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Thermo Fisher Scientific GeneChip HVT 3' IVT PLUS Reagent Kit Assay Automated Workflow User guide

  • Hello! I'm your chat assistant, and I've reviewed the GeneChip HT 3' IVT PLUS Reagent Kit user guide. This document provides detailed instructions for automated RNA sample preparation using the Biomek FXP workstation and GeneChip 3' Expression Arrays. The guide describes the use of a kit designed for up to 96 samples and including information for cDNA synthesis, cRNA purification, fragmentation, and hybridization setup. I'm here to answer your questions about the procedures and equipment outlined in this document.
  • What is the purpose of the GeneChip HT 3' IVT PLUS Reagent Kit?
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For Research Use Only. Not for use in diagnostic procedures.
GeneChip™ HT 3' IVT PLUS Reagent Kit
Assay Automated Workflow
USER GUIDE
for use with:
GeneChip™ 3' IVT PLUS Reagent Kit
GeneChip™ HT 3' IVT PLUS Reagent Kit
GeneChip™ 3' Expression Arrays
Beckman Coulter™ Biomek™ FXP Automated Workstation
Catalog Numbers 902416 and 902417
Publication Number MAN0018309
Revision B.0
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Products:
GeneChip™ HT 3' IVT PLUS Reagent Kit
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Keppel Logistics Building |
Singapore 629563
Products:
GeneChip™ 3' Expression Arrays
The information in this guide is subject to change without notice.
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Revision history: Pub. No. MAN0018309
Revision Date Description
B.0 21 December 2020 Add Bio-Rad™ white PCR plate option, Cat. No. HSP9601, to the consumables list.
Change some component volumes in GeneChip™ HT 3' IVT PLUS Reagent Kit.
A.0 21 December 2018 •Initial release in Thermo Fisher Scientific document control system
•Supersedes legacy Aymetrix™ publication number 703208.
•Updated to the current document template, with associated updates to trademarks, logos, licensing, and
warranty.
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GmbH. Bio-rad, Hardshell, and Microseal are trademarks of Bio-Rad Laboratories. Corning and Costar are trademarks of Corning,
Inc. FrameStar is a registered trademark of 4titude Ltd. Greiner Bio-One, and UV-Star are trademarks of Grenier Bio-One International
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©2020 Thermo Fisher Scientific Inc. All rights reserved.
Contents
■CHAPTER 1 GeneChip™ HT 3' IVT PLUS Reagent Kit ........................... 6
Product information ............................................................. 6
Purpose of the product ...................................................... 6
Assay workflow ............................................................. 8
Approximate times required ................................................. 10
Kit contents and storage ........................................................ 12
■CHAPTER 2 Required materials .................................................. 13
Required equipment, consumables, labware, and reagents .......................... 13
Equipment ................................................................ 13
Consumables and labware .................................................. 15
Reagents ................................................................. 20
Consumables and labware images ............................................... 20
Labware templates ......................................................... 26
Reagent and labware requirements for Part 1 through Part 4 ......................... 27
Reagent and labware required for Part 1 ...................................... 27
Reagent and labware required for Part 2 ...................................... 29
Reagent and labware required for Part 3 ...................................... 30
Reagent and labware required for Part 4 ...................................... 33
■CHAPTER 3 RNA preparation .................................................... 34
Prepare control RNA ............................................................ 34
Prepare HeLa positive Control RNA .......................................... 34
Prepare poly-A RNA controls ................................................ 35
Prepare total RNA .............................................................. 36
Evaluate RNA quality ....................................................... 36
Evaluate RNA integrity ...................................................... 37
Determine RNA quantity .................................................... 37
Prepare total RNA/Poly-A RNA control mixture ................................ 38
Prepare total RNA plates for processing on automated systems ................. 38
GeneChip™ HT 3' IVT PLUS Reagent Kit Assay Automated Workflow on Biomek™ FXP User Guide 3
■CHAPTER 4 Biomek™ FXP Target Prep Express Automated
Workstation setup and target preparation ...................................... 39
Run setting options and method preferences in the 3' IVT PLUS target prep method ... 40
Run setting options ........................................................ 40
Method preferences ........................................................ 42
Part 1—First- and Second-strand cDNA Synthesis, IVT cRNA Synthesis .............. 43
Approximate time required for Part 1 ......................................... 43
Reagent and labware required for Part 1 ...................................... 44
Clean the 'P' Pad in the autosealing microplate lid ............................. 46
Prepare the Biomek™ FXP Target Prep Express Automated Workstation ........... 47
Breaking the light curtain while running the assay ............................. 52
Prepare the samples and reagents ........................................... 52
