation Master Mix is prepared in two stages.
Ligation Master Mix: Stage 1
To Begin Preparing the Ligation Master Mix:
Place the 15 mL conical tube marked Lig on ice.
2. Use appropriate single-channel pipettes to add reagents to the 15 mL tube labeled Lig in the order
shown in Table 4.34.
1.
Table 4.34 Ligation Master Mix Preparation: Stage 1
Reagent
per Array
Master Mix 96+
To the tube marked Lig, add:

Axiom Ligate Buffer
22.1 μL
2778.3 μL

Axiom Ligate Soln 1
4.4 μL
551 μL

Axiom Ligate Soln 2
1.1 μL
132.3 μL
27.5 μL
3.46 mL
Subtotal
Mix well by vortexing the tube for 3 seconds.
4. Place the tube marked Lig back on ice.
3.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
82
Ligation Master Mix: Stage 2
To Finish Preparing the Ligation Master Mix:
Remove the Axiom Ligate Enzyme from the –20°C freezer and place in a cooler chilled to –20°C.
2. Use appropriate serological and single-channel pipettes to add reagents to the 15 mL tube labeled Lig
in the order shown in Table 4.35.
Gently flick the Axiom Ligate Enzyme tube 2-3 times, then perform a quick spin immediately prior
to adding the enzyme to the Master Mix.
1.
Table 4.35 Ligation Master Mix Preparation: Stage 2
Reagent
per Array
Master Mix 96+

Ligation Master Mix from Stage 1
27.5 μL
3.46 mL

Axiom Probe Mix 1
3.5 μL
441 μL

Axiom Probe Mix 2
3.5 μL
441 μL

Axiom Ligate Enzyme
0.53 μL
66.4 μL
35 μL
4.4 mL
Total
Gently invert 10 times to mix. Do not vortex.
4. Place on ice and protect from direct light (e.g., cover with aluminum foil or ice bucket lid).
3.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
83
3: Aliquot Master Mixes and Axiom Hold Buffer into Trays
Label the Trays
Gather the scan tray and the stain trays and covers from the Axiom® 384 HT High Volume
GeneTitan® Consumables Kit.
2. When preparing the hybridization and reagent trays to be loaded onto the GeneTitan MC
Instrument, you will need to mark the front of each tray in a way that identifies its contents.
Obtain the stain trays and label each specific tray as listed on Table 4.36:
1.
IMPORTANT: It is critical that you write only on the proper location of the stain/reagent
trays (on the edge in front of wells A1 to F1) as illustrated in Figure 3.7 on page 38.
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.
Table 4.36
Stain Tray Type
Label Color
Label the Tray
384 Layout GeneTitan® Stain Tray, P/N 501279
White
Stain 1-1
384 Layout GeneTitan® Stain Tray, P/N 501279
White
Stain 1-2
Blue
Stain 2
384 Layout Axiom® Ligation Tray, P/N 501398
Yellow
Lig
384 Layout Axiom® Stabilization Tray, P/N 501396
Green
Stbl
384 Layout Axiom® Stain2 Tray, P/N 501394
About Aliquoting Reagents to Trays
Stain Trays: Only fill Quadrant 1 of the stain trays with ligation, staining, and stabilization reagents.
Figure 4.8 Quadrant 1 Wells of a 384 Stain Tray
1
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
84
Scan Tray: It is important to fill all 96 wells with Hold Buffer. The scan tray has an open-bottom design,
so it is very important that all 96 wells of the scan tray receive 170 μL of Axiom Hold Buffer.
IMPORTANT: Immediately load the reagent trays onto the GeneTitan MC Instrument.
For all trays, pipet into trays on the bench top. If the trays are not being used immediately, protect them
from light by covering with foil or placing in a cabinet.
When aliquoting ligation, staining, and stabilization reagents to the trays, it is not necessary to spread
the reagent to each corner of the well. The reagent will spread evenly when the array plate is inserted into
the reagent tray during processing with the GeneTitan MC Instrument.
Stain 1 Master Mix
To Aliquot the Stain 1 Master Mix:
Pour the S1 Master Mix into the reagent reservoir marked S1, placed on the bench top at room
temperature.
2. Load a P200 12-channel pipette with 12 new pipette tips and aliquot 35 μL per Q1 well to both S1
trays. Dispense to the first stop only to avoid creating bubbles.
You do not need to change pipette tips between additions of the Stain 1 Master Mix.
3. If:
 Bubbles are present, puncture them with a pipette tip.
 Droplets of liquid splashed onto the well dividers, place a Kimwipe on top of the tray to blot and
remove. (Figure 4.9).
1.
Figure 4.9 Well Dividers in Stain Trays (partial tray view)
Example of a droplet of liquid
that has splashed onto the well
divider of a stain tray during
reagent aliquoting.
Ensure no droplets of liquid are
on top of the wells dividers. Blot
with a Kimwipe to remove.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
4.
85
Place covers on the S1 trays. Orient cover correctly on the tray with the notched corners
together (Figure 4.10).
Figure 4.10 Placing Cover on Stain Tray
Notched corners of
Stain Tray and Lid
Notched corners
should face the front
5.
Protect the trays from light if not immediately loading onto the GeneTitan MC Instrument.
Stain 2 Master Mix
To Aliquot the Stain 2 Master Mix:
1.
2.
3.
4.
5.
Carefully pipet or pour the Stain 2 Master Mix into the reagent reservoir marked S2, placed on the
bench top at room temperature.
Load a P200 12-channel pipette with 8 new pipette tips and aliquot 35 μL per Q1 well to the S2 tray.
Dispense to the first stop only to avoid creating bubbles.
You do not need to change pipette tips between additions of the Stain 2 Master Mix.
If:
 Bubbles are present, puncture them with a pipette tip.
 Droplets of liquid splashed onto the well dividers, place a Kimwipe on top of the tray to blot
and remove.
Place a cover on the S2 tray. Orient the cover correctly on the tray with the notched corners
together (Figure 4.10).
Protect the tray from light if not immediately loading onto the GeneTitan MC.
Stabilization Master Mix
To Aliquot the Stabilization Master Mix:
Carefully pipet or pour the Stabilization Master Mix into the reagent reservoir marked Stbl, placed
on the bench top at room temperature.
2. Load a P200 12-channel pipette with 12 new pipette tips and aliquot 35 μL per Q1 well to the Stbl
tray. Dispense to the first stop only to avoid creating bubbles.
You do not need to change pipette tips between additions of the Stabilization Master Mix.
3. If:
 Bubbles are present, puncture them with a pipette tip.
 Droplets of liquid splashed onto the well dividers, blot the top of the tray with a Kimwipe.
4. Place a cover on the tray. Orient cover correctly on the tray with the notched corners together
(Figure 4.10).
1.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
86
Ligation Master Mix
To Aliquot the Ligation Master Mix:
1.
2.
3.
4.
5.
Carefully pipet or pour the Ligation Master Mix into the reagent reservoir marked Lig, placed on the
bench top at room temperature.
Load a P200 12-channel pipette with 12 new pipette tips and aliquot 35 μL per Q1 well to the Lig
tray. Dispense to the first stop only to avoid creating bubbles.
You do not need to change pipette tips between additions of the Ligation Master Mix.
If:
 Bubbles are present, puncture them with a pipette tip.
 Droplets of liquid splashed onto the well dividers, place a Kimwipe on top of the tray to blot and
remove.
Place a cover on the tray. Orient cover correctly on the tray with the notched corners
together (Figure 4.10).
Protect the tray from light if not immediately loading onto the GeneTitan MC.
Axiom Hold Buffer
To Aliquot the Axiom Hold Buffer to the Scan Tray:
Ensure that the Axiom Hold Buffer has equilibrated to room temperature. Vortex and then pour the
Axiom Hold Buffer into the reagent reservoir labeled Hold, placed on the bench top at room
temperature.
2. Remove the scan tray from its pouch.
3. Remove the scan tray cover, but leave the scan tray on its protective black base.
4. Place the cover as shown in Figure 4.12 on page 87 to prevent dust or static from accumulating on
the bottom of the cover.
1.
Use a 12-channel P200 pipette with new pipette tips to aliquot 170 μL to EACH of the 96 wells of
the 384 Layout GeneTitan Scan Tray. Dispense to the first stop and avoid touching the bottom of
the tray.
 You do not need to change pipette tips between additions of the Hold buffer.
5. If droplets of liquid splashed onto the well dividers, place a Kimwipe on top of the tray to blot and
remove.
6. Cover the tray by orienting the notched corner of the scan tray cover over the notched edge of the
tray and the flat side of the cover against the scan tray (Figure 4.11).

