Thermo Fisher Scientific CytoScan XON Assay User guide

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

CytoScan™ XON Assay

USER GUIDE

24-Array Format Manual Workflow

Catalog Numbers 931310

Publication Number 703456

Revision 1

Multiple Life Technologies Corporation manufacturing sites are responsible for manufacturing the products associated with the workflow

covered in this guide.

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

DISCLAIMER

TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE,

MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Important Licensing Information

This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept the terms and conditions of all applicable Limited

Use Label Licenses.

Corporate entity

Life Technologies | Carlsbad, CA 92008 USA | Toll Free in USA 1 800 955 6288

TRADEMARKS

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

Tough-Spots is a registered trademark of Diversified Biotech.

CytoScan™ XON Assay Manual Workflow User Guide 3

Contents

CHAPTER 1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Assay warnings and precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

CHAPTER 2 CytoScan™ XON Assay target preparation . . . . . . . . . . . 10

Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Stage 1: Genomic DNA preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Stage 2: DNA amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

1: Prepare for DNA amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

2: Prepare the Denaturation Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

3: Add the Denaturation Master Mix to samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

4: Add the Neutralization Solution to samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

5: Preparation of Amplification Master Mix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

6: Add Amplification Master Mix to samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

7: Freeze or proceed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Stage 3: Stop the DNA amplification reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Thaw DNA samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Stage 4: Fragmentation and precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Input required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

1: Prepare for fragmentation and precipitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

2: Preparation of Fragmentation Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

3: Add the Fragmentation Master Mix to samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

4: Addition of Stop Solution to the samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

5: Preparation of Precipitation Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

6: Prepare and add isopropanol to the Precipitation Plate. . . . . . . . . . . . . . . . . . . . . . . . . 24

7: Freeze the Precipitation Plate overnight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

8: Freeze reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Stage 5: Centrifuge and drying, resuspension and hybridization preparation, and

sample QC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Input required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Stage 5A: Centrifuge precipitation tubes and dry the DNA pellet . . . . . . . . . . . . . . . . . . . . . 26

Contents

4CytoScan™ XON Assay Manual Workflow User Guide

Stage 5B: Resuspension and labeling preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

1: Resuspension of WGA pellets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

2: OD measurement and calculation of mass for labeling . . . . . . . . . . . . . . . . . . . . . . . . . 29

Stage 5C: Fragmentation QC checks and preparing Labeling Plate . . . . . . . . . . . . . . . . . . . 32

1: Perform fragmentation QC check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

2: Preparation of the Labeling Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Stage 6: Labeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Labeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Stage 7: Target hybridization via AGCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Important information about this stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Prepare the equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Prepare the arrays and create a Batch Registration file . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Prepare the reagents and consumables. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Prepare the Hybridization Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Add the Hybridization Master Mix and denature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Load the samples onto arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Stage 8: Washing, staining, and scanning arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Prime the fluidics station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Washing and staining arrays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Scanning arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Adding arrays during an AutoLoader run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Shutting down the fluidics station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

APPENDIX A Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

APPENDIX B Workflow and practices. . . . . . . . . . . . . . . . . . . . . . . . . 51

Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

Specific laboratory practices for CytoScan™ XON Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

Reagent handling and thawing instructions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

Seals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

Spin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

Vortexing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Thermal cycler setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

Pipetting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

Aliquot volumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

Contents

CytoScan™ XON Assay Manual Workflow User Guide 5

APPENDIX C Thermal cycler protocols. . . . . . . . . . . . . . . . . . . . . . . . 57

CYTOXON AMP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

CYTOXON STOP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

CYTOXON FRAG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

CYTOXON LABEL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

CYTOXON HYB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

APPENDIX D Required equipment, consumables, and reagents . . . . 59

From Thermo Fisher Scientific . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Array and reagent bundles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

From other suppliers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

Pre-PCR Clean Room–equipment required but not provided . . . . . . . . . . . . . . . . . . . . . . 62

Post-PCR Room – equipment required but not provided. . . . . . . . . . . . . . . . . . . . . . . . . . 63

Consumables required but not provided . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

Symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

APPENDIX E In-process quality control . . . . . . . . . . . . . . . . . . . . . . . 65

Total DNA yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Fragmented product . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

APPENDIX F Array quality control threshold. . . . . . . . . . . . . . . . . . . . 67

