Infinium Karyomapping Protocol Guide Part - Support

Transcription

Infinium® Karyomapping Assay
Protocol Guide
For Research Use Only. Not for use in diagnostic procedures.
ILLUMINA PROPRIETARY
Part # 15052710 Rev. B
June 2015
This document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended solely for the
contractual use of its customer in connection with the use of the product(s) described herein and for no other purpose. This
document and its contents shall not be used or distributed for any other purpose and/or otherwise communicated, disclosed,
or reproduced in any way whatsoever without the prior written consent of Illumina. Illumina does not convey any license
under its patent, trademark, copyright, or common-law rights nor similar rights of any third parties by this document.
The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order
to ensure the proper and safe use of the product(s) described herein. All of the contents of this document must be fully read
and understood prior to using such product(s).
FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN
MAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR OTHERS, AND
DAMAGE TO OTHER PROPERTY.
ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S)
DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE) OR ANY USE OF SUCH PRODUCT(S) OUTSIDE
THE SCOPE OF THE EXPRESS WRITTEN LICENSES OR PERMISSIONS GRANTED BY ILLUMINA IN CONNECTION
WITH CUSTOMER'S ACQUISITION OF SUCH PRODUCT(S).
FOR RESEARCH USE ONLY
In addition to all other terms and conditions, the use of Karyomapping products is subject to the following terms and
conditions. If you do not agree to these terms and conditions you are not permitted to use these products.
1
The Karyomapping products may only be used in compliance with all applicable laws and regulations, including without
limitation those laws and regulations concerning pre-implantation genetic diagnosis and pre-implantation genetic
screening, privacy rights, reporting of incidental findings (as further discussed below), and the storage, transfer, and
disposal of samples and materials. These laws and regulations vary significantly from jurisdiction to jurisdiction.
2
The Karyomapping products have not been cleared, approved, or otherwise reviewed by the U.S. FDA or any other
governmental regulatory agency or authority for use in pre-implantation genetic diagnosis, pre-implantation genetic
screening, or for any other use.
3
The Karyomapping products attempt to make a prediction of genetic phase in embryos, however, the user is responsible
for examining both the supporting and contrary evidence for this predicted phase before making a determination
concerning the true phase. Results obtained from the use of Karyomapping products should be confirmed using
alternative methods. The Karyomapping products may generate false positives, false negatives, or inconclusive results.
4
Use of the Karyomapping products may produce incidental findings about genetic relatives used to perform the analysis.
These incidental findings may include, without limitation:
a
Paternity issues
b
Copy number variations (CNVs) that may relate to disorders that have yet to manifest, or are of unknown
significance
c
Consanguinity between relatives
d
Sex chromosome abnormalities
The laws and regulations concerning the reporting of such incidental findings vary significantly from jurisdiction to
jurisdiction, and it is solely the user’s responsibility to ensure it uses Karyomapping products, including results obtained
through their use, in compliance with all applicable laws and regulations.
5
All users of the Karyomapping products should be skilled in the areas of genetics and genetic linkage.
© 2015 Illumina, Inc. All rights reserved.
Illumina, 24sure, BaseSpace, BeadArray, BlueFish, BlueFuse, BlueGnome, cBot, CSPro, CytoChip, DesignStudio,
Epicentre, GAIIx, Genetic Energy, Genome Analyzer, GenomeStudio, GoldenGate, HiScan, HiSeq, HiSeq X, Infinium,
iScan, iSelect, ForenSeq, MiSeq, MiSeqDx, MiSeq FGx, NeoPrep, Nextera, NextBio, NextSeq, Powered by Illumina,
SeqMonitor, SureMDA, TruGenome, TruSeq, TruSight, Understand Your Genome, UYG, VeraCode, verifi, VeriSeq, the
pumpkin orange color, and the streaming bases design are trademarks of Illumina, Inc. and/or its affiliate(s) in the U.S. and/or
other countries. All other names, logos, and other trademarks are the property of their respective owners.
Infinium Karyomapping Assay Protocol Guide
ii
Revision History
Part #
Revision
Date
Description of Change
15052710
B
June
2015
• Removed references to BlueGnome.
• Edited "Scan BeadChip" section. Removed iScan procedures and
referenced iScan System User Guide (part # 11313539). Added
reference to NextSeq 550 System User Guide (part # 15069765).
15052710
A
February
2014
iii
Initial release.
Part # 15052710 Rev.B
Table of Contents
Revision History
Table of Contents
List of Tables
Chapter 1 Overview
Intended Use
Tracking Tools
Chapter 2 Lab Setup and Maintenance
Introduction
Safety Precautions
Preventing Amplification Product Contamination
Preparing and Storing Reagents
Best Practices
Lab Maintenance
Chapter 3 karyomapping assay Protocol
karyomapping assay Workflow
Equipment, Materials, and Reagents
Amplification of biopsy samples with SureMDA
Prepare and Incubate the MSA3 Plate
Fragment the DNA
Precipitate the DNA
Resuspend the DNA
Hybridize DNA to the BeadChip
Wash the BeadChip
Extend and Stain (XStain) BeadChips
Scan BeadChip
Appendix I: Agarose Gel Electrophoresis
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Index
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Technical Assistance
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List of Tables
Table 1 Lab Equipment Maintenance
Table 2 Sample Vortexer Calibration Label
Table 3 User-Supplied Equipment
Table 4 User Supplied Materials
Table 5 User-Supplied Reagents
Table 6 Reagents
Table 7 Reagents—Supplied in the Infinium Analysis Kit
Table 8 Items from Illumina
Table 9 Preparation of Buffer D2
Table 10 Control DNA dilutions
Table 11 Amplification control reactions
Table 12 Preparation of Amplification Master Mix
Table 13 Illumina General Contact Information
Table 14 Illumina Customer Support Telephone Numbers
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Part # 15052710 Rev.B
Chapter 1 Overview
Intended Use
Tracking Tools
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1
Chapter 1
Overview
Overview
Intended Use
Karyomapping is a molecular karyotyping method that can be used on a single cell or few
cells from an embryo. The method provides a comprehensive test for preimplantation
genetic diagnosis (PGD) of single gene defects. It can be used where there is a risk of severe
genetic disorders being inherited from parents. A couple could have a family history of a
genetic disorder or have had a child with a genetic disease. PGD screening with
karyomapping can be used to identify embryos that do not carry defective genes and can be
safely implanted.
Infinium Karyomapping Assay Protocol
The Infinium Karyomapping Assay utilizes SNP array technology. It consists of whole
genome amplification using the SureMDA DNA Amplification System, BeadArray analysis
using HumanKaryomap-12 (v1.0) DNA Analysis Kit, scanning with iScan System, and
analysis/reporting using BlueFuse Multi software.
The first stage of the karyomapping assay uses the SureMDA DNA Amplification System
to amplify DNA from blastomere and trophectoderm biopsied cells to quantities suitable for
use in the Infinium protocol.
During the second stage of the karyomapping assay the SureMDA amplified DNA from the
embryo biopsies, together with parental and reference genomic DNA, is prepared and
hybridized to the HumanKaryomap-12 BeadChip.
The Infinium protocol uses the DNA Analysis Kit and features single-tube preparation and
whole-genome amplification, hybridization to the BeadChip, and a two- step allele
detection process using labeled nucleotides.
The complete Infinium Karyomapping Assay, which includes the SureMDA amplification
protocol together with the Infinium Assay, can be performed within 24 hours.
Audience and Purpose
This guide is for laboratory technicians running the Infinium karyomapping assay. The
guide documents the laboratory protocols associated with the assay. It is important that all
sections of this protocol are read before starting the assay. Follow all the steps in the
protocol in the order shown.
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Illumina provides the following tools for sample tracking and guidance in the lab:
} The Lab Tracking Form guides you through the protocols with minimal illustrations
and explanatory notes and includes user-entry fields for tracking progress and
recording lot numbers, barcodes, and time metrics.
These documents and tools are available for download and reference at
www.illumina.com/documentation.
3
Tracking Tools
Tracking Tools
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Part # 15052710 Rev.B
Chapter 2 Lab Setup and Maintenance
Introduction
Safety Precautions
Best Practices
Lab Maintenance
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13
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Chapter 2
Lab Setup and Maintenance
Introduction
This chapter describes the essential equipment and operating procedures for a
karyomapping assay laboratory. It explains how to equip and run a laboratory, providing
important information on the following topics:
} Safety precautions
} Preventing amplification product contamination
} Preparing and storing reagents
} Calibrating and using the vortexer
} Best practices
} Lab maintenance
The assay protocols assume that you are familiar with the contents of this chapter, have
implemented all the recommendations, and have obtained all the requisite equipment,
materials, and reagents.
RELATED TRAINING
For more guidance on setting up an Infinium laboratory, see the Infinium,
Lesson 1: Lab Setup and Best Practice training video on the Array Online
Training page of the Illumina website.
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Safety Precautions
Safety Precautions
CAUTION
Only qualified laboratory personnel can perform the protocols described in this
guide. Exercise caution when handling biological samples to avoid crosscontamination among pre-amp and post-amp samples.
WARNING
This protocol uses an aliphatic amide (formamide) that is a probable
reproductive toxin. Personal injury can occur through inhalation, ingestion,
skin contact, and eye contact. For more information, consult the safety data
sheet for this assay at support.illumina.com/sds.ilmn. Dispose of containers
and any unused contents in accordance with the governmental safety
standards for your region.
WARNING
Buffer DLB contains potassium hydroxide: corrosive, harmful. Risk and
safety phrases: R22-35, S26-36/37/39-45. When working with chemicals,
always wear a suitable lab coat, disposable gloves, and protective goggles.
For more information, consult the SDS sheets.
CAUTION
BeadChips are glass. Inspect for broken edges and handle with care.
7
The karyomapping assay protocol uses 2 whole genome amplification (WGA) processes,
the first amplifies DNA from single or few cells to quantities suitable for the Infinium
protocol (WGA1). The second is the first step of the Infinium protocol (WGA2). Unless
sufficient caution is exercised, amplification products can contaminate reagents,
instrumentation, and DNA samples, causing inaccurate and unreliable results.
Amplification product contamination can shut down lab processes and significantly delay
resumption of normal operations. For this reason, Illumina strongly encourages performing
the amplification processes in separate, dedicated laboratory spaces.
CAUTION
It is imperative to establish procedures for preventing amplification product
contamination before working in the lab.
Separate WGA1, WGA2, and Post-Amp Areas
The first whole genome amplification uses the SureMDA kit to amplify DNA from embryo
biopsies, which are single cells (blastomere - day 3) or a few cells (5-10 cells, trophectoderm
- day 5) taken from the developing embryo. The preparation for this amplification is
included in the Amplification of biopsy samples with SureMDA section and is performed
in the WGA1 area. DNA is amplified to quantities sufficient for use in the Infinium
protocol. It is critical that this step is performed in a clean area, ideally in a clean 'sample
protection' hood.
The second amplification - WGA2 - is the first step of the Infinium protocol. The
preparation for this amplification is included in the Prepare and Incubate the MSA3 plate
section. Ideally WGA2 is performed in a physically separate laboratory area from the
WGA1 area.
Perform the remaining Infinium protocol in a post-amp area that is a separate laboratory
space from the amplification areas. The 3 laboratory areas are referred to as WGA1, WGA2,
and post-amp.
Dedicated Equipment and Supplies
Dedicate separate sets of instruments (pipettes, centrifuge, Illumina Hybridization Oven,
heat block, etc.) to the pre-amp and post-amp areas. Never share instruments between
areas.