IMPORTANT: The scan tray has an open-bottom design, so it is very important that all 96
wells of the scan tray receive 170 μL of Axiom Hold Buffer.
CAUTION: Do not remove the scan tray from its protective black base until loading onto
the GeneTitan MC instrument. To avoid scratching, do not touch the bottom of the tray
with pipette tips. Dispense hold buffer to the first stop only.
See Stage 3: Ligate, Wash, Stain and Scan on page 118 for instructions on loading the reagent trays.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
87
Figure 4.11 Scan Tray with Cover on the Blue Base
Always leave the scan tray in its protective blue base.
Barcoded Scan Tray Cover
P/N 202757
Scan Tray
protective base
GeneTitan® Scan Tray
P/N 501006 or
P/N 500860
Notched corner of the cover is aligned
with the notched corner of the scan tray.
Figure 4.12 Loading the Scan Tray with Axiom Hold Buffer
Leave the scan tray in its protective blue base while loading with Axiom Hold Buffer.
Cover
Protective Blue Base
4: Store Remaining Reagents
Store remaining Module 4-1 and Module 4-2 reagents for future use. Follow the guidelines presented in
the section Freeze-Thaw Instructions on page 28.
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
Figure 4.13 Stage 5: GeneTitan Reagent Preparation, #1 of 3
GeneTitan Reagent Preparation
Labware and Reagents Needed
Axiom® 2.0:
Module 4-1
Module 4-2
QTY 1
QTY 5
QTY 5
QTY 4
Prepare Stain 1 Trays
Wash A
7392 μL
Stain
Buffer
154 μL
Stain 1A
77 μL
Prepare Stain 2 Tray
Stain 1B
Wash A
Stain
Buffer
Stain 2A
Stain 2B
77 μL
4200 μL
87.5 μL
43.8 μL
43.8 μL
Invert tube 10X to mix.
Do not vortex.
Invert tube 10X to mix.
Do not vortex.
Pour into reservoir
Pour into reservoir
35 μL/well to Q1
35 μL/well to Q1
Tray Names: S1
Fill two trays
Confirm there are no
droplets on well dividers
Tray Name: S2
Confirm there are no
droplets on well dividers
2 Trays
Continue to
GeneTitan Reagent Preparation
Page 2
GeneTitan Reagent Preparation – Page 1 of 3
88
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
89
Figure 4.14 Stage 5: GeneTitan Reagent Preparation, #2 of 3
GeneTitan Reagent Preparation
Prepare Stabilization Tray
Axiom
Water
Stabilize
Diluent
Stabilize
Soln
3914 μL
441 μL
55 μL
Continue from
GeneTitan Reagent Preparation
Page 1
Vortex
Pour into reservoir
35 μL/well to Q1
Tray Name: Stbl
Confirm there are no
droplets on well dividers
Continue to
GeneTitan Reagent Preparation
Page 3
GeneTitan Reagent Preparation – Page 2 of 3
Chapter 4 | Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation
Figure 4.15 Stage 5: GeneTitan Reagent Preparation, #3 of 3
GeneTitan Reagent Preparation
Continue from
GeneTitan Reagent Preparation
Page 2
Prepare Ligation Master Mix
Part 1
Ligate
Buffer
Ligate
Soln 1
Ligate
Soln 2
2778.3 μL
551 μL
132.3 μL
Aliquot Hold Buffer to Scan Tray
Ligation Master Mix:
Vortex to mix
Hold Buffer
Pour into reservoir
Prepare Ligation Master Mix Part 2
and Prepare Ligation Tray
Probe
Mix 1
441 μL
Probe
Mix 2
Ligate
Enzyme
441 μL
66.4 μL
170 μL/well
Confirm there are no
droplets on well dividers
Add reagents to
Ligation Master Mix tube
Invert tube 10X to mix.
Do not vortex.
Pour into reservoir
Process Plates on the
GeneTitan® MC Instrument
35 μL/well in Q1
Tray Name: Lig
Confirm there are no
droplets on well dividers
GeneTitan Reagent Preparation – Page 3 of 3
90
Chapter 5
Array Processing with the GeneTitan® Multi-Channel Instrument
The Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol is designed for processing 96 samples at
a time on 96 arrays simultaneously. The protocol is performed in two sets of steps:
 Target Preparation: See Chapter 4, Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation on
page 40.
 Array Processing: performed on the GeneTitan® Multi-Channel (MC) Instrument.
This chapter includes instructions for Part 2: Array Processing. These instructions are presented as
follows:
 Before Using the GeneTitan® MC Instrument on page 91
 Stage 1: Create and Upload Batch Registration File on page 100
 Stage 2: Hybridization on page 101
 Stage 3: Ligate, Wash, Stain and Scan on page 118
Before Using the GeneTitan® MC Instrument
Proper Tray Alignment and Loading
Proper alignment and loading of a tray and its cover is critical when using the GeneTitan MC
Instrument. Each tray and cover has one notched corner. The notched corner of the tray and its
corresponding cover or protective base 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 5.1
and Figure 5.2 on page 93).
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.
TIP: Mark the notched corner of each tray and cover with permanent marker to help ensure
proper alignment and loading onto the GeneTitan MC Instrument.
CAUTION: Take care not to damage the consumables or bend the blue cover posts or scan
tray posts.
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.
Chapter 5 | Array Processing with the GeneTitan® Multi-Channel Instrument
Figure 5.1 Proper Alignment and Loading of Plates, Covers and Trays in the GeneTitan® MC Instrument
Shipping Cover
(to be discarded)
IMPORTANT: Remove the plastic
protective shipping tray cover.
Array Plate
protective base
Array Plate
Notched corner of array plate aligned
with notched corner of blue base.
Tip
Mark the notched corner of each plate,
cover and tray with permanent marker to
help ensure proper alignment and loading.
Plates and trays must be seated in this groove.
The notched corner of all plates, bases, and
covers and must be seated in this corner of
the drawer per the Tray Alignment guide.
92
Chapter 5 | Array Processing with the GeneTitan® Multi-Channel Instrument
93
Figure 5.2 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
IMPORTANT: When you install the consumables, ensure that the fingers are retracted
(Figure 5.3). Do not lay the consumables on top of the drawer fingers - this indicates that the
instrument is not functioning correctly. Please 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.
Figure 5.3 Fingers retracted
Fingers retracted
Fingers retracted
Chapter 5 | Array Processing with the GeneTitan® Multi-Channel Instrument
94
Stain Trays and Covers
IMPORTANT: Always place the flat side of the cover against the stain tray.
Figure 5.4 Placement of Covers on Trays
Correct placement of cover on stain tray.
Incorrect placement of cover on stain tray.
Labeling GeneTitan Hybridization and Reagent Trays
When 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.
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.
Proper labeling for hyb trays and reagent trays is described in:
Labeling for Hyb Trays, on page 95
 Labeling for Stain Trays on page 95

IMPORTANT: Do not confuse hyb trays with stain trays.
Chapter 5 | Array Processing with the GeneTitan® Multi-Channel Instrument
95
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 5.5. The proper section for labeling is closest to the notched corner, corresponding to
the A1 and F1 wells.
Figure 5.5 Labeling Hyb Trays
Do NOT label trays on
the long side of the tray
Notched corner of the hyb tray
should face the front
Label the hyb tray here
CAUTION: Writing on the wrong side of the Hyb tray, or on the wrong part of the long side,
may interfere with the operation of sensors in the GeneTitan MC Instrument.
Labeling for Stain Trays
You may label the stain trays on the left side of the front of the tray as shown in Figure 5.6. The correct
side is closest to the notched corner, corresponding to the A1 through F1 wells.
Figure 5.6 Labeling Stain Tray (Stain Tray shown with Lid)
DO NOT label trays on
the long side of the tray
Label the stain tray here
Notched corner of the stain
tray should face the front
Chapter 5 | Array Processing with the GeneTitan® Multi-Channel Instrument
96
Email and Telephone Notifications from the GeneTitan® MC Instrument
We strongly recommend that you configure the Affymetrix GeneChip® Command Console (AGCC)
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. Refer to the AGCC user manual for
instructions.
The types of notifications available will let you know when a process:
 Starts
 Completes
 Aborts
 Encounters an error
Chapter 5 | Array Processing with the GeneTitan® Multi-Channel Instrument
97
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 two 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® Multi-Channel Instrument User Guide, P/N 08-0308.
Refer to the GeneTitan® MC Instrument User Guide 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. Please 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 5.7. A brief
description of each option is given below.
Figure 5.7 Setup Options for Processing Array Plates
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98
Hyb-Wash-Scan
This setup option enables you to hybridize, wash-ligate-stain-stabilize, and scan an array plate on the
GeneTitan MC Instrument.
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.


Hyb: the array plate is moved to the hybridization oven inside the instrument. Each denatured sample
in the hyb tray is hybridized to an array on the array plate.
 Duration for 96 samples = 23.5 hr
Wash: samples on arrays are ligated, washed, stained and stabilized.
 Duration for 96 samples = ~5 hr
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.

Scan: The array plate is moved to the imaging device in the GeneTitan MC Instrument and each array
is scanned.
 Duration for 96 samples = ~1.5 hr
Hyb-Wash
If 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:
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 plate. 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.
1.
When Ready to Scan the Array Plate:
Keeping the plate protected from light, bring the plate to room temperature for ~50 min.
2. Remove the aluminum foil and load onto the GeneTitan MC Instrument.
1.
Wash-Scan
Use this option if:
 It was necessary to hybridize the array plate in an oven separate from the GeneTitan MC Instrument.
 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 Resume
Use this option if:
 Fluidics processing has been interrupted (e.g., a power failure occurs at your facility).
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Scan
Use 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.
 If you want to queue a second plate for scanning. Using the Scan option allows you to start a second
scan workflow while another scan workflow is already running. See Queuing a Second Plate for Scanning
on page 114.
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 5.8.
If the instrument aborts a process, you can retrieve the array plate and related consumables as described
in Figure 5.8. An instrument-initiated abort may occur:
 Due to improper placement of plates
 If the UPS detects a long power interruption, draining the UPS to 75% power.
Figure 5.8 Manually Aborting an Array Plate
To abort array plate processing:
1.
2.
3.
4.
5.
Click the Stop button.
Select the array plate that you want to abort.
Click Abort.
Click Yes.
Wait until the status of the array plate in the WorkFlow
window changes from AbortRequest… to Aborted
(5A and 5B).
6. Once aborted, retrieve the array plate and other related
consumables by:
 Using Setup Option: Unload Plates
 Loading a new array plate.
1.
2.
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.
3.
4.
5A.
5B.
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Stage 1: Create and Upload Batch Registration File
You must create and upload a Batch Registration file in the AGCC software before you begin Stage 2:
Hybridization on page 101 (example shown in Figure 5.9). This file contains information critical for:
 Data file generation during scanning
 Tracking the experimental results for each sample loaded onto an array plate
1.
2.
3.
4.
5.
6.
7.
If you have not already created a batch registration file, create one now. (See Appendix C, Registering
Samples in Affymetrix GeneChip® Command Console® on page 156 for detailed instructions.)
In AGCC, select the array plate format (384 samples) and open a GeneTitan batch registration file
template.
Scan the array plate barcode into the yellow barcode field, column F.
Enter a unique name for each sample and any additional information.
Scan the barcode of the hybridization tray if your batch registration file template includes a column
for the hybridization tray barcode.
Save the file.
Upload the file.
NOTE: When creating the sample registration file, you have the ability to scan the barcode
of the hybridization tray to implement sample traceability. If you do not upload your
samples before scanning the array plate barcode, the software will assign names to your
sample.
IMPORTANT: It is very important to create and upload a batch registration file with your
sample information prior to starting Stage 2: Hybridization on page 101.
Figure 5.9 Example of a Batch Registration File for an Array Plate
Chapter 5 | Array Processing with the GeneTitan® Multi-Channel Instrument
101
Stage 2: Hybridization
Reagents Required
Reagents RequiredRequired
Table 5.1 Reagents Required from the Axiom® 2.0 Assay Mini 96 Reagent Kit
Reagent
Module
Axiom Wash Buffer A, P/N 901446
(both bottles; 1L)
Axiom Wash Buffer B, P/N 901447
Module 3,
Room Temperature
Axiom Water, P/N 901578

