Thermo Fisher Scientific KaryoStat™ Assay User guide

Brand: Thermo Fisher Scientific

Model: KaryoStat™ Assay

Type: User guide

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

KaryoStat™ Assay

USER GUIDE

KaryoStat™ Assay is equivalent to and comprised of CytoScan™ Optima

arrays and reagents

Catalog Number 905403

Publication Number MAN0017068

Revision A.0

Manufacturer: Affymetrix Inc. | 3450 Central Expressway | Santa Clara, CA 95051 | USA

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: 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. All other trademarks are

the property of their respective owners.

Contents

■CHAPTER 1 Product information ....................................... 6

Introduction .................................................................... 6

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

Precautions ................................................................ 7

Pre/post-PCR .............................................................. 7

Do not ..................................................................... 7

Do ........................................................................ 8

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

GeneChip™ System 3000 ......................................................... 9

Safety symbols and information ................................................... 9

■CHAPTER 2 KaryoStat™ Assay procedure ............................ 11

Before you begin ............................................................... 11

Prepare gDNA plate ............................................................ 12

Dilute stock gDNA to working concentration ................................... 12

Aliquot prepared gDNA into assay plate ....................................... 13

Set up work area .......................................................... 13

Restriction enzyme digestion .................................................... 14

Prepare reagents_ equipment_ and consumables ............................. 14

Prepare reagents .......................................................... 14

Load samples on thermal cycler ............................................. 17

Ligation ....................................................................... 18

Set up work area .......................................................... 18

Thaw reagents and digested samples ........................................ 18

Prepare digested samples and reagents ...................................... 19

Prepare ligation master mix ................................................. 20

Add ligation master mix .................................................... 20

Load samples on thermal cycler ............................................. 21

PCR .......................................................................... 22

Dilute ligated samples ...................................................... 22

Run PCR .................................................................. 23

Check PCR reaction ........................................................ 27

PCR product puriﬁcation ........................................................ 28

Prepare puriﬁcation wash buffer ............................................. 28

Prepare PCR products ...................................................... 28

KaryoStat

™

Assay User Guide

Quantitation ................................................................... 32

Prepare reagents, equipment, and consumables ............................... 33

Microplate spectro-photometer ............................................. 33

Fragmentation ................................................................. 34

Precautions ............................................................... 34

Preparation ............................................................... 35

Fragmentation ............................................................ 37

Labeling ...................................................................... 40

Preparation ............................................................... 40

Labeling .................................................................. 42

Target hybridization via AGCC .................................................... 43

Important information about this stage ....................................... 43

Prepare the equipment ..................................................... 43

Prepare the arrays and create a batch registration ﬁle ......................... 43

Prepare the reagents and consumables ...................................... 46

Prepare the hybridization master mix ........................................ 47

Add hybridization master mix and denature ................................... 47

Load the samples onto arrays ............................................... 48

Washing, staining, and scanning arrays ........................................... 49

Prime the ﬂuidics station ................................................... 49

Washing and staining arrays ................................................ 50

Scanning arrays ........................................................... 51

Adding arrays during an autoloader run ...................................... 53

Shutting down the ﬂuidics station ............................................ 53

In-Process quality control ....................................................... 54

PCR product .............................................................. 54

Fragmented product ....................................................... 56

Array quality control threshold .................................................. 57

■APPENDIX A Workﬂow and practices ................................. 58

Workﬂows .................................................................... 59

Speciﬁc laboratory practices .................................................... 61

Seals ..................................................................... 61

Vortex .................................................................... 61

Centrifuge ................................................................ 62

PCR practices ................................................................. 62

Samples .................................................................. 62

PCR product puriﬁcation .................................................... 62

Contents

KaryoStat

™

Assay User Guide

■APPENDIX B Thermal cycler programs .............................. 63

Pre-and post-PCR programs .................................................... 63

Thermal cycler programs ....................................................... 63

