CytoScan™ Assay Automated Workflow on Applied Biosystems™ NIMBUS™ SITE PREPARATION GUIDE
for 25 or 49 Samples
Catalog Numbers 901808, 901833, and 901858 Publication Number 703142 Revision 3
For Research Use Only. Not for use in diagnostic procedures. Manufacturer: Products: Affymetrix Pte Ltd CytoScan™ Arrays 7 Gul Circle #2M-01 Keppel Logistics Building Singapore 629563
Manufacturer: Products: Thermo Fisher Scientific Baltics UAB CytoScan™ Reagent Kit V.A. Graiciuno 8, LT-02241 Vilnius, Lithuania
The information in this guide is subject to change without notice. DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, Thermo Fisher Scientific AND/OR ITS AFFILIATE(S) will NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history: Pub. No. 703142 Revision Date Description
3 06 September 2018 Updated to the current document template, with associated updates to trademarks, logos, licensing, and warranty. Updated to include improved fragmentation protocol and reagents.
2 04 April 2013 Baseline for revision history.
Important Licensing Information
This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept the terms and conditions of all applicable Limited Use Label Licenses.
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CHAPTER 1 Introduction...... 5
About CytoScan™ Automated Target Preparation Solution on the NIMBUS™ Instrument . . . 5
CHAPTER 2 Safety precautions and hazards ...... 6
General precautions ...... 6 Operating the instrument ...... 6 Method programming ...... 6 Biohazard precautions ...... 7 Computer precautions ...... 7 Electrical Safety precautions ...... 7 Safety ...... 8 Pinch Points–Keep Hands Clear! ...... 10
CHAPTER 3 Pre-installation and site preparation ...... 11
Pre-installation information ...... 11 Site selection ...... 11 O-ring care ...... 13 Inventory of components and warranty registration ...... 13 Preparing the computer ...... 13 Preparing the power requirements ...... 13 Hamilton equipment, software, and labware ...... 14 Operation specifications for NIMBUS™ Instrument and devices ...... 14 Equipment...... 15 Set up CytoScan™ Assay-specific deck ...... 16 Secure the pedestals, thermoshake, and CPAC to the deck ...... 16 Assembling the trash chute ...... 24 Setting up the Thermoshake...... 27 Assembling the CPAC tube adapter and cover ...... 29 Placing the reagent template onto the CPAC device ...... 31 CytoScan™ Reagent Template ...... 32 Related Hamilton documentation ...... 32 Laboratory setup and recommendations ...... 33
CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument 3 Contents
Configuration 1—two separate rooms ...... 33 Configuration 2—one room ...... 36 Contamination prevention ...... 39 Equipment and calibration ...... 39 Reagent handling and storage ...... 40 Laboratory workflow ...... 41 Thermal cyclers, 96-well plate, and adhesive seals ...... 41 Hybridization oven ...... 42 Reagents, equipment, and consumables ...... 43 Equipment, software, and arrays required ...... 43 Reagents required ...... 45 Consumables required ...... 46 Optional equipment from Thermo Fisher Scientific ...... 47 Equipment required from other suppliers...... 48
4 CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument 1 Introduction
About CytoScan™ Automated Target Preparation Solution on the NIMBUS™ Instrument
IMPORTANT! The CytoScan™ Automated Target Preparation Assay protocol is optimized for processing from 25 or 49 samples at a time using the Applied Biosystems™ NIMBUS™ Target Preparation Instrument to obtain whole genome copy number and SNP information from Applied Biosystems™ CytoScan™ Arrays. Only the post-PCR steps from PCR purification to adding hybridization master mix are automated. This protocol is not intended for genome-wide association studies.
Cytogenetics research studies are performed to identify structural changes in DNA, such as copy number changes (CNV), loss of heterozygosity (LOH), and mosaicism. Individuals typically have two copies of the genome in each of their cells: one inherited from the mother and one inherited from the father. Chromosomal abnormalities, such as the ones listed below, are common in several disease states: • Deletions–When one or both copies of a particular chromosome region are lost. • Gains–When a chromosome or chromosomal region is duplicated or multiplied. • Uniparental Disomies (UPDs)–When two copies of a chromosome or chromosomal region are present, but both have been inherited from a single parent. Traditional cytogenetics techniques, such as karyotyping and fluorescent in situ hybridization (FISH) have been used to study chromosomal abnormalities for decades. However, karyotyping only detects abnormalities at low resolutions (larger than ~5 Mb), and FISH is a more focused and targeted approach without the benefit of genome-wide analysis. Further, these techniques are limited to only providing copy number information so that UPDs cannot be identified. Together, CytoScan™ Arrays and CytoScan™ Assay Automated Workflow, along with the Command Console and Chromosome Analysis Suite software, enable you to perform high-resolution genome-wide DNA copy number analysis. The Thermo Fisher Scientific solution for cytogenetics research also provides genotyping information with over 750,000 SNPs, enabling detection of loss of heterozygosity (LOH), which can be used to detect UPDs. The combined high resolution DNA copy number data and the ability to detect gains, losses, mosaicism, and LOH on a single array makes the CytoScan™ Solution a great tool for next generation cytogenetics research studies.
CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument 5 2 Safety precautions and hazards
General precautions ...... 6 Computer precautions ...... 7 Electrical Safety precautions ...... 7 Safety ...... 8 Pinch Points–Keep Hands Clear! ...... 10
General precautions
Applied Biosystems™ CytoScan™ Assay Kit as well as Applied Biosystems™ CytoScan™ Arrays are for research use only. All blood and other potentially infectious materials should be handled as if capable of transmitting infection and disposed of with proper precautions in accordance with federal, state, and local regulations. Some components required for this assay may pose significant health risks. Follow prudent laboratory practices when handling and disposing of carcinogens and toxins. Refer to the manufacturer’s Safety Data Sheet for additional information. Wear appropriate personal protective equipment when performing this assay. At a minimum, safety glasses and chemical resistant gloves should be worn.
Operating the Good Laboratory Practices (GLP) must be observed when using Applied Biosystems™ instrument NIMBUS™ Target Preparation Instrument. Wear suitable protective clothing, safety glasses, and protective gloves, particularly when dealing with an instrument malfunction where the risk of contamination from spilled liquids exists. During operation of the NIMBUS Instrument, stand clear of any moving parts or the working deck of the instrument. In general, never lean over the NIMBUS Instrument when working with it.
Method Perform test runs with tap water first and then with the final liquids prior to routine programming use, first perform test runs with tap water and then with the final liquids. Test all the liquid classes that will be used. A newly programmed test method must first be run on the NIMBUS Instrument with the final liquids prior to validation of the method and routine use. The method programmer should supervise this run. Before using any newly created or modified method for routine test purposes, a comparison study of the old and new method must be performed by the laboratory supervisor to ensure that the processing and data evaluation of both methods produces equivalent results.
6 CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument Chapter 2 Safety precautions and hazards Computer precautions 2
Biohazard If the NIMBUS Instrument becomes contaminated with biohazardous or chemical precautions material, it should be cleaned in accordance with the procedures in the Maintenance section of the MICROLAB® NIMBUS® User Guide (Pub. No. 62965-01). Failure to observe and carry out these maintenance procedures may impair the reliability and correct functioning of the NIMBUS Instrument. If working with biohazardous samples, pay particular attention to cleaning and decontamination procedures when performing the maintenance procedures. Wear gloves when handling the 96 pipetting arm, pedestals, containers, and tips. The NIMBUS Instrument drops used tips into a waste container. When the waste container becomes full, immediately empty it into a laboratory-supplied waste bin. Avoid contact with the discarded tips. Any surfaces on which liquid has been spilled must be decontaminated. Do not use disinfecting materials which contain hypochlorite (Javel water, Chlorox) or bleaching fluids.