Follow these rules to avoid contaminating the pre-amp area:
} Never use the same sink to wash pre-amp and post-amp reservoirs.
} Never share the water purification system for pre-amp and post-amp processes.
} Store all supplies used in the karyomapping assay protocols in the pre-amp area and
transfer to the post-amp area as needed.
Items Falling to Floor
The floor can be contaminated with amplification product transferred on the shoes of
individuals coming from the post-amp area. Therefore, treat anything falling to the floor as
if it were contaminated.
Throw away disposable items that have fallen to the ground at the completion of the assay.
Disposable items can include empty tubes, pipette tips, gloves, or lab coat hangers. During
the assay, never touch any items that have fallen to the ground.
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Use a 0.5% sodium hypochlorite (10% bleach) solution to clean any lab surface that has
contacted the contaminated item.
Individuals handling anything that has fallen to the floor, disposable or not, must throw
away their lab gloves and put on a new pair.
9
Preventing Amplification Product
Immediately and thoroughly clean nondisposable items that have fallen to the ground,
such as pipettes or important sample containers. Use a 0.5% sodium hypochlorite (10%
bleach) solution to remove product contamination.
To minimize errors in preparing user-supplied reagents each day, prepare large batches of
95% Formamide/1 mM EDTA. Then aliquot and store them as described in the following
sections.
It is important to keep a ‘first in, first out’ or FIFO system for reagents. Rotating the stock of
the remaining reagents helps to avoid accidentally using expired reagents.
Prepare Batches of 0.1N NaOH
Prepare 0.1 N NaOH fresh on the day of use from a liquid 10 N NaOH stock. Alternatively,
prepare it in large batches and aliquot it into 1 ml sealed tubes. These aliquots can be
stored for up to 12 months at 2°C to 8°C and used in the protocol as needed. When you
open an aliquot, use it on the same day that it was opened. Discard any reagent that is left
at the completion of the assay.
Prepare Batches of 95% Formamide/1 mM EDTA
To minimize errors in preparing 95% formamide/1 mM EDTA, prepare it in large batches
and aliquot it into sealed tubes. These aliquots can be stored up to 5 months at -25°C to
-15°C and used in the protocol as needed. When you open an aliquot, use it on the same
day that it was opened. Discard any reagent that is left at the completion of the assay.
WARNING
Prepare SureMDA reagents
Buffer DLB and Buffer D2 can be prepared before performing the SureMDA protocol and
stored in single use aliquots (see SureMDA buffer preparation on page 24).
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To optimize your data and minimize errors and waste, read and follow these best practices
whenever performing the karyomapping assay protocols.
General Laboratory Practice
Perform the protocols described in this guide with care while wearing appropriate personal
protective equipment, including laboratory coat, eye protection, and gloves.
Sample Mishandling
To reduce the risk of sample mishandling, it is important to witness and sign off critical
steps when following the laboratory protocol.
Preparation of SureMDA Controls
Positive controls
DNA controls must be prepared freshly on the day of use for each SureMDA reaction and
used promptly to reduce the risk of sample loss by adhesion to the plastic tube. Nucleasefree ‘LoBind’ tubes can be used to reduce this effect. Allow sufficient time for complete
resuspension when serially diluting the DNA. Do not vortex DNA solutions as vortexing
can shear the DNA. Control DNAs must be stored and handled away from test samples to
prevent contamination.
For control preparation instructions, see Preparation of amplification reaction controls on page
24.
Negative controls
Two types of negative control are recommended: a sample collection negative control
(buffer control, or BC) for each batch of cell isolations and an amplification reaction
negative control (no-template control, or NTC) per batch of amplification reactions.
Illumina recommends that amplification reactions are performed in this way to make sure
that no contamination of these solutions has occurred. As a result, the amplification
products seen are a result of the single cells only.
Cleaning and Calibrating Pipettes
Make sure that pipettes are properly calibrated, clean, and decontaminated.
Pipette Carefully
Perform all pipette dispenses carefully and slowly to avoid creating turbulence within the
plate wells and flow-through chambers.
Where possible, use a multichannel pipette to dispense reagents.
Handling Cap Mats
Orient the cap mat so that A1 on the cap matches A1 on the plate.
11
Best Practices
Best Practices
CAUTION
To prevent evaporation and spills, which could lead to assay variability and
cross-contamination, make sure that all 96 caps are securely seated in the wells.
When you remove a cap mat, do so carefully and slowly to avoid splashing the contents.
Set the cap mat aside, upside down, in a safe location for use later in the protocol. When
you place the cap mat back on the plate, be sure to match it to its original plate and orient
it correctly.
Reagent Reuse
Never reuse excess reagents after dispensing. Discard according to your facility
requirements.
Handling BeadChips
Avoid touching the BeadChip anywhere other than at the barcode end or on the edges.
Balancing the Centrifuge
Whenever you centrifuge plates or BeadChips, place a balance plate or rack with
BeadChips opposite each plate or BeadChip rack being centrifuged. Make sure that weights
are as similar as possible.
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Perform the following standard lab maintenance procedures for Infinium Assay labs.
Daily and Weekly Bleaching
CAUTION
To prevent sample or reagent degradation, make sure that all bleach vapors
have fully dissipated before starting any processes.
Post-amplification Area
Reducing the amount of product in the post-amp area reduces the risk of contamination in
the pre-amp areas (WGA1 and WGA2).
Identify post-amp area “hot spots” that pose the highest risk of contamination, and clean
these items daily with a 10% bleach solution.
Typical
•
•
•
•
•
•
•
•
hot spots include:
Bench space used to process amplified DNA
Door handles
Refrigerator/freezer door handles
Computer mouse
Keyboards
Centrifuges
Vortexers
Thermal cyclers
Every week, thoroughly bleach the entire post-amp area, including benchtops and
instruments that are not cleaned daily. Mop the floors with a 0.5% sodium hypochlorite
(10% bleach) solution as well.
Provide training for personnel responsible for cleaning the lab areas so that they know how
to prevent pre-amp product contamination.
Pre-amplification Area
Establish a daily and weekly bleaching schedule for the pre-amp areas (WGA1 and WGA2)
similar to the schedule for the post-amp area. Bleaching helps to eliminate product that
could have entered the pre-amp area.
Identify high-risk pre-amp items such as the items listed, and clean them with a 0.5%
sodium hypochlorite (10% bleach) solution each morning before beginning any pre-amp
processes:
• Benchtops
• Door handles
• Refrigerator/freezer door handles
• Computer mouse
• Keyboards
Every week, thoroughly clean all laboratory surfaces and instruments, including all the
benchtops and instruments that are not cleaned daily. Mop the floors with a 0.5% sodium
hypochlorite (10% bleach) solution as well.
Provide training for personnel responsible for cleaning the lab areas so that they know how
to prevent pre-amp product contamination.
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Lab Maintenance
Lab Maintenance
Cleaning the Glass Back Plates
The glass back plates are used in the flow-through chambers to control the flow of sample
and reagent onto the BeadChips. Clean the glass back plates when you open them for the
first time and between uses. In addition, perform a more thorough cleaning after every 7
uses or weekly (depending on the throughput of your lab). Both procedures are described in
the following sections.
Cleaning the Glass Back Plates after Every Use
When you open the glass back plates for the first time, and after every use, follow these
instructions to clean them.
1
Prepare a 1% dilution (2.5 g per 250 ml) of Alconox Powder Detergent in water.
2
Dip each glass back plate into the solution.
3
Remove the glass back plates from the detergent solution and wipe them with a
Kimwipe.
4
Hold the glass back plates under running DI water to remove any remaining detergent
completely.
5
Allow the glass back plates to dry in the storage rack or by leaning the glass back
plates at an angle against the black metal frames.
6
When the glass back plates are clean and dry, use a Kimwipe to wash with 70% EtOH.
7
Dry the glass back plates with a Whoosh duster or laboratory air gun.
8
To prevent surface damage to the glass back plates, store them in the Te-Flow Flow Cell
storage box.
Cleaning the Glass Back Plates Weekly
In addition to the cleaning procedure in the previous section, clean the plates periodically
after approximately every 7 uses and/or every week depending on individual lab
throughput.
CAUTION
Wear a lab coat, safety goggles, and gloves during this cleaning process.
Consumables
Item
Quantity
Bleach (5.25% sodium hypochlorite, NaOCl)
9“ x 13” Pyrex glass container
1
Fume hood
1
Backplate Tecan Storage rack
1
Deionized water
1L container for diluting bleach
CAUTION
Bleach is an irritant. Use caution when handling.
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Preparation
} Dilute bleach (5.25% sodium hypochlorite) with deionized water to 10% (0.525%
hypochlorite) in a 1L container. Mix thoroughly.
Example: Add 100 ml of bleach to 900 ml deionized water.
Cleaning Steps
1
Perform the following steps in the fume hood:
NOTE
Perform these steps in a location separate from the Infinium production lab.
a
b
c
Place glass container in the fume hood.
Place Pyrex glass back plates in the glass container with the plate reservoirs facing
up. Do not stack glass back plates on top of each other
Add enough 10% bleach solution to cover the glass back plates (fill the glass
container to approximately half full).
2
Let the glass back plates soak in bleach in the fume hood for 1 hour.
3
Fill the Backplate Tecan Storage Rack container with deionized water and transfer it to
the fume hood.
4
After the 1 hour bleach soak, individually transfer the glass back plates to the
Backplate Tecan Storage Rack that is submerged in deionized water. Be careful not to
chip the glass back plates.
NOTE
The Backplate Tecan Storage Rack container with glass back plates can now be
transferred from the fume hood to a nearby sink with deionized water.
5
Dip the rack containing the glass back plates up and down 20 times. Be careful not to
chip the glass back plates.
6
Remove the Backplate Tecan Storage Rack containing the glass back plates and rinse
with deionized water.
7
Dispose of the deionized water from the Backplate Tecan Storage Rack container; rinse
and refill with fresh deionized water.
8
Return the rack containing the glass back plates to the Backplate Tecan Storage Rack
container.
9
Dip rack containing glass back plates up and down 20 times and then let soak for 5
minutes.
10 Repeat 4 times—steps 7–9.
11 Dispose of the deionized water.
12 Allow the glass back plates to dry in the storage rack or by leaning the glass back
plates at an angle against the black metal frames.
13 When the glass back plates are clean and dry, us a Kimwipe to wash with 70% EtOH.
14 Dry the glass back plates with a Whoosh duster or laboratory air gun.
15
Lab Maintenance
WARNING
Do not perform this cleaning protocol in the BeadChip production lab.
Bleach fumes can degrade both the BeadChips and the fluorescent dyes used
in the Infinium Assay.
15 To prevent surface damage to the glass back plates, store them in the Te-Flow Flow Cell
storage box.
Maintaining and Calibrating Lab Equipment
Calibrate the following equipment on an annual basis, according to the specifications
listed.
Table 1 Lab Equipment Maintenance
Equipment
Specification
High-speed
Determine offset at 1600 rpm, 1800 rpm, 2000 rpm, and 2300 rpm
microplate shaker
using a calibrated stroboscope
Hybridization oven
±2°C at 40°C and 60°C
Heated cover heat
±1°C at 37°C and 95°C
block
Temperature probe
±1°C
for Te-Flow
Calibrating the Vortexer
The displayed speed of the vortexer can vary from the actual vortex speed. Use a digital
stroboscope to determine the actual vortex speed. When you have determined the actual
vortex speed, record it along with the displayed speed and use these measurements for
reference throughout the assay.