CytoScan™ Optima Digest ................................................... 63

CytoScan™ Optima Ligate ................................................... 64

CytoScan™ Optima PCR ..................................................... 64

CytoScan™ Optima Fragment ................................................ 64

CytoScan™ Optima Label .................................................... 65

CytoScan™ Optima Hyb ..................................................... 65

■APPENDIX C Required equipment, consumables, and reagents ... 66

Equipment and software required ................................................ 66

CytoScan™ Optima Assay reagents, required. ...................................... 67

From other suppliers ........................................................... 68

Reagents required but not provided .......................................... 68

Pre-PCR clean room – equipment required but not provided ..................... 68

Post-PCR room – equipment required but not provided ......................... 69

Consumables required but not provided ...................................... 70

Symbols ...................................................................... 71

■APPENDIX D Troubleshooting the KaryoStat™ Assay ................ 72

PCR gel QC step ............................................................... 72

Puriﬁcation yield QC step ....................................................... 74

Fragmentation QC step (gel) ..................................................... 76

.CEL ﬁle generation ............................................................ 79

DATA QC failures ............................................................... 80

■APPENDIX E Fluidics station care and maintenance ................ 82

General ﬂuidics station care ..................................................... 82

Fluidics station bleach protocol .................................................. 82

The bleach cycle ........................................................... 83

The rinse cycle ............................................................ 89

■Documentation and support ............................................. 91

Customer and technical support ................................................. 91

Limited product warranty ....................................................... 91

Contents

KaryoStat

™

Assay User Guide

Product information

■Introduction .......................................................... 6

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

■Controls ............................................................. 9

■GeneChip™ System 3000 ............................................... 9

■Safety symbols and information ......................................... 9

WARNING! Read the Safety Data Sheets (SDSs) and follow the handling

instructions. Wear appropriate protective eyewear, clothing, and gloves. Safety

Data Sheets (SDSs) are available from thermosher.com/support.

Introduction

KaryoStat™ Assay is intended to be used with genomic DNA in conjunction with

CytoScan™ Optima array, GeneChip™ System 3000, and Chromosome Analysis Suite

(ChAS).

KaryoStat™ Assay is equivalent to and comprised of CytoScan™ Optima arrays and

reagents. CytoScan™ Optima array consists of features covering control, copy number

(CN), and single-nucleotide polymorphism (SNP) probes. There are a total of 18,018

CN markers and 148,450 SNP markers uniformly spaced over the genome with

enhanced interrogation of 396 regions of prenatal interest. Cumulatively, through the

collection of SNPs and non-polymorphic probes the application provides the ability to

support detection of CNVs, enable the elucidation of allelic imbalance (i.e., the paern

of inheritance deviates from expected Mendelian inheritance paerns), identify copy

number neutral abnormalities such as absence (AOH) or loss of heterozygosity

(LOH), and characterize unbalanced translocation events in the samples of interest.

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 A, “Workow and practices“for specic setup

instructions, and equipment and technique illustrations, and Appendix B, “Thermal

cycler programs“ for specic thermal cycler program set ups.

For additional specic warnings, precautions, and procedures, see:

•GeneChip™ Command Console™ 3.2 User Guide (Cat. No. 702569)

•GeneChip™ Fluidics Station 450 User Guide for AGCC (Cat. No. 08-0295)

•GeneChip™ Hybridization Oven 645 User Guide (Cat. No. 08-0255)

KaryoStat

™

Assay User Guide

Visit our website for all user guides (CytoScan™ Optima Assay User Guide, Chromosome

Analysis Suite (ChAS) User Guide, GeneChip™ Command Console™ User Guide, GeneChip™

Fluidics Station 450 User Guide for AGCC, GeneChip™ Hybridization Oven 645 User

Guide), and training materials (containing training kit and positive control DNA), SDS

for all hazards in CytoScan™ Optima Assay reagents.

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

thoroughly investigated. Follow prudent laboratory practices and use general

laboratory safety equipment (eye protection, labcoat, 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 amplied 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 workow recommendations. PCR reactions

should be set up in the Pre-PCR Area only. Personnel should not reenter the Pre-PCR

Clean Area once exposed to PCR products without rst showering and changing into

clean clothes.

Carefully reading and following the protocol as wrien is essential. The KaryoStat™

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.

