Thermo Fisher Scientific HT-CMA Reference Guide
QUICK REFERENCE
CytoScan™ HT-CMA Assay 96-Array Format Automated Workflow
Catalog Numbers 906019 and 906024
Pub. No. MAN0018213 Rev. C.0
Note: For safety and biohazard guidelines, see the “Safety” appendix in the CytoScan™ HT-CMA Assay 96-Array Format Automated
Workflow User Guide—Applied Biosystems™ NIMBUS™ Instrument (Pub. No. MAN0018211). Read the Safety Data Sheets (SDSs) and
follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.
Introduction
The CytoScan™ HT-CMA Assay 96-Array Format Automated Workflow uses the HT Target Prep v1.0 method on the Applied Biosystems
NIMBUS™ Instrument for target preparation and GeneTitan™ reagent preparation to process 96 samples at a time.
Running the CytoScan™ HT-CMA Assay requires the following sets of steps:
1. Genomic DNA preparation, described in the CytoScan™ HT-CMA Assay 96-Array Format Automated Workflow User Guide—Applied
Biosystems™ NIMBUS™ Instrument (Pub. No. MAN0018211).
2. Target preparation of the samples, performed using the NIMBUS™ Target Preparation Instrument, described in this document.
3. Array processing, described in GeneTitan™ MC Protocol for Axiom™ Array Plate Processing Quick Reference (Pub. No.
MAN0017718).
This document describes the automated target preparation, performed using the NIMBUS™ Instrument.
IMPORTANT! This document contains an abbreviated set of instructions. Carefully read all the instructions in the target preparation
chapter of the CytoScan™ HT-CMA Assay 96-Array Format Automated Workflow User Guide—Applied Biosystems™ NIMBUS™ Instrument
before running the automated target preparation method.
™
Note: An option for a 3-hour DNA precipitation step is now available. See the user guide for details.
Assay notes
• This workflow allows you to run the CytoScan™ HT-CMA Assay for 96 samples one time using one HT Target Prep Reagent Kit 96F
(Cat. No. 906024).
• Remove seals from plates carefully and discard used seals. Do not reuse seals.
• Unless otherwise specified, all reagent modules are from the HT Target Prep Reagent Kit 96F. See the CytoScan™ HT-CMA Assay 96Array Format Automated Workflow User Guide—Applied Biosystems™ NIMBUS™ Instrument (Pub. No. MAN0018211) for a complete
list of equipment and consumables that are required for each stage.
• We recommend that you prepare the gDNA Sample Plate in a clean room. The clean room should be separate from the laboratory
where the CytoScan™ HT-CMA Assay is performed and be free of DNA amplified in other procedures.
Note: CytoScan™ HT-CMA Array Plates require a total of 100 ng of gDNA.
For Research Use Only. Not for use in diagnostic procedures.
Stage 1: Amplify the genomic DNA
Time required
Note: A 22–24 hour incubation is required at the end of this stage.
Activity Time
Hands-on time ~30 minutes
NIMBUS™ Instrument—DNA Amplification ~30 minutes
Incubation 23 ±1 hour
Total ~24 hours
Input required
The Amplification Sample Plate of genomic DNA samples in a round deepwell plate.
Equipment required
• Incubator/oven, temperature at 37°C
• Centrifuge, at room temperature
Reagent and sample plate handling
Module
From the HT Target Prep Reagent Kit 96F
Module 1
–20°C
Sample Plate
Thaw the gDNA Sample Plate at room temperature, then briefly centrifuge.
Note: Do not place a frozen sample plate directly on the NIMBUS™ Instrument deck.
Reagent and cap color Place at room temperature Deck loading instructions
10X Denat Solution Thaw at room temperature. Vortex and centrifuge.
Place in the cooling block.
Neutral Solution Thaw at room temperature water bath (~1 hour). Vortex for 30 seconds.
Pour in reservoir.
Amp Solution Thaw at room temperature water bath (~1 hour). Vortex for 30 seconds.
Pour in reservoir.
Water Thaw at room temperature water bath (~1 hour). Vortex for 30 seconds.
Pour in reservoir.
Amp Enzyme Do not thaw. Keep at −20°C until ready to use. Immediately before use: Gently flick the
tube 3 times, then centrifuge. Place in the
Vortex and centrifuge. Place in the cooling
block.