Follow the following instructions to calibrate the Signature* High-Speed Microplate Shaker
(VWR International, catalog # 13500-890).
1
Set the digital stroboscope display speed to 1600 rpm.
2
Turn on the vortexer and adjust the vortexer speed until the actual vortex speed
reaches 1600 rpm.
3
Record the displayed vortexer speed and note down that it represents an actual speed
of 1600 rpm.
4
Use the same method described in steps 1–3 to determine the displayed speed for the
actual vortex speed of 1800 rpm. These 2 vortex speeds are used in the karyomapping
assay.
5
Place a label on the vortexer with the calibration information. The following table
provides an example of a calibration label you can create and affix to your vortexer.
Table 2 Sample Vortexer Calibration Label
Display Speed
Actual Vortex Speed
1450 rpm
1600 rpm
1625 rpm
1800 rpm
Calibration Date
xx-xx-xx
xx-xx-xx
Calibrating the Hybridization Oven
Contact Illumina Technical Support to schedule yearly maintenance and calibration.
For more information, see the Hybridization Oven System Guide.
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Chapter 3 karyomapping assay Protocol
karyomapping assay Workflow
Fragment the DNA
Precipitate the DNA
Resuspend the DNA
Wash the BeadChip
Scan BeadChip
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Chapter 3
Karyomapping Assay Protocol
karyomapping assay Protocol
Karyomapping Assay Workflow
The following figure graphically represents the manual workflow for the Infinium
karyomapping assay.
Figure 1 Karyomapping Assay Manual Workflow
CAUTION
To avoid incidents of sample mishandling it is important to witness critical steps,
such as tube transfers in the protocol. It is recommended a witness signature is
included as part of the Lab Tracking Form.
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User-Supplied Equipment
Table 3 User-Supplied Equipment
Item
Catalog #
Air - clean, dry compressed source adjacent to
iScan: Either:
• Air tank with regulator and air hose, or
• House air with constant > 30 psi pressure
Check requirements with iScan engineer
General lab supplier
Sterile safety cabinet or UV-sterilized PCR
workstation
Dedicated for use in WGA1
General lab supplier - eg, Scanlaf Titan PCR or
Scanlaf Fortuna OR Bigneat PCR workstation
Microplate centrifuge with g-force range 8 to
3000 × g
(2, for pre-amp and post-amp use; post-amp
centrifuge must include refrigeration)
Centrifuge adapters for plates and tubes
Agarose gel electrophoresis equipment
General lab supplier - see Appendix I: Agarose
Gel Electrophoresis on page 67
Lidded thermal cycler
To hold microplates, temp range 4 °C to 95 °C. For
SureMDA step in WGA1 area
General lab supplier - eg, Applied
Biosystems:Veriti (4375786)
Tube vortexer
For pre- and post-amp
Pipettes
three separate sets, for WG1, WG2 and post-amp
use
• P-2, P-20, P-200, and P-1000
8-channel precision pipettes
Three separate sets, for WG1 [optional], WG2, and
post-amp use)
• 50 μL to 300 μL
Stop watch/timer
Three, for WG1, WG2 and post-amp use
Tube rack
VWR International catalog # 66023-526
Vacuum source
greater than 508 mm Hg (0.68 bar)
Vacuum gauge for vacuum desiccator
[recommended]
19
User-Supplied Materials
Table 4 User Supplied Materials
Item
Catalog #
Forceps
Powder-free gloves
Three separate stocks, for WG1, WG2 and post-amp
use
Lab coats
Safety glasses
15 ml conical tubes
0.8 ml storage plate (midi plate), conical
bottom
Abgene catalog # AB-0765
96-well PCR plate
96-well PCR plate seals
0.2 ml PCR tubes
Thin-walled, flip cap
1.5 ml LoBind tubes
Safe-Lock lid cap, nuclease-free
Eppendorf catalog # 0030108051
Heat Sealing foil sheets, Thermo-Seal
Rubber sealing roller
VWR International catalog #BRND701380 or
equivalent
96-well cap mats - pierceable, nonautoclavable
Absorbent pads
Kimwipes or any lint-free tissue
Aerosol filter pipette tips (3 separate stocks, for
WG1, WG2 and post-amp use)
• 2 μL, 20 μL, 200 μL, 1000 μL
Disposable pipetting troughs
Canned air, such as Aerosol Whoosh-Duster
User-Supplied Reagents
Table 5 User-Supplied Reagents
20
Item
Part #
Biopsied cells in 4 μL 1xPBS
N/A
Part # 15052710 Rev.B
Part #
Genomic DNA from Parents and Reference
Good quality genomic DNA - 50 ng/µl
N/A
Genomic control DNA
Promega G1521
Deionized water
N/A
EDTA, 0.5 M
Sigma-Aldrich catalog # E7889
Ethanol, 100%
Formamide, OmniPur
VWR International catalog # EM-4650
Isopropanol (2-propanol)
Other alcohols cannot substitute
Mild detergent, such as Alconox Powder
Detergent
Sodium Hydroxide solution NaOH, 10 M
Sigma-Aldrich catalog # T72068
PBS, 20X - dilute to 1x with nuclease-free water
For diluting DNA
Item
Cell Signaling Technologies 9808
Reagents
Table 6 Reagents
Item
Part #
SureMDA DNA Amplification System (96 rxns)
PR-40-405102-00
HumanKaryomap-12 DNA Analysis BeadChip
Kit (24 samples)
RH-103-1001
CAUTION
Download DMAP files before beginning the Infinium protocol. Scanning of
BeadChips cannot occur without the relevant associated DMAP file.
Table 7
Reagents—Supplied in the Infinium Analysis Kit
Item
Part #
HumanKaryomap-12 v1.0 (2x BeadChips)
15050055
MA1—Multi-Sample Amplification 1 Mix
11202880
MSM—Random Primer Mix
11203410
MA2—Multi-Sample Amplification Master Mix
11203401
FMS—Fragmentation solution
11203428
PM1—Precipitation solution
11203436
RA1—Resuspension, hybridization, and wash
solution
11292441
21
Item
Part #
PB2—Humidifying buffer used during
hybridization
11191130
PB1—Reagent used to prepare BeadChips for
hybridization. Also used to wash BeadChips
after hybridization and in XStain reaction.
11291245
XC1—XStain BeadChip solution 1
11208288
11208296
TEM—Two-Color Extension Master Mix
11208309
11208392
STM—Superior Two-Color Master Mix
11288046
ATM—Anti-Stain Two-Color Master Mix
11208317
11208430
NOTE
A DNA Analysis Kit includes the BeadChips and reagents. Reagents are supplied
in sufficient quantities to run all the BeadChips at the same time. It is important
that the frozen components do not go through more than 2 freeze-thaw cycles
from delivery.
The wash buffers (PB1 and XC4) are supplied in sufficient volume suitable for
single use. XC4 reagent (resuspended) can be reused up to 6 times over a twoweek period (maximum 24 BeadChips). PB1 and XC4 can be ordered separately
as a buffer kit (GT-201-1005).
Table 8 Items from Illumina
Item
Part #
iScan System (110 V/220 V) or NextSeq 550
System
SY-101-1001 or SY-415-1002
Infinium Option Starter Package (220 V)
WG-15-305
Infinium Option Starter Package (110 V)
WG-15-304
NOTE
New users, not already running the Infinium Assay, will require the Infinium Option Starter
Package. The Infinium Option Starter Package (220 V and 110 V) includes: hybridization oven
with rocker, high-speed microplate shaker, Hybex heatblock and accessories, heat sealer,
desiccator, hybridization chamber and gaskets, Tecan TeFlow wash chamber rack and
accessories, among other items.
22
Part # 15052710 Rev.B
These steps explain how to amplify the DNA from a single cell or few cells, taken from an
embryo biopsy, to quantities suitable for the Karyomapping assay. This procedure uses the
SureMDA kit and is performed in a dedicated clean laboratory area (WGA1).
Estimated Time
Hands-on time: 45 minutes
Incubation time: 2 hours
Consumables
Item
Quantity
SureMDA DNA Amplification System (96 reactions)
1 box
REPLI-g SC DNA Polymerase
4 tubes
REPLI-g SC Reaction Buffer
4 tubes
Buffer DLB
2 tubes
Stop Solution
1 tube
1x PBS SC
4 tubes
1M DTT
1 tube
Water, SC
4 tubes
0.2 ml PCR tubes or 96-well PCR plate
-
1.5 ml LoBind tubes
-
WARNING
Buffer DLB contains potassium hydroxide: corrosive, harmful. Risk and
safety phrases: R22-35, S26-36/37/39-45. For more information, consult the
safety data sheet at support.illumina.com/sds.html. When working with
chemicals, always wear a suitable lab coat, disposable gloves, and protective
eyewear. Dispose of containers and any unused contents in accordance with
the governmental safety standards for your region.
Preparation
} Prepare the starting materials, see Preparation of amplification reaction controls on page 24.
• Cell samples in 4 μl of 1x PBS in 0.2 ml tubes or a well of a PCR plate.
• Positive control DNA: 50 pg of genomic DNA in 4 μl of 1x PBS
• [Optional] Negative control 1: 4 μl of collection buffer (collection buffer control BC).
• Negative control 2: 4 μl of 1x PBS (no template control - NTC).
} Thaw SureMDA reagents - REPLI-g SC Reaction Buffer, Stop Solution, 1x PBS SC, 1M
DTT, and Water, SC at room temperature. Vortex briefly before use. To gather contents,
pulse centrifuge and then place on ice.
23
NOTE
Precipitate can form in REPLI-g SC Reaction Buffer after thawing. Dissolve the
precipitate by vortexing for 10 seconds.
} Prepare Buffer DLB and Buffer D2. For more information, see SureMDA buffer
preparation on page 24. Or if previously prepared, thaw sufficient Buffer D2 aliquots for
the number of whole genome amplification reactions.
} Preheat a PCR thermal cycler to 30°C with a 70°C heated lid for the SureMDA
Amplification.
} On the Lab Tracking Form, record:
• Date/Time
• Operator
• All SureMDA reagent tube lot numbers
SureMDA buffer preparation
Buffer D2 is made up from reconstituted Buffer DLB and 1M DTT. Both of these buffers can
be prepared in advance and stored in aliquots to minimize freeze-thawing of DTT.
Reconstituted Buffer DLB can be stored for 6 months at -20°C. Buffer DLB is pH-labile. Do
not store Buffer D2 for longer than 3 months. Keep Buffer D2 at -20°C.
1
Prepare Buffer DLB by adding 500 μl of Water, SC to the lyophilized buffer.
2
Mix thoroughly by vortexing, pulse centrifuge and retain on ice.
3
Prepare sufficient Buffer D2 for the total number of whole genome amplifications
required for the batch of samples being processed (Table 9).
Table 9 Preparation of Buffer D2
Component
Cap color Volume (4 reactions) Volume (28 reactions)
1M DTT
Reconstituted Buffer DLB
Total volume
4
1 μl
11 μl
12 µl
7 μl
77 μl
84 µl
Vortex to mix and pulse centrifuge to collect contents.
Preparation of amplification reaction controls
Positive controls
DNA controls must be prepared freshly on the day of use and used promptly to reduce the
risk of sample loss by adhesion to the plastic tube.
Negative controls
It is recommended that 2 negative controls are used: a sample collection negative control
(buffer control, or BC) for each batch of cell isolations and an amplification reaction
negative control (no-template control, or NTC) per batch of amplification reactions.
