Thermo Fisher Scientific Axiom 2.0 Assay 24-Array Format Owner's manual

Brand
Thermo Fisher Scientific
Model
Axiom 2.0 Assay 24-Array Format
Type
Owner's manual
Axiom™ 2.0 Assay 24-Array Format Manual Workflow
Catalog Number 902798
Pub.No. MAN0018147 Rev. D.0
Note: For safety and biohazard guidelines, see the “Safety” appendix in the Axiom™ 2.0 Assay 24-Array Format Manual Workflow User
Guide (Pub.No.703335). Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear,
clothing, and gloves.
Introduction
Running the Axiom™ 2.0 Assay 24-Array Format Manual Workflow requires the following sets of steps:
1. Genomic DNA preparation, described in the Axiom™ 2.0 gDNA Sample Preparation Quick Reference (Pub. No. MAN0017720), the
Axiom™ gDNA Sample Prep for Genome-Wide BOS 1 Array Plate Quick Reference (Pub. No. 702975), Chapter 2 in the Axiom™ 2.0
Assay 24-Array Format Manual Workflow User Guide (Pub No. 703335), or Chapter 2 in Section 1, in the Axiom™ Microbiome Assay
Protocol in the Axiom™ Microbiome Solution User Guide (Pub. No. 703408).
2. Manual target preparation of the samples, described in this document.
3. Array processing, described in GeneTitan™ MC Protocol for Axiom™ Array Plate Processing Quick Reference (Pub. No.
MAN0017718).
IMPORTANT! This document contains an abbreviated set of instructions used to perform target preparation. Carefully read all the
instructions in the target preparation chapter Axiom™ 2.0 Assay 24-Array Format Manual Workflow User Guide (Pub. No. 703335) before
performing manual target preparation.
Note: Array handling and processing protocols still require the use of a GeneTitan™ MC Instrument, as described in Chapter 5, Array
Processing with the GeneTitan™ Multi-Channel (MC) Instrument in the Axiom™ 2.0 Assay 24-Array Format Manual Workflow User Guide.
Assay notes
• This manual assay format allows the user to run the Axiom™ 2.0 Assay for 24 samples 4 times using 1 Axiom™ 2.0 Reagent Kit 4x24
Reactions (Cat. No. 902798) or 1 Axiom™ Microbiome Reagent Kit 4x24 Reactions (Cat. No. 902910). All samples are processed in
Columns 5, 7, and 9.
• This manual assay uses disposable divided reservoirs with a “trough-within-a-trough” design, which maximizes the amount of liquid
accessible to pipette tips when using small amounts of reagent. During the assay, dispense reagents into either the 8-channel,
4-channel, or both sides, as instructed.
• We recommend that you prepare your genomic DNA sample plate in a clean room.
• Remove seals from plates carefully and discard used seals. Do not reuse seals.
• Use 8-channel pipettes for all sample transfers and additions of reagents and master mixes to the samples and GeneTitan™ trays.
• Change pipette tips after each sample transfer or addition to the samples.
• Unless otherwise specified, all reagent modules are from the Axiom™ 2.0 Reagent Kit 4x24 Reactions or Axiom™ Microbiome Reagent
Kit 4x24 Reactions. See Chapter 4 of the Axiom™ 2.0 Assay 24-Array Format Manual Workflow User Guide (Pub. No. 703335) for a
complete list of equipment and consumables required for each stage.
QUICK REFERENCE
For Research Use Only. Not for use in diagnostic procedures.
Stage 1—DNA amplification
Prepare for Stage 1
Supplies required
Reagents from Axiom™ 2.0 Reagent Kit 4x24 Reactions (Cat. No. 902798) or Axiom™ Microbiome Reagent Kit 4x24 Reactions (Cat. No.
902910), Module1, –20°C, Part No. 901711.
Set up the instruments and prepare the reagents
1. Set an incubator/oven temperature at 37°C.
2. Set the centrifuge to room temperature.
3. Thaw and prepare reagents from Module1 of the Axiom™ 2.0 Reagent Kit 4x24 Reactions (Cat. No. 902798) or the Axiom™
Microbiome Reagent Kit 4x24 Reactions (Cat. No. 902910).
Module Reagent and cap color Treatment
Module1
Part No. 901711
–20°C
Axiom™ 2.0 Denat Soln10X Thaw, vortex, centrifuge, then keep at room temperature.
Axiom™ 2.0 Neutral Soln Thaw (see note below), vortex for 30 seconds to thoroughly mix, then keep at
room temperature.
Axiom™2.0 Amp Soln Thaw (see note below), vortex for 30 seconds to thoroughly mix, then keep at
room temperature.
Axiom™2.0 Amp Enzyme[1] Place in cooler and keep at −20°C until ready to use. Gently invert and flick
tube 3 times to mix, then centrifuge before use.
Axiom™ Water Thaw (see note below), vortex, then keep at room temperature.
[1] Leave at –20°C until ready to use.
Note: Allow ~1 hour for Axiom™2.0 Amp Soln to thaw on the benchtop at room temperature. If the solution is not thawed after 1
hour, vortex briefly and return to the benchtop to complete thawing. The bottles can also be thawed in a dish with ultra-pure water.
The Axiom™2.0 Amp Soln must be thoroughly mixed before use.
4. Thaw samples in the Sample Plate.
a. Bring the samples to room temperature on the benchtop.
b. Centrifuge the plate.
c. Leave at room temperature.
IMPORTANT!
·The samples must be brought to room temperature before proceeding with denaturation.
·The Sample Plate for genotyping studies must have 20 μL of each gDNA diluted to a concentration of 5ng/μL or 10ng/μL,
as required according to the sample type, in columns 5, 7, and 9 of an ABgene 96 square well storage plate, 2.2mL or
Eppendorf 96 Deepwell Plate, 2,00 μL.