Computer precautions
Take necessary precautions against software viruses and other malicious software. Only use the manufacturer’s original installation CD-ROM sets for the operating system and the original NIMBUS Instrument Software. Only CytoScan™ Post-PCR Software Methods for NIMBUS™ may be used to control the NIMBUS Instrument. Running other software concurrently with CytoScan™ Post-PCR Software Methods for NIMBUS™ may negatively affect the performance of the NIMBUS Instrument. Any manipulation of the NIMBUS Instrument data files or other information that determines or affects the NIMBUS Instrument functions can result in erroneous test results or instrument failure. Using an uninterrupted power supply (UPS) is recommended as the loss of power may cause data to be lost or corrupted. Ensure that the hard disk has sufficient available space for your needs. Data within the Log files directory (for example, traces and pipetting files) should be copied to the laboratory host device and deleted from the workstation computer at weekly intervals.
Electrical Safety precautions
Turn off the NIMBUS Instrument, then disconnect it from the main electrical supply and computer before removing a mechanical or electrical component.
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Safety
WARNING! Waste may contain biohazardous or chemical-contaminated material.
IMPORTANT! Use ESD control measures when working with instrument electronic assemblies.
IMPORTANT! Always make sure that the NIMBUS Instrument’s power is turned off and disconnected from the main electrical supply before installing or removing any electrical connections.
WARNING! Use good laboratory practice (GLP) and universal precautions, and wear appropriate personal protective equipment when working with and around the NIMBUS Instrument.
IMPORTANT! Examine the packaging for signs of damage. If the crate or instrument has been damaged, contact Thermo Fisher Scientific.
IMPORTANT! Verify that the contents of the package match the shipping lists. Contact Thermo Fisher Scientific if any items are missing.
Note: Instruments shipped outside the U.S. do not have the accessories carton strapped to the top of the crated instrument.
IMPORTANT! Clean exterior surfaces with soap and water. If bleach is used to disinfect, never allow bleach to remain on the deck or aluminum parts. Microcide SQ is an alternative broad-spectrum cleaner and disinfectant.
IMPORTANT! Follow safe laboratory practices in selecting and using a cleaning fluid to flush out contaminants. Do not use cleaning solutions that contain acetone or other ketones which can dissolve paint. Do not use caustic or acidic cleaning solutions.
IMPORTANT! Do not use an isopropyl alcohol-based solution to clean the safety shield. It can cause micro-fractures.
IMPORTANT! Do not wipe down areas near electrical connections with the 2 to 5% chlorine bleach solution. Bleach damages electrical connections.
WARNING! You may be required to disconnect a component or connection to completely remove the deck. Contact Thermo Fisher Scientific for additional information.
IMPORTANT! Preventive maintenance must only be performed by an authorized service technician. Before any preventive maintenance can be performed, you must empty and decontaminate the deck panel.
8 CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument Chapter 2 Safety precautions and hazards Safety 2
WARNING! Never move a fully assembled NIMBUS Instrument by yourself. Two or more people are required to move the instrument which weighs between 130 and 160 lbs, depending on its configuration. Always remove labware and pedestals prior to moving.
IMPORTANT! Do not overfill reagent containers, tubes, or other liquid containers.
WARNING! Promptly clean any spills around the NIMBUS Instrument.
WARNING! A chemical hazard could be created if labware is dropped from the labware gripper arm while it contains dangerous chemicals.
WARNING! Use caution during initialization and tip eject as the 96-head and its covers will leave the instrument “footprint” and travel out to the trash chute.
WARNING! Use caution during tip eject as the labware gripper arm will leave the instrument “footprint” and travel out to the trash chute.
WARNING! Use caution when removing package, installing, or moving instrument. The instrument could be dropped on a person or body part during transition to a new location. Always use the supplied lift straps.
WARNING! If the instrument experiences a crash and is user recoverable, re- calibrate instrument. If the instrument crashes and is not user-recoverable, contact Thermo Fisher Scientific.
WARNING! Liquid level detection must be explicitly tested when working with foaming liquids. Foam may affect the accuracy of liquid level detection.
WARNING! Contact Thermo Fisher Scientific if you believe the instrument has lost accuracy or precision in any motion axis.
WARNING! Do not remove the side shields under any circumstances.
IMPORTANT! The user is obligated to validate methods.
IMPORTANT! Place the NIMBUS Instrument on a stable bench or cart to prevent ripple or wave action in reagent troughs, otherwise liquid handling may be compromised.
IMPORTANT! Capacitance-based Liquid Level Detection (cLLD) does not function with the use of clear, non-conductive tips. Lack of cLLD can adversely affect fluid handling performance.
CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument 9 Chapter 2 Safety precautions and hazards 2 Pinch Points–Keep Hands Clear!
CAUTION! Keep your hands and head away from the NIMBUS Instrument while it is operating to avoid serious accidents related to CRASH INJURY. The robotic head moves rapidly and the tips can act as piercing agents. If you forgot to place a reagent or other item on the deck, pause the NIMBUS Instrument and then perform necessary actions.
Pinch Points–Keep Hands Clear!
WARNING! Do not place your hands in the way of moving parts while the instrument is operating.
IMPORTANT! While instrument is moving, keep hands, hair, and loose clothing away. Keep all other foreign objects away from the pinch point areas of the NIMBUS Instrument.
WARNING! It is not recommended to leave tips on the head or leave the head in its squeeze position for extended periods of time. This can lead to premature O-ring wear and poor system performance.
Potential pinch points of the NIMBUS Instrument include the: • Area between the gantry and the wing • Slots between the deck edge and the cosmetic cover • Area between the head or tips and the deck or labware • Area between the head and the shields during y-direction movement of the head • Front of the gantry between the gantry y-drive mechanism and the cosmetic covers • Area between the labware gripper arm and the 96-channel CO-RE head • Area between the labware gripper arm and the waste chute
NIMBUS™ Instrument arm NIMBUS™ Instrument head pinch point hazard area pinch point hazard area Figure 1 Pinch Point Hazard areas on the NIMBUS™ Instrument
10 CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument Pre-installation and site 3 preparation
Pre-installation information ...... 11 Set up CytoScan™ Assay-specific deck ...... 16 Laboratory setup and recommendations ...... 33 Reagents, equipment, and consumables ...... 43
Pre-installation information
IMPORTANT! Read the pre-installation information before unpacking or moving the instrument.
Site selection Note: Determine the NIMBUS Instrument location carefully. Moving the NIMBUS Instrument after installation requires re-calibration. See ʺConfiguration 1—two separate roomsʺ on page 33 and ʺConfiguration 2—one roomʺ on page 36 for suggested NIMBUS Instrument placement. Keep in mind the following considerations and requirements when preparing a location for the NIMBUS Instrument: • The NIMBUS Instrument comes with a laptop. • Keep the NIMBUS Instrument away from direct sunlight, drafts, excessive vibrations, and widely fluctuating temperatures or humidity. This is especially critical for low volume (< 10 µL) applications that require highly accurate and precise pipetting. • The NIMBUS Instrument must be placed on a stable bench, table, or hood that can support up to 200 lbs (89.6 kg) without bowing or swaying. • The NIMBUS Instrument must be oriented so that when facing the instrument, the waste disposal station is on the LEFT side of the instrument. • Make sure the bench/table surface is level as possible and affords at a minimum the following dimensions (Figure 2): – Width (front to rear) 20 in (50.8 cm) – Length (left to right) 52.5 in (133.4 cm) – Height 30 in (76.2 cm)
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Figure 2 The Applied Biosystems™ NIMBUS™ Target Preparation Instrument
For optimum serviceability, it is best to position the NIMBUS Instrument on an island location within the laboratory. Be sure to plan additional space for: • Laptop/PC, mouse and other accessories such as a printer. • Storage of manuals, tips, components, and labware. • Integrated off-deck accessories and third party devices. The NIMBUS Instrument has a solid waste chute for collection of used tips which is mounted on the front left-hand side of the instrument. The chute is designed with a slide such that the tips will be directed off the bench/table into a waste collection bag or bin. Accordingly, the front of the instrument should be approximately 1 inch (2.5 cm) from the front edge of the supporting table or bench.
