Quick Reference Card, Axiom Automated Target Prep Protocol

Quick Reference Card, Axiom Automated Target Prep Protocol
Quick Reference Card
Axiom™ Automated Target Prep Protocol
Stage 1. DNA Amplification
Introduction
Running the Axiom Assay requires the following sets of steps:
1. Genomic DNA Prep as described in Axiom™ gDNA Sample Prep Quick Reference Card (QRC) (P/N 702928).
2. Target Prep of the samples, performed using either:
… Automated Target Prep, described in this QRC.
… Manual Target Prep, described in Axiom™ Manual Target Prep Protocol QRC (P/N 702927).
3. Array Processing, described in GeneTitan® MC Protocol for Axiom™ Array Plate Processing QRC (P/N 702929).
This QRC describes the automated target prep, performed using the Biomek FXP Target Prep Express.
IMPORTANT: This QRC contains an abbreviated set of instructions. You must carefully read all the instructions in Chapter 3, Axiom GenomeWide Assay: Target Prep with Biomek of the Axiom Genome-Wide Human Assay User Manual (P/N 702830) before running the Automated
Target Prep Method.
The Axiom Genome-Wide Human Assay User Manual covers the assay steps in more detail and provides information on running multiple plates
per week through the automated target prep process.
WARNING: Make sure the PTC200 lid is closed before homing the axes or starting a method.
NOTE: The Biomek FXp should be homed before the first run of the day.
STAGE 1: DNA Amplification
Genomic DNA Plate Preparation
We recommend that you prepare your genomic DNA sample plate in a clean room. The clean room should be separate from the laboratory
where the Axiom Genome-Wide Human Assay is performed and should be free of DNA amplified in other procedures.
1. Performing DNA Amplification
1. Set the incubator/oven temperature at 30 °C.
2. Set the centrifuge temp at room temperature.
3. Prepare reagents from Module 1 (P/N 901275) as shown in Table 1.1:
Table 1.1. Reagents Preparation for Stage 1
Reagent
Axiom Amp Soln
Temp Out of Module*
Treatment
Thaw at Room Temp (~1 hr)
Vortex twice
Axiom Water
Thaw at Room Temp
Vortex
Axiom Denat Soln
Thaw at Room Temp
Vortex and spin
Axiom Neutral Soln
Thaw at Room Temp
Vortex and spin
Axiom Amp Enzyme Soln
Keep at -20 °C
Just before use, flick tube 3X, spin, and place in the cold block
*Temp Out of Module: temperature reagent is held at immediately after removal from module
4. Thaw Samples in gDNA Plate:
A. Bring your gDNA samples to room temperature on the bench top.
B. Vortex and spin.
C. Leave at room temperature.
5. Run Biomek method:
A. Select the array plate format and DNA Amplification step, then click OK.
B. Set up the deck as indicated in the deck setup prompt, then click OK.
NOTE: The deck setup is also shown in Figure 1.1 on the next page of this QRC.
6.
7.
8.
9.
When finished, remove the sample plate from the deck.
Blot the top of the plate with a Kimwipe.
Tightly seal the plate.
Place the sample plate in the preheated oven and incubate for 22 to 24 hr.
2. What to do Next
After the incubation period, do one of the following:
Proceed directly to Stage 2. Fragmentation and Precipitation.
■
Store the sample plate at –20 °C
■
1
P11
P10
BC P/N 717253
BC =Beckman Coulter
Thermal Cycler
P3
P2
P1
P1
P15
P5
P5
Samples
BC P/N 267007
P4
P16
Bio-Rad P/N HSP9631
P6
Bio-Rad P/N HSP9631
Figure 1.1. Deck Layout — DNA Amplification
Static Peltier
P9
P8
P7
P7
P10
W
A
S
H
P12
A1 - Axiom Denat Soln 10X
B1 - Axiom Neutral Soln 10X
C1 - Axiom Amp Enzyme
P14
P14
BC P/N 987925
P13
Cold block with template
Cold Block
Tip Disposal
Cold block with Template
L and R
BC P/N 372786 (75 mL)
P11
L: 2 BC P/N 372790 (40 mL)
R: 1 BC P/N 372786 (75 mL)
BC P/N 267007
Shaking Peltier
(with
deep well adaptor)
Axiom™ Automated Target Prep Protocol
Stage 1: DNA Amplification
For Research Use Only
Not for use in Diagnostic Procedures.