·The Sample Plate for Microbiome studies must have 20μL of stool gDNA diluted to a concentration of 2.5ng/μL and/or
17.5μL cDNA must be diluted with 2.5μL reduced EDTA TE buer in columns 5, 7, and 9 of the Eppendorf 96 Deepwell
Plate, 2,000μL. A no template (NTC) control must be plated in well G09 and Genomic DNA Standard (Ref 103) must be
plated in well H09.
5. Label the 1.7-mL microcentrifuge tube and the 15 mL conical tube as indicated in the following table.
Label Tube size Temperature Contents
D MM 1.7 mL microcentrifuge tube Leave tube at room temperature. Denaturation Master Mix
Amp MM 15 mL conical tube Leave tube at room temperature. Amplification Master Mix
2 Axiom™ 2.0 Assay 24-Array Format Manual Workflow Quick Reference
6. Label 3 solution basins (Matrix™ Reagent Reservoir with Divider, 25 mL) as indicated in the following table.
Label Temperature Contents Reservoir side
D MM Leave basin at room temperature. Denaturation Master Mix 8-channel
N Soln Leave basin at room temperature. Axiom™ 2.0 Neutral Soln 8-channel
Amp MM Leave basin at room temperature. Amplification Master Mix 8-channel
Prepare the Denaturation Master Mix
Prepare the Denaturation Master Mix at room temperature.
1. To the 1.7-mL microcentrifuge tube labeled “D MM”, use the following table to dilute the appropriate volume of Axiom™ 2.0 Denat
Soln10X using the Axiom™ Water.
Table1Denaturation Master Mix.
Reagent and cap color Per sample Master mix 24+
Axiom™ Water 18µL 1,044µL
Axiom™ 2.0 Denat Soln10X 2µL 116µL
Total volume 20µL 1,160µL
2. Vortex, centrifuge briefly, then leave at room temperature.
Add the Denaturation Master Mix to the samples
Perform these steps at room temperature.
1. Briefly centrifuge the Sample Plate, if needed.
Samples must be at room temperature for this step.
2. Using a P1000 pipette, gently pipet or pour the Denaturation Master Mix into the 8-channel side of the reagent reservoir labeled “D
MM”.
3. Carefully remove the seal from the sample plate, then discard the seal.
4. Using a P20 8-channel pipette, add 20 µL of Denaturation Master Mix to each sample in columns 5, 7, and 9.
• Pipet directly into the liquid of each well. Do not mix by pipetting up and down.
• Change tips between each addition.
• This plate is now known as the Denaturation Plate.
5. Seal, then vortex the Denaturation Plate. Start the timer for a 10-minute incubation.
6. Briefly centrifuge the Denaturation Plate in a room-temperature centrifuge by bringing the centrifuge speed to 1,000 rpm (takes
~1 minute).
The centrifuge time is included in the 10-minute incubation.
7. Visually examine the volume in each well (should be 40μL/well) and:
a. Keep a record of any wells that visually appear to have an unusually low or higher volume, as these samples might need to be
repeated.
b. Do not stop to measure volumes. Proceed without delay.
8. Complete the 10-minute incubation on the benchtop at room temperature.
While completing the incubation at room temperature, prepare the Axiom™ 2.0 Neutral Soln as described in step1 in “Add Axiom™
2.0 Neutral Soln to samples” on page4.
9. After incubation, immediately add the Axiom™ 2.0 Neutral Soln as described in “Add Axiom™ 2.0 Neutral Soln to samples” on
page4.
Axiom™ 2.0 Assay 24-Array Format Manual Workflow Quick Reference 3
Add Axiom™ 2.0 Neutral Soln to samples
Perform these steps at room temperature.
1. Measure 3.64 mL of Axiom™ 2.0 Neutral Soln, then slowly pipet the reagent into the 8-channel side of the reagent reservoir labeled
“N Soln”.
2. Carefully remove the seal from the Denaturation Plate, then discard the seal.
3. Use a P200 8-channel pipette to add 130μL of Axiom™ 2.0 Neutral Soln to each sample in columns 5, 7, and 9.
• Pipet down the wall of each well. Change tips between each addition.
• The plate is now known as the Neutralization Plate.
4. Seal, vortex, then briefly centrifuge the Neutralization Plate.
5. Visually examine the volume in each well. Total volume should be ~170μL/well.
a. Keep a record of any wells that visually appear to have an unusually low or higher volume as these samples might need to be
repeated.
b. Do not stop to measure volumes.
6. Proceed immediately to “Prepare the Amplification Master Mix” on page4.
Prepare the Amplification Master Mix
Perform these steps at room temperature.
1. Using the following table, pipette the appropriate amount of Axiom™2.0 Amp Soln into the 15-mL tube labeled “Amp MM”.
Table2Amplification Master Mix.
Reagent and cap color Per sample Master mix 24+
Axiom™2.0 Amp Soln 225µL 6.75mL
Axiom™2.0 Amp Enzyme 5µL 150 μL
Total volume 230µL 6.90mL
Note: The Axiom™2.0 Amp Soln is a viscous solution. To ensure that the reagent transfer is accurate:
·Pipet slowly.
·Allow bubbles that are generated from mixing to settle at the top before pipetting.
·Use a 10-mL serological pipette to transfer the Axiom™2.0 Amp Soln into the tube labeled “Amp MM”.
2. Remove the Axiom™2.0 Amp Enzyme from the freezer, then place in a portable cooler at –20°C.
a. Invert, then flick the Axiom™2.0 Amp Enzyme tube 3 times, then briefly centrifuge.
b. Using the table, add the appropriate amount of Axiom™2.0 Amp Enzyme to the tube labeled “Amp MM”.
c. Vortex the Amplification Master Mix well, invert the tube 2 times, then vortex again.
4 Axiom™ 2.0 Assay 24-Array Format Manual Workflow Quick Reference
Add the Amplification Master Mix to the samples
Perform these steps at room temperature.
1. Slowly pour the Amplification Master Mix to the 8-channel side of the reagent reservoir labeled “Amp MM”.
2. Carefully remove the seal from the Neutralization Plate, then discard the seal.
3. Using a P1200 8-channel pipette, slowly add 230μL of Amplification Master Mix to columns 5, 7, and 9 of the Neutralization Plate.
