CytoScanTM Optima Assay - Thermo Fisher Scientific · CytoScan® Optima Assay 5 Safety Information...

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

CytoScanTM

Optima Assay Site Preparation Guide

Information in this document is subject to change without notice.

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© 2017 Thermo Fisher Scientific Inc. All rights reserved.

P/N 703281

2 CytoScan® Optima Assay

Contents

Chapter 1 Introduction ....................................................................................................... 3

Safety.................................................................................................................................................................. 3

Text Alerts .................................................................................................................................................... 3

Contamination Prevention .................................................................................................................................. 3

Equipment and Calibration ................................................................................................................................. 4

Safety Information .............................................................................................................................................. 5

Reagent Handling and Storage ........................................................................................................................... 5

Thermal Cyclers, 96-Well Plate, and Adhesive Seals ........................................................................................ 5

Chapter 2 Laboratory Setup................................................................................................ 6

Before You Begin ............................................................................................................................................... 6

Configurations .................................................................................................................................................... 6

Two Separate Rooms .................................................................................................................................... 6

One Room with Single-Direction Workflow ................................................................................................ 9

GeneChipTM

System 3000 7G Installation ....................................................................................................... 10

GeneChipTM

System 3000 7G Specifications ................................................................................................... 10

GeneChipTM

Fluidics Station 450 ............................................................................................................... 10

Scanner ....................................................................................................................................................... 11

Oven ........................................................................................................................................................... 12

Chapter 3 Materials Required ........................................................................................... 13

Materials ........................................................................................................................................................... 13

Reagents from Other Suppliers ........................................................................................................................ 15

Pre-PCR Clean Area Equipment Required but Not Provided .......................................................................... 16

Post-PCR Room Equipment Required but Not Provided ................................................................................. 17

Consumables Required but Not Provided ........................................................................................................ 18

Thermal Cycler Programs ................................................................................................................................ 18

Documentation ................................................................................................................................................. 18

Labeling Symbols ............................................................................................................................................. 19

CytoScan® Optima Assay 3

Chapter 1 Introduction For information on the use of the CytoScan

TM Optima assay, refer to the CytoScan

TM Optima Assay User Guide (P/N

703280).

In this chapter:

Safety

Contamination Prevention

Equipment and Calibration

Reagent Handling and Storage

Thermal Cyclers, 96-Well Plate, and Adhesive Seals

Safety

IMPORTANT: Follow universal precautions in the laboratory. For waste disposal, follow federal, state, local, and within-country regulations.

Text Alerts

Text alerts draw your attention to a particular piece of information. There are five types of text alerts: Note, Important,

Caution, and Warning.

NOTE: Information that may be of interest or of help to a user but is not critical to the primary purpose of the text.

IMPORTANT: Information that is essential to the successful use of a product or the completion of a procedure and is not safety related.

CAUTION: An alert to the user of hazards that, if not avoided, can cause minor or moderate personal bodily injury and/or damage to an instrument or loss of data.

WARNING: Alerts the user to hazards that, if not avoided, can cause serious bodily injury or death, produce potentially incorrect data that could cause harm to a patient.

Contamination Prevention Proper laboratory practice is necessary because previously amplified PCR product is the most likely potential source

of contamination.

Set up the Pre-/Post-PCR areas for single-direction workflow

Use dedicated equipment for each area (e.g., thermal cyclers, microfuges, pipettes and tips, ice buckets, etc.)

Place all reagents and master stocks in use area. Do not move equipment between Pre- and Post-PCR Rooms, e.g.,

ice buckets, pipettes, etc.