P/N 702831 Rev. 1
© 2010 Affymetrix, Inc. All rights reserved. Affymetrix®, Axiom™, Command Console®, DMET™, GeneAtlas™, GeneChip®, GeneChip-compatible™, GeneTitan®, Genotyping Console™,
NetAffx®, and Powered by Affymetrix™ are trademarks or registered trademarks of Affymetrix Inc. All other trademarks are the property of their respective owners.
2
Quick Reference Card
Axiom™ Automated Target Prep Protocol
Stage 2. Fragmentation and Precipitation
Reagents and Samples Required
Reagents from Module 2, Box 1 (P/N 901528) and Module 2, Box 2 (P/N 901529); Isopropanol is user-supplied.
Prepare as shown in Table 2.1 and place in the appropriate place on the deck.
Table 2.1. Reagent Preparation for Stage 2
Reagent
Temp Out of
Module*
Treatment
Axiom 10X Frag Buffer
Thaw at Room Temp
Vortex
Axiom Frag Diluent
Place on ice
Vortex and spin
Axiom Frag Enzyme
Keep at -20 °C
Just before use, flick tube 3X, spin, and place in the cold block
Axiom Frag Rxn Stop
Room Temp
Vortex
Precip Soln 1
Place on ice
Vortex
Precip Soln 2
Thaw at Room Temp
Vortex and spin
Isopropanol
Not Applicable
Room Temp
Notes:
*Temp Out of Module: temperature reagent is held at immediately after removal from module
1. If frozen, thaw the Sample Plate
1.
2.
3.
4.
5.
6.
Place the deep well plate in a small bath of room temperature Millipore water for ~ 50 min (until all wells have thawed).
Spin at 1000 rpm for 30 sec.
Remove the seal and blot the top of the plate with a Kimwipe.
Tightly reseal the plate with a fresh seal.
Vortex the plate for 30 sec to thoroughly mix.
Spin at 1000 rpm for 30 sec.
2. Fragment Samples
1. Run Biomek method:
A. Select your array plate format and the Fragmentation step, then click OK.
B. Setup the deck as indicated the deck setup prompt, then click OK.
NOTE: The deck layout is also shown in Figure 2.1 on the next page of this QRC.
3. Precipitate Samples
1.
2.
3.
4.
When the Biomek method is finished, remove the sample plate (Precipitation Plate; position P8) from the deck.
Blot the top of the plate with a Kimwipe.
Tightly seal the plate.
Place the plate in a –20 °C freezer overnight to precipitate.
4. Centrifuge and Dry Pellets
1.
2.
3.
4.
5.
6.
Preheat the oven to 48 °C.
Centrifuge the plate at 3200 xg (or rcf) at 4 °C for 40 min.
Remove the seal, then invert the plate over a waste container to allow the liquid to drain.
While still inverted, gently press the top of the plate on a stack of Kimwipes.
Leave plate inverted on Kimwipes for 5 min.
Turn the plate right side up and place in the preheated oven for 20 min.
5. What to do Next
After the pellets have been dried, do one of the following:
■
Proceed directly to Stage 3. Resuspension and Hybridization Preparation (even if some droplets of liquid remain).
■
Tightly seal the sample plate and store at –20 °C.