• Pipet down the wall of the well (total volume: 400 µL/well). Do not mix by pipetting up and down.
• Change tips between each addition.
Note: After adding the Amplification Master Mix, the plate is now known as the Amplification Plate.
4. Blot the top of the plate with a laboratory tissue. Seal tightly, vortex twice, then centrifuge the Amplification Plate for 1 minute at
1,000 rpm.
5. Place the sealed Amplification Plate in an oven set at 37°C, then leave undisturbed for 23 ±1hours.
Note: If using a GeneChip™ Hybridization Oven, place the plate on the bottom of the oven. Plates do not rotate.
6. Gather all the reagents from Module 1, tighten all caps, then mark the reagent pouches, tubes, and bottles to track use. Store at
–20°C.
Freeze the plate or proceed to Stage 2
After the incubation finishes, do one of the following:
• Proceed to “Stage 2—Fragmentation and precipitation” on page6.
• Store the Amplification Plate at –20°C.
Note: If freezing, do not perform the stop amplification reaction step described in Stage 2 before you store the sample plate at –20°C. The
stop amplification reaction step is performed after thawing the frozen plate.
Axiom™ 2.0 Assay 24-Array Format Manual Workflow Quick Reference 5
Stage 2—Fragmentation and precipitation
Prepare for Stage 2
Supplies required
• Selected reagents from Axiom™ 2.0 Reagent Kit 4x24 Reactions or Axiom™ Microbiome Reagent Kit 4x24 Reactions.
– Module2‑1, –20°C, Part No. 901528
– Module2‑2, 2-8°C, Part No. 901529
• Isopropanol (supplied by user)
Instrument setup
• Prepare the following instruments for this stage before you begin the assay:
– One oven at 65°C
– One oven at 37°C
– One centrifuge at room temperature
Note: If the Amplification Plate was frozen at the end of Stage 1, thaw the plate before beginning Stage 2. See “Thaw and prepare the
amplified DNA samples and reagents” in Chapter 4 of the Axiom™ 2.0 Assay 24-Array Format Manual Workflow User Guide (Put No.
703335) for detailed instructions.
Note: Keep a balance plate ready to avoid delays during the fragmentation steps.
Thaw and prepare the reagents
Thaw and prepare the fragmentation and precipitation reagents from Module2‑1 and Module2‑2 of the Axiom™ 2.0 Reagent Kit 4x24
Reactions (Cat. No. 902798) or the Axiom™ Microbiome Reagent Kit 4x24 Reactions (Cat. No. 902910)..
Module Reagent and cap color Treatment
Module2‑1
Pouch1of2
Part No. 901528
–25°C to –15°C
Axiom™ Frag Enzyme Leave at –20°C until ready to use. Gently flick the tube 3 times to mix. Centrifuge
before use.
Axiom™ 10X Frag Buer Thaw on the benchtop to room temperature. When thawed, place on ice. Vortex
before use.
Axiom™ Precip Soln2 Thaw on the benchtop to room temperature. Vortex and centrifuge before use.
Module2‑2
Pouch2of2
Part No. 901529
2°C to 8°C
Axiom™ Frag Diluent Keep in refrigerator or place on ice.
Axiom™ Frag Rxn Stop Warm on the benchtop to room temperature. Vortex before use.
Axiom™ Precip Soln1 Warm on the benchtop to room temperature. Vortex before use.
N/A Isopropanol Keep in room temperature.
Label tubes and reagent reservoirs
1. Label the 15-mL conical tube as indicated in the following table.
Label Temperature Contents
Frag MM Place tube on ice Fragmentation Master Mix
Precip MM Place tube at room temperature Precipitation Master Mix
2. Label the 4 reagent reservoirs as indicated in the following table.
Label Size Temperature Contents
Frag MM 25mL Room temperature Fragmentation Master Mix
Stop 25mL Room temperature Frag Rxn Stop
Precip MM 25mL Room temperature Precipitation Master Mix
ISO 100mL Room temperature Isopropanol
6 Axiom™ 2.0 Assay 24-Array Format Manual Workflow Quick Reference
Incubate samples in preheated ovens
1. Stop the DNA amplification reaction.
a. Place the Amplification Plate in the 65°C oven.
• If proceeding directly from the end of Stage 1, transfer the Amplification Plate from the 37°C oven to the 65°C oven.
Ensure that the seal is still securely attached to the plate to minimize evaporation.
• If working with a frozen Amplification Plate, follow the guidelines in “Thaw and prepare the reagents” on page6 before
placing it in the 65°C oven.
b. Incubate for 20 minutes.
c. Prepare the fragmentation reagents as detailed in step1 in “Thaw and prepare the reagents” on page6 after starting the 65°C
incubation of the Amplification Plate.
2. Prepare for fragmentation.
a. Remove the Amplification Plate from the 65°C oven, then transfer the plate to the 37°C oven.
b. Incubate for 45 minutes.
Prepare the Fragmentation Master Mix
1. Start making the Fragmentation Master Mix when there are 5 minutes to the finish of the 37°C incubation.
2. Transfer the Axiom™ Frag Enzyme to a –20°C portable cooler until ready to use.
3. Use the appropriate single-channel pipettes to add the reagents to the 15-mL tube labeled “Frag MM” tube, in the order that is
shown in the following table.
Table3Fragmentation Master Mix.
Reagent and cap color Per sample Master mix 24+
Axiom™ 10X Frag Buer 45.7µL 1.69mL
Axiom™ Frag Diluent 10.3µL 381µL
Axiom™ Frag Enzyme 1.0µL 37µL
Total volume 57μL 2.11mL
a. Just before the end of the 45-minute 37°C incubation, flick the Axiom™ Frag Enzyme tube 2 to 3times, then centrifuge.
b. At the end of the 45-minute 37°C incubation, add the Axiom™ Frag Enzyme to the Fragmentation Master Mix.