APPENDIX G Troubleshooting the CytoScan™ XON Assay . . . . . . . . 68

APPENDIX H Fluidics station care and maintenance . . . . . . . . . . . . . 73

General fluidics station care . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

Fluidics station bleach protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

The bleach cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

The rinse cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

6CytoScan™ XON Assay Manual Workflow User Guide

1Introduction

CytoScan™ XON Assay has been optimized for the detection of exon-level copy

number changes. The CytoScan XON Assay is intended to be used with genomic DNA

from postnatal peripheral blood samples. CytoScan XON Assay is composed of the

CytoScan™ XON Array, the CytoScan™ XON Reagent Kit, in conjunction with

GeneChip™ System 3000 and Chromosome Analysis Suite (ChAS). Together, they

compose the complete solution for array exon-level copy number analysis. The

solution is a good complement to an exome sequencing approach to provide a

comprehensive variation analysis in disease-research. CytoScan XON Array consists of

approximately 6.85M probes of which 6.55M are non-polymorphic copy number

probes and approximately 300,000 are SNP markers. These amount of markers provide

extensive coverage for 453,076 exons in 17,974 genes, with increased sensitivity and

specificity in 4600 medically-relevant genes.

The product is for research use only. Not for use in diagnostic procedures.

Assay warnings and precautions

Follow universal precautions for laboratory and assay procedures, and waste disposal.

Follow federal, state, local, and within-country regulations.

Before you begin, go to Appendix B, ʺWorkflow and practicesʺ on page 51 for specific

setup instructions, and equipment and technique illustrations and Appendix C,

ʺThermal cycler protocolsʺ for specific thermal cycler protocol set ups.

For additional warnings, precautions, and procedures, read the specific

documentation for the assay or instruments being used. See ʺDocumentation and

supportʺ on page 80.

Precautions

The physical and toxicological properties of the products in this kit(s) have not been

thoroughly investigated. Follow prudent laboratory practices and use general

laboratory safety equipment (eye protection, lab coat, and lab gloves) and good

personal hygiene when working with these or any laboratory reagents. Refer to the

Safety Data Sheet for more information.

Proper laboratory practice is necessary as previously amplified PCR product is the

most likely potential source of contamination. We strongly recommend two separate

work areas be used to minimize the risk of cross contamination during the assay

procedure. It is essential to adhere to workflow recommendations. Personnel should

not re-enter the Pre-PCR Clean Area once exposed to PCR products without first

showering and changing into clean clothes.

Carefully reading and following the protocol as written is essential. The CytoScan™

XON Assay has been validated using the reagents and suppliers listed. Substitution of

reagents and not following detailed procedures are not recommended as your results

could be suboptimal.

CytoScan™ XON Assay Manual Workflow User Guide 7

Chapter 1 Introduction

Assay warnings and precautions 1

Pre-/Post-PCR

• Follow standard procedures and single-direction workflow for the Pre-PCR

laboratory area.

• When working with gDNA prior to amplification, avoid contamination with

amplified foreign DNA. Therefore, gDNA plating is to be performed in the pre-

amplification laboratory using dedicated pipettes and tips that are never exposed

to amplified material. Once plated, the sample plate can be moved to the main lab

for setting up of subsequent steps. 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.

• Use separate copy of assay procedure in Pre- and Post-PCR areas.

• Follow procedures for re-entry of Pre-PCR Clean Room from post-PCR.

• If pre-PCR work is done in a laminar flow hood or PCR cabinet, then additionally

ensure:

– laminar flow hood is always on

– UV lamp is on when not in use

Do not

• Use kit after its expiration date.

• Use reagents after more than four freeze-thaw cycles.

• Use any water other than nuclease-free water supplied with the CytoScan™ XON

Kit.

• Reuse a plate seal.

Do

Genomic DNA:

• Use 100 ng of double-stranded genomic DNA (gDNA) that is not degraded (size

10 kb by gel analysis), not contaminated, and free of PCR inhibitors.

• Verify concentration using quantitation method specific to dsDNA. The purity

ratio (A260/A280 ratio) of input DNA must be between 1.7-2.1.

• Use extraction methods that yield DNA compatible with DNA quality as

specified above.

Chapter 1 Introduction

Assay warnings and precautions

8CytoScan™ XON Assay Manual Workflow User Guide

1

Laboratory Practice and Equipment Handling:

• Follow procedures for gowning and Good laboratory practices.