• Follow standard procedures and single-direction workow for the Pre-PCR

laboratory area.

• Use dedicated equipment for each area (e.g., thermal cyclers, microfuges, pipees

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, pipees, 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 ow hood or PCR cabinet, then additionally

ensure:

– laminar ow hood is always on

– UV lamp is on when not in use

• 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™

Optima Kit.

• Reuse a plate seal.

Precautions

Pre/post-PCR

Do not

Chapter 1 Product information

Assay warnings and precautions

KaryoStat

™

Assay User Guide

• Use 250 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 specic to dsDNA. The purity ratio

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

• Follow procedures for gowning, etc.

• Use nuclease-free pipee tips with aerosol barriers for all pipeing steps.

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

specied above.

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

• Keep all tubes, master mixes, and working solutions in chilled cooling blocks on

ice.

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

cooler chilled to –15 to –25°C. Do not store enzymes at –80°C.

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

After thawing, immediately place on ice and use within 1.5 hours.

• Maintain sample consistency; ensure all transitions to incubation temperatures

are rapid and wellcontrolled. Enzyme activity is a function of temperature.

• Enzyme handling:

– Maintain tube in –15 to –25°C cooler

– Quick spin and vortex 1 to 3 seconds

– Place back in cooler for use

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

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

• Maintain 15 to 30°C (RT) 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 that plates are

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

new seal.

• Pipet accurately using calibrated pipeors.

• Use equipment calibrated according to manufacturer instructions.

• Use only specied assay stopping points.

• Check that your spectrophotometer or NanoDrop is accurately calibrated, and be

sure the OD measurement is within the linear range of the instrument as per the

manufacturer’s recommendations.

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

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

serviced at least once a year to ensure that they are operating within specication.

Chapter 1 Product information

Assay warnings and precautions

KaryoStat

™

Assay User Guide

Controls

• Use of a pre-qualied sample [e.g., Ref 103 (Cat. No. 900421)] as a positive control

is highly recommended. These controls are eective troubleshooting tools that

will help you conrm the successful completion of each stage of the assay.

• Use a negative control. Use a blank well with Low EDTA TE Buer (Cat. No.

902424). The absence of bands on your PCR gel for the negative control conrms

no previously amplied PCR product has contaminated your samples.

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

to the target samples prior to hybridization.

GeneChip™ System 3000

•GeneChip™ Fluidics Station 450 User Guide for AGCC (Cat. No. 08-0295).

•GeneChip™ Hybridization Oven 645 User Guide (Cat. No. 08-0255).

Safety symbols and information

Table 1

Reagent Contains Warnings/precautions

Hyb Buffer Part 1 Tetramethylamm

onium chloride

DANGER. Toxic if swallowed and in contact

with skin; causes skin irritation, serious eye

irritation; may cause respiratory irritation.

Avoid breathing and avoid contact with skin

or eyes; if contact occurs wash immediately.

If skin irritation or rash occurs, seek medical

advice.

TdT Buffer Cacodylic acid DANGER. Harmful if swallowed, may cause

cancer. Toxic if swallowed and in contact with

skin; causes skin irritation, serious eye

irritation; may cause respiratory irritation.

Avoid breathing and avoid contact with skin

or eyes; if contact occurs wash immediately.

If skin irritation or rash occurs, seek medical

advice.

BSA Glycerol WARNING. Causes minor skin irritation and

eye irritation. Avoid contact with skin or eyes;

if contact occurs wash immediately. If skin

irritation or rash occurs, seek medical

advice.

DNA Ligase Glycerol WARNING. Causes minor skin irritation and

eye irritation. Avoid contact with skin or eyes;

if contact occurs wash immediately. If skin

irritation or rash occurs, seek medical

advice.

Chapter 1 Product information

Controls

KaryoStat

™

Assay User Guide

Reagent Contains Warnings/precautions

Fragmentation Buffer Tris

[Tris(hydroxmethy

l) aminomethane]

WARNING. Causes minor skin irritation and

eye irritation. Avoid contact with skin or eyes;

if contact occurs wash immediately. If skin

irritation or rash occurs, seek medical

advice.