Start the Biomek™ Software ................................................. 53
Begin Part 1—First- and Second-strand cDNA Synthesis, IVT cRNA Synthesis .... 56
Part 2—cRNA Purification ....................................................... 62
Approximate time required for Part 2 ......................................... 62
Reagent and labware required for Part 2 ...................................... 62
Perform a prerun check of the Biomek™ FXP Target Prep Express Automated
Workstation ............................................................ 64
Prepare the samples and reagents ........................................... 64
Begin Part 2—cRNA Purification ............................................. 65
Part 3—cDNA Fragmentation and Hybridization Setup .............................. 80
Approximate time required for Part 3 ......................................... 80
Reagent and labware required for Part 3 ...................................... 81
Perform a prerun check of the Biomek™ FXP Target Prep Express Automated
Workstation ............................................................ 84
Prepare the samples and reagents ........................................... 84
Begin Part 3—cDNA Fragmentation and Hybridization Setup .................... 85
Part 4—GeneTitan™ MC Instrument Hybridization Preparation ....................... 97
Approximate time required for Part 4 ......................................... 97
Reagent and labware required for Part 4 ...................................... 97
Perform a prerun check of the Biomek™ FXP Target Prep Express Automated
Workstation ............................................................ 99
Label GeneTitan™ hybridization and reagent trays .............................. 99
Thaw and prepare the samples ............................................. 101
Deionization of GeneTitan™ trays and covers ................................. 101
Begin Part 4—GeneTitan™ MC Instrument Hybridization Preparation ............ 104
■APPENDIX A GeneChip™ HT 3' IVT PLUS workflow diagrams .............. 109
First-Strand cDNA synthesis ................................................... 110
Second-Strand cDNA synthesis ................................................ 111
In vitro cRNA synthesis ........................................................ 112
cRNA purification ............................................................. 113
Contents
4GeneChip™ HT 3' IVT PLUS Reagent Kit Assay Automated Workflow on Biomek™ FXP User Guide
Fragmentation ................................................................ 118
Cartridge hybridization setup ................................................... 119
Array plate hybridization setup .................................................. 121
GeneTitan™ hybridization preparation ............................................ 123
■APPENDIX B GeneChip™ HT 3' IVT PLUS labware and reagent setup
deck layouts ...................................................................... 125
Deck setup for 16 and 24 reactions using on-deck thermal cycler and plate reader .... 126
Deck setup for 16 and 24 reactions using on-deck thermal cycler and NanoDrop™
concentration measurement .................................................. 127
Deck setup for 16 and 24 reactions using o-deck thermal cycler and plate reader .... 128
Deck setup for 16 and 24 reactions using o-deck thermal cycler and
NanoDrop™ concentration measurement ....................................... 129
Deck setup for 96 reactions using on-deck thermal cycler and plate reader .......... 130
Deck setup for 96 reactions using on-deck thermal cycler and NanoDrop™
concentration measurement .................................................. 131
Deck setup for 96 reactions using o-deck thermal cycler and plate reader .......... 132
Deck setup for 96 reactions using o-deck thermal cycler and NanoDrop™
concentration measurement .................................................. 133
GeneTitan™ hybridization preparation ............................................ 134
■APPENDIX C GeneChip™ HT 3' IVT PLUS deck layout setup and cleanup 135
Part 1—First- and Second-strand cDNA Synthesis, IVT cRNA Synthesis ............. 136
Part 2—cRNA Purification ...................................................... 137
Part 3—cDNA Fragmentation and Hybridization Setup for cartridge arrays ........... 138
Part 3—cDNA Fragmentation and Hybridization Setup for array plates ............... 139
Part 4—GeneTitan™ MC Instrument Hybridization Preparation ...................... 140
■Safety ............................................................................... 141
Chemical safety .............................................................. 142
Biological hazard safety ....................................................... 144
■Documentation and support ...................................................... 145
Related documentation ........................................................ 145
Customer and technical support ................................................ 146
Limited product warranty ...................................................... 146
Contents
GeneChip™ HT 3' IVT PLUS Reagent Kit Assay Automated Workflow on Biomek™ FXP User Guide 5
GeneChip™ HT 3' IVT PLUS Reagent
Kit
■Product information .................................................................... 6
■Kit contents and storage .............................................................. 12
This manual describes the assay procedures recommended for target labeling for 3' expression analysis
using the Beckman Coulter™ Biomek™ FXP TPE System. The reagents and protocols have been
developed and optimized specifically for use with the Beckman Coulter™ Biomek™ FXP TPE System.