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O-ring care The o-rings on the head of the NIMBUS Instrument allow the instrument to pick up and manipulate the pipette tips loaded on the deck. When the head is left in a squeezed position for an extended period of time, the o-rings wear out and the head is stressed. WARNING! It is not recommended to leave tips on the head or leave the head in its squeeze position for extended periods of time. This can lead to premature O-ring wear and poor system performance.
Upon successfully completing a method, the head is normally parked with the o-rings in a relaxed position. Should a method be interrupted or stopped, it is possible that the head may stand still with the o-rings in the squeezed position (Figure 3). Should this happen, try “initializing” the instrument.
Figure 3 O-rings in “squeezed” and “relaxed” state
To help preserve the longevity of the O-rings, remember the following: • Never power down the instrument with any tips loaded on the head. • Never leave the instrument with the head in the squeezed position. • ‘Initializing” the instrument usually resolves the head remaining in the squeezed position. WARNING! It is not recommended to leave tips on the head or leave the head in its squeeze position for extended periods of time. This can lead to premature O-ring wear and poor system performance.
Inventory of Locate the NIMBUS Instrument Inventory List shipped with the unit. Use this list to components and check off each item as component are unpacked. Components that are kits will have warranty their own inventory list inside the kit box. registration
Preparing the The NIMBUS Instrument includes a laptop. The field application scientist (FAS) computer installs the CytoScan™ Post-PCR Software Methods for NIMBUS™.
Preparing the power The input power (Universal Supply) is 100-24 VAC, 50-60 Hz, 5/A. The power supply requirements is a UL/CSA/CE approved universal power supply with IEC connection. International power adapters will need to be supplied prior to installation A multi-port power strip is supplied with each instrument which can be plugged into a single outlet. If the power strip is not used, three power outlets are required: one for the NIMBUS Instrument, one for a laptop or PC, and one for the MTEC integration. UPS power backup for both the computer and the NIMBUS Instrument is recommended but not required.
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Hamilton equipment, software, and labware
Operation Table 1 provides operation specifications for the NIMBUS Target Preparation specifications for Instrument, and the Inheco Thermoshake and CPAC devices. NIMBUS™ Instrument and IMPORTANT! The NIMBUS Target Instrument is for indoor operation and use only. devices
Table 1 Operation specifications for the NIMBUS Instrument and Inheco devices
Specification
NIMBUS™ Target Preparation Instrument
Operating Temperature: 15°C to 35°C (59°F to 95°F) Relative Humidity: 30% to 85% R.H. non-condensing Altitude: 2,000 m (1.2 miles) above sea level
Inheco Thermoshake device
Limits for ambient operating temperature 15°C to 32°C (59°F to 90°F)
Tolerable relative humidity Max. 75%, non-condensing1
Inheco CPAC device
Temperature 15°C to 32°C (59°F to 90°F)
Tolerable relative humidity 30-80% relative (non-condensing)
1 Condensing can prevent the Thermoshake device to achieve the specifications.
WARNING! Condensate can prevent the devices from achieving the above operating specifications. Upon completion of Stage 5: Prepare Hyb Tray, be sure to click Finish in the final Cleanup window for that stage to prevent further condensation which may damage the devices.
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Equipment The Applied Biosystems NIMBUS Target Preparation Instrument (Cat. No. 00-0401) is required to run the CytoScan Assay Automated Workflow. This workstation includes the accessories, software and deck configuration listed in Figure 4.
CytoScan deck configuration for the NIMBUS™ Instrument 1. Variomag Thermoshake with adapter and plate collar 2. CPAC 3. Precision DWP Pedestal 4. DWP Pedestal 5. DWP Pedestal 6. Tip Adapter 7. Precision DWP Pedestal 8. DWP Pedestal 9. FTR Pedestal 10. FTR Pedestal 11. FTR Pedestal Figure 4 CytoScan™ Assay NIMBUS™ Instrument deck configuration
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Set up CytoScan™ The Multi TEC Controller is used to operate Thermoshake and CPAC Cooling Block. Assay-specific deck The Thermoshake is set up with the PCR adapter and plate positioning bracket. The CPAC cooling block is set up with tube rack and reagent template. There are two slot modules: one for CPAC and the other for Thermoshake.
Multi TEC Control (front view) Multi TEC Control (back view) Slot Module 12V Controls both devices
Figure 5 Multi TEC Control and Slot Module
Secure the The NIMBUS Target Preparation Instrument comes with a small accessory tool kit that pedestals, includes a 2 mm, 2.5 mm, and a 3 mm hex wrench (a.k.a., hex key, Allen key, or Allen thermoshake, and wrench) (Figure 6). Additionally, the INHECO base plate kit comes with a 1.5 mm hex CPAC to the deck wrench. These tools are required for securing pedestals and devices to the NIMBUS deck.
Figure 6 NIMBUS™ Target Preparation Instrument accessory tool kit
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Secure the pedestals The pedestals in deck positions 6, 9, 10, and 11 are secured to the NIIMBUS deck by tightening the set screws, located on the left and right side of each pedestal, with a 2 mm hex wrench. When securing the pedestals to the deck it is best to start with the pedestals in the back of the deck and work to those in the front to facilitate the ease of access to set screws. Thus, using a 2 mm hex wrench, follow the instructions below to place and lock down the pedestals in the following order: 1. Tip Adapter at deck position 6 2. FTR pedestal at deck position 9 3. FTR pedestal at deck position 10 4. Tip Isolator at deck position 11 Refer to Figure 4 on page 15 for specific deck position locations. The remaining pedestals are simply positioned by placing them on the locator pins found on the deck.
To Secure a pedestal to the NIMBUS Deck 1. Place pedestal on the deck, positioned using the two locator pins on the left and right side of the pedestal (Figure 7).
Locator pins
Set screw Figure 7 Pedestal placed on deck, positioned using locator pins (front view)
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2. Tighten the set screws on the left and right side of the pedestal using a 2 mm hex wrench (Figure 8).
Figure 8 Tightening set screws on the left and right side of pedestal
3. Using the locator pins on the deck, place and secure the remaining pedestals on deck in proper order, working from the back to the front of the deck (Figure 9).
Place and secure the pedestals onto the NIMBUS deck from back to front: 1. Deck position 6 2. Deck position 9 3. Deck position 10 4. Deck position 11
Note: Place a pedestal on the deck and secure by tightening the set screws. Be sure to secure each pedestal placed onto the deck before moving on to placing and securing the next pedestal.
Figure 9 Placing and securing the pedestals onto the deck in the proper order
4. Place the remaining pedestals onto the deck, positioning them using the locator pins on the left and right side of each pedestal. These remaining pedestals are not secured to the deck with set screws. Refer to Figure 4 on page 15 for deck configuration information (Figure 10).
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Figure 10 Place the remaining pedestals onto the deck according to the deck configuration.