3
P11
P10
BC =Beckman Coulter
BC P/N 717253
Thermal Cycler
P3
P2
P1
P1
P15
Bio-Rad P/N HSP9631
P6
Bio-Rad P/N HSP9631
P5
P5
Bio-Rad P/N HSP9631
P4
P16
Figure 2.1. Deck Layout — Fragmentation and Precipitation
Static Peltier
P9
ABgene Square Well Plate
P8
Samples in
BC Deep Well Titer Plate
P7
P7
P10
W
A
S
H
Cold Block
Tip Disposal
A2 - Axiom Frag Enzyme
B2 - Axiom Frag Diluent
C2 - Axiom Precip Soln 2
BC P/N 379503
P14
P14
BC P/N 987925
P13
Cold block with template
Cold block with Template
P12
L: BC P/N 372786 (75 mL)
R: BC P/N 372790 (40 mL)
P11
L: 2 BC P/N 372790 (40 mL)
Shaking Peltier
(with
deep well adaptor)
Axiom™ Automated Target Prep Protocol
Stage 2: Fragmentation and Precipitation
For Research Use Only
Not for use in Diagnostic Procedures.
P/N 702831 Rev. 1
© 2010 Affymetrix, Inc. All rights reserved. Affymetrix®, Axiom™, Command Console®, DMET™, GeneAtlas™, GeneChip®, GeneChip-compatible™, GeneTitan®, Genotyping Console™,
NetAffx®, and Powered by Affymetrix™ are trademarks or registered trademarks of Affymetrix Inc. All other trademarks are the property of their respective owners.
4
Quick Reference Card
Axiom™ Automated Target Prep Protocol
Stage 3. Resuspension and Hyb Preparation
Reagents and Samples Required
Reagents from Module 2, Box 1 (P/N 901528) and Module 2, Box 2 (P/N 901529); TrackIt reagents are user-supplied.
Table 3.1. Reagent Preparation for Stage 3
Reagent
Temp Out of Module*
Treatment
Axiom Hyb Buffer
Place on ice
Vortex
Axiom Hyb Soln 1
Thaw at Room Temp
Vortex and spin
Axiom Resusp Buffer
Warm to Room Temp (~ 1 hr)
Vortex
Axiom Hyb Soln 2
Place on ice
Vortex and spin
Nuclease-free water
Not Applicable
Not Applicable
TrackIt Gel Loading Buffer (diluted 1:1000)
Not Applicable
See Gel QC Instructions
TrackIt 25 bp DNA Ladder (diluted 1:15)
Not Applicable
See Gel QC Instructions
*Temp Out of Module: temperature reagent is held at immediately after removal from module
1. Prepare the Precipitation Plate (only if frozen)
Figure 3.1. Gel Image
1. Place the precipitation plate on the bench top and equilibrate to
room temperature 1 to 1.5 hr.
2. Resuspend the Samples
1. Label two of the Bio-Rad plates as follows:
… Hyb Ready <sample identifier>
… Gel QC
2. Run Biomek method:
A. Select your array plate format and the Resuspension and
Hybridization Preparation step, then click OK.
B. Setup the deck as indicated in the Biomek deck setup prompt,
then click OK.
NOTE: The deck setup is also shown in Figure 3.2 on the next
page.
3. Run Fragmentation QC Gels
1. Tightly seal the Gel QC plate, vortex and spin.
2. Onto a 4% agarose e-gel load:
… 20 μL from each well of the Gel QC plate.
… 15 μL diluted TrackIt 25 bp ladder to marker wells.
… 20 μL water to any unused wells.
3. Run for 22 min.
4. Review gel image (see Figure 3.1).
Example of good fragmentation QC gel. Fragments are
between 125 and 25 bp.
4. Quantitate the Resuspended Samples
1. Quantitate the samples prepared in the OD plate.
2. Assess the OD reading for each sample.
What to do Next
Do one of the following:
■
■
If the GeneTitan MultiChannel instrument is available, and if the
gel QC and quantitation results were acceptable, proceed to
Stage 4. Preparation for GeneTitan.
