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

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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Products:

GeneChip™ 3' Expression Arrays

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

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE

LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR

ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Revision history: Pub. No. 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.

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these

products, you accept the terms and conditions of all applicable Limited Use Label Licenses.

TRADEMARKS: All trademarks are the property of Thermo Fisher Scientiﬁc and its subsidiaries unless otherwise speciﬁed. Agilent and

Bioanalyzer are trademarks of Agilent Technologies, Inc. Alpaqua and Alpillo are trademarks of Alpaqua Engineering, LLC. Beckman

Coulter, Biomek, and Span are trademarks of Beckman Coulter, Inc. Biometra is a trademark of Biometra Biomedizinische Analytik

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

GmbH. LabChip is a trademark of Caliper Life Sciences, Inc., part of PerkinElmer, Inc. INHECO is a trademark of INHECO Industrial

Heating & Cooling GmbH. Microsoft, Windows, Internet Explorer, and Excel are either registered trademarks or trademarks of Microsoft

Corporation in the United States and/or other countries. Oligo is a trademark of Oligo LLC Limited Liability Company Delaware. Thomas

Scientiﬁc and ABgene are a trademarks of Thomas Scientiﬁc. Watlow is a trademark of Watlow Electric Manufacturing Company.

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

•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

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

•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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