Day 1
Make MSA2
Hands-on: ~45–60 min
Reagents
0.1N NaOH
MA1
MA2
MSM
Output
MSA2 Plate
Infinium
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Day 2
Fragment MSA2
Hands-on: 0.5 hours
Incubation: 1 hour
Reagents
FMS
Output
MSA2 Plate
Day 3
WashBC2
Hands-on: ~20–30 min
Reagents
WB1
PB1
Output
BeadChip
Incubate MSA2
Incubation: 20–24 hours
Output
MSA2 Plate with
Amplified DNA
Precip MSA2
Hands-on: ~1.5 hours
Dry Time: 1 hour
Reagents
2-propanol
PM1
Output
MSA2 Plate
Resuspend MSA2
Hands-on: ~30 min
Incubation: 1 hour
Reagents
RA1
Output
MSA2 Plate
XStain BC2
Hands-on: ~2.5 hours
Dry Time: 1 hour
Reagents
RA1
Formamide / EDTA
PB1
XC1
XC2
XC3
XC4
TEM
LTM
ATM
Output
BeadChip
Image BC2
Hands-on: 5 min/scan
Scan: 45 min/BeadChip
Output
Image and Data Files
Hyb Multi BC2
Hands-on: ~30–45 min
Incubation: 16–24 hours
Reagents
PB2
Output
BeadChip
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Part # 11208000 Rev. A
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(Pre-Amp)
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Make MSA2
Per 96 samples
Hands-on time:
45 minutes for 48 samples
60 minutes for 96 samples
20–24 hours
Incubation time:
Move DNA samples into the MSA2 plate. Denature and neutralize samples, and prepare them for amplification. Incubate overnight to amplify.
New Materials Quantity
96-well 0.2 ml microtiter plate
(MIDI or TCY)
WG#-DNA plate with 96 DNA samples, normalized to 50 ng/ml
0.1N NaOH
1 plate
1 plate
15 ml
MA2 2 tubes
Preparation
Preheat the Illumina hybridization oven in the post-amp area to 37°C.
Allow to equilibrate.
Enter the Sample_Name (optional) and Sample_Plate for each
Sample_Well in the Sample Sheet.
Thaw MA1, MA2, and MSM tubes to room temperature (22°C). Pulse centrifuge to 280 xg.
Thaw DNA samples to room temperature (22°C).
Apply a MSA2 barcode to a new MIDI or TCY plate.
Steps
1. (Optional) If you do not already have a WG#-DNA plate, dispense
DNA into either a:
•
MIDI plate: 20 µl to each WG#-DNA plate well
•
TCY plate: 10 µl to each WG#-DNA plate well
Apply a barcode label to the new WG#-DNA plate.
2. Dispense 20 µl MA1 to each MSA2 plate well that will contain sample.
3. Dispense 4 µl DNA to each well containing MA1.
4. Dispense 4 µl 0.1N NaOH to each well containing DNA and MA1.
5. Record the well for each DNA sample in the lab tracking worksheet.
6. Vortex the sealed MSA2 plate at 1600 rpm (actual vortex speed) for
1 minute.
7. Pulse centrifuge to 280 xg.
8. Incubate for 10 minutes at room temperature (22°C).
9. Dispense 68 µl MA2 to each well containing sample.
10. Dispense 75 µl MSM to each well containing sample.
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11. Seal MSA2 plate with cap mat.
12. Invert sealed plate at least 10 times to mix contents.
13. Pulse centrifuge to 280 xg.
14. Incubate in the Illumina Hybridization Oven for 20–24 hours at 37°C.
Good stopping point
Next step
Proceed to Fragment MSA2 .
This is the end of Pre-Amp. You may now remove these Experienced User
Cards from the Pre-Amp area and take them elsewhere. Do not return with them into the Pre-Amp area at any time.
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Fragment
MSA2
Hands-on time:
Incubation time:
0.5 hours
1 hour
Enzymatically fragment DNA, using end-point fragmentation to avoid overfragmentation.