Place separate copy of assay procedure in Pre- and Post-PCR areas

Follow procedures for re-entry of Pre-PCR Clean Area from post-PCR Area


Equipment in Pre-PCR Clean Area

Freezer, –15 to –25°C

Ice bucket with ice

Laminar flow cabinet or PCR cabinet

Microfuge

Pipettes on stand

Plate centrifuge

Refrigerator

Thermal cycler

Vortexer

Equipment in Post-PCR Area

Electrophoresis gel box

Electrophoresis power supply

Freezer, –15 to –25°C

Gel imager

GeneChipTM

System 3000 v.2

Computer, monitor, keyboard, and mouse

Fluidics station

Scanner

Hybridization Oven

Ice bucket

Magnetic stand

Pipettes on stand

Spectrophotometer, UV/VIS, single or multichannel

Vortexer

Microcentrifuge (for both strips and tubes)

Microfuge

Refrigerated plate centrifuge

Refrigerator

Thermal cycler(s)

Vortexer with foam tube adaptor

Equipment and Calibration

Set up, maintain, and calibrate all equipment according to the manufacturer’s instructions.


Safety Information

Warning: 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.

Caution: All chemicals should be considered as potentially hazardous. We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. Wear suitable protective clothing, such as lab coat, safety glasses and gloves. Care should be taken to avoid contact with skin and eyes. In case of contact with skin or eyes, wash immediately with water. See SDS (Safety Data Sheet) for specific advice.

Reagent Handling and Storage

Store all reagents at the indicated temperatures and conditions (see Chapter 3, Materials Required)

Do not store enzymes in a frost-free freezer

Store reagents used for digestion, ligation, and PCR only in the Pre-PCR Clean Area

Thermal Cyclers, 96-Well Plate, and Adhesive Seals Use the thermal cyclers, 96-well plate, and adhesive films listed in Chapter 3, Materials Required.


Chapter 2 Laboratory Setup In this chapter:

Before You Begin

Configurations

GeneChipTM

System 3000 7G Installation

GeneChipTM

System 3000 7G Specifications

Before You Begin

Read Chapter 1, Introduction.

Configurations Two setups are given following, one for two separate rooms and one for a single room with Pre- and Post- PCR

separation. Always set up for a single-direction workflow.

Two Separate Rooms

This configuration greatly reduces the risk of sample contamination due to previously-amplified PCR products. These

are the:

Pre-PCR Clean Room

Post-PCR Room

These rooms are set up to accommodate the steps in Table 2.1.

Table 2.1 Assay Workflow When Two Separate Rooms are Used

Room Template (Genomic DNA)

PCR Product

Pre-PCR Clean Room

Assay steps:

Genomic DNA preparation

Digestion

Ligation

PCR setup only

Post-PCR Room

Assay steps:

PCR thermal cycling and

purification

Fragmentation

Labeling

Hybridization

Washing and staining

Scanning


Pre-PCR Clean Room

The Pre-PCR Clean Room is a low-copy DNA template lab, and must be free of PCR product (amplicons). Setup and

major pieces of dedicated equipment required are shown in Figure 2.1.

Figure 2.1 Pre-PCR Clean Room


Post-PCR Room

Setup and major pieces of dedicated equipment required are shown in Figure 2.2.

Figure 2.2 Post-PCR Room


One Room with Single-Direction Workflow

One room with two distinctly separated areas: Pre-PCR Clean Area and Post-PCR Area (see Figure 2.3).

Figure 2.3 One Room Configuration

Equipment in Pre-PCR Clean Area


1. Laminar flow cabinet or PCR cabinet

2. Vortexer

3. Microfuge

4. Pipettes on stand

5. Ice bucket with ice

6. Thermal cycler

7. Plate centrifuge

8. Freezer, –15 to –25°C

9. Refrigerator


Equipment in Post-PCR Area


1. Vortexer

2. Microfuge

3. Pipettes on stand

4. Vortexer (with foam tube adaptor)

5. Ice bucket

6. Magnetic stand

7. Thermal cycler

8. Hybridization oven

9. Refrigerated plate centrifuge

10. Microcentrifuge

11. Spectrophotometer, UV/VIS, single or multichannel

12. Gel imager

13. Electrophoresis gel box

14. Computer, monitor, keyboard

15. Fluidics station

16. Scanner

17. Refrigerator

18. Freezer, –15 to –25°C

GeneChipTM

System 3000 7G Installation The system consists of the fluidics station, scanner, computer, monitor, and keyboard. Install in the Post- PCR

Room/Area on a bench that is free of possible vibration.