An Axiom Mini 96-Array Format 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:
A. 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.
WARNING: Do not remove the array plate from the protective base or touch the
surface of any arrays.
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 Stage 1: Create and Upload Batch Registration File on page 100).
A hybridization tray containing denatured samples is also required for this step. The denatured samples
should be transferred to the hyb tray only after the GeneTitan MC Instrument is ready for loading the
hyb tray in the Load Axiom® Array Plate and Hyb Tray Onto the GeneTitan ® MC Instrument section on
page 106.
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Setup the Instrument
To Setup the Instrument:
1.
Launch AGCC Launcher and select AGCC GeneTitan Control (Figure 5.10).
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
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.
IMPORTANT: Please 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 Shutting Down the GeneTitan® MC Instrument on page 126.
Figure 5.10 Launching AGCC and Initializing the GeneTitan® MC Instrument
System ready
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Figure 5.11 System Setup Tab and the Information Displayed in this Pane
System Setup tab
Setup Option: The various options you can
choose for processing Axiom array plates.
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.
Workflow Steps: This field
displays an overview of the user
actions required to process an
array plate based on the setup
option selected.
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.
Select the System Setup tab (Figure 5.11).
3. Configure the software as follows:
B. Setup Option: Hyb-Wash-Scan
Other options available are described under Setup Options for Array Plate Processing on page 97.
C. Click Next.
2.
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 eight Axiom array plates.

One Axiom Mini 96-array format plate requires ~7 GB
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104
D. Plate Information:

Barcode: Scan or manually enter the Axiom array plate barcode and click Next.
The first six 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.
Figure 5.12 Barcode Error Message
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.
Protocol Name: Select the protocol name and click Next.
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 5.13 on page 105)
1) 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 two plates on the same day.
Rinse: fill with Axiom Water—keep at 1L full
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 Assay Mini 96 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. 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 two array plates on the same day, fill the Wash B bottle
to the 1L mark (use both bottles from the Axiom® 2.0 Assay Mini 96 Reagent Kit).
2) Empty the waste bottle.
3) Press the Confirmation button on GeneTitan MC Instrument to continue. A fluidics check is
run (~1 min).
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105
Figure 5.13 Example of the Remaining Workflow Steps
Workflow step
Specific instructions for
each workflow step
B. Empty trash bin
1) Open the trash bin and empty.
If already empty, the trash bin remains locked and the Status pane reads “Trash bin is empty.”
2) Press the Confirmation button to continue.
C. Remove consumable trays and plates
1) Remove used trays and plates when drawers open.
If no consumables to remove, the Status window reads “Drawers are empty.”
2) Press the Confirmation button to continue.
D. Continue to Load Axiom® Array Plate and Hyb Tray Onto the GeneTitan ® MC Instrument on
page 106.
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106
Procedure to Clamp a Mini 96-Array Format Plate to Hybridization Tray
NOTE: Follow the procedure in Chapter 5, Stage 2: Hybridization of this manual to initiate the
hybridization step. Once the AGCC software prompts the user to load the array plate and
hybridization tray onto GTMC, follow the procedure below to complete this task
Load Axiom® Array Plate and Hyb Tray Onto the GeneTitan® MC Instrument
1.
When drawer 6 opens, load the array plate and hyb tray as follows:
A. Examine the wells of the hyb tray for bubbles; puncture any bubbles with a pipette tip.
IMPORTANT: Removing bubbles at this step greatly reduces the chance of bubbles
under the arrays when the hyb tray and the Axiom array plate are clamped. Bubbles
under an array can result in black spots on the array image.
B. Load the uncovered hyb tray on the right side of the drawer (Figure 5.15).
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. The array plate must be loaded on its protective blue base, as shown
in Figure 5.15 below. The white plastic cover on top of the array plate SHOULD NOT be loaded
in the GeneTitan MC Instrument (Figure 5.14).
D. Load the array plate with the protective blue base on the left side of the drawer (Figure 5.15).
Figure 5.14 Array Plate Packaging
Shipping Cover
(to be discarded)
Array Plate
protective base
Array Plate
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Figure 5.15 Array Plate with Protective Blue Base and the Hyb Tray Properly Loaded into Drawer 6
Array Plate with Protective Blue Base
Hyb Tray
IMPORTANT: Do not install a 3 plate stack of trays. Confirm that you have removed
the white plastic shipping cover.
CAUTION: The notched corner of each plate, cover and tray must be aligned as
indicated by the Tray Alignment guide in the drawer.
The error message shown in Figure 5.15 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.
E. Press the Confirmation button on the GeneTitan MC Instrument.
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Figure 5.16 Barcode Error Messages
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 5.16) you to load the correct plate with the proper orientation into the instrument (Figure 5.15).
 Check the loading of the array plate and click OK to retry; or
 Click Skip if the instrument has problems reading the barcode and after verifying that the trays have
been placed in the proper orientation.
F. Select the arrays to scan. By default, all arrays are selected.
2.
Click Next, then click OK in the Start Processing dialog box to begin processing the samples
(Figure 5.17).
Figure 5.17 Click OK to Start Processing the First Array Plate and Hyb Tray
Click OK to confirm that you wish to proceed with hybridization.
The plate stack is in the left position (the left side of the drawer).
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109
The array plate is placed on top of the hyb tray (now referred to as the plate stack). The software starts
the process for placing the array plate on to the hybridization tray. Press OK on the dialog shown in
Figure 5.18 and wait for the drawer to open completely before retrieving the array plate and hybridization
tray combo for manual clamping and inspection. The sandwich of the array plate and hybridization tray
needs to be manually clamped and inspected before the array processing can begin. Once clamping is
complete the dialog shown in Figure 5.19 on will be displayed. If you do not press OK in Figure 5.18 the
dialog box will go away without intervention and Figure 5.19 will be displayed.
Figure 5.18
3.
When drawer 6 opens and the prompt in Figure 5.19 is displayed:
Figure 5.19
CAUTION: At this stage, the array plate does not latch securely to the hyb tray. DO NOT grip
only the array plate to remove the plate stack from the drawer of the GeneTitan MC
Instrument.
Chapter 5 | Array Processing with the GeneTitan® Multi-Channel Instrument
1.
Follow the sequence of events in Figure 5.20 to clamp the array plate securely to the hyb tray.
Figure 5.20 Array Plate/Hyb Tray Clamping Procedure
1 Grip the body of the hyb tray by hand then
remove the plate stack from drawer 6 right
location of the GeneTitan MC Instrument.
Chamfer Corner
2 Place the plate stack on a flat surface of the table
or the lab bench. Position the plate stack to match
the orientation as shown in the picture.
3 Position the left and right thumb fingers on the
location indicated in the picture. Press the
array plate downward until the clicking sound
is detected and stopped.
4 While resting on the flat surface, rotate the plate
stack 90º clockwise direction. Position the left and
right thumb fingers on the location indicated in the
picture. Press the array plate downward until the
clicking sound is detected and stopped.
110
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111
G. Verify the plate stack to ensure the array plate is securely clamped to the hyb tray. Press the array
plate downward following the positions specified in Figure 5.21. No clicking sound indicates
proper clamping.
Figure 5.21 Clamping Verification Procedure
1
2
3
H. Keeping the plate level, inspect the bottom of the plate stack for bubbles under the arrays—do
NOT invert the plates.
I. If bubbles are present, gently tap the plate until the bubbles move out from under the arrays—
do NOT unclamp the plate stack.
J. Return the plate stack to the drawer with the notched corner facing you, and press the
Confirmation button to proceed.
The message in Figure 5.22 may be displayed again if plate orientation is incorrect or if the hyb
tray barcode cannot be read.
 Check the loading of the array plate and click OK to retry; or
 Click Skip if the instrument has problems reading the barcode and after verifying that the
correct trays have been placed in the proper orientation.
K. Click OK to proceed.
Figure 5.22 Verification Message
2.
Proceed to Load a Second Axiom® Array Plate and Hyb Tray onto the GeneTitan® MC Instrument on
page 112.
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112
Load a Second Axiom® Array Plate and Hyb Tray onto the GeneTitan® MC Instrument
When You Can Load a Second Array Plate and Hyb Tray
Once processing begins, you have a specific period of time during which you can load another Axiom
array plate and hyb tray. This period of time is displayed above the Hyb Oven Status pane (Figure 5.23).
You cannot load another hyb tray before or after this period of time.
IMPORTANT: You must load the next array plate and hyb tray during the period of time
displayed above the Hyb Oven Status. You cannot load another hyb 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 eight plate workflow and fewer than eight plates processed per week.
NOTE: 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 scan time of
the first plate (up to ~7.5 hrs.).
Figure 5.23 Loading a Second Hyb Tray based on Hybridization Oven Status Information
This pane displays the period of time during which
another array plate and hyb tray can be loaded.
Additional plates cannot be loaded before or after this
period of time while the instrument is operating.
In this figure, the system is currently available.
Position of plate stack in the hybridization oven. Only 1
plate being processed in this figure. As such, position 2
is blank.
Position 1: left side of oven
Position 2: right side of oven
Green indicates the current oven temperature is within
the target temperature range.
Yellow indicates oven temperature outside of target
temperature range.
1.
Select the System Setup tab.
Chapter 5 | Array Processing with the GeneTitan® Multi-Channel Instrument
2.
113
Load an Axiom array plate and hyb 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 and the hyb tray without the cover, 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 (Figure 5.24).
Figure 5.24 Confirm Resume Processing Prompt
Select the System Status tab to view Axiom array plate status in the WorkFlow window (Figure 5.25).
Figure 5.25 Example of the WorkFlow Window When Two Plates are Loaded and are in the Hybridization Oven
Left and Right positions = the position of the scan tray in drawer 2 (left or right side of the drawer).
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114
Queuing a Second Plate for Scanning
Using the Scan option in the System Setup tab, you can start a second scan workflow while another scan
workflow is already running.
Start the first Scan workflow in the GeneTitan Instrument. Wait until the first plate is loaded into
the imaging device and starts scanning.
2. Go to the System Setup tab and select Scan from Setup Option drop-down list (Figure 5.26).
The Setup Option drop-down list is active only after the first plate begins scanning.
1.
Figure 5.26 Scan Setup Option for Processing a Second Array Plate
System Setup Tab
Setup Option
3.
4.
5.
6.
7.
8.
9.
10.
Click Next in the lower left section of the window under the Status box.
Scan or manually enter the Axiom array plate barcode, then click Next.
Following the instructions in the Status box, empty the trash bin if necessary and then press the
GeneTitan Confirmation button to continue.
Place the array plate on top of a scan tray in the correct orientation such that notched corner of the
array plate and scan tray are aligned.
Load the array plate/scan tray combo in drawer 2 of the GeneTitan Instrument, on the left or right
side, as instructed in the Status box.
 Be sure to load the array plate/scan tray combo in the correct orientation in the drawer. If necessary,
refer to Figure 5.1 on page 92 for further information on the proper alignment and loading of
plates, covers and trays in the GeneTitan® MC Instrument.
Press the GeneTitan Confirmation button when ready.
Select the arrays to scan in the Array Selection section in the upper right corner of the window, then
click Next.
A Start Processing confirmation message appears (Figure 5.27). Click OK to continue.
Figure 5.27 Start Scan Confirmation Message
11. The second queued plate runs after the first scan finishes and the scanner is available.
Chapter 5 | Array Processing with the GeneTitan® Multi-Channel Instrument
115
Status Window Prompts and Actions Required
As a part of normal GeneTitan MC Instrument operations you may see the following status prompts.
Table 5.2, Table 5.3 Table 5.4 and explains the necessary actions required. Table 5.5 and Table 5.6
explain possible barcode error messages and the necessary action required.
Table 5.2 Refilling Buffer Bottles and Emptying the Waste Bottle
Status Window Prompt
Action Required
Buffer bottles have been depressurized.
Please refill buffer into the bottles.
Empty the waste bottle.