• Use nuclease-free pipette tips with aerosol barriers for all pipetting steps.

• Chill essential equipment such as cooling blocks and reagent coolers before use.

• Maintain the lab environment at 15 to 30°C (room temperature) throughout the

procedure.

• Always use freshly prepared master mixes.

• Use only nuclease-free water supplied with the kit.

• Follow instructions for sealing, vortexing, and centrifuging. Ensure plates are

tightly sealed to prevent sample loss and cross-well contamination. Always use a

new seal.

• Pipet accurately using calibrated pipettes.

•Use equipment calibrated according to manufacturer instructions.

• Use only specified assay stopping points.

• Check all thermal cyclers for accuracy of program.

• For all steps requiring thermal cyclers, turn on the machine prior to use to allow

sufficient time for the lid to preheat.

• Use a 12 strip tube to aliquot MM for assays 8 samples. See Appendix B for strip

tube set-ups for various batch sizes.

• Check that the Spectrophotometer or Nanodrop is accurately calibrated, and

ensure that OD measurement is within the linear range of the instrument per

manufacturer’s recommendations.

• Hybridization oven temperature is critical to the performance of the assay. Use

the GeneChip™ Hybridization Oven 645 only. Hybridization ovens should be

serviced at least once a year to ensure that they are operating within specification.

Reagent Handling:

• Keep enzymes at –25 to –15°C until needed, then immediately place in reagent

cooler chilled to –25 to –15°C. Do not store enzymes at –80°C or in a frost-free

freezer.

• For master mixes that contain enzymes, add all components except enzyme(s),

vortex, and spin down briefly (1-3 seconds). Add enzyme(s) last and only remove

them from the freezer when they are ready to be added to the master mix.

• Where indicated, keep reagents chilled at 2 to 8°C and place on ice during use.

• After thawing, either place on ice or at room temperature and use within the

recommended time frame as indicated in Appendix B.

• Maintain sample consistency; ensure all transitions to incubation temperatures

are rapid and well-controlled. Enzyme activity is a function of temperature.

• Because Fragmentation Enzyme activity can decline over time after dilution on

ice, add it to the samples as quickly as possible.

CytoScan™ XON Assay Manual Workflow User Guide 9

Chapter 1 Introduction

Controls 1

Controls

• Use of a pre-qualified sample [e.g., Applied Biosystems Ref 103 (Part No. 900421)]

as a positive control is highly recommended. These controls are effective

troubleshooting tools that will help you confirm the successful completion of each

stage of the assay. If using Ref 103 (Cat. No. 900421), directly plate 5 µL/well for

starting the assay.

• Oligonucleotide controls are included in the reagent kit. These controls are added

to the target samples prior to hybridization and act to confirm successful

hybridization, washing, staining, and scanning of the array.

• A negative control (i.e., water) is not required with this assay.

10 CytoScan™ XON Assay Manual Workflow User Guide

2CytoScan™ XON Assay target

preparation

Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Stage 1: Genomic DNA preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Stage 2: DNA amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Stage 3: Stop the DNA amplification reaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Stage 4: Fragmentation and precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Stage 5: Centrifuge and drying, resuspension and hybridization preparation, and

sample QC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Stage 5A: Centrifuge precipitation tubes and dry the DNA pellet . . . . . . . . . . . 26

Stage 5B: Resuspension and labeling preparation . . . . . . . . . . . . . . . . . . . . . . . . . 28

Stage 5C: Fragmentation QC checks and preparing Labeling Plate . . . . . . . . . . 32

Stage 6: Labeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Stage 7: Target hybridization via AGCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Stage 8: Washing, staining, and scanning arrays . . . . . . . . . . . . . . . . . . . . . . . . . . 43

The CytoScan™ XON Assay protocol is optimized for processing 8 to 24 samples at a

time. While master mix preparation at each step is described for 8, 16, and 24 samples,

the assay workflow in this section is outlined for 24 sample format.

Before you begin

Read Specific laboratory practices for CytoScan™ XON Assay in Appendix B.

Read all warnings and precautions and refer to Table 19 on page 60 for module and

component part number and labeling. Refer to Appendix A, ʺSafetyʺ on page 48.

Temperature definitions

Master mixes

The volume provided in the instructions for master mixes indicating the amount to be

dispensed into the strip tubes is only applicable when preparing 24 sample master

mixes and not less.