Note: Leave the Axiom™ Frag Enzyme at –20°C until ready to use.
4. Vortex twice, then place on ice.
5. Using a P1000 pipette, slowly transfer the Fragmentation Master Mix in the 8-channel side of the reagent reservoir labeled “Frag
MM” placed at room temperature.
Add the Fragmentation Master Mix to the samples
IMPORTANT! Work quickly to perform this set of steps to minimize the time that the Fragmentation Plate is out of the 37°C oven.
1. Carefully remove the Amplification Plate from the 37°C oven and place on the bench top at room temperature.
Do not place the Amplification Plate on ice.
2. Add 57μL of Fragmentation Master Mix to each sample, pipetting directly into the liquid. Do not mix by pipetting up and down.
The plate is now known as the Fragmentation Plate.
3. Seal the plate, then vortex twice.
4. Start the timer for 30 minutes.
5. Briefly centrifuge the Fragmentation Plate in the room temperature plate centrifuge.
Axiom™ 2.0 Assay 24-Array Format Manual Workflow Quick Reference 7
6. Quickly transfer the plate to a 37°C oven, then incubate for 30 minutes.
CAUTION! Be watchful for the end of the 30-minute incubation step. Fragmentation is an exact 30-minute incubation step.
Longer and shorter incubation times may lead to poor performance of the assay.
Add the stop solution to the samples
Perform the following steps at room temperature.
1. A few minutes before the end of the 30-minute incubation, carefully transfer 988 μL of the Axiom™ Frag Rxn Stop solution into the
8-channel side of the reagent reservoir labeled “Stop”.
Leave the stop solution reservoir on the benchtop at room temperature.
2. Remove the Fragmentation Plate from the oven and place on the bench top at room temperature.
3. At the end of the 30-minute fragmentation incubation period, carefully remove the seal from the Fragmentation Plate, then discard
the seal.
4. Using a P20 8-channel pipette, end the fragmentation reaction by adding 19μL of Axiom™ Frag Rxn Stop solution to each sample.
• Do not mix by pipetting up and down.
• Pipette directly into the liquid of each well,.
• Change tips after each addition.
• Proceed immediately to the next step.
5. Seal, vortex, then centrifuge at 1,000rpm.
6. Leave the Fragmentation Plate on the bench top at room temperature while you prepare the Precipitation Master Mix.
Prepare the Precipitation Master Mix
Perform the following steps at room temperature.
1. Prepare the Precipitation Master Mix in a 15-mL conical tube labeled “Precip MM”. Add the reagents in the order and volumes
shown in the following table.
Table4Precipitation Master Mix.
Reagent Per sample Master mix 24+
Axiom™ Precip Soln1 238µL 6.19mL
Axiom™ Precip Soln2 2µL 52µL
Total volume 240µL 6.24mL
Note: Use a 5-mL serological pipette to pipet Axiom™ Precip Soln1.
2. Vortex the tube labeled “Precip MM”, then place on the benchtop at room temperature.
3. Pour the Precipitation Master Mix into the 8-channel side of the reagent reservoir labeled “Precip MM”.
4. Carefully remove the seal from the Fragmentation Plate, then discard the seal.
5. Using a P1200 8-channel pipette, add 240μL Precipitation Master Mix to each sample. Rest each pipette tip against the wall of each
well while delivering.
• You do not need to mix up and down.
• Change tips after each addition.
• After adding the Precipitation Master Mix, the plate is now known as the Precipitation Plate.
6. Seal the Precipitation Plate, vortex, then centrifuge briefly.
8 Axiom™ 2.0 Assay 24-Array Format Manual Workflow Quick Reference
Prepare and add isopropanol to the Precipitation Plate
1. Remove the Precipitation Plate from the centrifuge and place on the benchtop at room temperature.
2. Pour 20 mL of isopropanol into both sides of the reservoir.
3. Carefully remove the seal from the Precipitation Plate, then discard the seal.
4. Use a P1200 8-channel pipette to add 600 µL isopropanol to each sample, then mix well by pipetting up and down 6 or 7 times
within the solution to ensure mixing.
Observe the solution while it is within the tips—it should look homogeneous after pipetting 5 to 7 times. If not, repeat mixing a few
more times until the solution looks homogeneous.
• Do not vortex the plate after isopropanol addition to avoid cross-contamination of the samples.
• Change the tips after each addition.
5. Blot the top of the plate with laboratory tissue, then seal tightly.
6. Carefully transfer the sample plate into the –20°C freezer, then incubate overnight (16–24hours).
7. Gather all the reagents from Module2‑1 and Module2‑2 and tighten all caps. Mark reagent pouches, tubes, and bottles to track use.
Store Module2‑1 at –20°C and Module2‑2 at 4°C.
8. After incubation, proceed to “Stage 3—Centrifuge and drying, resuspension and hybridization preparation, and sample QC” on
page10.
Axiom™ 2.0 Assay 24-Array Format Manual Workflow Quick Reference 9
Stage 3—Centrifuge and drying, resuspension and hybridization preparation, and sample QC
Prepare for Stage 3
CAUTION! Some of the steps in this stage should be performed under a fume hood.
Supplies required
• Selected reagents from the Axiom™ 2.0 Reagent Kit 4x24 Reactions or Axiom™ Microbiome Reagent Kit 4x24 Reactions (see Table
3.1):
Module2‑1, –20°C, Part No, 901528
Module2‑2, 2–8°C, Part No. 901529
• Other reagents required for QC steps (optional):
– Invitrogen™ TrackIt™ Cyan/Orange Loading Buer (Cat. No. 10482028)
– Applied Biosystems™ 25bp DNA Ladder (Cat. No. 931343)
– UltraPure™ DNase/RNase-Free Distilled Water, 5 mL (Cat. No. 10977023; for OD and Dilution QC Plate preparation)
– Invitrogen™ E‑Gel™ 48 Agarose Gels, 4% (Cat. No.G800804)
Instrument setup
• Prepare the following instruments for this stage:
– Oven preheated to 37°C
– Plate centrifuge set at 4°C
– Thermo Fisher Scientific microplate shaker or Jitterbug™
Prepare the reagents for Stage 3
1. Prepare the Gel Diluent for Sample QC (100-fold dilution of TrackIt™ Cyan/Orange Loading Buer): Add 500µL of TrackIt™
Cyan/Orange Loading Buer to 49.95 mL of nuclease-free water. Mix well.