*
Replenish the fluid in Wash Bottles A and B,
and the Rinse bottle*.
Empty the Waste Bottle.
Press the Confirmation button to continue.
Receptacle – Reagent
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.

Every time you are prompted to refill the buffer bottles, the system runs a fluidics check (duration ~1 min).
Table 5.3 Emptying the Trash Bin
Status Window Prompt
Action Required
Empty trash bin


Open and empty the trash bin.
Press the Confirmation button to continue.
Receptacle – Reagent
—
NOTE: If the trash bin is empty, you will not be able
to open it. Continue the process by pressing the
blue confirmation button
Table 5.4 Selecting Which Arrays to Scan
Status Window Prompt
Action Required
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.
Reagent and Receptacle
—
Chapter 5 | Array Processing with the GeneTitan® Multi-Channel Instrument
116
Table 5.5 Loading the Array Plate and Hyb Tray; Barcode Error Messages
Status Window Prompt
Action Required
Load array plate tray on
[Left/Right] side of drawer.
Load hyb tray without cover on
[Left/Right] side of drawer.
Load the array plate with the blue base and the hyb 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.
 Press the Confirmation button to continue.
Text version of the error message
WARNING: The system was not able to verify the array plate barcode.
Please 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 hyb 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.
Refer to the System Setup tab or the user guide provided with the assay or AGCC 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.
Reagent – Receptacle

Hyb Tray loaded with
denatured samples.
These messages are
displayed if:
 A plate has been loaded
improperly.
 The bar code is missing
or obscured
Chapter 5 | Array Processing with the GeneTitan® Multi-Channel Instrument
117
Table 5.6 Loading the Scan Tray and Stain Tray; Barcode Error Messages
Error Message
Action Required
Verify Scan Tray Load
The system was not able to verify that GeneTitan
Consumable tray using the barcode on the tray.



Verify that the tray in the drawer is a Scan Tray
Verify that the Scan Tray is placed in the drawer in
the correct orientation
The Scan Tray should have a cover or Array Plate,
as applicable, in the correct orientation
NOTE: When a tray has been correctly loaded but
the system is unable to read the barcode, a Skip
button is present in the error message allowing you
the option to proceed.
Wrong Stain Trays - Drawer 3
The system was not able to verify that GeneTitan
Consumable tray using the barcode on the tray.
 Verify that the trays in drawer 3 are:
 STAIN 1 on the Left, and
 LIGATION on the Right
 Verify that the trays are placed in the drawer in the
correct orientation
 Verify that the trays have covers and that the
covers are on the trays in the correct orientation
NOTE: When a tray has been correctly loaded but
the system is unable to read the barcode, a Skip
button is present in the error message allowing you
the option to proceed.
Wrong Stain Trays - Drawer 4
The system detected the wrong GeneTitan
Consumable Tray using the barcode on the tray.


Verify that the trays in drawer 4 are:
 STAIN 2 on the Left, and
 STABILIZING on the Right
Verify that the trays are placed in the drawer in the
correct orientation
NOTE: When a tray has been correctly loaded but
the system is unable to read the barcode, a Skip
button is present in the error message allowing you
the option to proceed.
Wrong Stain Tray - Drawer 5
The system detected the wrong GeneTitan
Consumable Tray using the barcode on the tray.


Verify that the tray in drawer 5 is:
 STAIN1 on the Left
Verify that the tray is placed in the drawer in the
correct orientation
NOTE: When a tray has been correctly loaded but
the system is unable to read the barcode, a Skip
button is present in the error message allowing you
the option to proceed.
Chapter 5 | Array Processing with the GeneTitan® Multi-Channel Instrument
118
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; Figure 5.28).
CAUTION: Do not remove the scan tray from its protective blue 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.
Figure 5.28 The Scan Tray with Cover on the Blue Base.
Always leave the scan tray in its protective blue base.
Barcoded Scan Tray Cover
P/N 202757
GeneTitan® Scan Tray
P/N 501006 or
P/N 500860
Scan Tray
protective base
Notched corner of the cover is aligned
with the notched corner of the scan tray.
Chapter 5 | Array Processing with the GeneTitan® Multi-Channel Instrument
119
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 (refer to Figure 5.29 and Figure 5.30).
Figure 5.29 You must rotate and load the trays so that the barcode faces into the instrument
Front of
Instrument
(facing you)
Notch
This faces out and left)
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.
Barcode
(This faces BACK TO THE REAR
of the instrument)
Figure 5.30 The proper loading of the GeneTitan® Tray Consumables is shown
(the image shows the Stain Tray and the Stain Tray cover as an example).
Barcode faces
in and back.
Notch faces out and left.
The Affymetrix logo and
“For Research Use Only” faces out
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.
Chapter 5 | Array Processing with the GeneTitan® Multi-Channel Instrument
120
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 (Figure 5.31) will alert you to resume
the workflow setup. Press OK and the software takes you directly back to the System Setup tab.
Figure 5.31 The Resume Workflow Setup Message
This prompt to continue into reagent load (Figure 5.31) 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 5.7).
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, and
reload the tray.
CAUTION:

Orient trays as indicated by the guide inside the drawer (Figure 5.33 on page 123).
Improper orientation may cause the run to fail.

Remove the protective blue base from the scan tray immediately prior to loading
Figure 5.32 on page 122).