Preparation of DNA dilutions
1
24
Dilute the stock solution of human genomic DNA to 2 ng/μl (2000 pg/μl) in 1x PBS
(provided in the SureMDA kit). Mix by gently flicking the tube (do not vortex), pulse
centrifuge to collect contents. This is DNA Tube 1 (Table 10).
Part # 15052710 Rev.B
Follow the dilution series in Table 10
Table 10 Control DNA dilutions
DNA tube
Vol. DNA
Vol. PBS
1
2
3
2 μl stock
4 μl Tube 1
4 μl Tube 2
As required
36 μl
60 μl
Dilution Factor
Final concentration
As required
1 in 10
1 in 16
2000 pg/μl
200 pg/μl
12.5 pg/μl
Amplification control reactions
1
For each set of test amplification reactions set up the following control reactions in
0.2 ml PCR tubes:
Table 11 Amplification control reactions
Control type Tube label
Contents
Positive
Negative 1
Negative 2
2
G+
BC
NTC
4 μl DNA Tube 3
4 μl collection Buffer Control
4 μl 1x PBS SC
Comment
50 pg control DNA
Collected at time of cell isolation
Negative control
Pulse centrifuge the control reactions to collect the contents and place on ice until
required.
NOTE
Make sure that the PBS source used for the preparation of the positive controls is
the same as the NTC negative control.
Make sure that the PBS source used to collect the biopsied cells is the same as the
BC negative control
SureMDA Amplification
NOTE
Throughout the SureMDA amplification protocol, only have 1 sample tube open
at any one time.
1
Collect cells in 4 μl of 1x PBS in 0.2 ml PCR tubes. If the sample is in a lower volume,
make up the volume to 4 μl with 1x PBS SC. Centrifuge the tube/plate at 200 × g for 3
minutes (at 4°C if possible). Keep the cells on ice during the protocol.
NOTE
Place the samples on ice in a 96-well tube rack or plate. Make sure that the ice is
in contact with the base of the tube and that they are not held above it.
2
Place the control reaction tubes (G+, BC, NTC) in the rack after all the test samples so
that the controls are processed last.
3
Add 3 μl Buffer D2 to each sample or control tube. Briefly centrifuge (200 × g) to
combine the 2 liquid volumes.
NOTE
When pipetting into tubes that contain solutions, eject the drop of liquid onto
the side of the PCR tube. Do not place the tip into or near the sample solution.
4
Incubate the samples for 65°C for 10 minutes with a 105°C heated lid using a WGA1
designated thermal cycler.
On the Lab Tracking Form, record the start and stop times.
25
2
5
During the 10 minute incubation, prepare the Amplification Master Mix according to
Table 12.
6
Add components in the order listed. Following combination of the Water, SC and
REPLI-g SC Reaction Buffer vortex briefly and pulse centrifuge. Thaw REPLI-g SC DNA
Polymerase tube on ice, gently flick tube to mix, briefly pulse centrifuge to gather
contents. Keep all reagents on ice and add the REPLI-g SC DNA Polymerase
immediately before use. Mix well by pipetting.
Table 12 Preparation of Amplification Master Mix
Component
Cap color
Volume/reaction
Water, SC
REPLI-g SC Reaction Buffer
REPLI-g SC DNA Polymerase
Total volume
9 μl
29 μl
2 μl
40 µl
7
At the end of the 10 minute 65°C incubation step transfer the tubes immediately to ice,
do not allow the PCR block to cool the reactions to 4°C.
8
Add 3 μl of Stop Solution (red cap) to the side of each reaction tube. Briefly centrifuge
to combine the droplet into the reaction. If large numbers of samples are processed,
gently tap the tubes at regular intervals during the pipetting process to combine the
buffers and neutralize the reaction.
NOTE
When pipetting into tubes that contain solutions, eject the drop of liquid onto
the side of the PCR tube. Do not place the tip into or near the sample solution.
9
Add 40 μl of Amplification Master Mix to the 10 μl reaction volume. Eject the solution
down the side of the tube; do not touch the denatured DNA solution with the tip.
10 Seal and briefly invert the tubes to mix, pulse centrifuge to collect the contents and
return the samples to ice. Immediately proceed to the prepared thermal cycler for
amplification.
NOTE
It is critical to proceed directly to the cycling step after the reagents are mixed as
quickly as possible, not doing so could result in poor amplifications.
11 Incubate the samples in a preheated thermal cycler with the heated lid at 70°C as
follows:
1 cycle
30°C
120 minutes
1 cycle
65°C
3 minutes
1 cycle
4°C
Hold
12 To determine the success of the amplification, load 2 μl of each amplified sample
(made up to 10 μl with water and gel loading buffer as required) on a 0.8% agarose 1x
TBE gel. Run until fully resolved (60 minutes). For detailed instructions on performing
agarose gel electrophoresis, see Appendix I: Agarose Gel Electrophoresis on page 67.
13 Perform 1 of the following:
• While agarose gel electrophoresis is running, proceed to - Prepare and Incubate the
MSA3 plate.
26
Part # 15052710 Rev.B
NOTE
Make sure that the next step Prepare and Incubate the MSA3 plate is performed in
a separate laboratory area - WGA2.
27
• Store amplified DNA at 4°C for short-term storage (< 1 week) or -20°C/-80°C for
longer term storage.
These steps explain how to prepare a DNA plate (MSA3) for the Infinium Karyomapping
Assay. Good quality parental and reference genomic DNA is required for karyomapping.
The DNA is added to the MSA3 DNA plate at this stage together with the amplified biopsy
samples and controls from the SureMDA procedure. This step denatures and neutralizes
the samples, preparing them for overnight incubation. During incubation, the genomic and
amplified DNA is further amplified and prepared for downstream procedures.
Figure 2 Denaturing and Neutralizing DNA
Estimated Time
Hands-on time: 15 minutes for 12 samples
Incubation time: 2 hours
Consumables
Item
Quantity
MA1
1 tube
MSM
1 tube
MA2
1 tube
0.1 N NaOH
1 ml
96-well 0.8 ml storage plate (MIDI)
1 plate
DNA plate with genomic DNA samples (50 ng/μl)
1 plate or in tubes
DNA plate with SureMDA amplified DNA
1 plate or in tubes
Preparation
} In preparation for the incubation process, preheat the heat block, with a midi plate
insert, in the post-amp area to 37°C and allow the temperature to equilibrate.
} Thaw MA1, MSM, and MA2 tubes to room temperature. Gently invert at least 10 times
to mix contents, pulse centrifuge 280 × g to gather contents.
} Thaw DNA samples to room temperature.
• Date/Time
• Operator
28
Part # 15052710 Rev.B
DNA plate barcode (if necessary)
MSA3 plate barcode (if necessary)
MA1 tube lot number
MA2 tube lot number
MSM tube lot number
Steps
1
Dispense 40 μl MA1 into the MSA3 plate wells of the midi plate.
Fill wells according to the plate layout diagram where columns 1 and 2 contain
samples for a single BeadChip.
2
Transfer 8 μl (50 ng/μl) of gDNA from parents and reference, followed by 8 μl of
amplified DNA from the SureMDA plate, to the corresponding wells of the MSA3 plate.
3
In the Lab Tracking Form, record the original DNA sample ID for each well in the
MSA3 plate.
4
Dispense 8 μl 0.1N NaOH into each well of the MSA3 plate that contains MA1 and
sample.
5
Seal the MSA3 plate with a 96-well cap mat.
NOTE
Orient the cap mat so that A1 on the cap matches A1 on the plate. To prevent
evaporation and spills, which could lead to assay variability and crosscontamination, make sure that all 96 caps are securely seated in the wells.
6
Vortex the plate at 1600 rpm for 1 minute.
7
Pulse centrifuge at 280 × g.
8
Incubate for 10 minutes at room temperature.
9
Carefully remove the cap mat.
NOTE
When you remove a cap mat, set it aside, upside down, in a safe location for use
later in the protocol.
10 Dispense 68 μl MA2 into each well of the MSA3 plate containing sample.
11 Dispense 76 μl MSM into each well of the MSA3 plate containing sample.
12 Reseal the MSA3 plate with the cap mat.
NOTE
When you place the cap mat back on the plate, be sure to match it to its original
plate and orient it correctly.
29
•
•
•
•
•
13 Vortex the sealed MSA3 plate at 1600 rpm for 1 minute.
14 Pulse centrifuge at 280 × g.
15 Incubate in the heat block with midi insert for 2 hours at 37°C.
16 Discard unused reagents in accordance with facility standards.
17 Proceed to Fragment the DNA.
NOTE
Perform the next step, Fragment the DNA, in a separate laboratory area - postamp.
30
Part # 15052710 Rev.B
Fragment the DNA
Fragment the DNA
These steps explain how to enzymatically fragment the DNA, using endpoint
fragmentation to avoid overfragmentation.
Figure 3 Fragmenting DNA
Estimated Time
Hands-on time: ~ 5 minutes for 12 samples
Incubation time: 30 minutes
Consumables
Item
Quantity
FMS
1 tube
Preparation
} Keep the heat block with the midi plate insert at 37°C.
} Thaw FMS tube to room temperature. Gently invert at least 10 times to mix contents,
pulse centrifuge 280 × g to gather contents.
} Remove the MSA3 plate from the heat block.
} Thaw RA1 to room temperature in preparation for the later step Resuspend the DNA.
• Date/Time
• Operator
• FMS tube lot number
Steps
1
Pulse centrifuge the MSA3 plate at 280 × g.
2
Carefully remove the cap mat.
NOTE
When you remove a cap mat, set it aside, upside down, in a safe location for use
later in the protocol.
3
Add 50 μl FMS to each well containing sample.
4
Seal the MSA3 plate with the 96-well cap mat.
NOTE
5
6
Pulse centrifuge the plate at 280 × g.
7
Place the sealed plate on the 37°C heat block for 30 minutes.
8
Discard unused reagents in accordance with facility standards.
9
Perform 1 of the following:
31
• Continue to the next step Precipitate DNA, prepare for the next step during the 37°C
heat block incubation.
• If you do not plan to proceed to the next step within the next 2 hours, store the
sealed MSA3 plate at -25°C to -15°C. Do not store for more than 24 hours.
SAFE STOPPING POINT
This is a good stopping point in the process.
32
Part # 15052710 Rev.B
These steps explain how to precipitate the DNA samples using 100% 2-propanol and PM1.
Figure 4 Precipitating DNA
Estimated Time
Hands-on time: ~10 minutes for 12 samples
Centrifugation: 20 minutes
Dry time: 15 minutes
Consumables
Item
Quantity
PM1
1 tube
100% 2-propanol
15 ml
Preparation
} Preheat heat block to 37°C.
} Perform 1 of the following
• If you froze the MSA3 plate after fragmentation, thaw it to room temperature, and
then pulse centrifuge the MSA3 plate at 280 × g.
• If you proceeded immediately from Fragment the MSA3 Plate, leave the plate in the
37°C heat block until setup is complete.
} Bring PM1 to room temperature. Gently invert at least 10 times to mix contents.
• Date/Time
• Operator
• PM1 tube lot number
• 100% 2-propanol lot number and date opened
Steps
1
Remove the 96-well cap mat and add 100 μl PM1 to each MSA3 plate well containing
sample.
2
Seal the plate with the cap mat.
NOTE
33
Precipitate the DNA
Precipitate the DNA
3
Vortex the sealed plate at 1600 rpm for 1 minute.
4
Place the sealed plate on the 37°C heat block for 5 minutes.
5
NOTE
Set centrifuge to 4°C in preparation for the next centrifuge step.
6
Add 310 μl 100% 2-propanol to each well containing sample.