Run the DNA amplification step
1. In the HT-CMA NIMBUS Target Preparation Setup window, select DNA Amplification, then click Run.
2. Set up the deck as indicated in the deck setup prompt and following screens, then click Run. See Figure 2.
3. When finished, remove the sample plate from deck position 3.
a. Blot the top of the plate with a laboratory tissue to remove any droplets present.
b. Tightly seal the plate.
c. Vortex the plate for 30 seconds, then centrifuge briefly.
d. Place in a preheated oven, then incubate at 37°C for 23 ±1 hour.
e. After 22–24 hours of incubation, do one of the following:
• Proceed directly to “Stage 2: Fragment and precipitate the DNA” on page 4.
• Store the amplified DNA sample plate at –20°C.
2 CytoScan
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HT-CMA Assay 96-Array Format Automated Workflow Quick Reference—Applied Biosystems™ NIMBUS™ Instrument
Stage 1 deck layout
1
2
3
4
5
6
7
8
11
1
2
3
4
5
6
7
8
9
10
11
Part No. 101179 Rev. 1
HT Target Prep Reagent Kit Template - NIMBUS
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Part No. 101179 Rev. 1
HT Target Prep Reagent Kit Template - NIMBUS
Figure 1 Stage 1: Amplify the genomic DNA deck layout.
Round deepwell plate
1
Cooling block and tube collar. Load reagent tubes only in the
2
green column of the reagent block. Use the template as guidance
for specific reagent placement.
Round deepwell plate (gDNA Samples)
3
4-column reservoir & Reservoir frame (Master Mix Reservoir)
4
4-column reservoir, Reservoir frame, & Alpillo™ Plate Cushion
5
• Column 1: Water
• Column 2: Neutral Solution
• Column 3: Amp Solution
• Column 4: (Empty)
(Empty)
6
96-well full-skirt plate & Plate collar
7
96-well full-skirt plate & Plate collar
8
CO-RE™ Filter Tips 1,000 µL
9
CO-RE™ Filter Tips 300 µL
10
CO-RE™ Filter Tips 300 µL & Square deepwell plate
11
CytoScan™ HT-CMA Assay 96-Array Format Automated Workflow Quick Reference—Applied Biosystems™ NIMBUS™ Instrument 3
Stage 2: Fragment and precipitate the DNA
Time required
Activity Time
Hands-on time ~25 minutes
~50 minutes if frozen amplified DNA from Stage 1
NIMBUS™ Instrument—Fragmentation
~1.5 hours
• Deactivation incubation—20 minutes to deactivate the amplification
reaction and 20 minutes to equilibrate to the fragmentation
temperature
• Fragmentation incubation—30 minutes
O-line precipitation incubation at –20°C 3 hours (optional), or
overnight (16—24 hours)
Total (does not include precipitation time) ~2—2.5 hours
Input required
The Amplification Plate of amplified DNA from Stage 1 in a round deepwell plate.
Reagent and Amplification Plate handling
Thaw and prepare reagents and the Amplification Plate according to the following table.
Module
Reagent and cap color
Reagents from the HT Target Prep Reagent Kit 96F
Frag Enzyme Do not thaw. Keep at −20°C until ready to use. Immediately before use: Gently flick
Module 2-1
–20°C
10X Frag Buer Thaw in a small water
bath
Thaw, then
place on ice
[1]
Place on
ice
Place at room
temp
Deck loading instructions
tube 3 times, centrifuge briefly. Place in
the cooling block.
Vortex. Pour in reservoir.
Precip Solution 2 Vortex, then centrifuge briefly. Place in
Precip Solution 1 Vortex. Pour in reservoir.
Module 2-2
Frag Diluent Vortex, then centrifuge briefly. Place in
2–8°C
Frag Reaction Stop Vortex. Pour in reservoir.
User-supplied
Isopropanol 99.5%, 70 mL Pour in reservoir.
Note: Estimated reagent thawing time is 30 minutes.
Amplification Plate
If the Amplification Plate was frozen after the DNA
amplification step.
Place the deepwell plate
in a small water bath
for 50 minutes until all
wells have thawed.
If the Amplification Plate was not frozen after the
DNA amplification step.
[1]
For example, on the benchtop at room temperature, pour ultra-pure water into a small tray.
Centrifuge at 1,000 rpm for
[1]
30 seconds.
Do not vortex.
the cooling block.
the cooling block.
4 CytoScan
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HT-CMA Assay 96-Array Format Automated Workflow Quick Reference—Applied Biosystems™ NIMBUS™ Instrument
Run the fragmentation step, then precipitate samples
1. In the NIMBUS Target Preparation Setup window, select Fragmentation, then click Run.
2. Set up the deck as indicated in the deck setup prompt and following screens, then click Run. See Figure 2.
3. After ensuring that the deck layout is correct, click Run, then click Yes in the confirmation window to start.
The fragmentation method starts. The sample plate is incubated at 65°C to inactivate amplification. When complete the
Fragmentation—Cleanup window.