New Materials
Per 96 samples FMS
Quantity
2 tubes
Preparation
Preheat the heat block with MIDI plate insert to 37°C.
Thaw FMS tubes to room temperature (22°C). Gently invert to mix contents, and then pulse centrifuge to 280 xg.
Steps
1. Centrifuge the MSA2 plate to 50 xg for 1 minute.
2. Dispense 50 µl FMS to each well containing sample.
3. Seal the plate with the cap mat.
4. Vortex the plate at 1600 rpm for 1 minute.
5. Centrifuge to 50 xg for 1 minute.
6. Incubate on heat block for 1 hour at 37°C.
Good stopping point
Next step
Do one of the following:
•
Proceed to Precip MSA2 . Leave plate in 37°C heat block until you have completed the preparatory steps.
•
Store the MSA2 plate at -20°C.
Pre-Amp
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Precip MSA2
Hands-on time:
Incubation and dry time:
0.5 hours
2 hours
Precipitate the DNA sample using 2-propanol and PM1.
New Materials Quantity
Per 96 samples
100% 2-propanol 40 ml
Preparation
Preheat the heat block to 37°C.
If frozen, thaw MSA2 plate to room temperature (22°C). Pulse centrifuge to 50 xg.
Thaw PM1 to room temperature (22°C). Centrifuge to 280 xg for
1 minute.
Set centrifuge to 4°C.
Steps
1. Dispense 100 µl PM1 to each MSA2 plate well containing sample.
2. Seal the plate with the cap mat.
3. Vortex at 1600 rpm for 1 minute.
4. Centrifuge to 50 xg for 1 minute at 22°C.
5. Incubate in heat block for 5 minutes at 37°C.
6. Centrifuge to 50 xg for 1 minute at 22°C.
7. Dispense 300 µl 2-propanol to each well containing sample.
8. Seal the plate with a new, dry cap mat.
9. Invert plate at least 10 times to mix contents.
10. Incubate plate in centrifuge for 30 minutes at 4°C.
11. Centrifuge to 3000 xg for 20 minutes at 4°C.
Perform the next step immediately, to avoid dislodging the blue pellet. If any delay occurs, repeat the 20-minute centrifugation before proceeding.
12. Remove the cap mat.
13. Decant supernatant by quickly inverting the MSA2 plate and smacking it down onto an absorbent pad.
Tap firmly several times for 1 minute or until all wells are devoid of liquid. Do not allow supernatant to pour into other wells.
14. Place inverted, uncovered plate on tube rack for 1 hour at 22°C to air dry the pellet.
Good stopping point
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Next step
Do one of the following:
•
Proceed immediately to Resuspend MSA2 .
•
Seal the MSA2 plate with a cap mat and store it at -20°C.
Hands-on time:
Incubation time:
0.5 hours
1 hour
Resuspend
MSA2
Resuspend the precipitated DNA using RA1.
New Materials
RA1
Quantity
Bottle (42 µl per sample well)
Preparation
Preheat the Illumina Hybridization Oven to 48°C.
Preheat the heat sealer.
Thaw RA1 to room temperature (22°C). Gently invert to re-dissolve solution.
Steps
1. Dispense 42 µl RA1 to each well containing a DNA pellet. Reserve any leftover reagent for Hyb Multi BC2 and XStain BC2.
2. Heat-seal the MSA2 plate with a foil seal.
3. Incubate in the Illumina Hybridization Oven for 1 hour at 48°C.
4. Vortex the sealed plate at 1800 rpm for 1 minute.
5. Pulse centrifuge to 280 xg.
If you stored the DNA pellets at -20°C for more than 72 hours after
Precip MSA2, you may need to repeat the vortexing and centrifugation steps until the pellets are completely resuspended.
Good stopping point
Next step
Do one of the following:
•
Proceed to Hyb Multi BC2 . If you proceed immediately, you may leave the RA1 reagent at room temperature temporarily.
•
Seal the MSA2 plate and store it at -20°C (-80°C if storing for more than 24 hours). Store RA1 at -20°C.