It is anticipated that by following the recommended procedures, you will be able to generate sucient
biotin-labeled complementary RNA (cRNA) target from each sample for hybridization to a single
GeneChip™ Cartridge Array or Array Plate. For information on RNA preparation, see Chapter 3, “RNA
preparation”.
Product information
Purpose of the product
The GeneChip™ HT 3' IVT PLUS Reagent Kit enables you to use automation to prepare RNA samples
for transcriptome expression analysis with GeneChip™ 3' Expression Arrays. The kit generates amplified
and biotinylated complementary RNA (cRNA) from poly(A) RNA in a total RNA sample without the need
for an up-front removal of ribosomal RNA. The kit is optimized for processing of up to 96 samples in a
single run with minimal hands-on time.
The GeneChip™ HT 3' IVT PLUS Reagent Kit uses a reverse transcription priming method that primes
the poly(A) tail junction of RNA to provide gene expression profiles from mRNA. RNA amplification
is based upon linear amplification and employs T7 in vitro transcription (IVT) technology. The kit is
comprised of reagents and a protocol for preparing hybridization ready targets from 50 to 500 ng of
total RNA (Figure 1 and Figure 2). The GeneChip™ HT 3' IVT PLUS Reagent Kit is designed for sample
types such as fresh or frozen tissues, cell lines, and globin-reduced whole blood.
The GeneChip™ HT 3' IVT PLUS Reagent Kit is developed for the Beckman Coulter™ Biomek™ FXP
Target Prep Express (TPE) System. The automation workstation eliminates many of the labor intensive
tasks required when preparing eukaryotic RNA for 3' expression analysis. The HT 3' IVT PLUS
automated target preparations include all stages beginning with cDNA synthesis from RNA samples
and finishing with hybridization ready target preparation.
Note: User prepared reagents are not recommended for use with the GeneChip™ HT 3' IVT PLUS
Reagent Kit. Use the Applied Biosystems™ GeneTitan™ Hybridization, Wash, and Stain Kit for 3' IVT
Arrays (Cat. No. 901530) or the Applied Biosystems™ GeneChip™ Hybridization, Wash, and Stain Kit
for Cartridge Arrays Cat. No. 900720) for all the reagents needed for the hybridization, wash, and stain
steps of this assay.
1
6GeneChip™ HT 3' IVT PLUS Reagent Kit Assay Automated Workflow on Biomek™ FXP User Guide
The Beckman Coulter™ Biomek™ FXP TPE System automates the preparation of targets that can be
hybridized to a:
•GeneChip™ 3' Expression Cartridge Array that can be processed using the GeneChip™
Hybridization Oven 645, GeneChip™ Fluidics Station 450, and GeneChip™ Scanner 3000 7G system
Target preparation for cartridge arrays uses the first 3 parts of the Biomek™ FXP method with an
optional stopping point at the completion of each part (Figure 1). At the end of target preparation,
the sample is ready to be denatured and hybridized onto a GeneChip™ 3' Expression Cartridge
Array.