Securing the Thermoshake and CPAC devices to the NIMBUS™ deck The Thermoshake and CPAC devices sit on the NIMBUS deck in deck positions 1 and 2. When fully assembled, they are found behind the trash chute attachment plate and trash chute mounting bracket. The Thermoshake and CPAC devices are secured to the deck using a 1.5 mm hex wrench and set screws. The trash chute attachment plate and trash chute mounting bracket are secured using a 2.5 mm hex wrench and M3 screws. The instructions that follow assume an initial setup. If the set screws for the Thermoshake and/or CPAC device(s) need to be tightened after the initial installation, the trash chute attachment plate and the trash chute mounting bracket need to be removed first to allow access to the device set screws.
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To secure the Thermoshake and CPAC devices to the NIMBUS™ deck 1. The Thermoshake and CPAC devices are placed in their proper locations onto the deck using the locator pins (Figure 11).
Locator pin
Thermoshake CPAC Deck position #1 Deck position #2 Figure 11 Place devices securely on the deck using the locator pins.
Only one set screw for each device. Close up view of set screw. Figure 12 Set screws on Thermoshake and CPAC devices
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2. Using a 1.5 mm hex wrench, tighten the set screws for each device to lock them into place on the NIMBUS deck (Figure 13).
Figure 13 Securing devices onto the deck by tightening set screws.
3. After securing the devices, place the trash chute attachment plate on the NIMBUS Instrument, first by sliding the left side of the trash chute attachment plate horizontally into position and then sliding the right side down into place (Figure 14).
Left side of trash chute attachment plate Sliding right side of trash chute attachment plate down into place Figure 14 Place trash chute attachment plate onto the NIMBUS Instrument
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4. With the trash chute attachment plate in place, insert the trash chute mounting bracket into position on the right side, as shown in Figure 15.
Figure 15 Inserting the trash chute mounting bracket
5. Using a 2.5 mm hex wrench and two M3 screws secure the trash chute mounting bracket onto the NIMBUS Instrument (Figure 16).
Figure 16 Tighten M3 screws to secure trash chute mounting bracket.
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6. Using a 2.5 mm hex wrench and two M3 screws secure the left side of the trash chute attachment plate onto the NIMBUS Instrument (Figure 17).
Figure 17 Tighten M3 screws to secure attachment place.
7. Snap end cover into place (Figure 18).
End cover Figure 18 Snap cover into place
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Assembling the This section provides instructions for the proper assembly of the trash chute and trash trash chute chute cover to the NIMBUS Instrument. Utilize these instruction to properly attach or remove the trash chute as needed for cleaning or other purposes. To remove the trash chute please follow these instructions in reverse. 1. Locate the trash chute mounting bracket on the left side of the NIMBUS Instrument (Figure 19).
Trash Chute Mounting Bracket–found on left side of the NIMBUS™ Instrument Figure 19 Trash chute mounting bracket
2. Hold trash chute with both hands and align the two securing pins on the right side of the trash chute with the two holes in the trash chute mounting bracket (Figure 20).
Align securing pins with holes in trash chute mounting bracket Figure 20 Trash chute securing pins
3. Insert securing pins into the holes in the trash chute mounting bracket and pull the trash chute forward toward the front of the NIMBUS Instrument. This will hold the trash chute in place (Figure 21).
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Figure 21 Insert securing pins into the two holes in trash chute mounting bracket, then pull trash chute forward.
4. After successfully attaching the trash chute to the NIMBUS Instrument, turn the leveling foot, found on the underside of the trash chute, to support and level the trash chute (Figure 22).
Figure 22 Leveling Foot–found on underside of trash chute.
IMPORTANT! It is essential that the trash chute be level. Trash chutes that are not level will result in problems with disposal of tips, plates or other materials from the deck (Figure 23).
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Example of trash chute Example of level trash chute. that is not level.
INCORRECT! CORRECT
Figure 23 Examples of trash chute alignments–incorrect and correct alignments
5. Once the trash chute has been installed and leveled, attach the trash chute cover (Figure 24).
Figure 24 Trash Chute successfully installed, level, with cover attached.
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Setting up the Make sure the Thermoshake and the CPAC orientations are correct as shown in Thermoshake Figure 25, below. The Thermoshake orientation is set as shown to prevent its exhaust directly blowing into the CPAC.
Thermoshake CPAC Exhaust Intake
Figure 25 Installation of Inheco devices
This section explains how to secure the Thick PCR Adapter and Fixture Retainer onto the Thermoshake. 1. Secure the Thick PCR Adapter (Figure 26) onto the Thermoshake using the three screws shown in Figure 27.
Figure 26 Inheco Thermoshake (left) and thick PCR adapter (right)
Figure 27 Screws for adaptor (left) and for the bracket (right).
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2. Tighten the 8 screws on the outer surface of the positioning brackets so that the tabs fit snugly against the Thick PCR Adapter (as shown by the yellow arrows in Figure 28).
Figure 28 Positioning brackets
3. Secure the fixture retainer (Figure 29) onto the Thermoshake fitted with the Thick PCR Adapter using the three screws shown on the right in Figure 27. Figure 30 shows the final configuration.
Figure 29 Fixture retainer
Figure 30 Thick PCR adaptor and fixture retainer final setup
28 CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument Chapter 3 Pre-installation and site preparation Hamilton equipment, software, and labware 3
Assembling the If the CPAC tube adapter and tube adapter cover is not properly aligned when CPAC tube adapter installed on the unit, the user may experience difficulty inserting the reagent tubes into and cover the block when instructed by the method. Tubes that are not correctly inserted may cause reagent tube labels to peel off, or may generate an error during the run. If you experience difficulties inserting the reagent tubes into the CPAC unit, please follow the instructions below to ensure proper alignment of the holes and adapter plates. 1. Gather required materials, as shown in Figure 31.
Securing screw & Hex Wrench (a.k.a. Hex Key, Allen Key, or Allen Wrench) 2 mL tube x 24 24-hole tube adapter 24-hole tube adapter cover Figure 31 Materials required
2. Place the 24-hole tube adapter over the CPAC unit, ensuring that it in the proper orientation with the bottom side down (Figure 32).
Top side Bottom side—The bottom side of the 24-hole tube adapter is easily identified by the ridge around the edge of the adapter.
The 24-hole tube adapter correctly placed on CPAC unit.
Figure 32 Properly placing the 24-hole tube adapter onto the CPAC unit
CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument 29 Chapter 3 Pre-installation and site preparation 3 Hamilton equipment, software, and labware
3. Place the 24-hole tube adapter cover over the 24 hole tube adapter (Figure 33), as shown below.
The 24-hole tube adapter The 24-hole tube adapter placed on CPAC unit. cover in place. Figure 33 24-hole tube adapter and 24-hole tube adapter cover in place
4. Insert 22 x 2 mL tubes into the cover as shown in Figure 34. After inserting the tubes, inspect the alignment of the holes and adjust the alignment if necessary. Figure 34 - A shows holes that are not properly aligned, while Figure 34 - B shows holes that are properly aligned.
AB
Holes not properly aligned. Holes properly aligned.
Figure 34 Hole Alignment
5. Complete the assembly of the CPAC unit (Figure 35): a. Carefully insert 2 x 2 mL tubes into the remaining empty holes. b. Insert the securing screw into the center hole. c. Using the Allen wrench, tighten the tube adapter and tube adapter cover to the CPAC unit.
30 CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument Chapter 3 Pre-installation and site preparation Hamilton equipment, software, and labware 3
ABC Figure 35 Completing the assembly of the CPAC unit
6. Remove all of the 2 mL tubes from the unit to complete the assembly.
Placing the reagent 1. Place the CytoScan™ reagent template directly on top of the CPAC with the 24- template onto the tube rack adapter (Figure 36). CPAC device
Figure 36 CPAC with 24-tube rack adaptor, with and without reagent template
2. Place reagent tubes as instructed before running a method.
Reagent tubes
Figure 37 Example of tubes in the 24-tube rack adaptor
CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument 31 Chapter 3 Pre-installation and site preparation 3 Hamilton equipment, software, and labware
Reagent tubes
Figure 38 Tube collar placed over reagent template with reagent tubes inserted.