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Post-Amp Page 6 of 15 Part # 11208000 Rev. A
Hyb Multi
BC2
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Hands-on time:
Incubation time:
30 minutes for 48 samples
40 minutes for 96 samples
16–24 hours
Dispense the fragmented, resuspended DNA samples onto BeadChips.
Incubate the BeadChips in the Illumina Hybridization Oven to hybridize the samples onto the BeadChips.
New Materials Quantity
Pre-Amp
Post-Amp
BeadChips 8
Per 96 samples Hyb Chambers
2
Hyb Chamber gaskets
Hyb Chamber inserts
2
8
Preparation
Preheat the heat block to 95°C.
Preheat the Illumina Hybridization oven to 48°C.
Steps
1. Place the Hyb Chamber gaskets into the Hyb Chambers.
2. Dispense 200 µl PB2 to each of the 8 humidifying buffer reservoirs in each Hyb Chamber.
3. Seal the lid of each Hyb Chamber. Keep on bench at room temperature (22°C) until ready to load BeadChips.
4. Incubate the MSA2 plate on the heat block for 20 minutes at 95°C.
5. Remove all BeadChips from their packages.
6. Slide each BeadChip into a Hyb Chamber insert so that the barcode lines up with the barcode symbol on the insert.
Handle BeadChips by the edges or by the barcode end.
7. Pulse centrifuge the MSA2 plate to 280 xg.
8. Dispense 12 µl of each DNA sample onto the appropriate BeadChip stripe, following the illustration below.
Place pipet tip directly onto the array surface to dispense sample.
Dispense A1 through F1 into the 6 stripes on the left side of
BeadChip 1, and then finish dispensing the first column (H1 and G1) into the first two stripes on the right side of the chip. Next, dispense
A2 through D2 into the remaining stripes on the right side of the chip.
Work your way down each plate column from A to H, dispensing samples to the left and then the right sides of each chip.
Tip : For an alternate loading pattern, see the illustration at the end of these steps.
Page 7 of 15 Part # 11208000 Rev. A
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G
H
E
F
C
D
A
B
9. Record the BeadChip barcodes in the lab tracking worksheet.
To avoid contamination or evaporation, proceed to the next step as soon as you finish dispensing samples to the arrays.
BC2 #1 BC2 #2 BC2 #3 BC2 #4
A1 E2 A4 E5
G1 C3 C6
G4
B1 F2 B4 F5
H1
D3 H4 D6
WG#-MSA2 Plate C1
A2
G2
E3
C4
A5
G5
E6
D1 H2 D4 H5
B2
F3 B5 F6
E1 A3 E4 A6
C2 G3 G6
C5
F1 B3 F4 B6
D2 H3 H6
D5
1123456789
1123456789
1123456789 1123456789
BC2 #5 BC2 #6 BC2 #7 BC2 #8
A7 E8 A10 E11
G7 C9 G10 C12
B7 F8 B10 F11
H7 D9
H10
D12
1 2 3 4 5 6 7 8 9 10 11 12
BC2 #1 & #2 BC2 #3 & #4 BC2 #5 & #6 BC2 #7 & #8
C7
D7
A8
G8
H8
E9
C10
D10
A11
G11
H11
E12
B8 F9 B11 F12
E7 A9 E10
A12
C8 G9
C11
G12
F7 B9 F10 B12
D8 H9
D11
H12
1123456789 1123456789
1123456789
1123456789
10. Visually inspect all stripes to ensure that DNA sample covers all sections on each stripe. Record any sections that are not completely covered.
11. Load the Hyb Chamber inserts into the Hyb Chambers.
12. Seal the Hyb Chamber lid by lowering the back of the lid first and then bringing it down in front, to avoid dislodging the inserts.
13. (Optional) Set the rocker speed to 5.
14. Incubate the Hyb Chamber(s) in the Illumina Hybridization Oven for
16 to 24 hours at 48°C.