GeneChipTM

System 3000 7G Specifications The GCS 3000 7G comes with these standard hardware components:

System Workstation (computer, monitor, and keyboard)

GeneChipTM

Fluidics Station 450

GeneChipTM

Scanner 3000 with AutoLoader

GeneChipTM

Hybridization Oven 645

GeneChipTM Fluidics Station 450

Table 2.2 Fluidics Station 450 Specifications

Specification Details

Height 15.8 inches (40.2 cm)

Width 28 inches (71.1 cm)

Depth 16.1 inches (41 cm)

Weight 80 pounds (36.3 kg)

Voltage 100-240 VAC, 3 A; 300 watts

Operating Temperature Range 15 to 30°C

Operating Humidity Range 10 to 90% RH (non-condensing)


Scanner

WARNING:

The GeneChipTM

Scanner 3000 7G contains an incorporated Class 3B laser with these specifications: Wavelength=532 nm ±1 nm, Beam Divergence (full angle)=<8 mrad, Output Duration=Continuous Wave, Maximum Power Output=500 mW.

Table 2.3 Scanner 3000 7G Specifications

Specification Details

Height without AutoLoader 31 inches (78.7 cm)

Height with AutoLoader (door closed) 44.5 inches (113 cm)

Width 22.5 inches (57.2 cm)

Depth 31 inches (78.7 cm)

Weight without AutoLoader 70 pounds (31.8 kg)

Weight with AutoLoader (fully loaded) 105 pounds (47.6 kg)

Line (Mains) Voltage 100-240 VAC, 50-60 Hz

Current 4-2 A

Excitation wavelength 532 nm (green) laser, 10 mW maximum

Emission Filter 570 nm, Long-Pass

Autoloader 48 microarrays per carousel

Cooled Environment Average temperature = 15°C


Oven

Table 2.4 GeneChipTM

Hybridization Oven 645

Item Specifications

Input Voltage 100 to 120 VAC, 5 A maximum or 220–240 VAC, 2.5 A maximum 50 to 60 Hz

Rotisserie Rotation Speed 10 to 80 RPM, programmable to 1 RPM

Oven Temperature Set Point

Programmable Range

30 to 70°C, programmable to 0.1°C

Time to Temperature 30 minutes from ambient to 60°C

Oven Temperature Accuracy ±1.0°C from 35 to 60°C

Communications (optional) Nine-pin RS-232 port, 9600 Baud rate

Monitors and reports oven temperature, rotisserie rotation rate, and oven status


Chapter 3 Materials Required In this chapter:

Materials

Reagents from Other Suppliers

Pre-PCR Clean Area Equipment Required but Not Provided

Post-PCR Room Equipment Required but Not Provided

Consumables Required but Not Provided

Thermal Cycler Programs

Documentation

Labeling Symbols

Materials

Table 3.1 Equipment and Software, Required

Item Part Number

Equipment

GeneChipTM

Fluidics Station 450 00-0079

Tubing, Silicone peristaltic for GeneChipTM

Fluidics Station 450 400110

GeneChipTM

Hybridization Oven 645 00-0331

GeneChipTM

Scanner 3000 7G 00-0213

Software

GeneChip Command Console Software Version 3.2.2 or higher

Chromosome Analysis Suite Software (ChAS 2.1 or higher) 901394

Upon receipt store all components as indicated in Table 3.2.


