24sure Microarray Dual Channel Summary Protocol
Experienced User Card
FOR RESEARCH USE ONLY
ILLUMINA PROPRIETARY
Part # 15056063 Rev. B
June 2015
Page 1 of 14
24sure Sample Preparation
Preparation and Materials
Table 1 Consumables and Equipment for Sample Preparation
Materials
Ordering Information
SurePlex DNA Amplification System
Illumina PR-40-415101-00
20x PBS (dilute to 1x with nuclease-free water)
Cell Signaling Technologies 9808
Genomic control DNA
Promega G1521
PCR tubes (0.2 ml, thin walled, flip cap) OR
Thermo Scientific AB-0620
96-well PCR plate and adhesive plate seals
Thermo Scientific AB-0600 (plate) AB-0558 (seals)
Microcentrifuge tube (1.5 ml, flip cap)
Sarstedt 72.690.001
Table 2 Starting Material for Sample Preparation
Component
Material
DNA
Single cell in 2.5 µl 1x PBS in 0.2 ml tubes or PCR plates
Positive control 1
15 pg genomic DNA in 2.5 µl 1x PBS
Positive control 2
60 pg genomic DNA in 2.5 µl 1x PBS
Negative control
2.5 µl of 1x PBS (0.2 ml tube/PCR plate)
Negative control
2.5 µl of collection buffer control (0.2 ml tube/PCR plate)
Table 3 SurePlex DNA Amplification System (50 Reactions) Kit Components
Component Name
Volume
Prepare
Cell extraction buffer
300 µl
Thaw on ice
Extraction enzyme dilution buffer
300 µl
Thaw on ice
Cell extraction enzyme
15 µl
Transfer to ice before use
SurePlex pre-amp buffer
275 µl
Thaw on ice
SurePlex pre-amp enzyme
15 µl
Transfer to ice before use
SurePlex amplification buffer
1.4 ml
Thaw on ice
SurePlex amplification enzyme
50 µl
Transfer to ice before use
1.8 ml
Thaw on ice
Nuclease-free water
Cap Color
Clear
Page 2 of 14
24sure Microarray Dual Channel Summary Protocol
Experienced User Card
Part # 15056063 Rev. B
Preparation of Amplification Reaction Controls
Estimated Time
Hands-on time: 30 minutes
Consumables
Item
Quantity
1x PBS
805 µl
Genomic DNA (100ng/µl)
5 µl
Steps
[_] 1
In a sterile containment cabinet (vertical laminar flow cabinet), label 4 sterile 1.5 ml
microcentrifuge tubes, as follows.
Control Tube
1
2
3
4
Label
1x PBS negative
2.5 ng/µl positive
25 pg/µl positive
6.25 pg/µl positive
1x PBS
100 µl
195 µl
495 µl
15 µl
Female gDNA/Stock
None
5 µl of 100 ng/µl stock
5 µl of 2.5 ng/µl stock (from tube 2)
5 µl of 25 pg/µl stock (from tube 3)
[_] 2
Add the appropriate volume of 1x PBS into each labeled tube.
[_] 3
Add 5 µl of female genomic DNA to tube 2. Invert to mix.
[_] 4
Dilute 5 µl of tube 2 into tube 3. Invert to mix.
[_] 5
Dilute 5 µl of tube 3 into tube 4. Invert to mix.
[_] 6
In the sterile containment cabinet, label 3 sterile 0.2 ml flat-capped PCR tubes, as follows.
PCR tube
1
2
3
Label
15.6 pos
62.5 pos
Neg
Contents
2.5 µl of Control tube 4 (6.25 pg/µl positive)
2.5 µl of Control tube 3 (25 pg/µl positive)
2.5 µl of Control tube 1 (1x PBS)
[_] 7
Add 2.5 µl from the appropriate control tube to the appropriate PCR tubes, and cap each
tube after transfer.
• Add 2.5 µl from control tube 4 to PCR tube 1.
• Add 2.5 µl from control tube 3 to PCR tube 2.
• Add 2.5 µl from control tube 1 to PCR tube 3.
[_] 8
Set aside the PCR tubes on ice in a 96-well rack.