7
Carefully seal the MSA3 plate with a new, dry cap mat, taking care not to shake the
plate in any way until the cap mat is fully seated.
8
Invert the plate at least 10 times to mix contents thoroughly.
9
Place the sealed MSA3 plate in the centrifuge opposite another plate of equal weight.
10 Centrifuge at 3000 × g at 4°C for 20 minutes.
CAUTION
Perform the next step immediately after the centrifuge stops to avoid dislodging
the blue pellet. If any delay occurs, repeat the 20 minute centrifugation before
proceeding.
11 Remove the MSA3 plate from centrifuge. Remove the cap mat and discard it.
12 Over an absorbent pad, decant the supernatant by quickly inverting the MSA3 plate.
Drain liquid onto the absorbent pad and then smack the plate down, avoiding the
liquid that was drained onto the pad.
RELATED TRAINING
To learn more, see the Decanting the Plate topic in the Infinium, Lesson 2: Assay
Techniques training video on the Array Online Training page of the Illumina
website
13 Tap firmly several times for 1 minute or until all wells are devoid of liquid.
CAUTION
Keep plate inverted. To ensure optimal performance, do not allow supernatant
in wells to pour into other wells.
14 Leave the uncovered, inverted plate on the tube rack for 15 minutes at room
temperature to air dry the pellet.
After drying, make sure that blue pellets are present at the bottoms of the wells.
15 On the Lab Tracking Form, enter the start and stop times.
Figure 5 Uncovered MSA3 Plate Inverted for Air Drying
34
Part # 15052710 Rev.B
• Continue to the next step, Resuspend the DNA.
• If you do not plan to proceed to the next step immediately, seal the MSA3 plate
with a new cap mat and store it at -25°C to -15°C. Do not store for more than 24
hours.
SAFE STOPPING POINT
35
Precipitate the DNA
Resuspend the DNA
These steps explain how to resuspend the precipitated DNA using RA1.
Figure 6 Resuspending DNA
Estimated Time
Hands-on time: ~5 minutes for 12 samples
Incubation time: 15 minutes
Consumables
Item
Quantity
RA1
17 μl per sample well
NOTE
Pour out only the recommended volume of RA1 needed for the suggested
number of samples listed in the consumables table. Additional RA1 is used later
in the Extend and Stain (XStain) the BeadChip step.
WARNING
sheet for this assay at support.illumina.com/sds.html. Dispose of containers
Preparation
} If you plan to continue to the hybridization step immediately after resuspension, begin
preheating the heat block to 95°C now.
} Preheat the Illumina Hybridization Oven to 48°C.
} Turn on the heat sealer to preheat. Allow 10 minutes.
} Invert the previously thawed RA1 several times to redissolve the solution.
NOTE
RA1 might form visible precipitates or crystals. Before use, hold the bottle in
front of a light and visually inspect, invert several times to redissolve the
solution. [Optional] Heat at 48°C for 15 minutes until RA1 is dissolved.
} On
•
•
•
36
the Lab Tracking Form, record:
Date/Time
Operator
RA1 bottle lot number
Part # 15052710 Rev.B
Steps to Resuspend the DNA
1
Add 17 μl RA1 to each well of the MSA3 plate containing a DNA pellet.
Reserve leftover reagent in the bottle for the Extend and Stain (XStain) the BeadChip step
later in this protocol.
2
Apply a foil heat seal (with the dull side facing down) to the MSA3 plate by firmly and
evenly holding the heat sealer sealing block down for 5 seconds.
3
Immediately remove the MSA3 plate from the heat sealer and forcefully roll the rubber
plate sealer over the plate until you can see all 96 well indentations through the foil.
Repeat application of the heat sealer if all 96 wells are not defined.
4
Place the sealed plate in the Illumina Hybridization Oven and incubate for 15 minutes
at 48°C.
5
6
NOTE
If you store the pellets at -25°C to -15°C for extended periods of time after the
precipitate process, you might need to resuspend the pellets. Repeat the
vortexing and centrifugation in the previous steps until pellets are resuspended.
7
Discard unused reagents in accordance with facility standards.
8
Perform 1 of the following:
• Continue to the next step, Hybridize the DNA to the BeadChip. If you plan to do so
immediately, it is safe to leave the MSA3 plate at room temperature for up to 1
hour.
• If you do not plan to proceed to the next step, immediately:
— Store the sealed MSA3 plate at -25°C to -15°C for no more than 24 hours. Store
at -80°C if storing for more than 24 hours. Do not store the MSA3 plate for
longer than one week at -80°C.
— Store RA1 at room temperature for no more than 24 hours. Store at -25°C to 15°C if storing for more than 24 hours.
SAFE STOPPING POINT
37
Resuspend the DNA
NOTE
Use fresh RA1 for each protocol step where it is required. RA1 that has been
stored properly and has not been dispensed for use in either the XStain or
Resuspension step is considered fresh RA1.
These steps explain how to dispense the fragmented, resuspended DNA samples onto
BeadChips and incubate the BeadChips in the Illumina Hybridization Oven to hybridize
the samples onto the BeadChips.
RELATED TRAINING
To learn more, see the Hybridization topic in the Infinium, Lesson 2: Assay
Techniques training video on the Array Online Training page of the Illumina
website.
Figure 7 Hybridize Multi BeadChip
Estimated Time
Hands-on time: ~10 minutes for 1 BeadChip (12 samples)
Incubation: 20 minutes
Cool-down: 5 minutes
Incubation: 12 hours
Consumables
Item
Quantity
PB2
1 tube
BeadChips
1
Hyb Chambers
1
Hyb Chamber gaskets
1
Hyb Chamber inserts
1
EtOH
330 ml
CAUTION
BeadChips are glass. Inspect BeadChips for broken edges and handle with care.
Preparation
} Make sure that DMAP files for each BeadChip have been downloaded, scanning
cannot proceed without a DMAP file.
} If frozen, thaw MSA3 plate to room temperature, and then pulse centrifuge the MSA3
plate to 280 × g.
} If the heat block is not already heated, preheat the heat block to 95°C.
38
Part # 15052710 Rev.B
Prepare the Hybridization Chambers
1
Place the resuspended MSA3 plate on the heat block to denature the samples at 95°C
for 20 minutes.
On the Lab Tracking Form, enter the start and stop times.
2
During the 20 minute incubation, prepare the Hyb Chambers.
Place the following items on the benchtop for use in this procedure:
Figure 8 BeadChip Hyb Chamber Components
A
B
C
Hyb Chambers
Hyb Chamber Gaskets
Hyb Chamber Inserts
39
} Preheat the Illumina Hybridization Oven to 48°C and set the rocker speed to 5.
} Prepare the Illumina Hybridization Oven as follows:
a Preheat the oven to 48°C:
— Press the "F" button one time to change the display to TSET.
— Press the "S" button to enter the set-temperature mode, and then use the
Increment/Decrement dial to set the oven to 48°C.
— Press the "S" button again to set 48°C as the temperature.
b Set the rocker speed to 5:
— Press the "F" button twice until SPd is indicated on the display.
— Press the "S" button to enter the rocker speed mode.
— Use the Increment/Decrement dial to set the rocker speed to "5".
— Press the "S" button again.
• Date/Time
• Operator
• PB2 tube lot number
NOTE
To ensure optimal results from Hyb Chambers, keep the Hyb Chamber lids and
bases together. Adopt a labeling convention that keeps each Hyb Chamber base
paired with its original lid. Check Hyb Chamber lid-base pairs regularly to
make sure that the fit remains secure. Check hinges regularly for any signs of
abnormal wear or loose fittings. It is important that the hinges provide
adequate clamping strength to ensure an airtight seal between the lid and the
base. Record the Hyb Chamber that was used for each BeadChip, so that Hyb
Chambers can be investigated and evaluated in the event of sample evaporation
or other lab processing anomalies.
a
b
Remove the BeadChips from 2°C to 8°C storage, leaving the BeadChips in their
ziplock bags and mylar packages until you are ready to begin hybridization.
Place the Hyb Chamber gaskets into the Hyb Chambers.
— Match the wider edge of the Hyb Chamber gasket to the barcode-ridge side of
the Hyb Chamber.
Figure 9 Hyb Chamber and Gasket
A
B
C
D
Humidifying Buffer Reservoirs
Barcode Ridges
Narrower Edges
Wider Edges
— Lay the gasket into the Hyb Chamber, and then press it down all around.
Figure 10 Placing Gasket into Hyb Chamber
— Make sure that the Hyb Chamber gaskets are properly seated.
40
Part # 15052710 Rev.B
c
Dispense 400 μl PB2 into the required humidifying buffer reservoirs in the Hyb
Chamber, 2 for each BeadChip.
Figure 12 Dispensing PB2 into Hyb Chamber Reservoir
d
e
3
After filling the Hyb Chamber reservoirs with PB2, place the lid on the Hyb
Chamber right away to prevent evaporation. You do not need to lockdown the lid.
Leave the closed Hyb Chambers on the bench at room temperature until the
BeadChips are loaded with DNA sample. Load BeadChips into the Hyb Chamber
within 1 hour.
After the 20 minute incubation, remove the MSA3 plate from the heat block and place
it on the benchtop at room temperature for 5 minutes to cool.
41
Figure 11 Hyb Chamber with Gasket in Place
Load BeadChips
1
After the 5 minute cool down, pulse centrifuge the MSA3 plate to 280 × g.
2
Just before loading DNA samples, remove all BeadChips from their ziplock bags and
mylar packages.
CAUTION
Hold the BeadChip by the ends with your thumb and forefinger (thumb at the
barcode end). Do not hold the BeadChip by the sides near the sample inlets.
Avoid contacting the beadstripe area and sample inlets.
CAUTION
BeadChips are glass. Inspect BeadChips for broken edges and handle with care.
3
Place each BeadChip in a Hyb Chamber insert, orienting the barcode end so that it
matches the barcode symbol on the Hyb Chamber insert.
Figure 13 Placing BeadChips into Hyb Chamber Inserts
4
Remove the foil seal from the MSA3 plate, and then, using a multichannel precision
pipette, dispense 15 μl of each DNA sample onto the appropriate BeadChip section.
Figure 14 Loading Samples
a
b
c
42
Load samples A1–F1 from the MSA3 plate into the left side of the BeadChip.
Make sure that the pipette tip is in the sample inlet before dispensing.
Load samples A2–F2 from the MSA3 plate into the right side of the BeadChip.
Continue in this manner, following the color-coded sections in the figure, until all
samples are loaded.
Part # 15052710 Rev.B
5
On the Lab Tracking Form, record the BeadChip barcode for each group of samples
6
After loading all DNA onto the BeadChip, wait for the sample to disperse over the
entire surface.
7
Inspect the loading port to see if a large bolus of liquid remains.
Excess sample volume in the BeadChip loading port helps prevent low-intensity areas
resulting from evaporation.
Figure 16 Bolus of Liquid at Loading Port
If no excess liquid is visible, it is acceptable to add additional sample from the leftover
volume in the amplification plate until there is a large bolus around the loading port.
NOTE
Do not add RA1 (sample Hyb buffer) as this will dilute the sample.
8
Record the additional added sample on the Lab Tracking Form.
9
Proceed immediately to the next section, Set up the BeadChips for Hybridization.
Set up the BeadChips for Hybridization
CAUTION
For optimal performance, take care to keep the Hyb Chamber inserts
containing BeadChips steady and level when lifting or moving. Avoid shaking
and always keep parallel to the lab bench. Do not hold by the sides near the
sample inlets.