Table 3.2 CytoScanTM

Optima Assay Reagents, Required

Module P/N Module Item/Reagent Qty Part Number

902527 CytoScanTM

Optima – Module 1: Pre-

Lab Restriction, Ligation, and

Amplification

Nsp I

Nsp I Buffer

100X BSA

DNA Ligase

DNA Ligase Bfr

CytoScan Taq Bfr

CytoScan Taq DNA

Polymerase

dNTP

5M Betaine

Adaptor, Nsp I

PCR Primer

1

1

1

1

1

1

1

1

1

1

1

902421

902422

902423

902379

902378

902582

902581

902401

902402

902426

902427

902528 CytoScanTM

Optima – Module 2: Pre-

Lab TE Buffer and Water Low EDTA TE Buffer

Water, Nuclease free

1

1

902424

902425

Module P/N Module Item/Reagent Qty Part Number

902529 CytoScanTM

Optima – Module 3: Main

Lab Fragmentation, Labeling, and

Hybridization

Fragmentation Reagent

Fragmentation Buffer

TdT Enzyme

TdT Buffer

DNA Labeling Reagent

Oligo Control Reagent

Hyb Buffer Part 1

Hyb Buffer Part 2

Hyb Buffer Part 3

Hyb Buffer Part 4

1

1

1

1

1

1

1

1

1

1

902428

902301

902429

902430

902431

902432

902433

902434

902435

902436


902530 CytoScanTM


Lab Stain, Holding Buffer, Beads, and

Water

Stain Buffer 1

Stain Buffer 2

Array Holding Buffer

Purification Beads

Water, Nuclease free

1

1

1

1

1

902440

902441

902442

902443

902425

902531 CytoScanTM


Lab Elution Buffer, and Purification

Wash Buffer

Elution Buffer

Purification Wash

Buffer

1

1

902444

902437

Wash A 4 902438

Wash B 4 902439

Arrays Required

902507 CytoScan Optima Array Kit, 6 pack

Reagents from Other Suppliers Reagents Required but not provided.

Table 3.3 Reagents Required from Other Suppliers

✓ Item

• Bleach, 6.15% sodium hypochlorite

• PCR gel DNA ladder: 50 to 2000 bp

• Fragmentation gel DNA ladder: 25 to 2600 bp

• Ethanol, absolute

• Gel-loading buffer


Pre-PCR Clean Area Equipment Required but Not Provided

Table 3.4 Pre-PCR Clean Room Equipment, Required

✓ Item

If assay is to be performed in one room:

• Laminar Flow Cabinet, 6 foot

Or

• PCR Cabinet

• Benchtop Cooler, with the capacity to hold 8 to 32 tubes (1.5 mL) and ability to maintain temperature below –

15°C for 2 hours.

• Centrifuge, plate, multipurpose, 330 μL capacity

• Cooling chamber, double-block, with the capacity to hold 96 well plates with a maximum volume capacity of

330 μL

• Freezer, –15 to –25°C; deep freeze; manual defrost; 17 cu ft

• Microfuge (for tubes and strip tubes)

Pipettors:

• 12-channel, 2–20 μL

• 12-channel, 20–200 μL

• single-channel, 100–1000 μL



• Rectangular Ice Tray Large – 9L (16 x 13 in, 41 x 33 cm)

• Storage Racks, Tube, 96-well

• Thermal cycler: capable of holding 200 μL volume and 96-well plate; heat block capable of holding temperature

of 4–99.9°C; temperature accuracy of ±0.25°C (at 35 to 99.9°C); heating and cooling block ramp rate of ≤4°C

per second; thermal uniformity of ±0.5°C.

• Vortexer, 60 Hz, 75 W, 600 to 3200 RPM


Post-PCR Room Equipment Required but Not Provided

Table 3.5 Post-PCR Room Equipment Required

✓ Item

• Adhesive film applicator (hard plastic)

• Anti-vibration pad, used with vortexer to prevent movement during operation

• Cooler, benchtop, with capacity to hold 8 to 32 tubes (1.5 mL) and maintain temperature below –15°C for 2

hours

• Cooling chamber, double-block, with capacity to hold 96-well plates with a maximum volume capacity of 330 μL

• Electrophoresis supplies

• Freezer, –15 to –25°C; deep freeze; manual defrost; 17 cu ft

• Gel imager

• Magnetic rack with magnet on the side and capable of holding 8 to 12 tubes of 1.5 to 2 mL capacity

• Microcentrifuge, non-refrigerated with capacity to hold 24 tubes and maximum rotation speed of 16,200xg

• Microfuge (for tubes and strip tubes)

• Microtube foam insert

Pipettors:

• 12-channel, 100–1200 μL

• 12-channel, 2–20 μL

• 12-channel, 20–200 μL




• Plate centrifuge, refrigerated multipurpose, plate carriers for 4 x 96-well assay plates

• Platform Head, 6-inch, for microtube foam insert

• Rectangular ice tray, large - 9L (16 x 13in; 41 x 33cm)

• Refrigerator, 2-8°C, 6 cu ft

• Spectrophotometer, UV/VIS, single or multichannel

• Storage racks, tube, 96-well with lid, 1.5mL/2.0mL

• Thermal cycler: capable of holding 200 μL volume and 96-well plate; heat block capable of holding temperature

of 4 to 99.9°C; temperature accuracy of ±0.25°C (at 35-99.9°C); heating and cooling block ramp rate of ≤4°C per

second; thermal uniformity of ±0.5°C.