•GeneChip™ 3' Expression Array Plate that can be processed using the GeneTitan™ MC Instrument
Target preparation for array plates uses all 4 parts of the Biomek™ FXP method with an optional
stopping point at the completion of each part (Figure 2). At the end of target preparation, the
denatured sample is ready to be hybridized onto a GeneChip™ 3' Expression Array Plate.
Chapter 1 GeneChip™ HT 3' IVT PLUS Reagent Kit
Product information 1
GeneChip™ HT 3' IVT PLUS Reagent Kit Assay Automated Workflow on Biomek™ FXP User Guide 7
Assay workflow
Workflow diagrams
Figure 1 Overview of the HT 3' IVT PLUS amplification and labeling process for cartridge arrays.
Chapter 1 GeneChip™ HT 3' IVT PLUS Reagent Kit
Product information
1
8GeneChip™ HT 3' IVT PLUS Reagent Kit Assay Automated Workflow on Biomek™ FXP User Guide
Figure 2 Overview of the HT 3' IVT PLUS amplification and labeling process for array plates.
Chapter 1 GeneChip™ HT 3' IVT PLUS Reagent Kit
Product information 1
GeneChip™ HT 3' IVT PLUS Reagent Kit Assay Automated Workflow on Biomek™ FXP User Guide 9
Approximate times required
The following tables provide the time required to perform each part of the assay. Depending on
the selected parameters, target preparation takes about 11 to 23 hours, depending on the length of
IVT incubation, and requires 4 human user interventions. Additional time is required for hybridization,
washing, staining, and scanning, and is dependent on the number of samples processed and the array
type.
Approximate time required for Part 1
Table 1 Approximate time required for part 1.
Part 1—First- and Second-strand cDNA Synthesis, IVT
cRNA Synthesis
16 reactions
in minutes
24 reactions
in minutes
96 reactions
in minutes
Deck setup 15 15 15
Biomek™ FXP Target Prep Express Automated Workstation
pipetting steps: First-strand synthesis
5 5 13
Thermal cycler: first-strand method 125 125 125
Biomek™ FXP Target Prep Express Automated Workstation
pipetting steps: Second-strand synthesis
7 7 16
Thermal cycler: second-strand method 76 76 76
Biomek™ FXP Target Prep Express Automated Workstation
pipetting steps: IVT
7 7 19
Thermal cycler: IVT method 240 or 960 240 or 960 240 or 960
Part 1 total time—4-hour IVT incubation 475
7.9 hours
475
7.9 hours
504
8.4 hours
Part 1 total time—16-hour IVT incubation 1,195
19.9 hours
1,195
19.9 hours
1,224
20.4 hours
Approximate time required for Part 2
Table 2 Approximate time required for part 2.
Part 2—cRNA Purification 16 reactions
in minutes
24 reactions
in minutes
96 reactions
in minutes
Deck setup 15 15 15
Biomek™ FXP Target Prep Express Automated Workstation
pipetting steps: cRNA purification
65 65 76
Part 2 total time 80
1.3 hours
80
1.3 hours
91
1.5 hours
Chapter 1 GeneChip™ HT 3' IVT PLUS Reagent Kit
Product information
1
10 GeneChip™ HT 3' IVT PLUS Reagent Kit Assay Automated Workflow on Biomek™ FXP User Guide
Approximate time required for Part 3
Table 3 Approximate time required for part 3.
Part 3—cDNA Fragmentation and Hybridization Setup 16 reactions in
minutes
24 reactions
in minutes
96 reactions
in minutes
Deck setup 10 10 10
Biomek™ FXP Target Prep Express Automated Workstation
pipetting steps: cRNA fragmentation
4 4 5
Thermal cycler: fragmentation method 40 40 40
Biomek™ FXP Target Prep Express Automated Workstation
pipetting steps: Hybridization setup
GeneChip™ cartridge array hybridization setup
or
GeneChip™ array plate hybridization setup
11
or
11
12
or
12
24
or
17
Part 3 total time for cartridge array 65
1.1 hours
66
1.1 hours
79
1.3 hours
Part 3 Total time for array plate 65
1.1 hours
66
1.1 hours
72
1.2 hours
Approximate time required for Part 4
Table 4 Approximate time required for part 4.