1
Load the Hyb Chamber inserts containing BeadChips into the Illumina Hyb Chamber.
Position the barcode end over the ridges indicated on the Hyb Chamber.
43
Figure 15 Distributing Sample in MSA3 Plate
Figure 17 Placing Hyb Chamber Inserts into the Hyb Chamber 2
Place the back side of the lid onto the Hyb Chamber and then slowly bring down the
front end to avoid dislodging the Hyb Chamber inserts.
Figure 18 Seating Lid onto Hyb Chamber 3
Close the clamps on both sides of the Hyb Chamber so that the lid is secure and even
on the base (no gaps). It is best to close the clamps in a kitty-corner fashion, closing
first the top left clamp, then the bottom right, then the top right followed by the bottom
left.
4
Place the Hyb Chamber in the 48°C Illumina Hybridization Oven so that the Illumina
logo on top of the Hyb Chamber is facing you.
CAUTION
After loading the BeadChips into the Hyb Chambers, place the Hyb Chambers
into the Illumina Hybridization Oven immediately. Do not modify the
hybridization environment by adding additional fixtures or humidifying
elements. Leave the Hyb Chambers in the oven at the correct orientation and
temperature until hybridization is complete. Changes to the hybridization
environment can have unexpected effects on data quality.
44
Part # 15052710 Rev.B
Figure 19 Hyb Chamber Correctly Placed in Hyb Oven
Figure 20 Incorrectly Placed Hyb Chamber
NOTE
If you are stacking multiple Hyb Chambers, make sure the feet of the top Hyb
Chamber fit into the matching indents on top of the bottom Hyb Chamber. This
will hold the Hyb Chambers in place while they are rocking. You can stack up to
3 Hyb Chambers per row for a maximum total of 6 Hyb Chambers in the Hyb
Oven.
5
[Optional] Start the rocker, setting the speed to 5.
6
Incubate at 48°C for 12 hours or overnight but no more than 24 hours.
7
On the Lab Tracking Form, enter the start and stop times.
8
Place RA1 at 4°C for use the next day.
9
Discard the MSA3 plate.
10 After the overnight incubation, proceed to Wash the BeadChip.
45
Resuspend XC4 Reagent in Preparation for XStain
Keep the XC4 in the bottle in which it was shipped until you are ready to use it. Follow
these steps to resuspend the XC4 reagent in preparation for the Extend and Stain (XStain)
BeadChips on page 56
46
1
Add 330 ml fresh 100% EtOH to the XC4 bottle. The final volume will be 350 ml.
Each XC4 bottle has enough solution to process up to 24 BeadChips.
2
Shake the XC4 bottle vigorously to ensure complete resuspension.
When it is resuspended, use XC4 at room temperature. You can store it at 2°C to 8°C
and reuse up to 6 times over a two-week period for a maximum of 24 BeadChips.
Part # 15052710 Rev.B
Wash the BeadChip
Wash the BeadChip
These steps explain how to prepare the BeadChips for the staining process.
Figure 21 Washing BeadChip
Estimated Time
Hands-on time: ~5 minutes for 1 BeadChip
Consumables
Item
Quantity
PB1
550 ml (up to 4 BeadChips)
Multi-Sample BeadChip Alignment Fixture
1 (per 4 BeadChips)
Te-Flow flow-through chambers (with Black Frames,
Spacers, Glass Back Plates, and Clamps)
1 (per BeadChip)
Wash Dish
2
Wash Rack
1
CAUTION
Pour only the recommended reagent volume needed for the suggested number
of samples listed in the Consumables table of each section. Some reagents are
used later in the protocol.
WARNING
Preparation
} Make sure that the water circulator is filled to the appropriate level.
} Turn on the water circulator. Set it to a temperature that brings the Chamber Rack to
44°C at equilibrium.
47
} Remove bubbles trapped in the Chamber Rack.
} Test three or more locations on the Chamber Rack, using the Illumina Temperature
Probe. Make sure that all locations are at 44°C ± 0.5°C. If the temperature on the probe
is not within ± 0.5°C, adjust the water circulator control knob to obtain 44°C ± 0.5°C on
the temperature probe.
} In preparation for the wash step, remove each Hyb Chamber from the Illumina
Hybridization Oven.
} Have ready on the lab bench:
• Two wash dishes:
— Containing 200 ml PB1, and labeled as such
• Multi-Sample BeadChip Alignment Fixture
— Using a graduated cylinder, fill with 150 ml PB1
• Te-Flow flow-through chamber components:
— Black frames
— Spacers (separated for ease of handling)
— Clean glass back plates (see Cleaning the Glass Back Plates on page 14)
— Clamps
• Date/Time
• Operator
• PB1 bottle lot number
In preparation for XStain BeadChip step
} Thaw XC1, XC2, TEM, STM, ATM, and 95% Formamide / 1mM EDTA to room
temperature. Gently invert the reagent tubes and bottles at least 10 times to mix
contents.
} Shake the XC4 bottle vigorously to ensure complete resuspension. If necessary, vortex
until dissolved.
Steps to Wash BeadChip
1
Attach the wire handle to the rack and submerge the wash rack in the first wash dish
containing 200 ml PB1.
Figure 22 Wash Rack in Wash Dish Containing PB1
48
Part # 15052710 Rev.B
Remove a BeadChip from the Hyb Chamber and then remove its cover seal.
RELATED TRAINING
To learn more, see the Removing the IntelliHyb Seal topic in the Infinium, Lesson 2: Assay
Techniques training video on the Array Online Training page of the Illumina website.
NOTE
To make sure that no solution splatters on you, Illumina recommends removing
the cover seal over an absorbent cloth or paper towels, preferably in a hood.
CAUTION
Inspect BeadChips for broken edges and handle with care.
a
b
Using powder-free gloved hands, hold the BeadChip securely and by the edges in
one hand. Avoid contact with the sample inlets. Make sure that he barcode is
facing up and is closest to you, and that the top side of the BeadChip is angled
slightly away from you.
Remove the entire seal in a single, continuous motion. Start with a corner on the
barcode end and pull with a continuous upward motion away from you and
towards the opposite corner on the top side of the BeadChip.
Figure 23 Removing the Cover Seal
c
Discard the cover seal.
CAUTION
Do not touch the arrays!
3
Immediately and carefully slide the BeadChip into the wash rack, making sure that the
BeadChip is submerged in the PB1.
Figure 24 Submerging BeadChips in Wash Dish Containing PB1
49
Wash the BeadChip
2
4
Repeat steps 2 and 3 if necessary until all BeadChips are transferred to the submerged
wash rack.
5
When all the BeadChips are in the wash rack, move the wash rack up and down for
1 minute, breaking the surface of the PB1 with gentle, slow agitation.
6
Move the wash rack to the other wash dish containing clean PB1. Make sure the
BeadChips are submerged.
7
Move the wash rack up and down for 1 minute, breaking the surface of the PB1 with
gentle, slow agitation.
8
When you remove the BeadChips from the wash rack, inspect them for remaining
residue.
NOTE
Residue that can adversely affect results is sometimes left on BeadChips after
seals are removed. If there is residue left on the BeadChips after the second PB1
wash, use a 200 μl pipette tip for each BeadChip. Slowly and carefully scrape off
the residues outward (away) from the bead-sections under PB1. Use a new
pipette tip for each BeadChip. Then, continue with the protocol.
Assemble Flow-Through Chambers
RELATED TRAINING
To learn more about and assembling the flow-through chambers, see the flow-through
chamber topic in the Infinium, Lesson 2: Assay Techniques training video on the Array Online
1
If you have not done so, fill the BeadChip Alignment Fixture with 150 ml PB1.
2
For each BeadChip to be processed, place a black frame into the BeadChip Alignment
Fixture.
Figure 25 Placing Black Frames into BeadChip Alignment Fixture, prefilled with PB1
3
50
Place each BeadChip into a black frame, aligning its barcode with the ridges stamped
onto the Alignment Fixture. Make sure that each BeadChip is fully immersed in PB1.
Part # 15052710 Rev.B
Wash the BeadChip
Figure 26 Placing BeadChip into Black Frame on Alignment Fixture
4
Place a clear spacer onto the top of each BeadChip. Use the Alignment Fixture grooves
to guide the spacers into proper position.
NOTE
Be sure to use the clear plastic spacers, not the white ones.
Figure 27 Placing Clear Plastic Spacer onto BeadChip
5
Place the Alignment Bar onto the Alignment Fixture.
Make sure that the groove in the Alignment Bar fits over the tab on the Alignment
Fixture.
51
Figure 28 Placing Alignment Bar onto Alignment Fixture
6
Place a clean glass back plate on top of the clear spacer covering each BeadChip. Make
sure that the plate reservoir is at the barcode end of the BeadChip, facing inward to
create a reservoir against the BeadChip surface.
Figure 29 Placing Glass Back Plate onto BeadChip
A
B
7
52
Reservoir at Barcode End of Glass Back Plate
Glass Back Plate in Position
Attach the metal clamps onto each flow-through chamber as follows:
a Gently push up the glass back plate against the Alignment Bar with one finger.
b Place the first metal clamp around the flow-through chamber so that the clamp is 5
mm from the top edge.
c Place the second metal clamp around the flow-through chamber at the barcode
end, 5 mm from the bottom of the reagent reservoir.
Part # 15052710 Rev.B
Wash the BeadChip
Figure 30 Securing Flow-Through Chamber Assembly with Metal Clamps
A
B
C
8
One Stripe Shows Between First Clamp and Alignment Bar
Glass Back Plate Pressed Against Alignment Bar
No Stripes Show Between Second Clamp and Barcode
Remove the assembled flow-through chamber from the alignment fixture and, using
scissors, trim the spacer at the nonbarcode end of the assembly. Slip the scissors up
over the barcode to trim the other end.
Figure 31 Trimming Spacer Ends from Flow-Through Chamber Assembly
A
B
9
Trim spacer at nonbarcode end of flow-through chamber
Trim spacer at barcode end of flow-through chamber
Continue to the next step, Extend and Stain (XStain) BeadChip.
CAUTION
Keep assembled flow-through chambers on the lab bench in a horizontal
position while you perform the preparation steps for the Extend and Stain
(XStain) BeadChip step in this protocol. Do not place the assembled chambers on
absorbent paper. Do not place the flow-through chambers in the Chamber Rack
until the preparation is complete.
10 Wash the Hyb Chamber reservoirs with DiH2O.
53
CAUTION
It is important to wash the hybridization chamber reservoirs immediately and
thoroughly to make sure that no traces of PB2 remain in the wells.
Set Up the Chamber Rack
NOTE
To learn more about removing bubbles from the Chamber Rack, see the flow-through
chamber topic in the Infinium, Lesson 2: Assay Techniques training video on the Array Online
1
Make sure that the water circulator reservoir is filled with water to the appropriate
level.
2
If not already done so, turn on the water circulator and set it to a temperature that
brings the Chamber Rack to 44°C at equilibrium.
This temperature can vary depending on facility ambient conditions.
Figure 32 Water Circulator Connected to Chamber Rack
A
B
C
54
Chamber Rack
Water Circulator with Programmable Temperature Controls
Reservoir Cover
3
The temperature displayed on the water circulator LCD screen can differ from the
actual temperature on the Chamber Rack. Confirm the actual temperature using the
temperature probe for the Chamber Rack.
4
Remove bubbles trapped in the Chamber Rack each time you run this process.
5
Use the Illumina Temperature Probe in several locations to make sure that the
Chamber Rack is at 44°C. Make sure that all locations are at 44°C ± 0.5°C.