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.
Chapter 5 | Array Processing with the GeneTitan® Multi-Channel Instrument
Table 5.7 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
3
Scan Tray with cover—do not load the protective blue base
(left side of drawer as indicated in Status window)
Figure 5.32 on page 122
Stain Tray with Stain 1
Ligation Tray
Figure 5.34 on page 123
4
Stain Tray with Stain 2
Stabilization Tray with Stabilization Reagent
Figure 5.35 on page 123
121
Chapter 5 | Array Processing with the GeneTitan® Multi-Channel Instrument
Table 5.7 Sequence for Loading the Trays with Reagents (Continued)
Loading
Sequence by
Drawer
Number
Left
Right
5
Stain Tray with Stain 1
Empty
Figure 5.36 on page 124
Figure 5.32 Scan Tray Loaded in Drawer 2
Scan tray with cover
loaded in drawer 2.
Do NOT load the protective blue
base packaged with the scan tray.
122
Chapter 5 | Array Processing with the GeneTitan® Multi-Channel Instrument
Figure 5.33 Loading the plates or trays
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.
Tab or Finger
Figure 5.34 Stain 1 Tray and Ligation Tray Loaded in Drawer 3
Drawer 3
Stain 1 Tray (left, white label)
and
Ligation Tray (right, yellow label)
Figure 5.35 Stain 2 Tray and Stabilization Tray Loaded in Drawer 4
Drawer 4
Stain 2 Tray (left, blue label)
and
Stbl Tray (right, green label)
123
Chapter 5 | Array Processing with the GeneTitan® Multi-Channel Instrument
124
Figure 5.36 Stain 1 Tray Loaded in Drawer 5
Drawer 5
Stain 1 Tray (left, white label)
3.
At the prompt shown in Figure 5.37, click Yes to load another Axiom array plate and hyb tray.
Figure 5.37 Prompt asking to load another plate. Right or left position
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.
D. When drawer 6 opens:
1) Remove the blue cover from the previous Axiom array plate.
2) Load a new Axiom array plate and new blue base on the left; load a new hyb tray on the right.
3) Press the Confirmation button.
E. Click OK when prompted (Figure 5.38).
Figure 5.38 Confirm Resume Processing Message
F. When drawer 6 opens, confirm that the plate stack is securely clamped by following the procedure
in Figure 5.20, then press the Confirmation button.
Chapter 5 | Array Processing with the GeneTitan® Multi-Channel Instrument
125
The following is a description of array plate movements in the GeneTitan MC Instrument as users
execute a multi-plate workflow.
The plate stack which has finished hybridization is moved from the Hyb oven to drawer 1
(temporarily).
2. The new plate stack in drawer 6 is moved to the Hyb 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.
1.
NOTE: At the end of a Hyb-Wash-Scan run, all plate and tray covers and the stabilization tray
cover should be in the trash.
Figure 5.39 is an example of how the System Status Workflow window will appear when three Axiom
array plates are being processed.
Figure 5.39 Example of the System Status Window—Three Axiom® Array Plates are Being Processed
Workflow indicates the
number of plates being
processed and where they are in
the instrument. In this example,
three Axiom array plates are
being processed: 2 in the Hyb
Oven and 1 in Fluidics.
Estimated Completion Time
is for the current process.
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.
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.
Chapter 5 | Array Processing with the GeneTitan® Multi-Channel Instrument
126
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 5.40 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 AGCC 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 5.40 Scan Control Window
This window must remain open
while scanning is in progress.
If you close this window,
scanning will stop and delay
sample processing.
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.
WARNING: Do not attempt to shut down the GeneTitan MC Instrument while array plates
are being processed.
To Shutdown the GeneTitan MC Instrument:
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 AGCC software if it does not close automatically.
1.
NOTE: If the instrument is processing an array plate, the software will not allow you to shut
down the system.
Chapter 6
Manual Target Preparation for Processing Three Axiom® Array Plates
per Week
When using the Axiom® 2.0 Assay for Mini 96-Array Format Manual Protocol, one person can process
up to three Axiom® Mini 96-array format plates in one forty-hour work week.
This chapter describes the timing of the steps for each sample and array plate that are required to perform
this workflow.
IMPORTANT: Experienced users and careful timing are critical for the successful execution of
this workflow.
The three plate per week workflow is described in the following sections:
 Overview of the Three-plate Workflow for Manual Target Preparation
 Thawing Frozen Plates of Amplified DNA on page 130
 Manual Target Preparation and Array Processing on page 130
Detailed instructions for the manual target preparation protocol and the array plate processing are given in:
 Chapter 4, Axiom® 2.0 Assay for Mini 96-Array Manual Target Preparation on page 40
 Chapter 5, Array Processing with the GeneTitan® Multi-Channel Instrument on page 91
Overview of the Three-plate Workflow for Manual Target Preparation
The table below displays the timing and duration of the hands-on processing necessary for performing
the three plate workflow by one person.
Figure 6.1 Three Plate per Week Manual Target Preparation Workflow
Full Week Activities for the Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol Three Plate Workflow
Plate
AM
Day 1
PM
Day 2
AM
PM
Day 3
AM
Day 4
PM
AM
Day 5
PM
AM
PM
A
B
C
User Activities
Freeze
Background Activities
Amplification Incubation
Hybridization Setup (Denature & Transfer to Hyb Tray)
Hybridization in the GeneTitan® MC Instrument
DNA Amplification Setup
Fluidics Processing in the GeneTitan® MC Instrument
GeneTitan® Reagent Tray Prep & Loading
Imaging in the GeneTitan® MC Instrument
Fragmentation & Precipitation
Off-deck Centrifugation & Drying Pellets
Resuspension and Hybridization Preparation
Sample QC
OD
Run Gel QC
Chapter 6 | Manual Target Preparation for Processing Three Axiom® Array Plates per Week
128
The three plates are referred to as Plates A, B, and C in the manual target preparation and in the
GeneTitan Array Processing.
In order to process three plates during a 40-hour week, the steps must be performed in the order and with
the timing described in this chapter.
Table 6.1 Daily Steps for Manual Target Preparation Workflow
Day
Activities
Plates
1

Amplify 3 plates of genomic DNA.
A, B, and C
2

Fragment and precipitate two plates amplified on Day 1.
Freeze one plate of amplified DNA for fragmentation later in the week.

Fragment and precipitate one plate.
Centrifuge, dry, resuspend and QC two plates precipitated on Day 2.
Denature and begin hybridization for one plate on the GeneTitan MC Instrument



Centrifuge, dry, resuspend and QC plates precipitated on Day 3
Denature and begin Hybridization for two plates on the GeneTitan MC Instrument
GeneTitan reagent trays preparation and loading

C
B, C
A

GeneTitan Reagent Trays Preparation and Loading

B, C

3



4


5




A, B
C
C
A, B
A
The timing of these steps is critical because of constraints on both the target preparation, done on the lab
bench, and the array processing, done using the GeneTitan MC Instrument.
These constraints are described in more detail in:
 Timing Issues for Manual Target Preparation on page 128
 Timing Issues for GeneTitan® MC Array Processing on page 129
Timing Issues for Manual Target Preparation
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.
.
Table 6.2 Time Required for Manual Target Preparation
Manual Preparation
Hands-on Time
Required
Total Prep
Time*
Incubation/Hybridization/
Processing
0.5 hr
1.5 hr
23 ±1 hr
2 hr
2 hr
Overnight Precipitation
Stage 1: DNA Amplification
Stage 2: Fragmentation and Precipitation
Stage 3: Centrifuge and Drying, Resuspension and Hybridization
Preparation, and Sample QC
*

Stage 3A: Centrifuge Precipitation Plate and Dry the DNA Pellet
30 min
1 hr 20 min
N/A

Stage 3B: Resuspension and Hybridization Preparation
25 min
25 min
N/A

Stage 3C: Sample QC
45 min
45 min
N/A
Stage 4: Denaturation and Hybridization
25 min
45
23.5 - 24 hr hybridization
Stage 5: GeneTitan® Reagent Preparation
1 hr
1.5 hr
Additional time for processing:
96 arrays: 12.5 hr
Total Preparation Time includes reagent thawing time and hands-on time.
Chapter 6 | Manual Target Preparation for Processing Three Axiom® Array Plates per Week
129
Timing Issues for GeneTitan® MC Array Processing
IMPORTANT: Maintaining consistent timing during the set up of the GeneTitan MC
Instrument is critical to containing the user interventions of the three plate workflow within
a work day. Once one process begins late, there is little opportunity to catch up until the end
of the workflow.
The hybridization time for the Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol on the
GeneTitan MC Instrument is 23.5 to 24 hr (Table 6.3). This provides a 30 min window during which you
are prompted by the instrument control software to load the reagents required for washing and staining.
Table 6.3 Time Required for Array Plate Processing on the GeneTitan MC Instrument
Steps on the GeneTitan MC Instrument
Time Required
Hybridization of two plates in one day
First plate loaded at 9:30 a.m.
 Second plate loaded at 5:00 p.m.
23.5 hr each plate

Loading Reagent Trays
15 min
Fluidics
5 hr each plate
Imaging*
*
up to 7.5 hr depending on array format
For labs that run several array plate formats, imaging times may vary.
Changing Oven Temperatures for the Three Plate Workflow
Multiple ovens are required for manual target preparation. If you are running the three plate/week
workflow, three ovens are recommended. Table 6.4 lists the different temperatures required for each step.
Though only two ovens are strictly required, we recommend maintaining separate 37°C ovens for the
amplification and fragmentation stages to avoid confusion of plates and to minimize excess opening and
closing of oven doors during incubation periods. Table 6.5 provides a list of suggested settings for three
ovens when performing the three plate/week workflow.
Table 6.4 Oven Temperatures Needed for Each Step of the Workflow
Workflow step
*
Oven Temp
Amplification
37°C
Stopping Amplification
65°C
Pre-Fragmentation Incubation
37°C
Fragmentation Incubation
37°C
Drying
37°C
Hybridization*
48°C
For preheating of the 96-well metal chamber for hyb transfer
Table 6.5 Suggested Settings for Ovens When Performing Three Plate/Week Manual Target Preparation Workflow
Day of Workflow
Oven 1
Oven 2
Oven 3
Day 1
37°C
N/A
N/A
Day 2
37°C
65°C
37°C
Day 3
48°C
65°C
37°C
Day 4
48°C
65°C
37°C
Day 5
N/A
N/A
N/A
Chapter 6 | Manual Target Preparation for Processing Three Axiom® Array Plates per Week
130
Thawing Frozen Plates of Amplified DNA
To Thaw Frozen Plates of Amplified DNA:
Place the deep well plate in a small water bath.
For example, pour Millipore water into a small tray. Place the frozen plate in the water in the tray.
2. Leave the plate in the water bath for ~50 min until all wells have thawed.
3. Spin down at 1000 rpm for 30 sec.
4. To avoid cross-contamination of wells during vortexing:
1.
A. Remove the seal and blot the top of the plate with a Kimwipe.
B. Tightly reseal the plate with a fresh seal.
Vortex the plate for 30 sec to thoroughly mix (refer to guidelines described in Seal, Vortex, and Spin
on page 25).
6. Spin at 1000 rpm for 30 sec.
5.
Manual Target Preparation and Array Processing
Day 1