Fragmentation

Reagent

Glycerol WARNING. Causes minor skin irritation and

eye irritation. Avoid contact with skin or eyes;

if contact occurs wash immediately. If skin

irritation or rash occurs, seek medical

advice.

Nsp I Glycerol WARNING. Causes minor skin irritation and

eye irritation. Avoid contact with skin or eyes;

if contact occurs wash immediately. If skin

irritation or rash occurs, seek medical

advice.

CytoScan DNA

Polymerase

Glycerol WARNING. Causes minor skin irritation and

eye irritation. Avoid contact with skin or eyes;

if contact occurs wash immediately. If skin

irritation or rash occurs, seek medical

advice.

TdT Glycerol WARNING. Causes minor skin irritation and

eye irritation. Avoid contact with skin or eyes;

if contact occurs wash immediately. If skin

irritation or rash occurs, seek medical

advice.

These materials contain ≤0.1% sodium azide: Array Holding Buer, Stain Buer 1,

Stain Buer 2, and Purication Beads. Exercise caution in disposal: buildup of sodium

azide in lead and copper plumbing can form highly explosive metal azides.

Chapter 1 Product information

Safety symbols and information

KaryoStat

™

Assay User Guide

KaryoStat™ Assay procedure

■Before you begin ..................................................... 11

■Prepare gDNA plate .................................................. 12

■Restriction enzyme digestion .......................................... 14

■Ligation ............................................................. 18

■PCR ................................................................ 22

■PCR product purication .............................................. 28

■Quantitation ......................................................... 32

■Fragmentation ....................................................... 34

■Labeling ............................................................ 40

■Target hybridization via AGCC ........................................ 43

■Washing, staining, and scanning arrays ................................. 49

■In-Process quality control ............................................. 54

■Array quality control threshold ........................................ 57

Before you begin

Read all warnings and precautions and refer to “CytoScan™ Optima Assay reagents,

required.“ on page 67 for module and component part number and labeling.

KaryoStat

™

Assay User Guide

Prepare gDNA plate

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

gDNA samples

Low EDTA TE Buffer

1 2 3 4 5 6 7 8

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

2. Place the gDNA at 15 to 30°C (RT) until thawed (≤30 minutes); then place in the

cooling block on ice.

IMPORTANT! Use within 1 hour.

3. Vortex the gDNA samples for 3 seconds.

4. Centrifuge 650 × g for 1 minute; then place in the cooling block.

5. Dilute each sample to 50 ng/μL with Low EDTA TE in separate wells of the 96-

well plate or in 1.5 mL Eppendorf™ tubes.

Note: Use double-strand-specic quantitation method to determine sample

concentration.

6. Tightly seal the plate.

7. Centrifuge 650 × g for 1 minute.

8. Place on cooling block.

Dilute stock gDNA

to working

concentration

Chapter 2 KaryoStat™ Assay procedure

Prepare gDNA plate

KaryoStat

™

Assay User Guide

1. Mark 96-well plate (Table 2, Figure 2).

Table 2 Temperature range deﬁnitions.

Temperature Range

Freeze –15 to –25°C

Ice 2 to 8°C

RT (Room Temperature) 15 to 30°C

Figure 2 Plate marking.

2. Place the plate on the lower half of the cooling block.

3. Vortex gDNA 3 seconds; centrifuge.

Aliquot prepared

gDNA into assay

plate

Set up work area

Chapter 2 KaryoStat™ Assay procedure

Prepare gDNA plate

KaryoStat

™

Assay User Guide

4. Transfer 5 μL of rst sample from gDNA stock plate to well A1 of DIG-LIG plate;

transfer all samples in same way (Figure 3).

Diluted gDNA samples

(at 50 ng/μL concentration)

96-well plate labeled for

digestion and ligation

Figure 3 Transfer samples from gDNA plate to DIG-LIG plate.

5. For the controls, aliquot 5 μL of:

• Positive Control gDNA to well A7.

• Low EDTA TE buer [negative control] to well A8.