Part # 15052710 Rev.B
Wash the BeadChip
NOTE
Do not leave the temperature probe in the first 3 rows of the Chamber Rack.
Reserve this space for BeadChips.
Figure 33 Illumina Temperature Probe and Temperature Probe in Chamber Rack
6
For accurate temperature measurement, make sure that the Temperature Probe is
touching the base of the Chamber Rack.
55
These steps explain how to: wash unhybridized and nonspecifically hybridized DNA
samples from the BeadChips, add labeled nucleotides to extend the primers hybridized to
the DNA, stain the primers, disassemble the flow-through chambers, and coat the
BeadChips for protection.
Figure 34 Extending and Staining BeadChip
Estimated Time
Hands-on time: ~3 hours
Dry time: 1 hour
Consumables
Item
Quantity
RA1
5 ml (per 4 BeadChips)
XC1
1 tube (per 4 BeadChips)
XC2
TEM
XC3
25 ml (per 4 BeadChips)
STM (make sure that all STM tubes indicate the same
stain temperature on the label)
ATM
PB1
XC4
Alconox Powder Detergent
as needed
EtOH
as needed
95% formamide/1 mM EDTA
CAUTION
Pour out only the recommended reagent volume needed for the suggested
number of samples listed in the Consumables table of each section.
56
Part # 15052710 Rev.B
Preparation
} Place the reagent tubes in a rack in the order in which they will be used: RA1, XC1,
XC2, TEM, 95% Formamide / 1mM EDTA, STM, XC3, ATM.
Figure 35 XStain BeadChip Reagent Tubes and Bottles
A
B
C
D
E
F
G
H
I
J
RA1
XC3
XC1
XC2
TEM
95% Formamide / 1mM EDTA
STM
ATM
PB1
XC4
NOTE
It is important to use fresh RA1 for each protocol step in the assay where it is
required. RA1 that has been stored properly and has not been dispensed
previously is considered fresh RA1. After RA1 has been poured out into a
reservoir and exposed to room temperature air for extended periods of time, it
is no longer fresh.
NOTE
RA1 might form visible precipitates or crystals. Before use, hold the bottle in
front of a light and visually inspect to make sure that all precipitates have
dissolved. Heat to dissolve, if necessary.
} On
•
•
•
the Lab Tracking Form, record:
Date/Time
Operator
RA1 lot number
57
WARNING
•
•
•
•
•
•
•
•
XC3 lot number
XC1 lot number
XC2 lot number
TEM lot number
STM lot number
ATM lot number
PB1 lot number
XC4 lot number
Single-Base Extension
CAUTION
The remaining steps must be performed without interruption.
1
When the Chamber Rack reaches 44°C ± 0.5°C, quickly place each flow-through
chamber assembly into the Chamber Rack.
2
Make sure that each flow-through chamber is properly seated on its rack to allow
adequate heat exchange between the rack and the chamber.
3
Into the reservoir of each flow-through chamber, dispense:
a 150 μl RA1. Incubate for 30 seconds. Repeat 5 times.
CAUTION
Do not allow pipette tips to contact BeadChip surface. Touch off in the reservoir
of the glass back plate.
Figure 36 Dispensing RA1 into Each Flow-Through Chamber
b
c
d
e
f
g
h
58
450 μl XC1. Incubate for 10 minutes.
450 μl XC2. Incubate for 10 minutes.
200 μl TEM. Incubate for 15 minutes.
450 μl 95% formamide/1 mM EDTA. Incubate for 1 minute. Repeat one time.
Incubate 5 minutes.
Begin ramping the Chamber Rack temperature to the temperature indicated on the
STM tube ± 0.5°C.
450 μl XC3. Incubate for 1 minute. Repeat one time.
Part # 15052710 Rev.B
Wait until the Chamber Rack reaches the correct temperature.
Stain BeadChip
1
Into the reservoir of each flow-through chamber, dispense:
a 250 μl STM. Incubate for 10 minutes.
b 450 μl XC3. Incubate for 1 minute. Repeat one time.
c Wait 5 minutes.
d 250 μl ATM. Incubate for 10 minutes.
e 450 μl XC3. Incubate for 1 minute. Repeat one time.
f Wait 5 minutes.
g 250 μl STM. Incubate for 10 minutes.
h 450 μl XC3. Incubate for 1 minute. Repeat one time.
i
Wait 5 minutes.
j
250 μl ATM. Incubate for 10 minutes.
k 450 μl XC3. Incubate for 1 minute. Repeat one time.
l
Wait 5 minutes.
m 250 μl STM. Incubate for 10 minutes.
n 450 μl XC3. Incubate for 1 minute. Repeat one time.
o Wait 5 minutes.
2
Immediately remove the flow-through chambers from the Chamber Rack and place
horizontally on a lab bench at room temperature.
Wash and Coat BeadChips
Before starting the Wash and Coat process, read these important notes:
} Take the utmost care to minimize the chance of lint or dust entering the wash dishes,
which could transfer to the BeadChips. Place wash dish covers on wash dishes when
stored or not in use. Clean wash dishes with low-pressure air to remove particulates
before use.
} Place a clean tube rack on top of several layers of Kimwipes or on an absorbent pad.
You will place the staining rack containing BeadChips on this tube rack after removing
it from the XC4 wash dish.
} Prepare an additional clean tube rack that fits the internal dimensions of vacuum
desiccator for removal of the BeadChips. Allow one rack per 8 BeadChips. No
Kimwipes are required under this tube rack.
} Wash the tube racks and wash dishes thoroughly after use. Rinse with DI water.
Immediately following wash, place racks and wash dishes upside down on a wash
rack to dry.
Equipment Needed
Place the following items on the bench:
} 1 staining rack and wash dishes
} 1 vacuum desiccator
} 1 tube rack
} Self-locking tweezers
} Large Kimwipes
} Vacuum hose
59
4
Steps
1
Set up 2 top-loading wash dishes, labeled "PB1" and "XC4".
2
To indicate the fill volume before filling wash dishes with PB1 and XC4, pour 310 ml
water into the wash dishes and mark the water level on the side. Empty the water from
the wash dish. This enables you to pour reagent directly from the PB1 and XC4 bottles
into the wash dishes, minimizing contaminant transfer from labware to wash dishes.
Figure 37 PB1 and XC4 Wash Dishes with Staining Rack
A
B
Wash Dishes
Staining Rack
3
Pour 310 ml PB1 into the wash dish labeled “PB1.”
4
Submerge the unloaded staining rack into the wash dish with the locking arms and tab
facing towards you. This orients the staining rack so that you can safely remove the
BeadChips. Let the staining rack sit in the wash dish. You will use it to carry the
BeadChips after disassembling the flow-through chambers.
Figure 38 Staining Rack Locking Arms and Tab
A
60
Locking Arms
Part # 15052710 Rev.B
5
Tab
One at a time, disassemble each flow-through chamber:
a Use the dismantling tool to remove the 2 metal clamps.
CAUTION
It is important to use the dismantling tool to avoid chipping the glass back
plates.
Figure 39 Removing the Metal Clamps from Flow-Through Chamber
b
Remove the glass back plate by lifting the glass straight up.
CAUTION
Do not slide the glass along the BeadChip. This can damage the BeadChip.
c
d
Set the glass back plate aside. When you finish the XStain protocol, clean the glass
back plates. See Cleaning the Glass Back Plates on page 14 for instructions.
Remove the spacer. To avoid damaging the stripes on the BeadChip, pull the
spacer out so that the long sides slide along the sides of the BeadChip.
CAUTION
Do not remove the spacer before removing the glass. This can cause the glass
and BeadChip to come into contact and can damage the BeadChip.
e
Remove the BeadChip from the black frame.
CAUTION
Do not touch the face of the BeadChips. Handle them by the barcode end or by
the edges.
6
Place the BeadChips in the staining rack while it is submerged in PB1. Make sure that
the BeadChip barcodes are facing away from you and that the locking arms on the
handle are facing towards you.
If necessary, briefly lift the staining rack out of the wash dish to seat the BeadChip.
Replace it immediately after inserting each BeadChip.
7
Make sure that the BeadChips are submerged.
CAUTION
Do not allow the BeadChips to dry. Submerge each BeadChip in the wash dish
as soon as possible.
8
Slowly move the staining rack up and down 10 times, breaking the surface of the
reagent.
NOTE
If the top edges of the BeadChips begin to touch during either PB1 or XC4
washes, gently move the staining rack back and forth to separate the slides. It is
important for the solution to circulate freely between all BeadChips.
61
B
Figure 40 Washing BeadChips in PB1
9
Soak for 5 minutes.
10 Shake the XC4 bottle vigorously to ensure complete resuspension. If necessary, vortex
until dissolved.
11 Pour 310 ml XC4 into the dish labeled “XC4,” and cover the dish to prevent any lint or
dust from falling into the solution.
CAUTION
Do not let the XC4 sit for longer than 10 minutes.
12 Remove the staining rack from the PB1 dish and place it directly into the wash dish
containing XC4.
Figure 41 Moving BeadChips from PB1 to XC4
13 Slowly move the staining rack up and down 10 times, breaking the surface of the
reagent.
NOTE
If the top edges of the BeadChips begin to touch during either PB1 or XC4
washes, gently move the staining rack back and forth to separate the slides. It is
important for the solution to circulate freely between all BeadChips.
14 Soak for 5 minutes.
15 Remove the staining rack in one smooth, rapid motion and place it directly on the
prepared tube rack.
62
Part # 15052710 Rev.B
To ensure uniform coating, place the staining rack on the center of the tube rack,
avoiding the raised edges.
Figure 43 Staining Rack in Correct Orientation
16 For each of the BeadChips, working top to bottom:
a Continuing to hold the staining rack handle, carefully grip each BeadChip at its
barcode end with self-locking tweezers.
NOTE
The XC4 coat is slippery and makes the BeadChips difficult to hold. The selflocking tweezers grip the BeadChip firmly and help prevent damage.
b
Place each BeadChip on a tube rack with the barcode facing up and towards you.
63
Figure 42 Moving the Staining Rack from XC4 to Tube Rack
Figure 44 BeadChips on Tube Rack
TIP
Remove the staining rack handle if it facilitates removal of the BeadChips from
the staining rack. Holding the top of the staining rack in position, gently remove
the staining rack handle by grasping the handle between the thumb and
forefinger. Push up the tab with your thumb and push the handle away from
you (unlocking the handle), then pull up the handle and remove.
CAUTION
To prevent wicking and uneven drying, do not allow the BeadChips to rest on
the edge of the tube rack or to touch each other while drying. Do not use the
bottom level of the tube rack. The back of the BeadChips will not dry as
effectively and dye protectant could pool.
17 Place the tube rack in the vacuum desiccator. Each desiccator can hold 1 tube rack (8
BeadChips).
CAUTION
Make sure that the vacuum valve is seated tightly and securely.
18 Remove the red plug from the three-way valve before applying vacuum pressure.
19 Start the vacuum, using at least 675 mm Hg (0.9 bar).
20 To make sure that the desiccator is properly sealed, gently lift the lid of the vacuum
desiccator. Make sure that the lid does not lift off the desiccator base.
21 Dry under vacuum for 50–55 minutes.
Drying times can vary according to room temperature and humidity.
22 Release the vacuum by turning the handle slowly.
WARNING
Make sure that air enters the desiccator slowly to avoid disturbing the
contents. Improper use of the vacuum desiccator can result in damage to the
BeadChips. This is especially true if you remove the valve plug while a
vacuum is applied. For detailed vacuum desiccator instructions, see the
documentation included with the desiccator.