On this day you start amplification of the three plates: each plate must incubate 23 1 hours after
amplification begins.
All amplifications should be set up on Day 1 to allow for a 23 ±1 hr amplification incubation for each
plate and to minimize movement between pre-amplification and post-amplification areas.
Begin thawing the amplification reagents, particularly the Axiom 2.0 Amp Soln, 60 min prior to the
start of each reaction.
IMPORTANT: Amplification preparation should take place in an Amplification Staging Room
or dedicated area such as biosafety hood with dedicated pipettes, tips, vortex, etc. See
Amplification Staging Area on page 22 for more information.
Table 6.6 Day 1 Activities for the Axiom 2.0 Assay Mini 96-Array Format Manual Protocol
Approximate Times*
Activity
*
Plate
Start Time
End Time
Duration
DNA Amplification
A
9:30 a.m.
11:00 a.m.
30 min
DNA Amplification
C
10:30 a.m.
12:00 p.m.
30 min
DNA Amplification
B
1:30 p.m.
3:00 p.m.
30 min
Approximate start time indicates start of thawing of reagents.
See Stage 1: DNA Amplification on page 41 for more information on the protocol.
Chapter 6 | Manual Target Preparation for Processing Three Axiom® Array Plates per Week
131
Figure 6.2 Day 1 Activities for the Axiom 2.0 Assay Mini 96-Array Format Manual Protocol
Day 1 Activities for the Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol Three Plate Workflow
8
9
10
Plate #
A
11
12
1
2
3
Amp
Amp
B
Amp
C
User Activities
Thaw and Prepare Reagents for DNA Amplification
DNA Amplification Setup
Background Activities
Amplification Incubation
4
5
6
Chapter 6 | Manual Target Preparation for Processing Three Axiom® Array Plates per Week
132
Day 2



Table 6.7 shows the steps that need to be performed on the second day.
Plates A and B are fragmented and precipitated on Day 2 without freezing to preserve a 23 hr amplification incubation.
Precipitation is carried out at –20°C overnight.
IMPORTANT: Store Plate C at –20°C immediately after the end of the 23 hr Amplification reaction
(without performing the 65°C Stop Amplification Reaction step).
Table 6.7 Day 2 Activities for the Axiom 2.0 Assay Mini 96-Array Format Manual Protocol
Approximate Times
Activity
Plate
Start Time
End Time
Duration
Fragment and Precipitate
A
10:00 a.m.
12:00 p.m.
2 hours
Freeze (–20°C)
C
11:00 a.m.
—
Overnight
Fragment and Precipitate
B
2:00 p.m.
4:00 p.m.
2 hours
Figure 6.3 Day 2 Activities for the Axiom 2.0 Assay Mini 96-Array Format Manual Protocol
Day 2 Activities for the Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol Three Plate Workflow
8
9
10
Plate #
A
11
12
1
2
3
Frag & Precip
Frag & Precip
B
C
User Activities
Freeze
Prepare Reagents for Fragmentation
Fragmentation & Precipitation
Background Activities
Amplification Incubation
4
5
6
Chapter 6 | Manual Target Preparation for Processing Three Axiom® Array Plates per Week
133
Day 3






Centrifuge, dry, resuspend and QC Plates A and B.
Thaw Plate C (see Thawing Frozen Plates of Amplified DNA on page 130).
Fragment (including the 65°C Stop Amplification Reaction step) and precipitate Plate C.
Perform Denaturation on Plate A.
Transfer Plate A samples to Hyb Tray A
Load Hyb Tray A and array plate into GeneTitan MC Instrument and begin hybridization.
WARNING: The Hybridization Tray preparation should take place under a running fume hood.
IMPORTANT: Amplified plates that are frozen must be thawed and thoroughly mixed by
following the procedure under Thawing Frozen Plates of Amplified DNA on page 130.
Table 6.8 Day 3 Activities for the Axiom 2.0 Assay Mini 96-Array Format Manual Protocol
Approximate Times
Activity
Plate
Start Time
End Time
Duration
Centrifuge and Dry
A, B
9:00 a.m.
10:20 a.m.
1 hour 20 min
Resuspension and Hyb Preparation
A, B
10:20 a.m.
10:45 a.m.
25 min
Sample QC
A, B
10:45 a.m.
11:05 a.m.
20 min
Sample Quantitation (OD)*
A, B
11:05 a.m.
11:10 a.m.
5 min
Frag Gel QC Run
A, B
11:05 a.m.
11:30 a.m.
25 min
Thaw Plate C
C
12:00 p.m.
1:00 p.m.
1 hour
Fragment and Precipitate
C
1:00 p.m.
3:00 p.m.
2 Hours
Denature and Hybridization
A
4:15 p.m.
5:00 p.m.
45 min setup,
23.5 to 24 hours Hyb
*Sample
Quantitation runs concurrently with Frag Gel QC Run. Load the Gel QC Plate first, then read the OD QC Plate.
Chapter 6 | Manual Target Preparation for Processing Three Axiom® Array Plates per Week
134
Figure 6.4 Day 3 Activities for the Axiom 2.0 Assay Mini 96-Array Format Manual Protocol
Day 3 Activities for the Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol Three Plate Workflow
8
9
10
Plate #
A
Centrifuge & Dry
B
Centrifuge & Dry
11
12
1
2
3
4
5
Denature
& Hyb
Frag & Precip
C
User Activities
Prepare Reagents for Resuspension and Hyb Prep
Prepare Reagents for Fragmentation
Warm Array Plate to Room Temperature
Thaw DNA Amplification Plate
Fragmentation & Precipitation
Centrifugation & Drying Pellets
Resuspension and Hybridization Preparation
Sample QC
Sample Quantitation - OD
Fragmentation Gel QC Run
Denature & Hybridiztion
Background Activities
Hybridization in the GeneTitan® MC Instrument
6
Chapter 6 | Manual Target Preparation for Processing Three Axiom® Array Plates per Week
135
Day 4



Denaturation of Samples/Load array plate and hyb tray in the GeneTitan MC Instrument for Plates B
and C
Centrifuge, dry, resuspend, and QC Plate C
GeneTitan reagent trays preparation and loading for Plate A
WARNING: The Hybridization Tray preparation should take place under a running fume hood.
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.
Table 6.9 Day 4 Activities for the Axiom 2.0 Assay Mini 96-Array Format Manual Protocol
Approximate Times
Activity
Plate
Start Time
End Time
Duration
Denature and Hybridization
B
8:45 a.m.
9:30 a.m.
45 min setup,
23.5 to 24 hours Hyb
Centrifuge and Dry
C
9:30 a.m.
10:50 a.m.
1 hour 20 min
Resuspension and Hyb Preparation
C
10:50 a.m.
11:15 a.m.
25 min
Sample QC
C
11:15 a.m.
11:35 a.m.
20 min
Sample Quantitation (OD)*
C
11:35 a.m.
11:40 a.m.
5 min
Fragmentation Gel QC Run
C
11:35 a.m.
12:00 p.m.
25 min
GeneTitan Reagent Prep and Loading
A
3:30 p.m.
5:00 p.m.
1 hour
Denature and Hybridization
C
4:15 p.m.
5:00 p.m.
45 min setup,
23.5 to 24 hours Hyb
*Sample
Quantitation runs concurrently with Frag Gel QC Run. Load the Gel QC Plate first, then read the OD QC Plate.
Chapter 6 | Manual Target Preparation for Processing Three Axiom® Array Plates per Week
136
Figure 6.5 Day 4 Activities for the Axiom 2.0 Assay Mini 96-Array Format Manual Protocol
Day 4 Activities for the Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol Three Plate Workflow
8
9
10
11
12
1
2
Plate #
A
B
3
4
5
GT Reagent Prep
Denature
& Hyb
C
Denature
& Hyb
Centrifuge & Dry
User Activities
Warm Array Plate to Room Temperature
Prepare Reagents for Resuspension and Hyb Prep
Prepare Reagents for GeneTitan Reagent Preparation
Warm Array Plate to Room Temperature
Load Array Plate into GTMC, begin Wash-Scan
Centrifugation & Drying Pellets
Resuspension and Hybridization Preparation
Sample QC
Sample Quantitation - OD
Fragmentation Gel QC Run
GeneTitan® Reagent Tray Prep & Loading
Hybridization Setup (Denature & Transfer to Hyb Tray)
Background Activities
Hybridization in the GeneTitan® MC Instrument
Fluidics Processing in the GeneTitan® MC Instrument
6
Chapter 6 | Manual Target Preparation for Processing Three Axiom® Array Plates per Week
137
Day 5

GeneTitan reagents preparation and loading for Plates B and C.
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.
Table 6.10 Day 5 Activities for the Axiom 2.0 Assay Mini 96-Array Format Manual Protocol
Approximate Times
Activity
Plate
Start Time
End Time
Duration
GeneTitan Reagent Tray Prep and Loading
B
8:00 a.m.
9:30 a.m.
1 hour
GeneTitan Reagent Tray Prep and Loading
C
3:30 p.m.
5:00 p.m.
1 hour
Figure 6.6 Day 5 Activities for the Axiom 2.0 Assay Mini 96-Array Format Manual Protocol
Day 5 Activities for the Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol Three Plate Workflow
8
9
10
11
12
1
2
3
4
5
6
Plate #
B
GT Reagent Prep
C
GT Reagent Prep
User Activities
Thaw Reagents for GeneTitan® Reagent Tray Prep
Background Activities
Hybridization in the GeneTitan® MC Instrument
Load Array Plate into GTMC, begin Wash-Scan
Fluidics Processing in the GeneTitan® MC Instrument
GeneTitan Reagent Tray Prep & Loading
Imaging in the GeneTitan® MC Instrument
Chapter 7
Troubleshooting
GeneTitan® Multi-Channel Instrument
Refer to the GeneTitan® Multi-Channel Instrument User Guide, P/N 08-0306 for further troubleshooting
information.
Table 7.1 GeneTitan® MC Instrument Troubleshooting Guidelines for the Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol
Problem
Possible Causes
Plate trapped in GeneTitan MultiChannel Instrument.