Tightly seal the Hyb Ready plate and store at –20 °C. (This plate
is referenced as Hyb Rxn in the Biomek software.)
5
P10
BC =Beckman Coulter
BC P/N 717253
Thermal Cycler
Static Peltier
BC P/N 379503
P14
P14
BC P/N 987925
P13
Cold block with template
Cold Block
Tip Disposal
A3 - Axiom Hyb Soln 1
B3 - Axiom Hyb Soln 2
Cold block with Template
OD Quantitation Plate
Bio-Rad P/N HSP9631
BC P/N 717253
P12
P9
P6
Gel QC Plate
Bio-Rad P/N HSP9631
P11
L: 3 BC P/N 372790 (40 mL)
R: 1 BC P/N 372792 (19 mL)
P10
W
A
S
H
P3
P8
Bio-Rad P/N HSP9631
P7
P7
Shaking Peltier
(with
deep well adaptor)
Bio-Rad P/N HSP9631
P5
P5
P4
P16
Hyb Ready Plate
Bio-Rad P/N HSP9631
P2
Pelleted Samples
ABgene Square Well Plate
P1
P1
P15
Figure 3.2. Deck Layout — Resuspension and Hyb Prep
Axiom™ Automated Target Prep Protocol
Stage 3: Resuspension and Hyb Preparation
For Research Use Only
Not for use in Diagnostic Procedures.
P/N 702831 Rev. 1
© 2010 Affymetrix, Inc. All rights reserved. Affymetrix®, Axiom™, Command Console®, DMET™, GeneAtlas™, GeneChip®, GeneChip-compatible™, GeneTitan®, Genotyping Console™,
NetAffx®, and Powered by Affymetrix™ are trademarks or registered trademarks of Affymetrix Inc. All other trademarks are the property of their respective owners.
6
Quick Reference Card
Axiom™ Automated Target Prep Protocol
Stage 4. Preparation for GeneTitan®
Important Guidelines for this Stage
Begin this stage 45 min prior to when the array plate currently in the GeneTitan® will finish hybridization. This stage takes approximately 40 min
to run.
The Preparation for GeneTitan Stage has two different sets of steps:
■
Denature samples and Transfer denatured samples to hyb tray
■
Prepare GeneTitan reagent plates
The sets of steps are selected in the Axiom Target Prep dialog box.
Figure 4.1. Axiom Target Prep Step Selection
You can perform each part of the stage separately, or you can run both parts at the same time for the high-throughput workflow. For the highthroughput workflow, you are preparing reagent plates for the Array Plate that is currently finishing the Hybridization step in GeneTitan MC,
while preparing another hyb tray that will be loaded into GeneTitan MC with a new Array Plate to begin the Hybridization step.
Denaturation and Hyb Sample Transfer
1. Prepare the Hyb Ready Plate (sample plate):
Vortex briefly; spin at 1000 rpm for 30 sec; then place on ice.
2. Run Biomek method:
A. Select your array plate format, the Preparation for GeneTitan step, and the
following sub-steps:
… Denature samples
… Transfer denatured samples to hyb tray
B. Click OK.
C. Setup the deck as indicated in the deck setup prompt, then click OK. The deck setup is also shown in Figure 4.2 on the next page of this
QRC.
IMPORTANT: Clean the metal lid and pad by wiping with 70% ethanol.
3. Prepare the GeneTitan™ Multi-Channel (MC) Instrument.
4. Once GeneTitan is ready, return to Biomek and press OK at prompt to transfer samples from thermocycler to Hyb Tray.
5. Load the Hyb Tray and Array Plate in the GeneTitan MC.
See GeneTitan™ MC Protocol for Axiom™ Array Plate Processing QRC.