2. Thaw and prepare the reagents from Module2‑1 and Module2‑2 of the Axiom™ 2.0 Reagent Kit 4x24 Reactions (Cat. No. 902798) or
the Axiom™ Microbiome Reagent Kit 4x24 Reactions (Cat. No. 902910)..
Module Reagent and cap color Treatment
Module2‑1
Pouch1of2
Part No. 901528
–25°C to –15°C
Axiom™ Hyb Buer Warm to room temperature. Vortex before use.
Axiom™ Hyb Soln1 Thaw on the benchtop at room temperature. Vortex and centrifuge
before use.
Module2‑2
Pouch2of2
Part No. 901529
2°C to 8°C
Axiom™ Hyb Soln2 Warm to room temperature. Vortex and centrifuge before use.
Axiom™ Resusp Buer Warm to room temperature (1 hour). Vortex before use.
CAUTION! Some of the steps in this stage should be performed under a fume hood.
Stage 3A—Centrifuge the precipitation plate and dry the DNA pellet
Note: Keep the centrifuge ready at 4°C.
1. Transfer the Precipitation Plate from the –20°C freezer to a prechilled centrifuge.
2. Centrifuge the plate at 4°C at 3,200 x g for 40 minutes.
10 Axiom™ 2.0 Assay 24-Array Format Manual Workflow Quick Reference
3. Immediately after the 40-minute centrifugation time, empty the liquid from the plate.
CAUTION! During this step, handle the plate gently to avoid disturbing the pellets. Do not bump or bang the plate.
a. Carefully remove the seal from the Precipitation Plate, then discard the seal.
b. Invert the plate over a clean waste container and allow the liquid to drain. Collect liquid, then discard the liquid according to
local, state, and federal regulations.
c. While still inverted, gently press the plate on a pile of laboratory tissues on a bench, allow the plate to drain for 5 minutes.
Transfer the plate 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. After 20 minutes, remove the plate from the oven, even if some droplets of liquid remain, then do one of the following:
• Proceed directly to “Stage 3B—Resuspension and hybridization preparation” on page11, even if some droplets of liquid
remain. Leave the sample plate at room temperature. It is helpful to start preparing reagents for stage 3A and 3B while
centrifuging and drying pellets.
• Tightly seal the plates and store the plates for resuspension later in the same day.
– If resuspension is carried out 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 (2–8°C).
• Store the plates for resuspension on another day. Tightly seal the plate and store at –20°C.
Stage 3B—Resuspension and hybridization preparation
Prepare for resuspension and hybridization
• If a plate was stored at –20°C after drying the pellets, allow the plate to sit at room temperature for 1.5 hour before carrying out
resuspension.
• Make sure the Axiom™ Resusp Buer has equilibrated to room temperature before adding to the dry pellets in Step 1.
• Perform these steps at room temperature.
1. Pipet 1.4mL of Axiom™ Resusp Buer into the 8-channel side of a reagent reservoir. Transfer 35µL Axiom™ Resusp Buer to each
well of the sample plate with a dry pellet. Avoid touching pellets with the pipette tips.
After adding resuspension buer, the plate is known as the Resuspension Plate.
2. Seal the Resuspension Plate, then place the plate on one of the following shakers:
• Thermo Scientific™ Compact Digital Microplate Shaker: at speed 900 rpm for 10 minutes
• Jitterbug™: at speed 7 for 10 minutes
CAUTION! Perform the rest of the steps in this stage under a fume hood.
3. While the Resuspension Plate is shaking, prepare the Hybridization Master Mix in a 15mL tube as shown in the following table.
Vortex well to mix and pour contents in the 8-channel side of a reagent reservoir.
Table5Hybridization Master Mix.
Reagent and cap color Per sample Master mix 24+
Axiom™ Hyb Buer 70.5µL 2.26mL
Axiom™ Hyb Soln1 0.5µL 16µL
Axiom™ Hyb Soln2 9µL 288µL
Total Volume 80µL 2.56mL
4. Inspect the Resuspension Plate from the bottom. If the pellets are not dissolved, repeat Step 2 on page11. Briefly centrifuge.
5. Select a PCR plate appropriate to the type of approved thermal cycler that you will use in Stage 4, then label the plate “Hyb Ready
Plate [plate ID]”.
Axiom™ 2.0 Assay 24-Array Format Manual Workflow Quick Reference 11
6. Transfer the entire contents of each well in columns 5, 7, and 9 of the Resuspension Plate to the corresponding wells of the labeled
Hyb-Ready Plate.
7. Add 80 µL of the Hybridization Master Mix to each well in columns 5, 7, and 9 of the Hyb-Ready Plate.
8. Seal tightly, vortex, then briefly centrifuge.
9. Gather all the reagents from Module2‑1 and Module2‑2 and tighten all caps. Mark reagent pouches, tubes, and bottles to track use.
Store Module2‑1 at –20°C and Module2‑2 at 4°C.
Stage 3C—Recommended: Perform quantitation and fragmentation quality control checks
Before proceeding to Stage 4, we recommend that you perform quantitation and fragmentation QC checks.
1. Prepare the Dilution QC Plate.
a. Add 33 µL nuclease-free water to columns 5, 7, and 9 of a PCR plate labeled “Dil QC”.
b. Transfer 3 µL of the hybridization-ready sample from each well of the Hyb-Ready Plate to the corresponding well of the Dil QC
plate.
c. Seal, vortex, and briefly centrifuge.