Cell Lysis/Extraction
Estimated Time
Hands-on time: 30 minutes
Incubation time: 15 minutes
24sure Microarray Dual Channel Summary Protocol
Experienced User Card
Part # 15056063 Rev. B
Page 3 of 14
Consumables
Item
Quantity
1x PBS
2.5 µl per sample
Cell extraction buffer (green cap)
2.5 µl per sample
Extraction enzyme dilution buffer (purple cap)
4.8 µl per sample
Cell extraction enzyme (yellow cap)
0.2 µl per sample
NOTE
When pipetting into tubes that contain solutions, eject the drop of liquid onto the side of the PCR
tube. Do not place the tip into or near the sample solution.
NOTE
Throughout the SurePlex amplification protocol, only open 1 sample tube at a time.
Steps
[_] 1
Collect samples in 2.5 µl of 1x PBS.
[_] 2
Line up the sample tubes with prepared control tubes in a 96-well rack. Keep on ice until
required.
[_] 3
Centrifuge the tubes/plate at 200 × g for 3 minutes (at 4°C, if possible).
[_] 4
Add 2.5 µl of cell extraction buffer (green cap) to each sample (including controls) and store
at 2°C to 8°C.
[_] 5
Combine the Extraction Cocktail master mix components as described in the following table.
Invert to mix.
Extraction Cocktail Master Mix
Cap
color
Extraction enzyme dilution buffer
Cell extraction enzyme
Total Volume
Per Single
Sample
4.8 µl
0.2 µl
5 µl
[_] 6
For every sample or control, add 5 µl of freshly prepared Extraction Cocktail.
[_] 7
Briefly centrifuge samples.
[_] 8
Place on a thermal cycler and run the following program.
• 75°C for 10 minutes
• 95°C for 4 minutes
• Hold at room temperature
Per 5
Samples
24 µl
1 µl
25 µl
NOTE
Always use a heated lid on a PCR machine. Do not perform PCR reactions with oil.
Pre-amplification
Consumables
Item
Quantity
SurePlex pre-amp buffer (red cap)
4.8 µl per sample
SurePlex pre-amp enzyme (white cap)
0.2 µl per sample
Page 4 of 14
24sure Microarray Dual Channel Summary Protocol
Experienced User Card
Part # 15056063 Rev. B
Steps
[_] 1
Combine the Preamp Cocktail components as described in the following table. Invert to mix.
Cap
Color
Pre-Amp Cocktail
SurePlex pre-amp buffer
SurePlex pre-amp enzyme
Total Volume
Per Single
Sample
4.8 µl
0.2 µl
5 µl
Per 5
Samples
24 µl
1 µl
25 µl
[_] 2
For each 10 µl sample prepared in Cell Lysis/Extraction, add 5 µl of SurePlex Preamp
Cocktail. Briefly centrifuge.
[_] 3
Place on a thermal cycler and run the following program.
• 95°C for 2 minutes
• 12 cycles of:
— 95°C for 15 seconds
— 15°C for 50 seconds
— 25°C for 40 seconds
— 35°C for 30 seconds
— 65°C for 40 seconds
— 75°C for 40 seconds
• Hold at 4°C
[_] 4
Set aside on ice.
Amplification
Estimated Time
Hands-on time: 10 minutes
Incubation time: 50 minutes
Consumables
Item
Quantity
SurePlex amplification buffer (orange cap)
25 µl per sample
SurePlex amplification enzyme (blue cap)
0.8 µl per sample
Nuclease-free water (clear cap)
34.2 µl per sample
Steps
[_] 1
Combine the Amplification Cocktail components in and mix.
Amplification Cocktail
SurePlex amplification buffer
SurePlex amplification enzyme
Nuclease-free water
Total Volume
24sure Microarray Dual Channel Summary Protocol
Experienced User Card
Part # 15056063 Rev. B
Cap
Color
Clear
Page 5 of 14
Per Single
Sample
25 µl
0.8 µl
34.2 µl
60 µl
Per 5
samples
125 µl
4 µl
171 µl
300 µl
NOTE
Sample amplification efficiency can be analyzed using a real-time thermal cycler and adding
SYBR Green I dye (Invitrogen, catalog # S7563) at 0.125x final concentration in the
Amplification Cocktail.
[_] 2
Add 60 µl freshly prepared Amplification Cocktail to 15 µl pre-amplification reaction
product. Invert to mix, and then centrifuge briefly.