4. Remove the Fragmentation Plate from deck position 8.
The Fragmentation Plate is now known as the Precipitation Plate.
a. Blot the top of the plate with a laboratory tissue, then seal tightly.
b. Place the plate in a –20°C freezer overnight to precipitate the DNA.
Note: A 3-hour precipitation workflow option is also available. See the CytoScan™ HT-CMA Assay 96-Array Format Automated
Workflow User Guide—Applied Biosystems™ NIMBUS™ Instrument , Pub. No. MAN0018211.
5. Save or discard the labware as instructed.
6. Click Finish when deck cleanup is complete. Click Yes in the confirmation window.
7. After the incubation period, proceed directly to “Stage 3: Centrifuge and dry pellets” on page 7.
CytoScan™ HT-CMA Assay 96-Array Format Automated Workflow Quick Reference—Applied Biosystems™ NIMBUS™ Instrument 5
Stage 2 deck layout
1
3
4
5
6
7
8
11
1
2
3
4
5
6
7
8
9
10
11
Part No. 101179 Rev. 1
HT Target Prep Reagent Kit Template - NIMBUS
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Part No. 101179 Rev. 1
HT Target Prep Reagent Kit Template - NIMBUS
Figure 2 Stage 2: Fragment and precipitate the DNA deck layout.
Round deepwell plate (Amplified gDNA)
1
Cooling block and tube collar. Load reagent tubes only in the
2
yellow column of the reagent block. Use the template as guidance
for specific reagent placement.
4-column reservoir & Reservoir frame (precipitation reagents)
3
• Column 1: (Empty)
• Column 2: Isopropanol
• Column 3: Isopropanol
• Column 4: Precip Solution 1
4-column reservoir & Reservoir frame (fragmentation reagents)
4
• Column 1: 10X Frag Buer
• Column 2: (Empty)
• Column 3: (Empty)
• Column 4: Frag Reaction Stop
Square 1.2-mL plate & Alpillo™ Plate Cushion
5
(Empty)
6
96-well full-skirt plate & Plate collar
7
Square deepwell plate
8
CO-RE™ Filter Tips 1,000 µL
9
CO-RE™ Filter Tips 300 µL
10
CO-RE™ Filter Tips 300 µL & Square deepwell plate
11
6 CytoScan
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HT-CMA Assay 96-Array Format Automated Workflow Quick Reference—Applied Biosystems™ NIMBUS™ Instrument
Stage 3: Centrifuge and dry pellets
Time required
Activity Time
Hands-on time ~10 minutes
Centrifugation 40 minutes
Drying 25 minutes
Total ~75 minutes
Input required
One plate of precipitated samples from Stage 2 in a square deepwell plate.
Equipment required
• Incubator/oven, temperature at 37°C
• Centrifuge, at 4°C
Centrifuge and dry the pellets
Note: Keep the centrifuge ready at 4°C.
1. Transfer the Precipitation Plate from the –20°C freezer to a pre-chilled centrifuge.
2. Centrifuge the plate for 40 minutes at 4°C at 3,200 x g.
3. Immediately after the 40-minute centrifugation time, empty the liquid from the plate using the following steps:
a. Remove the seal.
b. Invert the plate over a waste container, then allow the liquid to drain.
Note: It is normal for the intensity of the blue color between pellets to vary and the color variation does not indicate any
significant dierences in the yield of precipitated DNA.
c. While still inverted, gently press the plates on a pile of laboratory tissues on a bench, then allow them to drain for 5 minutes.
Transfer the plates to a new pile of tissues twice during the 5-minute time frame.
4. Turn the plate right side up and place in an oven for 20 minutes at 37°C to dry.
5. Do one of the following:
• Proceed directly to “Stage 4A: Prepare the resuspension buer” on page 8, even if some droplets of liquid remain. Leave the
sample plate at room temperature. It is helpful to start preparing reagents for Stage 4A and 4B while centrifuging and drying
pellets.
• Store the plates for resuspension later in the same day. Tightly seal the plates.
– If resuspension is carried within 4 hours, keep the plates at room temperature.
– If resuspension is carried out in more than 4 hours, store the plates in a refrigerator (2 to 8°C).
• Store the plates for resuspension on another day. Tightly seal the plate and store at –20°C.
CytoScan™ HT-CMA Assay 96-Array Format Automated Workflow Quick Reference—Applied Biosystems™ NIMBUS™ Instrument 7
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