• Vortexer, 60 Hz, 75 W, 600-3200 RPM


Consumables Required but Not Provided

Table 3.6 Consumables from other Suppliers, Required

✓ Item

• Adhesive film, clear, PCR-certified, 96-well plates

• Adhesive label dot, 1/2-inch and 3/8-inch

• Agarose gel, 2%

• Agarose gel, 4%

• Microcentrifuge tubes, nuclease-free, sterile, 1.5 mL polypropylene

• Microcentrifuge tubes, nuclease-free, sterile, 2.0 mL polypropylene

• Microcentrifuge tubes, nuclease-free, sterile, non-stick, 1.5 mL amber polypropylene

• Microcentrifuge tubes, nuclease-free, sterile, non-stick, 1.5 mL blue polypropylene

• Microcentrifuge tubes, nuclease-free, sterile, non-stick, 1.5 mL natural polypropylene

• Microcentrifuge tubes, nuclease-free, sterile, non-stick, 50 mL polypropylene microcentrifuge tubes

• Pipette tips with aerosol barriers, 20 μL, 200 μL, and 1000 μL

• Plate, OD for UV, 96-well (required only if using spectrophotometer, UV/VIS, single or multichannel)

• Plates, unskirted PCR with a maximum volume of 330 μL

• Reagent reservoir, 25 mL

• Tube strips, nuclease-free, sterile, 8-well, 0.2 mL polypropylene

Thermal Cycler Programs Do not make alterations to the thermal cycler programs documented in the CytoScan

TM Optima Assay User Guide (P/N

703280).

Documentation The following documentation is available supporting the CytoScan

TM Optima assay.

CytoScanTM

Optima Assay User Guide (P/N 703280)

CytoScanTM

Optima Assay 8-, 16- and 24 Samples Quick Reference Card (P/N 703282)

Related Documentation

GeneChipTM

Command ConsoleTM

User Guide (P/N 702569)

Chromosome Analysis Suite Software User Guide (P/N 702943)


Labeling Symbols

Table below shows the legend of the graphic symbols used for product label, Package Inserts and User Guides.

Table 3.7 Labeling Symbols

Symbol / Label Statement / Meaning

Part/Catalog Number

Lot Number

Expiration Date

YYYY–MM Kit will expire on the last day of the month.

Temperature Limitation


Documentation and support

Obtaining support

Technical support For the latest services and support information for all locations,

visitwww.thermofisher.com.

At the website, you can:

Access worldwide telephone and fax numbers to contact Technical Support and Sales

facilities

Search through frequently asked questions (FAQs)

Submit a question directly to Technical Support (thermofisher.com/support)

Search for user documents, SDSs, vector maps and sequences, application notes,

formulations, handbooks, certificates of analysis, citations, and other product support

documents

Obtain information about customer training

Download software updates and patches

Safety Data Sheets (SDS)

Safety Data Sheets (SDSs) are available at thermofisher.com/support.

Limited product warranty

Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in

the Life Technologies’ General Terms and Conditions of Sale found on Life

Technologies’ website at www.thermofisher.com/us/en/home/global/terms-and-

conditions.html. If you have any questions, please contact Life Technologies at

www.thermofisher.com/support.

http://www.thermofisher.com/

http://www.thermofisher.com/support


http://www.thermofisher.com/us/en/home/global/terms-and-conditions.html

http://www.thermofisher.com/us/en/home/global/terms-and-conditions.html


For support visit thermofisher.com/support or [email protected]

thermofisher.com

23 January 2017

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