Figure 4 on page 15 shows the CytoScan deck configuration after device setup is complete.
CytoScan™ Reagent The CytoScan™ Reagent Template has been updated to accommodate the new Template fragmentation protocol and reagents. Use the reagent template Part No. 100981 Rev. 2 for the CytoScan™ Assay.
Figure 39 CytoScan™ Reagent Template, revision 2
Related Hamilton Documents that are shipped with the system documentation • EU Declaration of Conformity, Pub. No. 57704-01 • NIMBUS™ Instrument Assembly & Packing Checklist, Thermo Fisher Scientific, Pub. No. F-570 • NIMBUS™ Instrument Process Checklist, Pub. No. F-557 • Gravimetric Report, Pub. No. TL-61668-XX • DoQ Head, Pub. No. 608637/02 • DoQ Gripper, Pub. No. 608612/00 • Certificate of Conformance (document will also be emailed with the ASN), Pub. No. F-378
Documents that are shipped within the deck hardware kit: • MICROLAB® NIMBUS® User Manual, Pub. No. 62965-01 • Procedure for Installation and IQ/OQ Thermo Fisher Scientific, Pub. No. 67778-01 • Checklist, NIMBUS™ Accessories Box, Thermo Fisher Scientific, Pub. No. F-571
32 CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument Chapter 3 Pre-installation and site preparation Laboratory setup and recommendations 3
Laboratory setup and recommendations
This section provides an overview of two laboratory setups that can be used when performing CytoScan™ Assay Automated Workflow.
IMPORTANT! We strongly recommend using 2 separate rooms when performing this protocol, if possible.
Configuration 1— The use of two separate rooms greatly reduces the risk of sample contamination due to two separate rooms previously-amplified PCR products. These rooms are referred to as the: • Pre-PCR Clean Room • Post-PCR Room The high-level steps performed in each room are presented in Table 2.
Table 2 Assay workflow when 2 separate rooms are used Room Template PCR product (Genomic DNA)
Pre-PCR Clean Room Assay steps: – Genomic DNA preparation –Digestion – Ligation – PCR setup only
Post-PCR Room Assay steps: – PCR thermal cycling – Fragmentation – Labeling – Hybridization – Washing and staining – Scanning
CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument 33 Chapter 3 Pre-installation and site preparation 3 Laboratory setup and recommendations
Pre-PCR Clean Room The Pre-PCR Clean Room should be a low copy DNA template lab, and should be free of PCR product (amplicons). The major pieces of equipment required for this room are shown in Figure 40. The Post-PCR Room has airborne contamination with PCR product and template. After entering the Post-PCR Room, do not re-enter the Pre-PCR Clean Room without first showering and changing into freshly laundered clothes. Activities that take place in this room include: • Preparation of non-amplified genomic DNA. • Digestion and ligation reactions. • Preparation of PCR reactions.
12 3 5
6
4
Equipment shown 1. Vortexer 2. Microfuge 3. Pipettes on stand 4. Ice bucket 7 8 5. Thermal cycler 6. Plate centrifuge 7. Freezer 8. Refrigerator
Figure 40 Pre-PCR Clean Room
To help prevent sample contamination: • All of the reagents and master stocks required for the steps performed in the Pre- PCR Clean Room should be stored in this room under the appropriate conditions. • All of the equipment required for the steps performed in this room should be dedicated. Do not move any equipment including ice buckets and pipettes between the Pre- and the Post-PCR Rooms. • Always wear a fresh gown, booties, and gloves to prevent PCR carryover, and to minimize the risk of trace levels of contaminants being brought into the room.
34 CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument Chapter 3 Pre-installation and site preparation Laboratory setup and recommendations 3
Post-PCR Room Activities that take place in this room include: • PCR amplification. • PCR product purification and quantitation. • PCR product fragmentation and labeling. • Sample hybridization onto arrays. • Washing and staining of arrays. • Scanning of arrays. The major pieces of equipment required for this room are shown in Figure 41.
12 3 5 9 98 10 6 78 4 11 12
Equipment shown 1. Vortexer 2. Microfuge 3. Pipettes on stand 13 4. Ice bucket 17 5. Thermal cycler 6. GeneChip™ Hybridization Oven 645 7. Refrigerated plate centrifuge 8. Microcentrifuge 9. Plate spectrophotometer 10. Gel Imager 14 11. Electrophoresis gel box 12. Electrophoresis power supply 16 13. The NIMBUS Instrument and laptop 14. Fluidics Station 15. Scanner 15 16. Refrigerator 17. Freezer
Figure 41 Post-PCR Room
• To help prevent sample contamination: – All of the reagents and master stocks required for the steps performed in the Post-PCR Room should be stored in this room under the appropriate conditions. – All of the equipment required for the steps performed in this area should be dedicated. Do not move any equipment including ice buckets and pipettes between the Pre- and Post-PCR Rooms. – Always wear a fresh gown and gloves to minimize sample contamination. • The GeneChip™ System 3000 should be installed on a bench free of possible vibration.
CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument 35 Chapter 3 Pre-installation and site preparation 3 Laboratory setup and recommendations
Configuration 2— One room with 2 distinctly separated areas: Pre-PCR Clean Area and Post-PCR Area. one room
1 Laminar Flow or PCR Cabinet 5
23 4 7 7 Storage Area
6
We strongly recommend the use of a laminar flow cabinet or a PCR cabinet when the entire assay is to be performed in one Pre-PCR Clean Area room.
9 8
MARKING ON FLOOR TO DELINEATE PRE-PCR CLEAN AREA FROM POST-PCR AREA
17 2 3
1
Post-PCR Area 16
14 15 4 1210 13 11 12 13 9 8 7 6 5
Figure 42 One room configuration
Pre-PCR Clean Area For the best results, adhere to the following guidelines. • Keep the Pre-PCR Clean Area free of PCR amplicons. • If both pre- and post-PCR operations are performed in the same room and a laminar flow cabinet is used, keep it turned on at all times. • Keep the UV light in the laminar flow or PCR cabinet turned on when not in use. • Always wear a gown, booties, and gloves to prevent PCR carryover, and to minimize the risk of trace levels of contaminants being brought into this area.
36 CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument Chapter 3 Pre-installation and site preparation Laboratory setup and recommendations 3
Equipment in Pre-PCR Clean Area The equipment shown for the Pre-PCR Clean Area in Figure 42 on page 36 is listed below. 1. Laminar flow cabinet or PCR cabinet 2. Vortexer 3. Microfuge 4. Pipettes on stand 5. Ice bucket with ice 6. Thermal cycler 7. Plate centrifuge 8. Freezer 9. Refrigerator
About laminar flow cabinets The air curtain from the laminar flow cabinet prevents the introduction of contaminants from the surrounding air into work area, particularly PCR products from the Post-PCR Area. Open master stocks of PCR primer and adaptor only in the laminar flow cabinet. IMPORTANT! We strongly recommend that each pre-PCR step be performed in a laminar flow or PCR cabinet, including reagent and master mix preparation. The use of this cabinet is essential for preventing sample contamination due to the introduction of PCR products from the Post-PCR Area and DNA template. All of the equipment required for the pre-PCR steps should be dedicated for pre-PCR and kept in the laminar flow or PCR cabinet. This equipment includes pipettes and tips, the thermal cycler, and vortexer.