23 Store the desiccator with the red valve plug in the three-way valve of the desiccator to
stop accumulation of dust and lint within the valve port.
24 Touch the borders of the chips (do not touch the stripes) to make sure that the etched,
barcoded sides of the BeadChips are dry to the touch.
64
Part # 15052710 Rev.B
CAUTION
Do not touch the stripes with the wipe or allow EtOH to drip onto the stripes.
• Proceed to Scan the BeadChip.
• Store the BeadChips in the Illumina BeadChip Slide Storage Box at room
temperature. Scan the BeadChips within 72 hours.
27 Clean the glass back plates. See Cleaning the Glass Back Plates on page 14 for
instructions.
65
25 If the underside feels tacky, manually clean the underside of the BeadChip to remove
any excess XC4 using a Kimwipe sprayed with 70% EtOH. The bottom 2 BeadChips
are most likely to have some excess.
a Hold the BeadChip at a downward angle to prevent excess EtOH from dripping
from the wipe onto the stripes.
b Wipe along the underside of the BeadChip 5 or 6times, until excess XC4 is
removed.
Scan BeadChip
Scan BeadChips using either the iScan system or the NextSeq 550 system. To perform a
scan, you need Decode Map (DMAP) files, a manifest file, and a cluster file for the
BeadChip you are using.
} Use the Decode File Client to download the required DMAP folder. DMAP folders are
unique for each BeadChip type. For more information, see the DMAP Decode File Client
User Guide (part # 11337856).
} Download the manifest and cluster files from the Illumina support website. Each
manifest and cluster file is unique for each BeadChip type. If you are using the NextSeq
550 system, make sure to use files that include NS550 in the file name.
} Scan your BeadChip using the iScan or the NextSeq system. For more information, see
the iScan System User Guide (part # 11313539) or the NextSeq 550 System User Guide (part
# 15069765). In the lab tracking form, record the Scanner ID and scan date for each
BeadChip.
} Output files are generated during the scan and then queued for transfer to the specified
output folder.
Perform analysis using BlueFuse Multi software, which requires that scanning data are
available in a genotype call (GTC) file format. By default, the NextSeq 550 system generates
normalized data and associated genotype calls in the format of a GTC file. The iScan can
be configured to produce GTC files, in addition to the standard file types (such as IDAT
files).
Alternatively, you can use the Illumina Beeline software to convert IDAT files to GTC files
after scanning. For more information, see Beeline Software User Guide (part # 15016340).
66
Part # 15052710 Rev.B
This is a detailed protocol of how to prepare and carry out an agarose gel electrophoresis to
resolve the amplified DNA products following the SureMDA protocol. Agarose gel
electrophoresis of all the amplified samples and controls is required to make sure that the
amplification has been successful and the controls present as expected.
Estimated Time
Hands-on time: 30 minutes
Gel run timeline: 30 minutes - 1 hour
Equipment
Equipment item
Supplier
Gel running equipment, to include:
eg, VWR link or Scie-Plas link
gel mold and comb, gel tank, power
supply
Gel visualization, to include:
eg, VWR or UVP link
UV transilluminator, camera, printer
Glass bottle (for gel use)
eg, VWR, 215-1594
Microwave
General Lab Supplier
P10 pipette (for gel use)
Heat proof gloves
OR Precast gel system
eg, Lonza FlashGel DNA system link or Invitrogen eGel
system link
Materials required
Material
Supplier
Catalog#
Comments/hazards
Agarose
SigmaAldrich
A9539100G
R36/37/38: Irritating to eyes, respiratory system, and skin.
Gel Red
(10,000x
solution)
Cambridge
Biosciences
BT41003
There are no known hazards - safety report available with
SDS.
TBE
buffer
SigmaAldrich
93290-1L
Supplied 10x solution, dilute to 1x solution before use. eg, 50
ml 10x buffer, 450 ml water.
Loading
buffer
SigmaAldrich
G25265ML
Supplied 4-8x solution, add 5 ml water to the 5 ml supplied
for a 2x solution.
1 Kb
DNA
extension
ladder
Life
Technologies
10511012
Dilute to 0.05 μg/μl in loading buffer (loading buffer final
concentration of 1x). Aliquot into appropriate volumes and
store at -20°C until required. Load approximately 0.1 μg of
ladder per mm lane width.
67
Workflow
Estimated total
time
Estimated hands on
time
1. Making agarose gel solution and preparing gel
mold
30 minutes
15 minutes
2. Loading samples into gel and running gel
30 min - 1 hour
5 minutes
3. Taking images and cleaning
10 minutes
10 minutes
Procedure
Steps
Gel mold and comb preparation
1
Select the gel mold size suitable for the gel to be run. Common sizes available are 50
ml, 100 ml, 150 ml, and 400 ml.
2
Using autoclave tape, carefully tape the ends of the mold. If necessary, make sure that
there are no gaps for the solution to leak through.
3
Select the correct sized comb for the gel mold and place into position.
4
Place the agarose mold on a flat horizontal surface.
Preparation of agarose gel solution
1
Weigh out agarose powder and measure the required amount of 1xTBE buffer required
for the gel. (e.g 0.8% agarose gel – 0.8 g agarose powder in 100 ml of 1x TBE buffer)
2
Add the 1xTBE buffer to the ‘gel bench’ bottle followed by the agarose powder, swirl to
mix, place the lid on loosely.
3
Heat the agarose and buffer in the microwave for 1–2 mins, watching the solution to
make sure that it does not bubble over. Remove using heat proof gloves and swirl to
mix 2–3x during the 1–2 minutes. Heat until there are large bubbles arising to the
surface. Make sure that solution is homogeneous by swirling gently.
4
Using heat proof gloves remove the bottle from the microwave and place on the gel
bench to allow cooling for ~10–15 minutes. Make sure that the solution is cool enough
to touch. Swirl gently from time to time to prevent localized setting of gel.
5
When the solution is cooled, add the GelRed™ and gently swirl to mix. Add 5 μl
GelRed to every 50 ml of agarose gel solution.
6
Gently pour the agarose solution into prepared gel mold, pouring slowly to avoid air
bubbles. If air bubbles occur, use a pipette tip to remove/move to the side. Leave to set
for 1 hour before using.
Loading and running the gel
1
68
When the gel is set, remove the tape from the edges and place the mold into the gel
tank containing enough 1x TBE running buffer to cover the gel. Carefully remove the
combs.
Part # 15052710 Rev.B
Make sure that the gel is placed in the correct orientation in the gel tank – DNA is
negatively charged, so samples will migrate towards the positive electrode, which is
typically red – run to red!
3
The gel is now ready for samples to be loaded into the gel wells. Prepare the samples
by mixing 5 μl 2x loading buffer with 3 μl nuclease free water and 2 μl amplified DNA
from the SureMDA protocol. Load the prepared samples slowly and carefully into the
wells to avoid the samples floating out of wells.
4
Load 0.4 – 0.5 μg of a 0.05 μg/μl dilution of 1 kb DNA Extension Ladder either side of
samples to enable size estimation of bands. Close lid of tank. Record which sample has
been loaded into which well.
5
Connect the black and red leads to the black and red connections on the conductor
(again ensure the correct orientation - run to red!). Set voltage to 120 V (or around
4V/cm) and run for 5 minutes (to check for product) then a further 35 minutes or 55
minutes to check for fragment length.
6
Check there is a current flowing by observing for bubbles arising from the electrodes at
the bottom of the tank.
Gel imaging (exact instructions will depend upon local setup)
1
Place the gel mold onto a sheet of paper towel, wipe the bottom of the mold dry, and
place the gel directly on the UV transilluminator. Handle the tray with care as the gel
can easily slip off.
2
On the live setting, position the mold so the samples are visible on the screen, zoom
in/out as required. Close the door and turn on the UV source.
3
Adjust the focus settings to gain an ideal picture of your samples.
4
When happy with the image press the capture button on the UV transilluminator, then
print or save the image.
5
Note any weak or failed reactions, contaminated negative controls, and any products
that do not resemble the positive control. See Figure 45 on page 70
69
2
Figure 45 SureMDA samples electrophoresed for 5 (A), 40 (B) and 60 (C) minutes at 120 V in a
0.8% 1xTBE agarose gel. The lane contents are labeled as follows: 1–6: aDNA from a
lymphoblastoid cell line. 7: BC (buffer control), cell collection negative control. 8: NTC (notemplate control), amplification negative control. 9: 50pg male genomic DNA, amplification
positive control. L: 1 kb DNA Extension Ladder.
NOTE
Dispose of liquid and solid waste contaminated with DNA staining agents
correctly. Waste disposal and decontamination depends upon local rules.
70
Part # 15052710 Rev.B
B
balance centrifuge 12
BeadChips
glass back plates 14
best practices 11, 13
bleach
daily and weekly in lab 13
post-amplification area 13
pre-amplification area 13
vapor, effect of 13
C
calibrate
pipettes 11
cap mats 11
handling techniques 11
centrifuge
balance 12
clean
bleach, daily and weekly 13
glass back plates 14
pipettes 11
post-amplification area, schedule 13
pre-amplification area schedule 13
contamination
cap mats 11
high-risk areas 13
pipettes, clean 11
customer support 73
D
DNA
track 3
documentation 73
E
equipment
manual protocol
user-supplied 19
pre- and post-amp 8
experienced user cards 3
F
Flow-Through Chambers
single-base extension 58
stain 59
formamide warning 7
Index
Index
M
materials
automated protocol
user-supplied 20
multi-channel pipettes 11
P
pipettes
dispense technique 11
multi-channel 11
plates
cap mats 11
post-amplification
cleaning schedule 13
water purification 8
pre-amplification
cleaning schedule 13
protocols
bleach, daily and weekly 13
experienced user cards 3
R
reagents
batches, prepare in advance 10
bleach vapor 13
dispense technique 11
manual protocol
Illumina-supplied 21
user-supplied 20
S
safety
cap mats, handling 11
contamination 7
formamide 7
sample sheet 3
Signature* High-Speed Microplate
Shaker 16
sinks, separate pre- and postamplification 8
spin. See centrifuge 12
supplies, dedicated pre- and postamp 8
T
G
technical assistance 73
track sample DNA 3
glass back plates
clean 14
W
H
help, technical 73
71
Index
X
XStain BeadChips
single-base extension 58
stain 59
72
Part # 15052710 Rev.B
For technical assistance, contact Illumina Technical Support.
Table 13 Illumina General Contact Information
Website
Email
www.illumina.com
[email protected]
Table 14 Illumina Customer Support Telephone Numbers
Region
Contact Number
Region
North America
1.800.809.4566
Italy
Australia
1.800.775.688
Netherlands
Austria
0800.296575
New Zealand
Belgium
0800.81102
Norway
Denmark
80882346
Spain
Finland
0800.918363
Sweden
France
0800.911850
Switzerland
Germany
0800.180.8994
United Kingdom
Ireland
1.800.812949
Other countries
Contact Number
800.874909
0800.0223859
0800.451.650
800.16836
900.812168
020790181
0800.563118
0800.917.0041
+44.1799.534000
Safety Data Sheets
Safety data sheets (SDSs) are available on the Illumina website at
support.illumina.com/sds.html.
73
Illumina
5200 Illumina Way
San Diego, California 92122 U.S.A.
+1.800.809.ILMN (4566)
+1.858.202.4566 (outside North America)
[email protected]
www.illumina.com

Infinium Karyomapping Protocol Guide Part - Support

Transcription

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