[_] 3
Place on a thermal cycler and run the following program:
• 95°C for 2 minutes
• 14 cycles of:
— 95°C for 15 seconds
— 65°C for 1 minute
— 75°C for 1 minute
[_] 4
Load 5 µl of each amplified sample and 5 µl gel loading buffer (2X) onto a 1.5% agarose 1x
TBE gel until resolved.
[_] 5
Set aside amplified SurePlex products on ice.
Page 6 of 14
24sure Microarray Dual Channel Summary Protocol
Experienced User Card
Part # 15056063 Rev. B
24sure Dual Channel Labeling
Starting Materials
Table 4 Starting materials for labeling
Starting materials
Part Number
Amount
Amplified sample DNA
N/A
8 µl per hybridization
SureRef male reference DNA
Illumina PR-40-415203-00
8 µl per hybridization
SureRef female reference DNA
Illumina PR-40-415204-00
8 µl per hybridization
Table 5 Materials required for labeling
Materials required
Amount
Part number
PCR tubes (0.2 ml, thin walled, flip cap OR
32
Thermo Scientific AB-0620
96-well PCR plate and plate seals
1
Thermo Scientific AB-0600, AB-0558
Fluorescent Labeling System [dCTP]
1
Illumina PR-30-413103-00 inc. in PR10-408702-PK
COT Human DNA
400 µl
Illumina PR-40-413510-00 inc. in PR10-408702-PK
3M sodium acetate (pH 5.2)
200 µl
Sigma S7899
Absolute ethanol
5 ml
70% ethanol
5 ml
Microcentrifuge tube (1.5 ml, flip cap)
Sarstedt 72.690.001
Labeling
Estimated Time
Hands-on time: 30 minutes
Incubation time: 2–4 hours (may be increased up to 18 hours, if necessary)
Steps
NOTE
Perform all steps on ice unless otherwise indicated.
[_] 1
Thaw components from Fluorescent Labeling System. To collect the contents, vortex briefly
and then centrifuge. Retain on ice.
[_] 2
Prepare the labeling master mixes by adding the components in the quantities and order
listed in Table 6, retain on ice.
24sure Microarray Dual Channel Summary Protocol
Experienced User Card
Part # 15056063 Rev. B
Page 7 of 14
Table 6 Labeling master mix components and quantities
Component
Cap
color
Cy3
labeling
mix - 1 rxn
Cy3 labeling
master mix (*n +
5%)
Cy5
labeling
mix - 1 rxn
Reaction buffer
5 µl
5 µl
dCTP-labeling mix
5 µl
5 µl
Cy3 dCTP
1 µl
Cy5 dCTP
Cy5 labeling
master mix (*n +
5%)
1 µl
Klenow enzyme
1 µl
1 µl
Total
12 μl
12 μl
[_] 3
In a PCR tube or plate, combine 5 µl of primer solution (black cap) with 8 µl of amplified
sample DNA from 24sure Sample Preparation or SureRef reference DNA. Refer to the 96-well/8well plan for layout of samples.
[_] 4
Cap/seal and denature the combined primer solution and DNA in a prewarmed lidded
thermal cycler for 5 minutes at 94°C. Then transfer immediately to ice or to a precooled
thermal cycler for 5 minutes at 4°C.
[_] 5
Add 12 µl of Cy3 labeling master mix to the 13 µl of the sample DNA/primer solution. Add
12 µl of Cy5 labeling master mix to the 13 µl of SureRef DNA/primer solution. To spin down
the contents, seal, invert to mix, and then centrifuge.
[_] 6
Incubate in a prewarmed lidded thermal cycler for 2–4 hours at 37°C. Incubate for up to 18
hours, if necessary.
[_] 7
Place on ice.
Combination
There are two alternative protocols for combination and volume reduction of the labeled
material: the standard ethanol precipitation protocol, and a centrifugal evaporation protocol.
Combination and Ethanol Precipitation
[_] 1
Centrifuge the PCR tubes or plate from 24sure Dual Channel Labeling. Combine sample and
control DNA into numbered microcentrifuge tubes by adding the Cy5 labeling product to the
Cy3 labeling product for each hybridization area.
[_] 2
Add 25 µl COT Human DNA and 7.5 µl 3 M sodium acetate to each tube and vortex.