Temperature Range

Freeze –25 to –15°C

Ice 2 to 8°C

Room temperature 15 to 30°C

CytoScan™ XON Assay Manual Workflow User Guide 11

Chapter 2 CytoScan™ XON Assay target preparation

Stage 1: Genomic DNA preparation 2

Stage 1: Genomic DNA preparation

Dilute stock gDNA to working concentration

1. Place a 96-well plate in the upper half of the cooling block on ice.

2. Place the gDNA at room temperature until thawed (<15 minutes), then place in

the cooling block on ice.

3. Vortex the gDNA samples for (3x/1seconds each), pulse-spin for 1-3 seconds and

place the tube back on ice. Immediately proceed to dilution.

4. Dilute each sample to 20 ng/µL with Low EDTA TE.

5. Vortex the diluted sample (3x/1seconds each), pulse-spin for 1-3 seconds and

place on ice.

6. Transfer 5 µL of each diluted sample to the respective wells on the plate.

7. Tightly seal the plate. Spin down at 2,000 rpm for 60 seconds. Freeze the plate or

place on ice. Use within 1.5 hours.

8. The plate can be moved to the post-lab at this step.

Figure 1 96-well plate in cooling block, on ice

1

2

3

4

5

6

7

8

12345678910

11 12

13 14 15 16 17 18 19 20 21 22 23 24

gDNA samples

1

2

Low EDTA TE Buffer

1 2

Chapter 2 CytoScan™ XON Assay target preparation

Stage 2: DNA amplification

12 CytoScan™ XON Assay Manual Workflow User Guide

2

Stage 2: DNA amplification

The following sets of steps are necessary to perform DNA amplification:

•ʺ1: Prepare for DNA amplificationʺ on page 12

•ʺ2: Prepare the Denaturation Master Mixʺ on page 13

•ʺ3: Add the Denaturation Master Mix to samplesʺ on page 15

•ʺ4: Add the Neutralization Solution to samplesʺ on page 16

•ʺ5: Preparation of Amplification Master Mixʺ on page 17

•ʺ6: Add Amplification Master Mix to samplesʺ on page 18

•ʺ7: Freeze or proceedʺ on page 18

1: Prepare for DNA

amplification

To prepare for DNA amplification

1. Turn on the thermal cycler in the Post-PCR area to pre-heat the lid.

2. Thaw and prepare the reagents and Sample Plate.

To thaw and prepare the Sample Plate and reagents:

1. Thaw the Sample Plate on the benchtop at room temperature and pulse-spin

(2,000 rpm for 60 seconds). Leave on bench for 10 minutes to allow the Sample

Plate to come to room temperature.

2. Thaw the following reagents on the benchtop at room temperature for 30-

45 minutes before amplification set-up. Leave Amplification Enzyme in the

freezer until ready to use. Place nuclease-free water at room temperature.

• Denaturation Soln—use within 1 hour of thawing

• Neutralization Soln—use within 1 hour of thawing

• Amplification Soln—use within 1 hour of thawing

3. Vortex all reagents (except Amplification Enzyme), then place back at room

temperature.

• Denaturation Soln: Vortex at high speed 3 times, 1 second each time

(3x/1 second each) and pulse-spin 1-3 seconds before use.

• Nulcease-free Water: Vortex at high speed 3 times, 1 second each time

(3x/1 second each) to thoroughly mix.

• Neutralization Soln: Vortex at high speed for 3 times, 10 seconds each time

(3x/10 seconds each) to thoroughly mix.

IMPORTANT! Before proceeding to DNA Amplification, perform the gDNA

preparation described in ʺStage 1: Genomic DNA preparationʺ on page 11.

IMPORTANT! Amplification preparation should take place in a dedicated area such

as a biosafety hood with dedicated pipettes, tips, vortex, etc.

IMPORTANT!

• gDNA samples must be brought to room temperature (keep on bench for

10 minutes) before proceeding with denaturation.

• gDNA samples must be 5 µL volume of each gDNA at a concentration of

20 ng/µL (see ʺStage 1: Genomic DNA preparationʺ on page 11).

CytoScan™ XON Assay Manual Workflow User Guide 13

Chapter 2 CytoScan™ XON Assay target preparation

Stage 2: DNA amplification 2

• Amplification Soln: Vortex at high speed for 3 times, 10 seconds each time

(3x/10 seconds each) to thoroughly mix.