Part 4—GeneTitan™ MC Instrument Hybridization
Preparation
16 reactions
in minutes
24 reactions
in minutes
96 reactions
in minutes
Deck setup 10 10 10
Biomek™ FXP Target Prep Express Automated Workstation
pipetting steps and thermal cycler: denaturation method
18 18 18
Part 4 total time 28 28 28
Chapter 1 GeneChip™ HT 3' IVT PLUS Reagent Kit
Product information 1
GeneChip™ HT 3' IVT PLUS Reagent Kit Assay Automated Workflow on Biomek™ FXP User Guide 11
Kit contents and storage
Table 5 GeneChip™ HT 3' IVT PLUS Reagent Kit contents and storage.
Component
30-reaction kit for 24-
reaction automated use
(Cat. No. 902416)
96-reaction kit for 96-
reaction automated use
(Cat. No. 902417)
Storage
3′ IVT PLUS Amplification Kit Module 1
3′ First-Strand Enzyme 50 µL 150 µL
–20°C
3′ First-Strand Buer 500 µL 1.58 mL
3′ Second-Strand
Enzyme
70 µL 250 µL
3′ Second-Strand Buer 180 µL 650 µL
3′ IVT Enzyme 210 µL 750 µL
3′ IVT Buer 1.58 mL 2 x 1.58 mL
3′ IVT Biotin Label 140 µL 500 µL
Control RNA (1 mg/mL
HeLa total RNA)
5 µL 5 µL
Nuclease-free Water 2 x 1.0 mL NA Any temperature[1]
3′ IVT PLUS Amplification Kit Module 2
3′ Fragmentation Buer 1 mL 1.8 mL Room temperature or 4°C
Purification Beads 3.3 mL 11 mL 4°C
Do not freeze.
GeneChip™ Eukaryotic Poly-A RNA Control Kit
Poly-A Control Stock 16 µL 16 µL
–20°C
Poly-A Control Dil Buer 3.8 mL 3.8 mL
GeneChip™ Hybridization Control Kit
20X Hybridization
Controls
450 µL 2 x 1.125 mL
–20°C
3 nM Control Oligo B2 150 µL 750 µL
Tubes Organizer: Plastic vinyl template for organization and storage of components in 9 x 9 array, 81-places
square wells, 5-1/4 inch x 5-1/4 inch (for example, Nalgene™ Polycarbonate 9 x 9 CryoBox™, Cat No.
5026-0909 , or equivalent).
[1] Store the Nuclease-free Water at –20°C, 4°C, or room temperature.
Chapter 1 GeneChip™ HT 3' IVT PLUS Reagent Kit
Kit contents and storage
1
12 GeneChip™ HT 3' IVT PLUS Reagent Kit Assay Automated Workflow on Biomek™ FXP User Guide
Required materials
■Required equipment, consumables, labware, and reagents ................................ 13
■Consumables and labware images ..................................................... 20
■Reagent and labware requirements for Part 1 through Part 4 ............................... 27
This chapter provides a check list of required equipment, consumables, labware, and reagents.
Required equipment, consumables, labware, and reagents
Equipment
Unless otherwise indicated, all materials are available through thermofisher.com. "MLS" indicates that
the material is available from fisherscientific.com or another major laboratory supplier.
Table 6 Equipment required for target preparation.
Item Quantity Source
Biomek™ FXP Target Prep Express Automated Workstation 1 Beckman Coulter™
A83103
24-Position Tube Rack 1 Beckman Coulter™
373661
24-Position Tube Rack Insert, 11 mm, for 1.5-mL Microfuge Tubes Beckman Coulter™
373696
Reagent block
Blue Heater/Chiller 24 Well Block (referred to as Reagent Cold Block, 24
Well Block in this document)
1 Beckman Coulter™
A83054
Thin PCR adaptor (installed on the Peltier Shaking Device) 1 Beckman Coulter™
A19709
Centrifuge (for tube and plate) 1 MLS
Pipettes 1 MLS
Spectrophotometer (plate, single-channel, or multi-channel) 1 MLS
Thermal Cycler (optional for o-deck incubation) 1 MLS
Vortex Mixer 1 MLS
2
GeneChip™ HT 3' IVT PLUS Reagent Kit Assay Automated Workflow on Biomek™ FXP User Guide 13
Table 7 Installation kits required for Biomek™ FXP Target Prep Express.