[_] 3
Add 206 µl of absolute ethanol to each tube and invert twice to mix.
[_] 4
Precipitate in the dark for 20 minutes at -80°C, or on dry ice.
[_] 5
Pellet labeled DNAs at full speed (centrifuge at ≥ 13,000 × g) for 10 minutes, discard
supernatant.
[_] 6
Add 500 µl of 70% ethanol, mix by inversion, and centrifuge at full speed, for 5 minutes.
[_] 7
Decant supernatant. Keeping tube inverted, gently tap out remaining droplets onto folded
tissue.
[_] 8
Pulse tube in centrifuge and remove remaining ethanol with a P10 tip.
Page 8 of 14
24sure Microarray Dual Channel Summary Protocol
Experienced User Card
Part # 15056063 Rev. B
[_] 9
Allow pellet to air dry for 3–5 minutes at room temperature.
[_] 10 Proceed to Hybridization.
Combination and Centrifugal Evaporation
[_] 1
Prewarm a centrifugal evaporator, rotor, and PCR plate racks (if using) to 75°C for 30
minutes, or until warm.
[_] 2
Pulse spin PCR tubes or plate from the labeling step to collect the contents.
[_] 3
Combine Cy3 and Cy5 labeled DNA into numbered microcentrifuge tubes or PCR plate wells
by adding the Cy5 labeling product to the Cy3 labeling product for each hybridization area.
[_] 4
Add 25 µl COT Human DNA to each tube or PCR plate well containing combined Cy3/Cy5
labeling products.
[_] 5
Transfer the tubes or plate to the prewarmed centrifugal evaporator. Evaporate under
centrifuge at 75°C (or high) until around 3 µl remains in each tube/well.
[_] 6
Proceed to Hybridization.
24sure Microarray Dual Channel Summary Protocol
Experienced User Card
Part # 15056063 Rev. B
Page 9 of 14
24sure Hybridization
Materials required
Table 7 Materials required for hybridization
Materials required
Part number
DS Hybridization Buffer (15% dextran sulphate)
Illumina, part of FLS inc. in PR-10-408602-PK
24sure+ slides
Illumina, inluded in PR-40-408602-PK
22x22 mm glass cover slips
Fisher Scientific FB58633, inc. in ClearPack Lite
ClearHyb Hybridization System
Illumina PR-70-432101-00 (230 V)/ PR-70-43210200 (115 V)
Preparation
[_] 1
Place Hybridization Buffer in a heat block at 75°C to thaw and prewarm.
[_] 2
Prewarm the chambers of the ClearHyb Hybridization system at 47°C for at least 30 minutes.
Hybridization
Estimated Time
Hands-on time: 30 minutes
Incubation time: 3 hours (can extend to 16 hours, if necessary)
Steps
[_] 1
Resuspend each pellet of combined labeled samples/references/COT in 21 µl of prewarmed
DS Hybridization Buffer at 75°C, ensuring that pellet is dissolved. Pulse centrifuge to collect
contents.
CAUTION
Add Hybridization Buffer in a fume cabinet.
[_] 2
Denature at 75°C for a further 10 minutes, centrifuge for 20 seconds, allow to cool to room
temperature.
[_] 3
In a fume cabinet, apply 18 µl of labeled DNA solution to each coverslip and lower the slide
barcode-down for each hybridization area.
[_] 4
Use the hybridization template to position coverslips and confirm which labeled DNAs are
loaded on to each hybridization area.
[_] 5
Disassemble the preheated hybridization chambers and insert the slides into the racks at the
open end. Take care to keep the loaded 24sure slides flat and handle the slides by the edges
(see Figure 1).
Page 10 of 14
24sure Microarray Dual Channel Summary Protocol
Experienced User Card
Part # 15056063 Rev. B
Figure 1
[_] 6
Clip the loaded racks into the base of the chamber by placing the racks on the base with the
key holes over the keys. Then lock clips into place (see Figure 2).
Figure 2
[_] 7
Pipette 0.25 ml of distilled water onto a clean and dry absorbent pad (supplied with the
ClearHyb Hybridization Kit) secured inside the lid (see Figure 3). Allow the distilled water to
absorb into the absorbent pad.