2. Prepare and read the OD Plate.
a. Add 90 µL nuclease-free water to columns 5, 7, and 9 of the OD Plate (96-well UV-Star™ plate).
b. Transfer 10 µL of each Dilution QC Plate sample to the OD Plate and mix by pipetting up and down.
c. Read absorbance on a plate reader.
See Appendix B, Sample Quantitation after Resuspension of the Axiom™ 2.0 Assay 24-Array Format Manual Workflow User
Guide.
3. Prepare and run Gel QC samples.
a. Add 120µL Gel Diluent (100-fold dilution of TrackIt™ Cyan/Orange Loading Buer) to columns 5, 7, and 9 of the Gel QC Plate.
b. Transfer 3 µL of each Dilution QC Plate sample to the Gel QC Plate.
c. Seal, vortex, and briefly centrifuge.
d. Run the Gel:
See Appendix A, Fragmentation Quality Control Gel Protocol of the Axiom™ 2.0 Assay 24-Array Format Manual Workflow User
Guide.
Freeze or proceed to Stage 4
At this point you can:
• Proceed to “Stage 4—Denaturation and hybridization” on page13, or
• Store the hybridization-ready samples at –20°C.
12 Axiom™ 2.0 Assay 24-Array Format Manual Workflow Quick Reference
Stage 4—Denaturation and hybridization
Prepare for Stage 4
Supplies required
• Reagents from the Axiom™ 2.0 Reagent Kit 4x24 Reactions or Axiom™ Microbiome Reagent Kit 4x24 Reactions, Module3, Wash
Buer A (Part No. 901446), Wash Buer B (Part No. 901447), Axiom™ Water (Part No. 901578).
• Axiom™ 24-array plate in a protective base.
• Hybridization tray from the Axiom™ GeneTitan™ Consumables Kit (Cat. No. 901606)
Instruments and setup
• GeneTitan™ MC Instrument
• Approved thermal cycler.
– Must be programmed with the Axiom™ 2.0 Denature protocol of 95°C for 10 minutes; 48°C for 3 minutes; 48°C for hold.
– Use the heated lid option when setting up or running protocols.
• Hybridization-ready samples in plate appropriate to the thermal cycler model used.
• 96-well metal chamber pre-heated in a 48°C oven
CAUTION! Some of the steps of this stage should be performed under a fume hood.
Prepare the hybridization-ready samples stored at –20°C
1. Warm up the Hyb-Ready Plate at room temperature for 5 minutes.
It is not necessary to equilibrate the plate for a longer duration.
2. Ensure sure that the Hyb-Ready Plate is sealed well. If the plate is not sealed well:
a. Centrifuge the plate, then carefully remove the old seal.
b. If condensation is on the top of the plate, blot dry gently with a laboratory tissue.
c. Use a fresh seal to tightly reseal the plate.
3. Vortex the Hyb-Ready Plate briefly, then centrifuge at 1,000rpm for 30seconds.
4. Place the Hyb-Ready Plate at room temperature.
Prepare the GeneTitan™ MC Instrument and denature the hybridization-ready sample plate
1. Warm up the array plate on the bench top for a minimum of 25 minutes before setting up hybridization on the GeneTitan™ MC
Instrument.
2. At the end of the array warm up time, open the pouch and scan the array plate barcode into the Batch Registration file.
3. Before you denature the hybridization ready samples:
a. Prepare the reagents from Module3 by inverting the bottles 2 to 3 times to mix.
b. Upload the Batch Registration File.
See Appendix B, Register samples in GeneChip™ Command Console™, of the Axiom™ 2.0 Assay 24-Array Format Manual
Workflow User Guide.
c. Set up the GeneTitan™ MC Instrument.
See GeneTitan™ MC Protocol for Axiom™ Array Plate Processing Quick Reference (Pub. No. MAN0017718).
See Chapter 5, Array Processing with the GeneTitan™ MC Instrument of the Axiom™ 2.0 Assay 24-Array Format Manual
Workflow User Guide (P/N 703335).
4. Place the Hyb-Ready Plate in the thermal cycler block, secure the lid, and start the Axiom 2.0 Denature protocol.
Axiom™ 2.0 Assay 24-Array Format Manual Workflow Quick Reference 13
Prepare the hybridization tray and load into the GeneTitan™ MC Instrument
The following instructions assume familiarity with techniques and procedures that are associated with loading and operating the
GeneTitan™ MC Instrument. If needed, review procedures before starting.
CAUTION! Perform these steps under a fume hood.
1. After the Axiom 2.0 Denature protocol has completed, remove the Hyb-Ready Plate from the thermal cycler and place it into a
96-well metal chamber that has been prewarmed in an oven at 48°C.
2. Move the metal chamber containing the denatured Hyb-Ready Plate to a fume hood.
3. Remove the seal from the Hyb-Ready Plate, then discard.
4. Remove the hybridization tray from packaging.
5. Label the hybridization tray.
IMPORTANT! It is critical that you write only on the proper location of the hybridization tray (on the edge in front of wells A1 and
B1). Do not write on any other side, because the writing can interfere with sensors inside the GeneTitan™ MC Instrument and result in
experiment failure.
1
3
2
Figure1Label the GeneTitan™ hybridization tray.
• Do not label trays on the long side of the tray.
• Notched corner of the hybridization tray should face the front.
• Label the hybridization tray in this area.
6. Place the hybridization tray under the fume hood.
7. Using a P200 8-channel pipette set at 105 µL, slowly transfer the denatured samples in columns 5, 7, and 9 from the Hyb-Ready
Plate into the corresponding wells of the hybridization tray. Dispense to the first stop to avoid creating bubbles.
• Change pipette tips after each transfer. Discard the tip even if it shows some remaining volume.
• Ensure that there are no air bubbles present in the hybridization tray. Puncture any air bubbles that you see using a clean pipette
tip.
• There is no need to spread the sample on the bottom of the hybridization tray wells. Sample distribution across wells occurs
when the array plate is stacked together with the hybridization tray by the GeneTitan™ MC Instrument.