Post-PCR Area The Post-PCR Area has airborne contamination with PCR product and template. After entering the Post-PCR Area it is inadvisable to re-enter the Pre-PCR Clean Area without first showering and changing into freshly laundered clothes. The equipment shown for the Post-PCR Area in Figure 42 on page 36 consists of:
1. The NIMBUS Instrument and laptop 10. Refrigerated plate centrifuge 2. Fluidics station 11. Microcentrifuge 3. Scanner 12. Plate spectrophotometer 4. Ice bucket 13. Gel imager 5. Vortexer 14. Electrophoresis gel box 6. Microfuge 15. Electrophoresis power supply 7. Pipettes on stand 16. Refrigerator 8. Thermal cycler (one to three) 17. Freezer 9. GeneChip™ Hybridization Oven 645
CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument 37 Chapter 3 Pre-installation and site preparation 3 Laboratory setup and recommendations
Single-direction workflow To keep the Pre-PCR Clean Area as free from PCR amplicons and other contaminants as possible, always maintain a single direction workflow.
1 Laminar flow or PCR cabinet 5
23 4 7 7 Storage Area
6
Enter laboratory Pre-PCR Clean Area Note: Entering and exiting through the Pre-PCR Clean Area only is permissible as long as the Post-PCR Area has not been entered. 9 8
18 2 34
1
19 Post-PCR Area
Exit laboratory
16 17 5 6 1212 15 13 14 13 10 11 9 8 7
Figure 43 Single-direction workflow
38 CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument Chapter 3 Pre-installation and site preparation Laboratory setup and recommendations 3
Contamination prevention Care should be taken to minimize possible sources of contamination that could interfere with copy number and genotyping analysis. To reduce the possibility of cross-contamination, Affymetrix strongly recommends that you maintain a single direction workflow: from the Pre-PCR Clean Area to the Post-PCR Area. Do not re-enter the Pre-PCR Clean Area from the Post-PCR Area. Previously amplified PCR product is the most likely potential source of contamination for CytoScan™ Assay Automated Workflow. Precautions that you can take to minimize contaminating pre-PCR steps with amplified PCR product include the following: • Each area should contain dedicated equipment such as thermal cyclers, microfuges, pipettes and tips, ice buckets, etc. • Once you enter the Post-PCR Area, do not return to the Pre-PCR Clean Area until you have showered and changed into freshly laundered clothing. • Maintain an ambient laboratory environment throughout the procedure. • Store reagents under appropriate conditions according to the box label and reagent kit insert. • Use proper gowning procedures. • Print separate copies of the protocol for each room. •User filter tips for all pipetting steps.
Equipment and Keep dedicated equipment in each of the areas used for this protocol, including calibration pipettors, ice buckets, coolers, etc. It is critical to use equipment that conforms to the guidelines and specifications detailed in this manual. To avoid contamination, do not move equipment back and forth from the Post-PCR Room to Pre-PCR Clean Room. Lab instrumentation plays an important role in the successful execution of this assay. To help maintain consistency across samples and operators, all equipment must be well maintained and routinely calibrated per manufacturer recommendations, including: • All thermal cyclers • GeneChip™ Hybridization Oven 645 • GeneChip™ Fluidics Station • GeneChip™ Scanner 3000 7G • Plate spectrophotometer or NanoDrop™ • All single and multi-channel pipettes
CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument 39 Chapter 3 Pre-installation and site preparation 3 Laboratory setup and recommendations
Reagent handling and storage IMPORTANT! Always use the 24-reaction CytoScan™ Reagent Kit (Cat. No. 901808) for this protocol. You can freeze/thaw the reagents in the 24-reaction kit 5 times.
Proper storage and handling of reagents is essential for robust performance. Follow these guidelines to ensure best results: • Use reagents from the recommended vendors only. • Store all reagents at the recommended temperatures and conditions. Do not use reagents that have been improperly stored. Storage methods can profoundly impact activity. • Upon receipt of the reagent kit, store the nuclease-free water at 4°C and the Low EDTA TE Buffer at room temperature for your convenience. • Do not use expired reagents or reagents that have undergone more than the recommended number of freeze-thaw cycles. • Seal all vials and bottle caps well after use to prevent evaporation. • Do not store enzymes in a frost-free freezer. • Store the reagents used for digestion, ligation, and PCR only in the Pre-PCR Clean Area.
Using reagents at the lab bench • Properly chill essential equipment such as cooling blocks and reagent coolers before use. • Unless otherwise indicated, keep all reagents (except enzymes) on ice, or in a cooling block that has been chilled to 4°C and placed on ice during use. • Ensure that enzymes are kept at –20°C until needed. When removed from the freezer, immediately place in a bench top reagent cooler that has been chilled to –20°C. • Keep all tubes, master mixes and working solutions in chilled cooling blocks on ice. • Since enzyme activity is a function of temperature, ensure that all temperature transitions to incubation temperatures are rapid and/or well-controlled to help maintain consistency across samples.
Master mix preparation Carefully follow each master mix recipe. Use pipettes that have been calibrated as per the manufacturer’s specifications. Use only the nuclease-free water that is supplied with the kit. Do not use any other water. The enzymatic reaction in Stage 6: Fragmentation is particularly sensitive to pH and metal ion contamination. If you run out of master mix during any of these procedures, a volume error has been made or the pipettes are not accurate. We recommend that you stop and repeat the experiment.
40 CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument Chapter 3 Pre-installation and site preparation Laboratory setup and recommendations 3
Laboratory • Maintain a single direction workflow. Do not re-enter the Pre-PCR Clean Area workflow after entering the Post-PCR Area until you have showered and changed into freshly laundered clothing. • Never bring amplified products into the Pre-PCR Clean Area. • Keep dedicated equipment in each room or area used for this protocol. To avoid contamination, do not move equipment between the Pre-PCR Clean Area and the Post-PCR Area.
Thermal cyclers, 96- The CytoScan™ Assay Automated Workflow has been optimized using the following well plate, and thermal cyclers, 96-well plate, and adhesive films. adhesive seals IMPORTANT! Use only the 96-well plate and adhesive seals listed in Table 3, and only the thermal cyclers listed in Table 4 or Table 5. Using other plates and seals that are incompatible with these thermal cyclers can result in loss of sample or poor results.
Table 3 96-well plate and adhesive seals optimized for use with CytoScan™ Assay
Item Vendor Cat. No.
Multiplate 96-well unskirted PCR plate Bio-Rad MLP-9601
MicroAmp™ Clear Adhesive Film Thermo Fisher Scientific 4306311
Table 4 Thermal cyclers validated for use with CytoScan™ Assay Laboratory Thermal cyclers validated for use
Pre-PCR Clean Area Applied Biosystems units: Use one of these units • 2720 Thermal Cycler, Cat. No. 4359659 • GeneAmp™ PCR System 9700, Gold, Cat. No. 4314878 • GeneAmp™ PCR System 9700, Silver, Cat. No. N8050001
Post-PCR Area Applied Biosystems units: • GeneAmp™ PCR System 9700, Gold, Cat. No. 4314878 • GeneAmp™ PCR System 9700, Silver, Cat. No. N8050001
Table 5 Alternate thermal cyclers for use with the CytoScan™ Assay Laboratory Alternate Thermal Cyclers1
Post-PCR Area • Applied Biosystems Veriti™ 96-Well Thermal Cycler, Cat. No. 4375786 • Applied Biosystems Veriti™ Dx 96-Well Thermal Cycler, Cat. No. 44523002 •Bio-Rad® DNA Engine® PTC-200 Thermal Cycler • Eppendorf® Mastercycler® Pro S Thermal Cycler, Cat. No. 950030020 or Cat. No. 950040025
1 The FAST model (Applied Biosystems Veriti Thermal Cycler, Cat. No. 4375305) is not compatible with the CytoScan™ Assay. 2 This thermal cycler is classified as US FDA Class I Medical Device for in vitro diagnostic use.
CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument 41 Chapter 3 Pre-installation and site preparation 3 Laboratory setup and recommendations
No alterations to the thermal cycler protocols documented in the CytoScan™ Assay Automated Worflow on Applied Biosystems™ NIMBUS™ User Guide (Pub. No. 703141) are necessary. However, each thermal cycler has instrument-specific settings that should be programmed as follows:
Table 6 Instrument-specific settings for alternate thermal cyclers Thermal cycler
Setting Applied Bio-Rad® Eppendorf® Biosystems DNA Engine® Mastercycler® Veriti™ 96-well PTC-200 Pro
Lid temperature 103°C 103°C 103°C
Temperature mode N/A Calculated Safe
TSP heated lid N/A N/A Yes
Switch off lid at a low block N/A N/A No temperature
Program your thermal cyclers Use only calibrated thermal cyclers. We recommend that thermal cyclers be serviced at least once per year to ensure that they are operating within the manufacturer’s specifications. The thermal cycler programs listed in Table 7 and Table 8 are used in this protocol. Enter and store these programs on the appropriate thermal cycler in the Pre-PCR Clean Area and the Post-PCR Area.
Table 7 Pre-PCR Clean Area Number of thermal cyclers required Program name
CytoScan Digest 1 CytoScan Ligate
Table 8 Post-PCR Area Number of thermal cyclers required Program name
CytoScan PCR
CytoScan Fragment 1 CytoScan Label
CytoScan Hyb
Hybridization oven Confirm that the GeneChip™ Hybridization Oven 645 is calibrated before starting the hybridization step. Accurate hybridization temperature is critical for this assay. We recommend servicing hybridization ovens at least once per year to ensure that they are operating within the manufacturer’s specifications.
42 CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument Chapter 3 Pre-installation and site preparation Reagents, equipment, and consumables 3
Reagents, equipment, and consumables
This section provides the vendor and part number information for the reagents, equipment and consumables that have been validated for use with the CytoScan™ Assay Automated Workflow. IMPORTANT! This protocol has been optimized using the equipment, consumables and reagents listed in this user guide. For the best results, we strongly recommend that you adhere to the protocol as described. Do not deviate from the protocol; do not substitute reagents.
Unless otherwise indicated, all materials are available through thermofisher.com. MLS: Fisher Scientific (fisherscientific.com) or other major laboratory supplier.
Equipment, software, and arrays required Table 9 Equipment required Item Cat. No.
GeneChip™ Fluidics Station 450 00-0079
Tubing, Silicone peristaltic for GeneChip™ Fluidics Station 450 400110
GeneChip™ Hybridization Oven 645 00-0331
GeneChip™ 3000 Scanner with 7G upgrade Contact Thermo Fisher Scientific
Table 10 Software required Item Cat. No.
GeneChip™ Command Console Version 3.2.2 or higher
GeneChip™ Chromosome Analysis Suite Software (ChAS) 901394 Requires 64 bit workstation, Windows® operating system, 8 GB RAM
CytoScan™ Post-PCR Software Methods for NIMBUS™ 600732
CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument 43 Chapter 3 Pre-installation and site preparation 3 Reagents, equipment, and consumables
Table 11 CytoScan™ 750K reagents and array kits Item Cat. No.
CytoScan™ Reagent Kit (24 rxns per kit) 901808
CytoScan™ 750K Array Kit, 6 pack 901858
CytoScan™ 750K Array and Reagent Kit Bundle (24 arrays/rxns) 901859
CytoScan™ 750K Training Kit 901860
CytoScan™ 750K Kit Plus 24 905924
CytoScan™ 750K Kit Plus 96 905996
Table 12 CytoScan™ HD reagents and array kits
Item Cat. No.
CytoScan™ Reagent Kit (24 rxns per kit) 901808
CytoScan™ HD Array Kit, 6 pack 901833
CytoScan™ HD Training Kit 901834
CytoScan™ HD Array and Reagent Kit Bundle (24 arrays/rxns) 901835
CytoScan™ HD FAS Onsite Training 000.802
CytoScan™ HD Kit Plus 24 905824
CytoScan™ HD Kit Plus 96 905896
Table 13 NIMBUS™ CytoScan™ Starter Kit
Item Part Number
NIMBUS CytoScan Starter Kit 902167 – 96S Super Magnet Plate • 90-0929 – Micro Plate 96 PCR Adapter • 100987 – PCR Tube Racks • 100988 – CytoScan Reagent Template • 100981 – Plate Retainer • 11-1705
44 CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument Chapter 3 Pre-installation and site preparation Reagents, equipment, and consumables 3
Reagents required
Table 14 CytoScan™ Reagent Kit–24 Reactions components (Cat. No. 901808)
Item Part number Storage
CytoScan™ Module 1: Pre-Lab Restriction and Ligation 904004 –25°C to –15°C Nsp I – 901718 Nsp I Buffer – 901719 100X BSA – 901720 Adaptor, Nsp I – 902702 DNA Ligase Buffer – 901722 DNA Ligase – 901723 REF DNA 103 (50 ng/μL) – 900421 PCR Primer – 902674
CytoScan™ Module 2: Pre-Lab TE Buffer and Water 904001 2°C to 8°C Low EDTA TE Buffer – 902979 Water, Nuclease-Free – 902976
CytoScan™ Module 3: Post-Lab Fragmentation, Labeling, and Hybridization 904002 –25°C to –15°C Fragmentation Reagent – 902428 Fragmentation Buffer – 903001 TdT Enzyme – 902675 TdT Buffer – 902676 DNA Labeling Reagent – 902677 Oligo Control Reagent – 902678 Hyb Buffer Part 1 – 901725 Hyb Buffer Part 2 – 901726 Hyb Buffer Part 3 – 901727 Hyb Buffer Part 4 – 901728
CytoScan™ Module 4: Post-Lab Stain, Holding Buffer, Beads, and Water 904005 2°C to 8°C Stain Buffer 1 – 901751 Stain Buffer 2 – 901752 Array Holding Buffer – 901733 Purification Beads – 901807 Water, Nuclease-Free – 901781
CytoScan™ Module 5: Post-Lab Elution Buffer and Purification Wash Buffer 903000 15°C to 30°C Elution Buffer – 901738 Purification Wash Buffer – 901372
Wash A 901680 15°C to 30°C Wash B 901681
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Table 15 Other reagents required Item Source Cat. No.
DNA Marker, PCR Markers 50-2,000 bp Thermo Fisher Scientific 76710
TBE Buffer, 5X Solution Thermo Fisher Scientific 75891
TITANIUM™ DNA Amplification Kit (300 rxn) Clontech 639240 Sufficient for three 24 rxn CytoScan™ Kits1
TITANIUM™ DNA Amplification Kit (400 rxn) Clontech 639243 Sufficient for four 24 rxn CytoScan™ Kits1
Absolute ethanol Sigma-Aldrich 459844
Bleach (6.15% sodium hypochlorite) MLS various
1 Not required if 905924, 905996, 905824, or 905896 are used.
Consumables CytoScan™ Consumables Kit for Applied Biosystems NIMBUS™, (Labware sufficient required for 4x25 or 2x49 rxn runs), Cat. No. 902168.