Figure 3
[_] 8
Place the lid over the base and tighten the screws using the tool provided, starting with one
corner, then the one diagonally opposed to keep the pressure even (see Figure 4). To avoid
stripping threads, take great care not to tighten the screws too tightly. Fingertip pressure is
enough as the O ring provides a good seal.
24sure Microarray Dual Channel Summary Protocol
Experienced User Card
Part # 15056063 Rev. B
Page 11 of 14
Figure 4
[_] 9
Load the hybridization unit into the ClearHyb Heating base unit for 3–16 hours at 47°C.
Page 12 of 14
24sure Microarray Dual Channel Summary Protocol
Experienced User Card
Part # 15056063 Rev. B
24sure Washing
Materials and Equipment
Table 8 Materials and equipment required for washing
Materials and equipment required
Part number
Coplin or Hellendahl jar
Solmedia SJ320/SJ340
Slide staining rack (2), 400 ml lidded square glass
staining dishes (3), magnetic stir bars (3)
Illumina in ClearPack Lite PR-70-431001-00
Magnetic three-position stirrer
eg, Bibby Stuart SB161-3
ClearHyb Wash System
Illumina PR-70-432201-00 (230 V)/ PR-70-43220200 (115 V)
Tween 20
Sigma, P9416
20x SSC
Sigma, S6639
Prepare the following buffers:
Table 9 24sure wash buffers
Buffer
Wash
Recipe
1000 ml 2x SSC/0.05%
Tween 20
Remove cover slips and
Wash 1
100 ml 20x SSC [pH 7.0], 899.5 ml H2O, 0.5
ml Tween 20
500 ml 1x SSC
Wash 2
25 ml 20x SSC [pH 7.0], 475 ml H2O
1000 ml 0.1x SSC
Wash 3 and 4
5 ml 20x SSC [pH 7.0], 995 ml H2O
Prepare the following equipment:
Table 10 24sure wash equipment
Item
Number
required
Coplin jar/Hellendahl jar
1
Containing 2x SSC/0.05% Tween 20 at room
temperature for removal of cover slips.
400 ml lidded square glass staining
dishes
3
Washes 1 (2x SSC/0.05% Tween 20), 2 (1x SSC)
and 4 (0.1x SSC) at room temperature.
ClearHyb Wash System, ensure the
ClearHyb unit is calibrated.
1
Wash 3—Prefill the waterbath insert with 0.1x
SSC and preheat to 60°C for 30 minutes
Comments
NOTE
Glass containers are recommended for washing. For room temperature washes, use foil
covers over glass jars to protect slides from light.
24sure Microarray Dual Channel Summary Protocol
Experienced User Card
Part # 15056063 Rev. B
Page 13 of 14
Wash Procedures
Estimated Time
Hands-on time: 30 minutes
Steps
[_] 1
Prepare buffer for wash 1. Add 400 ml of 2xSSC/0.05% Tween 20 to a staining dish at room
temperature. Add a 2.5 cm stir bar and the 24 position stainless steel rack.
[_] 2
When hybridization incubation time is complete, disassemble the hybridization units and
remove the slides from racks by pushing from behind, taking care to handle the slides only
by the edges.
[_] 3
Remove the coverslips from each slide by manually agitating in 2xSSC/0.05% Tween 20 in a
Coplin jar at room temperature. Transfer immediately to the stainless steel rack sitting in the
dish prepared in step 1. Repeat for all slides.
[_] 4
When rack is fully loaded, replace lid, turn on stirrer, start timer, cover with foil, and
complete wash 1 and subsequent washes as summarized in Table 11.
Table 11 Wash Summary
[_] 5
Wash
Volume
Temp
Times
Agitation
Buffer
1
400 ml
Room Temperature
10 min
2.5 cm stir bar
2x SSC/0.05% Tween 20
2
400 ml
Room Temperature
10 min
2.5 cm stir bar
1x SSC
3
500 ml
60°C in ClearHyb Wash
5 min
None
0.1x SSC
4
400 ml
Room Temperature
1 min
2.5 cm stir bar
0.1x SSC
Dry slides by centrifugation at 170 × g for 3 minutes and store in the original blue box.
Page 14 of 14
24sure Microarray Dual Channel Summary Protocol
Experienced User Card
Part # 15056063 Rev. B