6. Tightly seal DIG-LIG plate with new seal; centrifuge 650 × g for 1 minute.

Restriction enzyme digestion

Turn on the thermal cycler to preheat lid. Leave block at RT.

Set up work area

1. Place the cooling block and water on ice.

Note: For more than 8 samples, place 8-12 tube strip on upper half of cooling

block.

2. Label a 1.5 mL tube “Dig” and place it in the cooling block.

3. Cut adhesive lm into strips wide enough to seal 8 or 12 strip tubes.

1. Thaw the following reagents at RT (≤30 minutes), immediately place on ice:

•Nsp I Buer

• 100X BSA

Prepare reagents_

equipment_ and

consumables

Prepare reagents

Chapter 2 KaryoStat™ Assay procedure

Restriction enzyme digestion

KaryoStat

™

Assay User Guide

IMPORTANT! Set up reaction within 1 hour.

Water,

Nuclease free

Strip tubes to aliquot

Digestion Master Mix

Nsp I Buffer

100X BSA

Digestion Master Mix

tube “ Dig”

1 2 3 4 5 6 7 –

Figure 4 Digestion setup (Nsp I enzyme not pictured, still at –15 to –25°C).

2. Prepare Nsp I Buer and 100X BSA:

a. Vortex 3 times for 1 second each.

b. Quick spin 3 seconds.

c. Place in cooling block on ice.

3. Place Nuclease-free Water on ice.

Chapter 2 KaryoStat™ Assay procedure

Restriction enzyme digestion

KaryoStat

™

Assay User Guide

Prepare digestion master mix

Prepare and store all reagents, tubes, and cooling block on ice. Dispense within

10 minutes.

1. To the 1.5 mL Eppendorf™ tube (“Dig”) add per Table 3:

• Water, Nuclease free, chilled (2 to 8°C)

• Nsp I Buer

• 100X BSA

Table 3 Digestion master mix (≥ 8 samples, 20% overage).

Reagent 1 sample 8 samples 16 samples 24 samples

Water,

Nuclease free

11.55 μL 110.9 μL 221.8 μL 332.6 μL

Nsp I Buffer 2.00 μL 19.2 μL 38.4 μL 57.6 μL

100X BSA 0.20 μL 1.9 μL 3.8 μL 5.8 μL

Nsp I 1.00 μL 9.6 μL 19.2 μL 28.8 μL

Total 14.75 μL 141.6 μL 283.2 μL 424.8 μL

2. Vortex 3 times for 1 second each.

3. Quick spin 3 seconds.

4. Place Digestion Master Mix in chilled cooling block (2 to 8°C).

5. Remove Nsp I enzyme from the freezer and immediately place in a chilled cooler

(–15 to –25°C).

6. Mix Nsp I.

1. Quick spin 1 second.

2. Vortex 1 second.

3. Quick spin 3 seconds.

7. Return to –15 to –25°C cooler while preparing Digestion Master Mix.

8. Immediately add Nsp I to Digestion Master Mix.

9. Return enzyme to –15 to –25°C cooler.

10. Vortex Digestion Master Mix 3 times for 1 seconds each.

11. Quick spin 3 seconds.

12. Place in chilled cooling block (2 to 8°C).

Chapter 2 KaryoStat™ Assay procedure

Restriction enzyme digestion

KaryoStat

™

Assay User Guide

Add digestion master mix to samples

1. Divide the Digestion Master Mix equally into 8- or 12-strip tubes on cooling

block on ice.

2. Seal strip tube with adhesive lm strip (or strip caps).

3. Centrifuge 650 × g for 1 minute and place on cooling block on ice.

4. Remove and discard seal.

5. Unseal the plate and discard seal.

6. Use multichannel P20 pipee and add 14.75 μL of the Digestion Master Mix to

each sample in row A (Figure 5).

Figure 5 Addition of digestion master mix to samples.

7. Seal the plate tightly with a new seal.

1. Vortex the plate 1 second all corners and center.

2. Centrifuge 650 × g for 1 minute.

Load samples on

thermal cycler

Chapter 2 KaryoStat™ Assay procedure

Restriction enzyme digestion

KaryoStat

™

Assay User Guide

3. Load the plate on thermal cycler, ensure thermal cycler lid is preheated, and run

CytoScan™ Optima Digest program.