Note: Allow ~30 minutes for Amplification Soln to thaw on the benchtop at room

temperature. If the solution is not completely thawed after 30 minutes, vortex

briefly and return to the benchtop to complete thawing. The Amplification Soln

must be thoroughly mixed before use.

4. Amplification Enzyme: Do not vortex. Gently invert and flick the tube 3 times

to mix and pulse-spin for 3 seconds just before use.

5. Label the 1.5 mL microcentrifuge tube and the 2.0 mL tube as indicated in the

table below:

2: Prepare the

Denaturation

Master Mix

To prepare the Denaturation Master Mix (carry out the following steps at room temperature):

Note: Ensure individual reagents have been vortexed and spun before aliquoting as

described in Step 1 on page 12.

Table 1 Labeling tubes

Label Tube size Temperature Contents

•D MM 1.5 mL Leave tube at room

temperature

Denaturation Master Mix

•Amp MM 2.0 mL Leave tube at room

temperature

Amplification Master Mix

1

1 2 3

Nuclease-free water

2 3

Denaturation Solution D-MM

Chapter 2 CytoScan™ XON Assay target preparation

Stage 2: DNA amplification

14 CytoScan™ XON Assay Manual Workflow User Guide

2

1. Per Table 2, dilute the appropriate volume of Denaturation Soln using the

nuclease-free water.

2. Vortex at high speed (3x/1 second each), pulse-spin for 1-3 seconds and leave at

room temperature.

Table 2 Preparing the Denaturation Master Mix (D MM)

(>8 samples, 35% overage)

Reagent 1 Reaction 8 Samples 16 Samples 24 Samples

Nuclease-free water 4.5 µL 48.6 µL 97.2 µL 145.8 µL

Denaturation Soln 0.5 µL 5.4 µL 10.8 µL 16.2 µL

Total volume 5 μL 54 μL 108 μL 162 μL

CytoScan™ XON Assay Manual Workflow User Guide 15

Chapter 2 CytoScan™ XON Assay target preparation

Stage 2: DNA amplification 2

3: Add the

Denaturation

Master Mix to

samples

To add the Denaturation Master Mix to your samples (carry out the following steps

at room temperature)

1. Aliquot 13 µL of the denaturation master mix into 12 wells of strip tube at room

temperature.

2. Carefully remove the seal from the Sample Plate and discard the seal.

3. Using a P-20 multi-channel pipette, add 5 µL of Denaturation Master Mix to each

sample (Total volume: 10 µL/well).

• Pipet directly into the liquid of each well.

•Do not mix by pipetting up and down.

• This plate is now known as the Denaturation Plate.

4. Seal and vortex (1x, 3 seconds/5 sectors) the Denaturation Plate. Start the timer for

a 10 minute incubation immediately after vortexing.

5. Quickly spin the plate at room temperature by centrifugation at 2,000 rpm for

60 seconds.

Note: The quick-spin time is included in the 10 minute incubation.

6. 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.

7. 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 16.

8. After incubation immediately add the Neutralization Soln as described in ʺ4:

Add the Neutralization Solution to samplesʺ on page 16.

IMPORTANT! All aliquot dispense volumes are for 12 strip tubes for 24 samples

workflow. See Table 12 in Appendix B for volumes for 8- and 16- sample workflows.

IMPORTANT! It is critical that the correct volume of Denaturation Solution is

dispensed.

Chapter 2 CytoScan™ XON Assay target preparation

Stage 2: DNA amplification

16 CytoScan™ XON Assay Manual Workflow User Guide

2

4: Add the

Neutralization

Solution to samples

To add the Neutralization Solution to your samples (carry out the following steps at

room temperature)

1. Vortex the Neutralization Solution (3x/10 seconds each), wait for 1 minute and

then aliquot 75 µL into 12 wells of the strip tubes at room temperature.

2. Carefully remove the seal from the Denaturation Plate and discard the seal.

3. Using a P-200 multi-channel pipette, add 32.5 µL of Neutralization Soln to each

sample (Total volume: 42.5 µL/well).

• Pipet down the wall of each well.

•Do not mix by pipetting up and down.

• The plate is now known as the Neutralization Plate.

4. Seal, vortex (1x, 3seconds/5sector), and spin down the Neutralization Plate at

2,000 rpm for 60 seconds.

5. Visually examine the volume in each 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. Place plate on bench and immediately proceed to ʺ5: Preparation of Amplification

Master Mixʺ on page 17.