Computer frozen.


Possible Actions
Plate (or plate with lid) not properly loaded 1. Restart the GeneTitan Multi-Channel Instrument.
in drawer.
2. Run the setup option Unload Plates.
Notched edge of lid and plate not aligned. 3. If the plate remains trapped in the instrument, call
Gripper failed to retrieve plate.
Affymetrix support.
System requires adjustment.
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 AGCC
transfer utility, move any data associated
with the current plate that is being processed/
scanned.
Hybridization aborted:
 System-initiated abort
 User-initiated abort
System-initiated abort:
 Power loss
User-initiated abort:
 User error
 Other
FAILED messages
See GeneTitan® MC Instrument Messages
that Appear When the Instrument has a
Fluidics Problem on page 140
FLUIDIC DIAGNOSTIC messages
See Fluidic Diagnostic Messages on page 140.
Fluidics aborted:
System-initiated abort
 User-initiated abort
System-initiated abort:
 Power loss

User-initiated abort:
 Incorrect protocol selected
Array plate and hyb 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.
Follow the recommendations and instructions under
Wash/Scan Resume on page 143.
Chapter 7 | Troubleshooting
139
Miscellaneous Messages
Table 7.2 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 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—covers
The 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 2
HtaHyb = Clamped Hyb Tray and Array Plate
Drawer(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 hyb tray or Drawer2L_ScanHta_Pk_DOWN indicating
it is an array plate with a scan tray
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
two 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.
Chapter 7 | Troubleshooting
140
Fluidic Diagnostic Messages
Table 7.3 GeneTitan® MC Instrument Messages that Appear When the Instrument has a Fluidics Problem
Problem and Possible Causes
If this message is displayed:
during a water wash step, array processing has been
compromised.
 during cleanup, array processing is OK, 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
Mini-96 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.

Rinse bottle—fluid level too low or bottle empty.
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 make sure bottle caps are secure.
 Replenish fluid level in Wash Bottle A to 2L.
 Secure the bottle cap.
 Replace the filter
Instructions for filter replacement in the GeneTitan® MultiChannel Instrument User Guide, P/N 08-0306.
If the problem persists, call Affymetrix 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® MultiChannel Instrument User Guide, P/N 08-0306.
Chapter 7 | Troubleshooting
141
Table 7.3 GeneTitan® MC Instrument Messages that Appear When the Instrument has a Fluidics Problem (Continued)
Problem and Possible Causes
When the instrument experiences a loss in Clean Dry Air (CDA) pressure,
the software will display the warning message.
Possible Causes
Please verify that the facility CDA or the portable CDA
compressor is in working condition. Refer to 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 application specialist and notify the
engineer about the error message.
When the pressure is detected again, a dialog message confirming the
availability of CDA pressure is displayed.
Leak Detected
Leak 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 Affymetrix field support. The system cannot be used
for any fluidic processing until this is resolved.
Chapter 7 | Troubleshooting
142
Table 7.3 GeneTitan® MC Instrument Messages that Appear When the Instrument has a Fluidics Problem (Continued)
Problem and Possible Causes
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 clicking 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 Affymetrix Field Service 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
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 three filters when a warning is displayed
for any one of the three filters.
Filter Error Message: Fill related check
Chapter 7 | Troubleshooting
143
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.
System Setup tab: Select Wash/Scan Resume
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.
1.
2.
To help ensure that the samples are processed correctly, we recommend that you:
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.
1.
Resume Step
You 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 hr 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 three minutes if a plate is in the Fluidics station. Status window Abort Requested
changes to Abort Completed.
Clamped Array-Plate-Hyb 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-initiated
Can abort processing of individual array plates.
If multiple plates are being processed, the gripper may continue to process the remaining array plates.
Appendix A
Fragmentation Quality Control Gel Protocol
Protocol for Running a Fragmentation Quality Control Gel
Equipment Required
Table A.1 Equipment Required
Item
Supplier
Part Number
Gel Imager
Your choice
—
Pipette, multi- or single-channel P20
Your choice
—
Plate centrifuge
Your choice
—
Vortexer
Your choice
—
E-Gels and Reagents
Table A.2 E-Gel and Reagents Required
Item
Supplier
Part Number
Mother E-Base™ Device
EB-M03
Daughter E-Base™ Device
(optional for running multiple gels in parallel)
EB-D03
Life Technologies
(formerly Invitrogen)
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
—
Consumables
Table A.3 Gel and Reagents Required
Item
Supplier
Adhesive film – use one of the following:
®
 MicroAmp Clear Adhesive Film


Microseal® 'B' Film
Pipette Tips

Life Technologies
(formerly Applied Biosystems)
Bio-Rad
Same brand as pipette
Part Number