Item Quantity Source
GeneChip™ HT 3' IVT PLUS and WT PLUS Core Installation Kit,
consists of:
•Alpaqua™ Magnetic Plate
•Alpillo™ Plate Cushion
•Arched autosealing lids with wide tabs
•1
•1
•package of 4
902453
GeneChip™ HT 3' IVT PLUS Installation Kit, consists of:
•HT 3' IVT PLUS Reagent Cold Block Template
•HT 3' IVT PLUS Reservoir Stickers
•PCR Tube Holder
•1
•1
•1
902455
3' IVT PLUS ATP Biomek™ method file (and support files) Installed by FAS
Table 8 Equipment required for array processing.
Instrument Source
GeneChip™ system for cartridge arrays, consists of
GeneChip™ Hybridization Oven 645 00-0331
GeneChip™ Fluidics Station 450 00-0079
GeneChip™ Scanner 3000 7G 00-0210
GeneChip™ AutoLoader with External Barcode Reader 00-0090 or 00-0129
GeneTitan™ system for array plates, consists of:
GeneTitan™ Multi-Channel (MC) Instrument 00-0372 (110 V)
00-0373 (220 V)
Chapter 2 Required materials
Required equipment, consumables, labware, and reagents
2
14 GeneChip™ HT 3' IVT PLUS Reagent Kit Assay Automated Workflow on Biomek™ FXP User Guide
Consumables and labware
Consumables
Unless otherwise indicated, all materials are available through thermofisher.com. "MLS" indicates that
the material is available from fisherscientific.com or another major laboratory supplier.
Item Quantity Source
HT 3' IVT PLUS Consumables for Biomek™ FXP Target Prep Express
Consists of labware sucient for 4 x 24 or 4 x 96 reaction runs.
•96-well deepwell microplate, polypropylene, 2 mL, nonsterile
•Greiner Bio-One™ U-bottom (round) plate, polypropylene
•Bio-Rad™ Hard-shell PCR plate, clear well
•Greiner Bio-One™ UV-Star™ plate, nonsterile, clear, flat bottom
•Corning™ 96-well Clear Round Bottom Polystyrene Treated
Microplate
•24-well plate, 10 mL, square well, pyramid bottom, polypropylene
•Microseal™ 'P' Pad for autosealing microplate lid
One 'P' Pad is used per run. The 'P' Pad can be reused after
cleaning.
•FrameStar™ Break-A-Way plate
•4
•4
•40
•4
•8
•4
•2
•4
902519
Nuclease-free 1.5 mL tubes As needed MLS
Labware
Unless otherwise indicated, all materials are available through thermofisher.com.
Table 9 Biomek™ tips and universal labware for 1 full run of the GeneChip™ HT 3' IVT PLUS Reagent Kit on
the Biomek™ FXP Target Prep Express Automated Workstation.