7
BC P/N 717253
BC =Beckman Coulter
Thermal Cycler
Hyb Tray
P3
Hyb-ready samples
Bio-Rad P/N HSP9631
P2
Bio-Rad P/N MSL-2032
Clean with 70% Ethanol
before use
P1
P1
P15
P6
P5
P5
P4
P16
Figure 4.2. Deck Layout — Denaturation and Transfer to Hyb Plate
P9
P8
P7
P7
Shaking Peltier
(with
deep well adaptor)
P12
P11
P10
W
A
S
H
P14
P14
P13
Static Peltier
Tip Disposal
Axiom™ Automated Target Prep Protocol
Stage 4: Preparation for GeneTitan
8
Axiom™ Automated Target Prep Protocol
Stage 4: Preparation for GeneTitan
Prepare Reagent Trays for GeneTitan
IMPORTANT: The reagent plates prepared are for use with an Axiom Array Plate that is already in the GeneTitan MC instrument and is
completing the hybridization stage.
1. Prepare the reagents from Module 4 as shown in Table 4.1, below:
Table 4 1. Reagent Prep
Reagent
Temp Out of Module*
Treatment
Module four, box 1 of 2 (P/N 901278)
Axiom Ligate Buffer
Thaw at Room Temp
1. Place on bench top at room temp for 30 min
2. Examine for precipitate
3. Vortex twice
4. Examine for precipitate
If any:
■ Warm bottle with your hands and vortex again for thirty seconds
Axiom Ligate Enzyme
Keep at –20 °C until ready to use
Just before use:
1. Flick 2 to 3 times to mix
2. Spin
3. Place in the cold block
Axiom Ligate Soln 1
Thaw at Room Temp
Vortex and Spin
Axiom Probe Mix 1
Thaw at Room Temp
Vortex and Spin
Axiom Stain Buffer
Thaw at Room Temp
Vortex and Spin
Axiom Stabilize Soln
Thaw at Room Temp
Vortex and Spin
Module four, box 2 of 2 (P/N 901276)
Axiom Ligate Soln 2
Thaw at Room Temp (do not place on ice!)
Vortex and Spin
Axiom Probe Mix 2#
Place on Ice
Flick 2 to 3 times to mix, then spin
Axiom Wash A
Leave on bench
1. Vortex twice
2. Place on Bench for 30 min
3. Look for precipitate
4. Vortex again if necessary
Axiom Stain 1-A#
Place on ice
Flick 2 to 3 times to mix, then spin
Axiom Stain 1-B#
Place on ice
Flick 2 to 3 times to mix, then spin
Axiom Stain 2-A#
Place on ice
Flick 2 to 3 times to mix, then spin
Axiom Stain 2-B#
Place on ice
Flick 2 to 3 times to mix, then spin
Axiom Stabilize Diluent
Place on ice
1. Vortex and Spin
2. Look for precipitate
If any:
■ Warm tube to room temperature and vortex again
Axiom Water
Place on ice
N/A
Axiom Hold Buffer#
Room Temp
Vortex
Notes:
* Temp Out of Module: temperature the reagent is held at immediately after removal from module.
# These solutions are light sensitive. Keep tubes out of direct light for a prolonged period of time.
N/A: not applicable in this case
2. Run Biomek method:
A. Select your array plate format, the Preparation for GeneTitan step, and the
following sub-step:
… Prepare GeneTitan reagent plates.
B. Click OK.
C. Setup the deck as indicated in the deck setup prompt, then click OK. The deck setup is also shown in Figure 4.3 of this QRC.
IMPORTANT: Label the stain trays and treat them with the antistatic gun.
3. Prepare the GeneTitan™ Multi-Channel Instrument.
See GeneTitan™ MC Protocol for Axiom™ Array Plate Processing QRC.
4. Treat the stain and scan tray lids with the antistatic gun.
5. Cover the reagent plates and Scan Tray with lids.
6. Examine each tray to ensure that all appropriate wells contain reagents (manually add if not present) and puncture any bubbles with a clean
pipette tip.