4306311

MSB1001
—
Appendix A | Fragmentation Quality Control Gel Protocol
145
Diluting the TrackIt™ Cyan/Orange Loading Buffer and 25 bp Ladder
The following recipe is for preparing a large batch of the Gel Diluent, a 1000-fold dilution of the TrackIt
Cyan-Orange Loading Buffer:
To Dilute the TrackIt Cyan/Orange Loading Buffer:
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.
1.
The following recipe is for preparing a 15-fold dilution of the Invitrogen TrackIt 25 bp DNA Ladder:
To Dilute the TrackIt 25bp Ladder (P/N 10488-022, Thermo Fisher Scientific):
In a 1.7 mL microcentrifuge tube, add 6 μL of TrackIt 25 bp DNA Ladder to 84 μL nuclease-free
water. Total volume: 90 μL.
2. Vortex tube to mix well. Pulse-spin to get droplets down.
1.
NOTE: The recipe has enough volume to fill 4 marker wells of one E-Gel® 48 4% agarose
gel. Scale up as needed if running multiple gels.
Fragmentation QC Gel Protocol
Running one E-Gel® 48 4% agarose gel to sample a 96 well plate is recommended. A suggested sampling
pattern is to load the gel with the following wells from the 96 well Gel QC Plate:
 Row A, C, E, G, or
 Row B, D, F, H
If processing multiple plates, sampling different wells from each plate can be helpful in monitoring assay
processing performance.
To Run a Fragmentation QC Gel:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Tightly seal the Gel QC Plate produced during Stage 3C: Sample QC.
Vortex the plate for 1 sec each corner and 1 sec in the center at the maximum setting; spin at
1000 rpm for 30 sec.
Connect an E-Base™ device(s) to an electrical outlet.
Push the Power/Prg button on each to ensure the program is in EG mode (not EP mode).
Take the gel out of the pouch and remove the combs.
Place the E-Gel® 48 gel into an E-Base unit.
Load 20 μL from each well of the Gel QC plate onto the gels.
Load 15 μL of diluted TrackIt 25 bp ladder into the marker wells (M).
Load 20 μL nuclease-free water into any unused wells.
Run the gels for 22 min.
Image the gel.
Appendix A | Fragmentation Quality Control Gel Protocol
Fragmentation QC gel images should look similar to the gel shown in Figure A.1.
Figure A.1 Example of a Typical Fragmentation QC E-gel
25 bp ladder
Fragments should fall between
125 bp and 25 bp.
125 bp
25 bp
125 bp
25 bp
25 bp ladder
146
Appendix B
Sample Quantitation After Resuspension
Protocol for Sample Quantitation After Resuspension
Equipment Required
The following equipment is required for this protocol.
Table B.1 Equipment Required for Sample Quantitation After Resuspension
Quantity
1
Item
DTX 880 Multimode Detector with Genomic Filter Slide
Quantitate the Diluted Samples
During target prep, two plates of diluted samples are prepared: one for OD quantitation and one 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 Suggested
Protocol for OD Quantitation Using the DTX 880 on page 149 for more information.
To Quantitate the Diluted Samples Prepared for OD Quantitation:
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.x
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, two XML files are generated (Figure B.1). Open the ResultData file with Microsoft Excel
to view and assess the OD readings. RawData file information is included in the ResultData file.
1.
Figure B.1 List of files that are generated post DTX-880 scan.
Appendix B | Sample Quantitation After Resuspension
148
Assess the OD Readings
If using the formula provided in this appendix, the raw data is included in the final Result Data file.
Figure B.2 is an example of a Result Data file. Your OD readings should be similar to those displayed
below.
Figure B.2 Example of Result Data file with Acceptable OD Readings
OD Yield Assessment Guidelines
The measurement of the yield of DNA after resuspension of the pellets is an important QC checkpoint
in the Axiom 2.0 Mini 96 target prep samples. If the median yield for the plate is 525 μg DNA:
 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.
Appendix B | Sample Quantitation After Resuspension
Suggested Protocol for OD Quantitation Using the DTX 880
The formula suggested below requires six passes. The settings and formula are shown below.
Protocol Type: Analysis
Figure B.3 Protocol Type
General Settings: Enter a Name for the Protocol
Figure B.4 General Settings
149
Appendix B | Sample Quantitation After Resuspension
Technique Type: Select Absorbance
Figure B.5 Technique Type
Labware: x_Abs_Greiner 96 UV clear std (96 microplate format)
Figure B.6 Labware
Layout Settings: as Appropriate for 96-array Format Plates
Figure B.7 Layout Settings
150
Appendix B | Sample Quantitation After Resuspension
151
Method Selection: add (+) the three formulas created on the Data Reduction Page to the Group 1 box
Figure B.8 Method Selection
Data Reduction Page: create the formulas required for scans at 260, 280 and 320
This protocol consists of six passes. Click Add new Pass to create passes two through six, shown in these
figures below.
Figure B.9 Data Reduction Page—First Pass
Appendix B | Sample Quantitation After Resuspension
Figure B.10 Data Reduction Page—Second Pass
1
Figure B.11 Data Reduction Page—Third Pass
152
Appendix B | Sample Quantitation After Resuspension
k
Figure B.12 Data Reduction Page—Fourth Pass
Figure B.13 Data Reduction Page—Fifth Pass
153
Appendix B | Sample Quantitation After Resuspension
Figure B.14 Data Reduction Page—Sixth Pass
Output Settings: Select Export to Microsoft® Excel and Show Result Viewer
Figure B.15 Output Settings
Save the protocol.
154
Appendix B | Sample Quantitation After Resuspension
155
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 = 50 (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 “B” above.
The optical path length is dependent on the type of plate and may depend on the spectrophotometer
used. Check your manufacturer’s 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 Axiom® 2.0 Assay Mini 96-Array Format Manual Protocol
Site Preparation Guide, P/N 703435, can employ an automated path length detection system. Consult
this instrument’s user guide for more information.
The resulting yield calculations can be compared against the typical yields shown in column H of
Figure B.2 on page 148 and against OD Yield Assessment Guidelines on page 148.
Appendix C
Registering Samples in Affymetrix 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 AGCC Portal, open the Samples menu and select GeneTitan Array Plate Registration
(Figure C.1).
Figure C.1 Selecting GeneTitan® Array Plate Registration
Create a new template in AGCC that includes fields that will achieve sample traceability
3. Select the array plate to be processed on the GeneTitan MC Instrument (Figure C.2 on page 157).
2.
A. Select the newly created template that contains the fields required for sample traceability.
B. Select the array plate type.
C. Select the project where sample registration and all associated data files will be saved.
D. Click Download.
Appendix C | Registering Samples in Affymetrix GeneChip® Command Console®
157
Figure C.2 Selecting the Type of Array Plate to be Processed
4.
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, such as hybridization tray barcode (Figure C.3).
TIP: The AGCC template created in Step 2 must have a field that reads Hyb Tray
Barcode. The Excel file that will be downloaded will have a column header that reads,
“Hyb Tray Barcode:*:Text”. The Barcode of the hybridization tray can be scanned
into the “Hyb Tray Barcode” text field. This barcode will be stored in to the sample file
for each array.
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 into column F 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.
Figure C.3 Entering Sample Information into an Array Plate Registration File
5.
Save the file as follows:
A. Open File  Save As.
Appendix C | Registering Samples in Affymetrix GeneChip® Command Console®
158
B. Enter a name for the array plate registration file.
C. Click Save.
By default, the file is saved in the Affymetrix_Downloads folder.
6. 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 C.4), 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 C.5 is displayed.
Figure C.4 Uploading the Array Plate Registration File to AGCC
Figure C.5 Array Plate Samples Successfully Registered
Appendix D
GeneTitan® Multi-Channel Instrument Care
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
prevents 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.
IMPORTANT: Before performing maintenance turn off power to the instrument to avoid
injury in case of an electrical malfunction.
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 Six 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.
Servicing the Outer Enclosure Fan Filters
Cleaning Schedule
The GeneTitan fan filter cartridge (Figure D.1) 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:
Affymetrix P/N: 01-0669
Number of filters required per GeneTitan Instrument: 3
Appendix D | GeneTitan® Multi-Channel Instrument Care
160
Figure D.1 The GeneTitan® Filter Cartridge
Cleaning Procedure
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.
1.
Appendix D | GeneTitan® Multi-Channel Instrument Care
161
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 D.2 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 D.2) along with the information on the specific dispense operation. You should change all three
filters when a warning is displayed for any one of the three filters.
Figure D.2 Filter Change Required Messages
NOTE: The reagent bottles are depressurized when this warning message is displayed. It is
safe to change the filters in all three fluidic bottles when this message is displayed.
After changing the filters in all three bottles using the procedure described below, please 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 AGCC 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.
We recommend that your site keep three spare filters on hand in the event the filters need to be replaced.
The procedure for replacing the filters is simple.
Appendix D | GeneTitan® Multi-Channel Instrument Care
162
Affymetrix GeneTitan Reagent Bottle Filters Part details:
Affymetrix P/N: 01-0671
Figure D.3 Replacing the Filter
Buffer Supply Line
Filter Holder
Filter
Removing and Inspecting the Filter
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.
1.
Replacing the Filter
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.
1.
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 D | GeneTitan® Multi-Channel Instrument Care
163
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.
Affymetrix GeneTitan Xeon Lamp P/N 01-0740
IMPORTANT: Please DO NOT try to replace the lamp when a plate is being processed either
in the fluidics or scanner system.
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 D.4):
Figure D.4 Lamp Life Above Tolerance
It displays a red or yellow notice when the lamp life is getting short (Figure D.5):
Figure D.5 Lamp Life above Tolerance
It also displays a red notice when the Imaging Device is offline (Figure D.6):
Figure D.6 Imaging Device Offline
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.
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 D | GeneTitan® Multi-Channel Instrument Care
164
Removing the Xenon Lamp
1.
Unscrew the four retaining bolts. They should be finger tight (Figure D.7).
Figure D.7 Unscrewing the Bolts
Unscrew these four bolts.
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 D.8). You must use both hands to remove the lamp successfully. Apply equal pressure on each
side of the lamp and gently lift.
2.
Appendix D | GeneTitan® Multi-Channel Instrument Care
Figure D.8 Lifting out the Lamp
Replacing the Lamp
CAUTION: Ensure that you install the lamp in the correct orientation.
Hold the lamp by the blue plastic flanges. Ensure that the lamp bulb faces inward toward the
reflecting mirror (Figure D.9) and vertically insert the lamp (Figure D.10).
2. Replace the warning cover and hand tighten the bolts (Figure D.7).
1.
165
Appendix D | GeneTitan® Multi-Channel Instrument Care
Figure D.9 The Reflecting Mirror
Reflecting Mirror
Figure D.10 Inserting the Lamp
IMPORTANT: The lamp bulb faces away from
the fan and toward the reflecting mirror.
166
Appendix D | GeneTitan® Multi-Channel Instrument Care
167
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 D.11).
Figure D.11 Inserting the Lamp
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 D.12).
Appendix D | GeneTitan® Multi-Channel Instrument Care
168
Figure D.12 Are you Sure?
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 D.13).
Figure D.13 The counter is reset.
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 Affymetrix 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 AGCC 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
specialist (FAS), field service engineer (FSE), or the Affymetrix call center.
AGCC Log Files
The following files are generated by the GeneTitan Instruments. All the AGCC log files are from the
following path: C:\Command_Console\Logs. The different log files include:
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 D | GeneTitan® Multi-Channel Instrument Care
169
Other AGCC Files
Your FAS and/or FSE may request you to send the following files for troubleshooting:
1.
2.
3.
4.
5.
Library files (*.PARAMS, *.MASTER, *.WORKFLOW, *.SMD, *.MEDIA) located in
C:\Command_Console\Library, excluding the large analysis library files (CDF, PSI, GRC).
Provide a list of all sub folders and their contents under the library files folder located in
C:\Command_Console\Library. Please ensure there are no duplicate library files, as these can cause
problems.
AGCC system configuration file located at
C:\Command_Console\Configuration\Calvin.System.config
Pending job order files located in C:\Command_Console\Jobs
Other AGCC 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.
AGCC 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. Affymetrix 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)
1) 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).
2) 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.
GeneTitan MC Instrument Imaging Device
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.
1.
Problems and Solutions
This section provides instructions on how to identify and solve problems with the unit.
If problems arise with the instruments use the following tables to locate the description that matches the
problem. If you cannot find a solution call Affymetrix 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 entire unit and then restart.
Appendix D | GeneTitan® Multi-Channel Instrument Care
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 D.14) when:
 you first initialize the software and instrument.
 you select arrays for imaging.
Figure D.14 Insufficient Disk Space Notice
If you see this notice, you will need to free up sufficient disk space before imaging starts.
170
Appendix E
Contact Information
Technical Support
Affymetrix, Inc.
3420 Central Expressway
Santa Clara, CA 95051 USA
Email: support[email protected]
Tel: 1-888-362-2447 (1-888-DNA-CHIP)
Fax: 1-408-731-5441
Affymetrix UK Ltd
Voyager, Mercury Park,
Wycombe Lane, Wooburn Green,
High Wycombe HP10 0HH
United Kingdom
Email: [email protected]
UK and Others Tel: +44 (0) 1628 552550
France Tel: 0800919505
Germany Tel: 01803001334
Fax: +44 (0) 1628 552585
Affymetrix Japan, K. K.
ORIX Hamamatsucho Bldg, 7F
1-24-8 Hamamatsucho, Minato-ku
Tokyo 105-0013, Japan
Email: [email protected]
Tel: +81-3-6430-4020
Fax: +81-3-6430-4021
Please visit our website for international distributor contact information
www.affymetrix.com
For complete contact information and specific regional support contact information, please go to
www.affymetrix.com/browse/contactUs.jsp
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