15. Cover the residual sample in the MSA2 plate with a foil seal. You can store the plate indefinitely at -80°C.
Pre-Amp
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Alternate loading pattern
Dispense A1 to A6 to the stripes on the left side of BeadChip 1. Dispense B1 to B6 to the stripes on the right side of BeadChip 1. Continue dispensing six wells from each row into each side of the BeadChips, as shown in the diagram.
E
F
C
D
A
B
G
H
BC2 #1
BC2 #2
BC2 #3
WG#-MSA2 Plate
BC2 #5
BC2 #6
BC2 #7
BC2 #4 BC2 #8
1 2 3 4 5 6 7 8 9 10 11 12
A7
BC2 #5
B7
A8
B8
A9
B9
A10
B10
A11
B11
A12
B12
1123456789
A1
BC2 #1
B1
A2
B2
A3
B3
A4
B4
A5
B5
A6
B6
1123456789
C1
BC2 #2
D1
C2
D2
C3
D3
C4
D4
C5
D5
C6
D6
1123456789
BC2 #6
C7
D7
C8
D8
C9
D9
C10
D10
C11
D11
C12
D12
1123456789
E7
BC2 #7
F7
E8
F8
E9
F9
E10
F10
E11
F11
E12
F12
1123456789
E1
BC2 #3
F1
E2
F2
E3
F3
E4
F4
E5
F5
E6
F6
1123456789
G1
BC2 #4
H1
G2
H2
G3
H3
G4
H4
G5
H5
G6
H6
1123456789
G7
BC2 #8
H7
G8
H8
G9
H9
G10
H10
G11
H11
G12
H12
1123456789
Next step
Proceed to Wash BC2 .
Wash BC2
Hands-on time: 20 minutes for 4 BeadChips
30 minutes for 8 BeadChips
Prepare the BeadChips for the staining process.
New Materials Quantity
WB1 Bottle
Per 4 or 8 BeadChips
Multi-Sample BeadChip Alignment
Fixture
Te-Flow Flow-Through Chambers
(with black frames, spacers, glass back plates, and clamps)
Wash Dish
Wash Rack
1
4 or 8
2
1
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Preparation
Fill a wash dish with 200 ml WB1. Label the dish.
Fill a wash dish with 200 ml PB1. Label the dish.
Fill the BeadChip Alignment Fixture with 150 ml PB1.
Separate the clear plastic spacers from the white backs.
Thaw WB1 to room temperature (22°C) and allow to equilibrate. Ensure that the solution is completely redissolved.
Clean the glass back plates according to the directions in the Infinium II
Assay Lab Setup and Procedures Guide (Illumina part # 11207963).
Steps
1. Attach the wire handle to the wash rack. Submerge wash rack in the wash dish containing WB1.
2. Remove the BeadChips from the Hyb Chambers.
3. Using power-free gloves and pulling from the barcode end, slide the coverseal off the BeadChip in a downward direction.
4. Carefully slide each BeadChip into the wash rack. Make sure each one is completely submerged in the WB1.
5. When all the BeadChips are in the rack, move the wash rack up and down for 1 minute, breaking the surface of the reagent with gentle, slow agitation.
6. Move the wash rack to the wash dish containing PB1. Make sure the
BeadChips are completely submerged.
7. Move the wash rack up and down for 1 minute, breaking the surface of the reagent with gentle, slow agitation.
Assemble Flow-Through Chambers
1. For each BeadChip, place a black frame into the BeadChip
Alignment Fixture. The fixture should be pre-filled with 150 ml PB1.
2. Place each BeadChip into a black frame, aligning its barcode with the ridges stamped onto the fixture.
3. Place a clear spacer on top of each BeadChip. Use the Alignment
Fixture grooves to guide the spacers into position.
4. Place the Alignment Bar onto the Alignment Fixture.
5. Use a Whoosh duster or laboratory air gun to quickly remove any accumulated dust from the glass back plates.
6. Place a clean glass back plate onto the clear spacer over each
BeadChip. Put the plate reservoir at the barcode end of the
BeadChip, facing inward to create a reservoir against the surface.