Table 16 CytoScan™ Consumables Kit for Applied Biosystems™ NIMBUS™ (Cat. No. 9021681) Component per run per kit
25 sample 49 sample
96-well Semi-skirted Plate 5 7 20
Tube, Screw Cap, No Ribs 6 6 24
1.2 mL Square Well Round Bottom 11 4 Storage Plate
Reservoir 4 ROW PART PYR PP 73 mL 6 6 25
96-well Full Skirt PCR Plate, Clear 7 7 28
2.2 mL Storage Plate 3 3 12
1 This Consumable Kit does not include 96 Well UV plate. Users need to buy Costar 96 Well Clear Flat Bottom UV-Transparent Microplate from E&K Scientific (PN: EK-680845).
46 CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument Chapter 3 Pre-installation and site preparation Reagents, equipment, and consumables 3
Table 17 Additional consumables required Item Vendor Cat. No. Conductive 50 μL Filter Tips in Frames (Case of 5,760) Hamilton 235948 Conductive 300 μL Filter Tips in Frames (Case of 5,760) Hamilton 235903 Conductive 1,000 μL Filter Tips in Frames (Case of 3,840) Hamilton 235905 MicroAmp Clear Adhesive Film for 96-well plates Thermo Fisher Scientific 4306311 Pipette tips, 20 μL filter tips Rainin GP-L10F Pipette tips, 200 μL filter tips Rainin GP-L200F Pipette tips, 1,000 μL filter tips Rainin GP-L1000F Plates, 96-well unskirted PCR Bio-Rad MLP-9601 Plate, OD for UV spec, 96-well E & K Scientific EK-25801 (required only if using microplate spectrophotometer) Reagent Reservoir, 25 mL Diversified Biotech RESE-3000 TBE Gel, 4%, BMA Reliant precast Lonza Group LTD 54929 TBE Gel, 2%, BMA Reliant precast Lonza Group LTD 54939 TBE for electrophoresis Any vendor or house made Tough-Spots, 1/2” Diversified Biotech Spot 2200 Tough-Spots, 3/8” USA Scientific 9185-0000 Tube, Safe-Lock Tube 1.5 mL, amber Eppendorf 022363221 Tube, Safe-Lock Tube 1.5 mL, blue Eppendorf 022363247 Tube, Safe-Lock Tube 1.5 mL, natural Eppendorf 022363204 Tube, centrifuge 50 mL VWR 93000-036 Tube, centrifuge 15 mL VWR 21008-103 Tube strips, 8-well, 0.2 mL VWR 20170-004
Optional equipment • GeneChip™ System 3000Dx v.2, running AMDS v1.1 or higher (Cat. No 00-0334) from Thermo Fisher Scientific
CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument 47 Chapter 3 Pre-installation and site preparation 3 Reagents, equipment, and consumables
Equipment required Pre-PCR Clean Area equipment required from other suppliers When performing the pre-PCR stages of the CytoScan™ Assay Automated Workflow, great care should be taken to avoid sample contamination with PCR products. If the assay is to be run in a single room, we strongly recommend that the pre-PCR stages be performed in a laminar flow or PCR cabinet.
Table 18 Pre-PCR Clean Area equipment required
Item Vendor Cat. No.
Recommended if protocol is to be performed in one room only: Laminar Cabinet: ESCO SVE-6A or equivalent • Laminar Flow Cabinet, 6 ft (ESCO, SVE-6A) PCR Cabinet: C.B.S. Scientific P-048-02 or • PCR Cabinet equivalent
Benchtop Cooler, –20°C Agilent Technologies 401349
Biocooler aluminum block, 96-well Bio-Smith 81001 Required if processing > 8 samples. (1 for 9 to 16 samples, 2 for 17 to 24 samples)
Cooling Chamber, double block Diversified Biotech CHAM-1020
Freezer, –20°C; deep freeze; manual defrost; 17 cu ft MLS —
Rectangular Ice Tray Large – 9L (16 x 13 in, 41 x 33 cm) LabScientific RECB1202
Microfuge (for tubes and strip tubes) Any vendor —
96-well Tube Storage Racks GeneMate R-7909-2
Pipette, single-channel, 2–20 μL Rainin L-20
Pipette, single-channel, 20–200 μL Rainin L-200
Pipette, single-channel, 100–1,000 μL Rainin L-1000
Pipette, 12-channel, 2–20 μL Rainin L12-20
Pipette, 12-channel, 20–200 μL Rainin L12-200
Plate centrifuge, multipurpose Eppendorf 5804 or 5810
Vortex Genie 2 MLS —
Thermal cycler-select one of the following: • 2720 Thermal Cycler • Thermo Fisher Scientific • 4359659 • GeneAmp® PCR System 9700, Gold • Thermo Fisher Scientific • 4314878 • GeneAmp® PCR System 9700, Silver • Thermo Fisher Scientific • N8050001
Note: Electronic multichannel pipettes are not recommended.
48 CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument Chapter 3 Pre-installation and site preparation Reagents, equipment, and consumables 3
Post-PCR Area equipment required
Table 19 Post-PCR Area equipment required Item Vendor Cat. No.
Benchtop cooler, –20°C Agilent Technologies 401349
Cooling Chamber, double block Diversified Biotech CHAM-1020
Freezer, –20°C; deep freeze; manual defrost; 17 cu ft MLS —
E-Gel® iBase™ Power System Thermo Fisher Scientific G6400
Electrophoresis power supply VWR VWR105
Gel imager MLS —
Rectangular ice tray, large - 9L (16 x 13in; 41 x 33cm) LabScientific RECB1202
96-well tube storage racks GeneMate R-7909-2
MicroAmp® Adhesive Film Applicator Thermo Fisher Scientific 4333183
Microcentrifuge 5424 or 5424R Eppendorf 5424 Cat. No. 022620401 (120V) or 5424R Cat. No. 5404000537 (120V)
Microcentrifuge Standard Rotor F-45-24-11, 24 bores Eppendorf 22636502
Microfuge (for tubes and strip tubes) MLS —
Pipette, single-channel, 2–20 μL Rainin L-20
Pipette, single-channel, 20–200 μL Rainin L-200
Pipette, single-channel, 100–1000 μL Rainin L-1000
Pipette, 12-channel, 2–20 μL Rainin L12-20
Pipette, 12-channel, 20–200 μL Rainin L12-200
Pipette, 12-channel, 100–1200 μL Rainin L12-1200
Plate centrifuge, multipurpose Eppendorf 5804R or 5810R
Refrigerator, 4°C, 6 cu ft MLS —
Spectrophotometer—select one of the following: • SpectraMax Plate Spectrophotometer • Molecular Devices • Spectramax • NanoDrop ND-1000 Spectrophotometer • Thermo Fisher Scientific Plus384 • ND-1000
Thermal cycler—Select one of the following: • GeneAmp® PCR System 9700, Gold • Applied Biosystems • 4314878 • GeneAmp® PCR System 9700, Silver • Applied Biosystems • N8050001 (If processing > 8 samples, you may need to use additional thermal cyclers for PCR.)
CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument 49 Chapter 3 Pre-installation and site preparation 3 Reagents, equipment, and consumables
Table 19 Post-PCR Area equipment required (Continued) Item Vendor Cat. No.
Vortex Genie 2 USA Scientific 7404-5600
Richter Anti-vibration Pad ISC BioExpress S-7350-25 The microtube foam insert listed above will be attached to one of the vortexers. The vortex used with the foam insert may migrate across the bench top during operation. We recommend the use of a pad such as the one listed here to prevent movement.
50 CytoScan™ Assay Automated Workflow Site Preparation Guide—Applied Biosystems™ NIMBUS™ Instrument For support visit thermofisher.com/support or email [email protected] thermofisher.com
6 September 2018