IMPORTANT! All aliquot dispense volumes are for 12 strip tubes for 24 samples

workflow. See Table 12 in Appendix B for volumes for 8- and 16- sample workflows.

IMPORTANT! Neutralization Solution is frothy after vortexing. It is important

to let the reagent bottle settle for 1 minute before aliquoting.

IMPORTANT! It is critical that the correct volume of Neutralization Solution is

dispensed.

1Neutralization Solution

CytoScan™ XON Assay Manual Workflow User Guide 17

Chapter 2 CytoScan™ XON Assay target preparation

Stage 2: DNA amplification 2

5: Preparation of

Amplification

Master Mix

To prepare and add the Amplification Master Mix (carry out the following steps at

room temperature):

Note: Ensure individual reagents have been vortexed and spun before aliquoting as

described in Step 1 on page 12.

1. Per Table 3, slowly pipette the appropriate amount of Amplification Soln into the

2.0 mL tube labeled Amp MM at room temperature.

Note: Amplification solution is frothy and must be dispensed slowly.

2. Remove the Amplification Enzyme from the freezer and place in a portable cooler

at –25 to –15°C.

a. Invert and flick the Amplification Enzyme tube three times, then pulse-spin

for 3 seconds.

b. Per Table 3, add the appropriate amount of Amplification Enzyme to the tube

labeled Amp MM.

c. Vortex the Amplification Master Mix well, at high speed (3x, 1 second each).

d. Invert the mix two times and vortex again at high speed (3x, 1 second each).

e. Pulse-spin for 1-3 seconds.

Table 3 Amplification Master Mix (Amp MM) (>8 samples, 20% overage)

Reagent 1 Reaction 8 Samples 16 Samples 24 Samples

Amplification Soln 56.3 µL 540.5 µL 1081 µL 1,621.4 µL

Amplification Enzyme 1.3 µL 12.5 µL 25 µL 37.4 µL

Total volume 57.6 μL 553 μL 1,106 μL 1,658.8 μL

1

1 2

Amplification Soln AMP-MM

2

Chapter 2 CytoScan™ XON Assay target preparation

Stage 2: DNA amplification

18 CytoScan™ XON Assay Manual Workflow User Guide

2

6: Add Amplification

Master Mix to

samples

1. Aliquot 128 µL of Amplification Master Mix into each 12-strip tube at room

temperature.

2. Carefully remove the seal from the Neutralization Plate and discard the seal.

3.

Using a P-200 multi-channel pipette,

slowly

add

57.6 µL of Amplification Master

Mix

to each sample of the Neutralization Plate

(total volume: 100.1 µL/well).

•Do not mix by pipetting up and down.

• After adding the Amplification Master Mix, the plate is now known as the

Amplification Plate.

4. Seal tightly, vortex well (2x, 3 seconds/5 sectors), and spin the Amplification Plate

for one minute at 2,000 rpm at room temperature (as described in Appendix B,

ʺWorkflow and practicesʺ on page 51).

5. Place the sealed Amplification Plate in thermal cycler. Optionally, a compression

pad can be placed on the plate. Initiate amplification using the CYTOXON AMP

protocol (37°C for 18 hours, 37°C for 2 hours, 37°C for 2 hours, 37°C hold) for

20 2 hours.

6. Store remaining reagents for future use. Follow guidelines presented in

Appendix B, ʺWorkflow and practicesʺ on page 51.

7: Freeze or

proceed

After the incubation finishes, you can either:

• Proceed to ʺStage 3: Stop the DNA amplification reactionʺ on page 19.

or

• Store the Amplification Plate at –25 to –15°C for up to 10 days. (Optional stopping

point 1.)

Note: If freezing, do not perform the stop DNA amplification reaction step before

you store the Amplification Plate at –25 to –15°C. The stop DNA amplification

reaction step will be performed after thawing the frozen plate, as described in

ʺStage 3: Stop the DNA amplification reactionʺ on page 19.

IMPORTANT! All aliquot dispense volumes are for 12 strip tubes for 24 samples

workflow. See Table 12 in Appendix B for volumes for 8- and 16- sample workflows.

IMPORTANT! It is critical to vortex the plate twice after addition of

Amplification Master Mix to ensure proper amplification.