Item Packaging 16 reactions 24 reactions 96 reactions
Biomek™ Tips P50 (AP96) Sterile Filtered (magenta)[1], Beckman Coulter™, A21586
Plates 96/box
10 boxes/case
6 boxes 6 boxes 8 boxes
Cartridges 6 boxes 6 boxes 7 boxes
Biomek™ Tips P250 (AP96) Sterile with Filter (aqua), Beckman Coulter™, 717253
Plates 96/box
10 boxes/case
4 boxes 4 boxes 4 boxes
Cartridges 3 boxes 3 boxes 3 boxes
Biomek™ Tips P250 Span-8 Sterile Filtered
(green), Beckman Coulter™, 379503
96/box
10 boxes/case
1 box 1 box 1 box
Biomek™ Tips P1000 Span-8 Sterile Filtered
Conductive (yellow), Beckman Coulter™, 987925
96/box
5 boxes/case
1 box 1 box 1 box
Chapter 2 Required materials
Required equipment, consumables, labware, and reagents 2
GeneChip™ HT 3' IVT PLUS Reagent Kit Assay Automated Workflow on Biomek™ FXP User Guide 15
Table 9 Biomek tips and universal labware for 1 full run of the GeneChip HT 3' IVT PLUS Reagent Kit on
the Biomek FXP Target Prep Express Automated Workstation. (continued)
Item Packaging 16 reactions 24 reactions 96 reactions
Quarter Reservoir, Divided by Width, Beckman Coulter™, 372792
Plates
48 units/case
4 units 4 units 4 units
Cartridges 3 units 3 units 3 units
Quarter Reservoir, Beckman Coulter™, 372790
Plates
48 units/case
5 units 5 units 5 units
Cartridges 3 units 3 units 3 units
Reusable Frame for Modular Reservoirs,
Beckman Coulter™ 372795
1 each 4 4 4
[1] One fewer box of Biomek™ Tips P50 (AP96) Sterile Filtered (magenta) will be needed if selecting the NanoDrop quantitation option for Part 2
purification.
Pipette tip usage
The following tables describe pipette tip usage for 1 full run of the GeneChip™ HT 3' IVT PLUS Reagent Kit
on the Biomek™ FXP Target Prep Express Automated Workstation for 16-reaction, 24-reaction, and 96-reaction
sample formats.
All tips used for the MC pod are to be discarded after the run, unless being used for the Span-8 pod. Tips on
the Span-8 pod can be used for the next part if continuing without interruption. A new box of Span-8 tips is
always required when resuming a run after the method has been stopped.
Chapter 2 Required materials
Required equipment, consumables, labware, and reagents
2
16 GeneChip™ HT 3' IVT PLUS Reagent Kit Assay Automated Workflow on Biomek™ FXP User Guide
Table 10 Pipette tip usage for a 16-reaction sample format (in boxes).
Item Total[1] Part 1 Part 2
Part 3
Part 4[2]
Cartridge Plate
Boxes
AP96 P50 Tips (magenta) 53 1 N/A 1 0
AP96 P250 Tips (aqua) 40 2 N/A 1 1
Span-8 P50 Tips (magenta) 11 1 N/A 1 0
Span-8 P250 Tips (green) 11 1 N/A 1 0
Span-8 P1000 Tips (yellow) 11 1 N/A 1 1
Tip count
AP96 P50 Tips (magenta) 480 288 96[3] N/A 96 0
AP96 P250 Tips (aqua) 384 0 192 N/A 96 96
Span-8 P50 Tips (magenta) 56 24 16 N/A 16 0
Span-8 P250 Tips (green) 22 4 8 N/A 10 0
Span-8 P1000 Tips (yellow) 75 4 32 N/A 15 24
[1] Total for array plates with GeneTitan™ hybridization options. Fewer tips are required for cartridge array options.
[2] Not required for cartridge array options.
[3] No Biomek™ Tips P50 (AP96) Sterile Filtered are required for Part 2 if the NanoDrop option is selected for the method.
Chapter 2 Required materials
Required equipment, consumables, labware, and reagents 2
GeneChip™ HT 3' IVT PLUS Reagent Kit Assay Automated Workflow on Biomek™ FXP User Guide 17
Table 11 Pipette tip usage for a 24-reaction sample format (in boxes).
Item Total[1] Part 1 Part 2
Part 3
Part 4[2]
Cartridge Plate
Boxes
AP96 P50 Tips (magenta) 53 1 1 1 0
AP96 P250 Tips (aqua) 40 2 1 1 1
Span-8 P50 Tips (magenta) 11 1 0 1 0
Span-8 P250 Tips (green) 11 1 1 1 0
Span-8 P1000 Tips (yellow) 11 1 1 1 1
Tip count
AP96 P50 Tips (magenta) 480 288 96[3] 96 96 0
AP96 P250 Tips (aqua) 384 0 192 96 96 96
Span-8 P50 Tips (magenta) 72 24 24 0 24 0
Span-8 P250 Tips (green) 19 2 8 8 9 0
Span-8 P1000 Tips (yellow) 79 6 32 17 17 24
[1] Total for array plates with GeneTitan™ hybridization options. Fewer tips are required for cartridge array options.