14 Axiom™ 2.0 Assay 24-Array Format Manual Workflow Quick Reference
8. Load the array plate and hybridization tray into the GeneTitan™ MC Instrument.
The hybridization tray must be loaded on the right side. The array plate must be loaded on the left side on its protective blue base.
The clear plastic cover on top of the array plate should not be loaded into the GeneTitan™ MC Instrument.
12
Figure2Array plate on protective blue base and the hybridization tray properly loaded into drawer 6.
1Array plate on protective base. 2Hybridization tray.
IMPORTANT! After the GeneTitan™ MC Instrument has stacked the array plate and hybridization tray, the instrument extends the
drawer. Manually check the stacking by gently pressing the 6 latching points to ensure that the 2 parts are clamped properly, and
check underneath the arrays to ensure that there are no bubbles. If bubbles are found, gently tap the plate on top to eliminate the
bubbles. Do not tip/tilt the array plate/hybridization tray sandwich as you are inspecting the bottom for bubbles.
9. Follow the prompts in the software..
Hybridization continues on the GeneTitan™ MC Instrument for 23.5–24 hours before you can load the ligation, staining, and
stabilization reagent trays into the GeneTitan™ MC Instrument.
10. Near the end of the 23.5–24 hour hybridization in the GeneTitan™ MC Instrument and approximately 1.5 hours from completion
(22 hours after the start of hybridization), proceed to “Stage 5—GeneTitan™ reagent preparation” on page16.
Axiom™ 2.0 Assay 24-Array Format Manual Workflow Quick Reference 15
Stage 5—GeneTitan™ reagent preparation
Prepare for Stage 5
Equipment required
Quantity Item
Equipment
1 GeneTitan™ MC Instrument
1 GeneTitan™ ZeroStat AntiStatic Gun
1 Microcentrifuge
1 Electronic pipettor for serological pipettes
1 each Pipettes
• Single-channel P200
• Single-channel P1000
• Multichannel P200
As required Disposable 10-mL serological pipettes
1 Vortexer
1 Portable cooler for enzyme
Consumables required
Quantity Item
As required Aluminum foil (optional)
• 1
• 5
• 5
Axiom™ GeneTitan™ Consumables Kit (Cat. No. 901606)
• Scan Tray with top cover and protective base
• Stain Tray
• Covers for trays
As required Pipette tips
5 Matrix™ 25-mL Sterile Reagent Reservoir with Divider (Cat. No. 8095)
4 15-mL conical tube
1 5 mL serological pipette
16 Axiom™ 2.0 Assay 24-Array Format Manual Workflow Quick Reference
Thaw and prepare the reagents
Prepare the reagents that are required for Stage 5 according to the following table.
Module Reagent and cap color Thaw, then
place onice
Place on
ice
Place at room
temperature Reagent handling
Module 4-1
Pouch1of2
Part No. 901278
–20°C
Axiom™ Ligate Buer[1]
Place on bench top at room temp for 30
minutes. Vortex twice for 30 seconds.
Examine for precipitate. If any, warm bottle
with your hands and vortex again for 30
seconds.
Axiom™ Ligate Enzyme[2] Do not thaw. Keep at −20°C until ready
to use.
Immediately before use: Gently flick tube
3 times, then centrifuge briefly. Place in a –
20°C portable cooler until use.
Axiom™ Ligate Soln1 Vortex, then centrifuge briefly.
Axiom™ Probe Mix1 Vortex, then centrifuge briefly.
Axiom™ Stain Buer Vortex, then centrifuge briefly.
Axiom™ Stabilize Soln Vortex, then centrifuge briefly.
Module 4-2
Pouch2of2
Part No. 901276
2°C to 8°C
Axiom™ Ligate Soln2 Vortex, then centrifuge briefly. When thawed,
do not place on ice.
Axiom™ Probe Mix2[2] Gently flick tube 3 times, then centrifuge
briefly.
Axiom™ WashA[1]
for
30 minutes
Vortex well. Place on bench for 30 minutes.
Look for precipitate. Vortex again if
necessary.
Axiom™ Stain1‑A[2] Gently flick tube 3 times, then centrifuge
briefly.
Axiom™ Stain1‑B[2] Gently flick tube 3 times, then centrifuge
briefly.
Axiom™ Stain2‑A[2] Gently flick tube 3 times, then centrifuge
briefly.
Axiom™ Stain2‑B[2] Gently flick tube 3 times, then centrifuge
briefly.
Axiom™ Stabilize Diluent Vortex, then centrifuge briefly.
Look for precipitate. If any, warm tube to
room temperature and vortex again.
Axiom™ Water
Axiom™ Hold Buer[2], [3] Vortex for 30 seconds. Pour in reservoir.
Estimated reagent thawing time is ~1 hour.
[1] Check for precipitate. If precipitate is present, repeat the vortex and centrifuge step.
[2] These solutions are light sensitive. Keep tubes out of direct light for a prolonged period of time.
[3] Axiom™ Hold Buffer for preparing the scan tray for the 2nd, 3rd, and 4th plate are provided in Module5‑2, Pouch 2of 2.
Note: The presence of some precipitate in Axiom™ Ligate Buer will not adversely impact assay performance. Follow the instructions
above to resuspend any precipitate before use
Note: Occasionally, crystals are observed in Axiom™ WashA and Axiom™ Stabilize Diluent upon removal from 2-8°C storage. Before using
these solutions, the crystals should be dissolved by warming the solutions to room temperature and then vortexing.