CytoScan™ XON Assay Manual Workflow User Guide 19

Chapter 2 CytoScan™ XON Assay target preparation

Stage 3: Stop the DNA amplification reaction 2

Stage 3: Stop the DNA amplification reaction

• If proceeding directly from the end of ʺStage 2: DNA amplificationʺ on page 12,

do not remove the Amplification Plate from the thermal cycler. Initiate the stop

reaction by starting the CYTOXON STOP protocol (65°C for 20 minutes, 37°C for

20 minutes, 37°C hold).

• Following the completion of Amplification Stop, directly proceed to ʺStage 4:

Fragmentation and precipitationʺ.

• If working with amplified DNA samples that are frozen, follow the instructions

in the section ʺThaw DNA samplesʺ, below.

Thaw DNA samples (Skip this step if the Amplified Sample Plate was not frozen at the end of the previous

stage.)

1. Thaw PCR plate in a room temperature block and allow to equilibrate for

~30 minutes until all wells have thawed.

2. After thawing, spin plate down 2,000 rpm for 60 seconds at room temperature.

3. To avoid cross-contamination of wells during vortexing:

a. Remove the seal.

b. Tightly reseal the plate using a fresh seal.

4. Vortex the plate well (2x, 3 seconds/5 sector) to thoroughly mix.

5. Spin at 2,000 rpm for 60 seconds.

6. Transfer the Amplification Plate to a thermal cycler. Optionally, place a

compression pad. Stop the reaction using CYTOXON STOP protocol (65°C for

20 minutes, 37°C for 20 minutes, 37°C hold).

7. Following the completion of Amplification Stop, directly proceed to ʺStage 4:

Fragmentation and precipitationʺ.

Stage 4: Fragmentation and precipitation

The following sets of steps are necessary to perform fragmentation and precipitation:

•ʺ1: Prepare for fragmentation and precipitationʺ on page 20

•ʺ2: Preparation of Fragmentation Master Mixʺ on page 20

•ʺ3: Add the Fragmentation Master Mix to samplesʺ on page 21

•ʺ4: Addition of Stop Solution to the samplesʺ on page 22

•ʺ5: Preparation of Precipitation Master Mixʺ on page 23

•ʺ6: Prepare and add isopropanol to the Precipitation Plateʺ on page 24

•ʺ7: Freeze the Precipitation Plate overnightʺ on page 25

•ʺ8: Freeze reagentsʺ on page 25

Input required Amplification Plate from ʺStage 3: Stop the DNA amplification reactionʺ on page 19.

Chapter 2 CytoScan™ XON Assay target preparation

Stage 4: Fragmentation and precipitation

20 CytoScan™ XON Assay Manual Workflow User Guide

2

1: Prepare for

fragmentation and

precipitation

Thaw and prepare the fragmentation and precipitation reagents

1. Thaw the following reagents on the benchtop at room temperature just prior to

starting CYTOXON STOP protocol. Vortex reagents at high speed, 3 times,

1 seconds each (3x/1 second each). Pulse-spin Precipitation Soln2 for 1-3 seconds

then place both the reagents on ice.

• Fragmentation Buffer—use within 30 minutes of thawing

• Precipitation Soln 2—use within 1.5 hours of thawing

2. Leave the Fragmentation Enzyme at –25 to –15°C until ready to use. Just before

use, spin down for 1 second so that the contents of the tube are uniform, vortex at

high speed for 1 second, and pulse-spin for 3 seconds.

3. Keep the following reagents on ice. Vortex 3 times, 1 second each (3x/1 second

each) and pulse-spin the Fragmentation Diluent for 1-3 seconds before use.

• Precipitation Soln 1

• Fragmentation diluent

4. Place the following reagents at room temperature and leave them for duration of

step. Vortex 3 times, 1 second each (3x/1 second each) the Fragmentation Rxn Stop

before use. Swirl the Isopropanol 2-3 times before use.

• Isopropanol

• Frag Rxn stop

2: Preparation of

Fragmentation

Master Mix

To prepare the Fragmentation Master Mix:

1. When there are approximately 5 minutes remaining in the CYTOXON STOP

protocol, use Table 4 to prepare the non-enzymatic portion of the Fragmentation

MM in a 1.5 mL tube on ice. Leave the Fragmentation Enzyme at –25 to –15°C

until ready to use.

1

2

Fragmentation Diluent

Fragmentation-MM

1

2

3

4

3

4

Fragmentation Buffer

Amplified Sample Plate at room temperature

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Thermo Fisher Scientific CytoScan XON Assay User guide

Thermo Fisher Scientific CytoScan XON Assay User guide

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