[2] Not required for cartridge array options.
[3] No Biomek™ Tips P50 (AP96) Sterile Filtered are required for Part 2 if the NanoDrop option is selected for the method.
Chapter 2 Required materials
Required equipment, consumables, labware, and reagents
2
18 GeneChip™ HT 3' IVT PLUS Reagent Kit Assay Automated Workflow on Biomek™ FXP User Guide
Table 12 Pipette tip usage for a 96-reaction sample format (in boxes).
Item Total[1] Part 1 Part 2
Part 3
Part 4[2]
Cartridge Plate
Boxes
AP96 P50 Tips (magenta) 63 1 1 2 0
AP96 P250 Tips (aqua) 40 2 1 1 1
Span-8 P50 Tips (magenta) 21 1 0 0 0
Span-8 P250 Tips (green) 10 1 1 1 0
Span-8 P1000 Tips (yellow) 11 1 1 1 1
Tip count
AP96 P50 Tips (magenta) 576 288 96[3] 96 192 0
AP96 P250 Tips (aqua) 384 0 192 96 96 96
Span-8 P50 Tips (magenta) 120 24 96 0 0 0
Span-8 P250 Tips (green) 24 0 8 16 16 0
Span-8 P1000 Tips (yellow) 88 11 32 30 21 24
[1] Total for array plates with GeneTitan™ hybridization options. Fewer tips are required for cartridge array options.
[2] Not required for cartridge array options.
[3] No Biomek™ Tips P50 (AP96) Sterile Filtered are required for Part 2 if the NanoDrop option is selected for the method.
Chapter 2 Required materials
Required equipment, consumables, labware, and reagents 2
GeneChip™ HT 3' IVT PLUS Reagent Kit Assay Automated Workflow on Biomek™ FXP User Guide 19
Reagents
Table 13 Additional reagents required.
Item Source
Applied Biosystems™ GeneChip™ Hybridization, Wash, and Stain Kit
(30 reactions, for cartridge arrays)
900720
Applied Biosystems™ GeneTitan™ Hybridization, Wash, and Stain Kit for 3'
IVT Arrays (96 reactions, for array plates)
901530
Invitrogen™ DNAZap™ PCR DNA Degradation Solutions (or equivalent
product)
AM9890
Invitrogen™ RNaseZap™ products (or equivalent product) AM9780 250 mL
AM9782 6 x 250 mL
AM9784 4L
Water, Nuclease-free, Molecular Biology Grade, Ultrapure
For elution, dilution and preparing 80% Ethanol Wash Solution:
Approximately 25 mL for 1 x 16 reactions and 1 x 24 reactions, and
approximately 75 mL for 1 x 96 reactions.
71786
or MLS
100% Ethanol[1] (molecular biology grade or equivalent)
For preparing 80% Ethanol Wash Solution:
Approximately 10 mL for 1 x 16 reactions and 1 x 24 reactions, and
approximately 25 mL for 1 x 96 reactions.
MLS
[1] Before handling any chemicals, see the SDS provided by the manufacturer, and observe all relevant precautions.
Consumables and labware images
Table 14 shows the types of labware used in the GeneChip™ HT 3' IVT PLUS Assay. For labware
requirements specific to each part of the assay, see “Reagent and labware requirements for Part 1
through Part 4” on page 27.
Unless otherwise indicated, all materials are available through thermofisher.com.
Table 14 Consumables and labware used on the Biomek™ workstation deck.
Labware Source Labware image
Biomek™ Tips P50 (AP96) Sterile Filtered
(magenta)
Beckman Coulter™,
A21586
Chapter 2 Required materials
Consumables and labware images
2
20 GeneChip™ HT 3' IVT PLUS Reagent Kit Assay Automated Workflow on Biomek™ FXP User Guide
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