Axiom™ 2.0 Assay 24-Array Format Manual Workflow Quick Reference 17
Label master mix tubes and reagent reservoirs
1. Label side of each conical tube as indicated in the following table.
Tube size Label Contents
15mL S1 Stain 1 Master Mix
15mL S2 Stain 2 Master Mix
15mL Stbl Stabilization Master Mix
15mL Lig Ligation Master Mix
2. Place the 4 tubes on ice.
3. Label the five Matrix™ Reagent Reservoirs with Divider, 25-mL, as indicated in the following table.
Label Contents
S1 Stain 1 Master Mix
S2 Stain 2 Master Mix
Stbl Stabilization Master Mix
Lig Ligation Master Mix
Stop Axiom™ Hold Buer
Prepare the Stain, Ligation, and Stabilization Master Mixes
Prepare the Stain 1 Master Mix
1. Use appropriate serological and single-channel pipettes to add reagents to the 15-mL tube labeled “S1” in the order shown in the
following table.
This recipe provides enough for both S1 reagent trays.
Table6Stain 1 Master Mix.
Reagent and cap color Per array Master mix 24+
Axiom™ WashA 201.6µL 5.24 mL
Axiom™ Stain Buer 4.2µL 109.2 µL
Axiom™ Stain1‑A 2.1µL 54.6 µL
Axiom™ Stain1‑B 2.1µL 54.6 µL
Total 210 µL (105 µL x 2) 5.46 mL
2. Gently invert the tube 10 times to mix. Do not vortex.
3. Place on ice, then protect from direct light (for example, cover with aluminum foil or ice bucket lid).
18 Axiom™ 2.0 Assay 24-Array Format Manual Workflow Quick Reference
Prepare Stain 2 Master Mix
1. Use appropriate serological and single-channel pipettes to add reagents to the 15-mL tube labeled “S2” in the order shown in the
following table.
Table7Stain 2 Master Mix.
Reagent and cap color Per array Master mix 24+
Axiom™ WashA 100.8µL 2.62 mL
Axiom™ Stain Buer 2.1µL 54.6 μL
Axiom™ Stain2‑A 1.05µL 27.3 µL
Axiom™ Stain2‑B 1.05µL 27.3 µL
Total 105 µL 2.73 mL
2. Gently invert the S2 MM tube 10 times to mix. Do not vortex.
3. Place on ice, then protect from direct light (for example, cover with aluminum foil or ice bucket lid).
Prepare Stabilization Master Mix
1. Use appropriate serological and single-channel pipettes to add reagents to the 15-mL tube labeled “Stbl” in the order shown in the
following table.
Table8Stabilization Master Mix.
Reagent and cap color Per array Master mix 24+
Axiom™ Water 93.19µL 2.38 mL
Axiom™ Stabilize Diluent 10.50µL 268 μL
Axiom™ Stabilize Soln 1.31µL 33.4 µL
Total 105 µL 2.68 mL
2. Vortex the master mix at high speed for 3seconds.
3. Place on ice.
Prepare Ligation Master Mix—part 1
The Ligation Master Mix is prepared in 2 parts.
1. Place the 15-mL conical tube marked “Lig” on ice.
2. Use appropriate serological and single-channel pipettes to add reagents to the 15-mL tube labeled “Lig” in the order shown in the
following table.
Table9Ligation Master Mix—part 1.
Reagent and cap color Per array Master mix 24+
Axiom™ Ligate Buer 66.15µL 1.75 mL
Axiom™ Ligate Soln1 13.12µL 348 μL
Axiom™ Ligate Soln2 3.15µL 83 µL
Total 82.42 µL 2.18 mL
3. Vortex the master mix tube at high speed for 3seconds.
4. Place on ice.
Axiom™ 2.0 Assay 24-Array Format Manual Workflow Quick Reference 19
Prepare Ligation Master Mix—part 2
The Ligation Master Mix is prepared in 2 parts.
IMPORTANT! Module5‑1 of the Axiom™ 2.0 Reagent Kit 4x24 Reactions contains an extra tube of Axiom™ Ligate Enzyme, which has
been provided for back-up purposes. If there is insucient volume of the Axiom™ Ligate Enzyme for the preparation of the fourth (4th)
24-sample Ligation Master Mix, discard the tube and its contents and proceed to use the second tube of Axiom™ Ligate Enzyme. The
remaining contents of the second tube of Axiom™ Ligate Enzyme may be discarded or stored in the freezer for future use.
1. Remove the Axiom™ Ligate Enzyme from the –20°C freezer, then place in a cooler chilled to –20°C.
2. Use appropriate serological and single-channel pipettes to add reagents to the 15-mL tube labeled “Lig” in the order shown in the
following table.
Note: Gently flick the Axiom™ Ligate Enzyme tube 2—3 times, then centrifuge briefly immediately before adding the enzyme to the
master mix.
Table10Ligation Master Mix—part 2.
Reagent Per array Master mix 24+
Ligation Master Mix from Stage 1 82.42 µL 2.18 mL
Axiom™ Probe Mix1 10.5µL 278 μL
Axiom™ Probe Mix2 10.5µL 278 μL
Axiom™ Ligate Enzyme 1.58µL 42 µL
Total 105 µL 2.78 mL
3. Gently invert the master mix tube 10 times to mix. Do not vortex.
4. Place on ice, then protect from direct light (for example, cover with aluminum foil or ice bucket lid).
Aliquot master mixes and Axiom™ Hold Buer into trays
Note: It is not necessary to change pipette tips between additions of the same reagents to stain trays and scan trays.
Prepare trays and covers
1. Label 2 stain trays “Stain1” or “S1-1” and “S1-2” (for Stain 1 Master Mix).
2. Label the remaining stain trays:
• “Stain2” (for Stain 2 Master Mix)
• “Stbl” (for Stabilization Master Mix)
• “Lig” (for Ligation Master Mix)
3. Destatic the inside of each tray and cover.
See Deionization of GeneTitan™ trays and covers in Appendix A of the Axiom™ 2.0 Assay 24-Array Format Manual Workflow User
Guide for the recommended technique.
20 Axiom™ 2.0 Assay 24-Array Format Manual Workflow Quick Reference
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Thermo Fisher Scientific Axiom 2.0 Assay 24-Array Format Owner's manual

Brand
Thermo Fisher Scientific
Model
Axiom 2.0 Assay 24-Array Format
Type
Owner's manual
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