7. Immediately load the reagent plates and Scan Tray into the GeneTitan.
See GeneTitan™ MC Protocol for Axiom™ Array Plate Processing QRC
9
P12
P11
BC =Beckman Coulter
Tip Loader
Thermal Cycler
P3
P2
P1
P1
P15
Stain Tray – Lig
Stain Tray – Stain 1
Static Peltier
P9
Stain Tray – Stain 2
P8
P6
Stain Tray – Stabilization
P5
P5
Stain Tray – Stain 1
Scan Tray in Protective Base
P7
P7
P16
P10
W
A
S
H
BC P/N 379503
P14
P14
BC P/N 987925
P13
Cold block with template
Tip Disposal
Load reagents as indicated on the template
Cold block with Template
L: 3 BC P/N 372790 (40 mL)
P12
L: 3 BC P/N 372792 (19 mL)
P11
RT reagent block
Load Axiom Ligate Soln 2 in A6
Shaking Peltier
(with
deep well adaptor)
P4
Figure 4.3. Deck Layout — Reagent Tray Preparation
Axiom™ Automated Target Prep Protocol
Stage 4: Preparation for GeneTitan
10
Axiom™ Automated Target Prep Protocol
Stage 4: Preparation for GeneTitan
Stage 4 for High-Throughput Workflow
In the high-throughput workflow you:
1. Denature hyb-ready samples and transfer them to a Hyb Tray for loading into GeneTitan MC for the Hybridization stage.
2. Prepare Reagent trays for another Hyb Tray and Array plate that is finishing the Hybridization stage.
IMPORTANT: The reagent plates prepared in the high-throughput workflow are not for use with the Hyb Tray currently being prepared on the
Biomek workstation, but for an Axiom Array Plate that is already in the GeneTitan MC instrument and is completing the hybridization stage.
To perform Stage 4 for high-throughput:
1. Prepare the reagents from Module 4 as shown in Table 4.1 on page 9.
2. Prepare the Hyb Ready Plate (sample plate):
Vortex briefly; spin at 1000 rpm for 30 sec; then place on ice.
3. Run Biomek method:
A. Select your array plate format, the Preparation for GeneTitan step, and the
following sub-steps:
… Denature samples
… Transfer denatured samples to hyb tray
… Prepare GeneTitan reagent plates
B. Click OK.
C. Setup the deck as indicated in the deck setup prompt, then click OK. The deck setup is also shown in Figures 4.2 and 4.3 of this QRC.
IMPORTANT: Clean the metal lid and pad by wiping with 70% ethanol.
IMPORTANT: Label the stain trays and treat them with the antistatic gun.
4. Prepare the GeneTitan™ Multi-Channel Instrument
See GeneTitan™ MC Protocol for Axiom™ Array Plate Processing QRC.
5. Treat the stain and scan tray lids with the antistatic gun.
6. Cover the reagent plates and Scan Tray with lids.
7. Examine each tray to ensure that all appropriate wells contain reagents (manually add if not present) and puncture any bubbles with a clean
pipette tip.
8. Immediately load the reagent plates and Scan Tray into the GeneTitan — do NOT click OK until all reagents plates have been loaded into the
GeneTitan.
9. Return to the Biomek workstation and click OK when prompted to resume the method. Denatured samples are transferred to the Hyb Tray.
10. Load the Hyb Tray and Array Plate into the GeneTitan.
See GeneTitan™ MC Protocol for Axiom™ Array Plate Processing QRC.
For Research Use Only
Not for use in Diagnostic Procedures.
P/N 702831 Rev. 1
© 2010 Affymetrix, Inc. All rights reserved. Affymetrix®, Axiom™, Command Console®, DMET™, GeneAtlas™, GeneChip®, GeneChip-compatible™, GeneTitan®, Genotyping Console™,
NetAffx®, and Powered by Affymetrix™ are trademarks or registered trademarks of Affymetrix Inc. All other trademarks are the property of their respective owners.
11
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