Table 4 CytoScan™ optima digest.

Temperature Time

37°C 2 hours

65°C 20 minutes

4°C 5 minutes

4°C ∞

4. Discard the remaining Digestion Master Mix and return reagents to the freezer.

When done, leave the plate in thermal cycler. Process within 2.5 hours.

5. Ensure the plate is sealed tightly and centrifuge 650 × g for 1 minute.

6. Place the plate in cooling block on ice and immediately go to “Ligation“ on

page 18.

Ligation

Turn on the thermal cycler to preheat lid. Leave block at RT.

1. Place cooling block on ice.

2. Label a 1.5 mL tube “Lig” and place it in cooling block.

3. Place 8-tube strip on upper half of cooling block.

4. Cut adhesive lm into strips wide enough to seal 8 or 12 strip tubes.

1. Allow the following to thaw at RT (≤30 minutes):

•Adaptor, Nsp I

• DNA Ligase Bfr

Set up work area

Thaw reagents

and digested

samples

Chapter 2 KaryoStat™ Assay procedure

Ligation

KaryoStat

™

Assay User Guide

2. Immediately place on ice and use within 1 hour.

Strip tubes to aliquot

Ligation Master Mix

Adaptor, Nsp I

DNA Ligase Bfr

Ligation Master Mix tube “ Lig”

1 2 3 4 5 6 7 –

Figure 6 Ligation setup (DNA Ligase not pictured, still at –15 to –25°C).

1. Prepare samples:

a. Centrifuge 650xg for 1 minute.

b. Place in lower half of cooling block on ice.

2. Prepare reagents:

a. Vortex DNA Ligase Bfr and Adaptor, Nsp I 3 times for 1 second each.

Note: If precipitate is present in DNA Ligase Bfr, vortex until precipitate is

dissolved.

b. Quick spin 3 seconds.

c. Place in chilled cooling block (2 to 8°C).

Prepare digested

samples and

reagents

Chapter 2 KaryoStat™ Assay procedure

Ligation

KaryoStat

™

Assay User Guide

Prepare and store all reagents, tubes, and cooling block on ice. Dispense within

10 minutes.

1. Label a 1.5 mL tube “Lig” and add per Table 5:

• DNA Ligase Buer

• Adaptor, Nsp I

Table 5 Ligation master mix (≥8 samples, 25% overage).

Reagent 1 sample 8 samples 16 samples 24 samples

DNA Ligase Bfr 2.50 μL 25.0 μL 50.0 μL 75.0 μL

Adaptor, Nsp I 0.75 μL 7.5 μL 15.0 μL 22.5 μL

DNA Ligase 2.00 μL 20.0 μL 40.0 μL 60.0 μL

Total 5.25 μL 52.5 μL 105.0 μL 157.5 μL

2. Vortex 3 times for 1 second each.

3. Quick spin 3 seconds.

4. Remove the DNA Ligase from freezer and immediately place in chilled cooler (–

15 to –25°C).

5. Mix DNA Ligase.

a. Quick spin 1 second.

b. Vortex 1 second.

c. Quick spin 3 seconds.

6. Place in –15 to –25°C cooler.

7. Immediately add the DNA Ligase to the Ligation Master Mix; place back in –15

to –25°C cooler.

8. Vortex the Ligation Master Mix 3 times for 1 second each.

9. Quick spin 3 seconds.

10. Place the Ligation Master Mix in cooling block on ice.

11. Immediately go to next step.

1. Divide the Ligation Master Mix equally into 8 or 12 strip tubes on ice.

2. Seal strip tube with adhesive lm strip (or strip caps) and quick spin.

3. Place back in cooling block on ice.

4. Remove and discard the strip tube seal.

5. Unseal the digested sample plate and discard seal.

Prepare ligation

master mix

Add ligation

master mix

Chapter 2 KaryoStat™ Assay procedure

Ligation

KaryoStat

™

Assay User Guide

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Model: KaryoStat™ Assay

Type: User guide

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