7. Attach the metal clamps as follows: a. Gently push the glass back plate against the Alignment Bar to hold it steady. b. Place the first metal clamp around the Flow-Through Chamber so that one stripe shows between it and the Alignment Bar.
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XStain BC2
c. Place a second metal clamp on the Flow-Through Chamber so that no stripes show between it and the barcode.
8. With scissors, trim the spacer at the non-barcode end of the assembly. Slip scissors up over the barcode to trim the other end.
Next step
Proceed to XStain BC2 .
Hands-on time:
Dry time:
3 hours for 8 BeadChips
1 hour
Wash unhybridized and non-specifically hybridized DNA sample from the
BeadChips. Add labelled nucleotides to extend the primers hybridized to the
DNA. Stain the primers, disassemble the Flow-Through Chambers, and coat the BeadChips for protection.
New Materials Quantity
95% formamide/1 mM EDTA
RA1
15 ml
Bottle (10 ml)
Per 96 samples /
8 BeadChips
XC3
LTM
Make sure that all LTM tubes specify the same stain temperature.
Bottle (75 ml)
2 tubes
Pre-Amp
Post-Amp
Alconox
®
Powder Detergent
EtOH
95% formamide/1 mM EDTA
as needed as needed
15 ml
Preparation
Ensure the water circulator is filled to the appropriate level.
Turn on the water circulator. Set it to a temperature that brings the
Chamber Rack to 44°C at equilibrium.
Test several locations on the Chamber Rack, using the Illumina
Temperature Probe, to ensure that it is uniformly 44°C.
Remove bubbles trapped in the Chamber Rack according to the Te-Flow
(Tecan Flow Through Module) Operating Manual, Tecan Doc ID 391584.
Thaw all reagent tubes to room temperature (22°C). Centrifuge the thawed reagents to 3000 xg for 3 minutes.
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Steps
The remaining steps must be performed without interruption.
1. Remove the Hyb Chamber from the Illumina Hybridization Oven.
Quickly place each Flow-Through Chamber assembly into the
Chamber Rack.
For 4 BeadChips, place the BeadChips in every other position, starting at 1, in the first row. For larger numbers of BeadChips, fill all positions in the first row, then the second and third.
2. Immediately wash the Hyb Chamber reservoirs with dH
2
O, and scrub them clean with a small brush, ensuring that no PB2 remains.
Single-Base Extension
Into the reservoir of each Flow-Through Chamber, dispense:
1. 150 µl RA1. Incubate for 30 seconds. Repeat 5 times.
2. 450 µl XC1. Incubate for 10 minutes.
3. 450 µl XC2. Incubate for 10 minutes.
4. 200 µl TEM. Incubate for 15 minutes.
5. 450 µl 95% formamide/1 mM EDTA. Incubate for 1 minute. Repeat once.
6. Incubate for 5 minutes.
7. Begin ramping the Chamber Rack to the stain temperature indicated on the LTM tube. If none is indicated, ramp to 37°C.
8. 450 µl XC3. Incubate for 1 minute. Repeat once.
9. Wait until the Chamber Rack reaches the correct temperature.
Stain BeadChip
1. If you plan to image the BeadChip immediately after the staining process, turn on the Illumina BeadArray™ Reader to allow the lasers to stabilize.
Into the reservoir of each Flow-Through Chamber, dispense:
2. 250 µl LTM. Incubate for 10 minutes.
3. 450 µl XC3. Incubate for 1 minute. Repeat once.
4. Wait 5 minutes.
5. 250 µl ATM. Incubate for 10 minutes.
6. 450 µl XC3. Incubate for 1 minute. Repeat once.
7. Incubate for 5 minutes.
8. 250 µl LTM. Incubate for 10 minutes.
9. 450 µl XC3. Incubate for 1 minute. Repeat once.
10. Incubate 5 minutes.
11. 250 µl ATM. Incubate for 10 minutes.
12. 450 µl XC3. Incubate for 1 minute. Repeat once.
13. Incubate for 5 minutes.
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14. 250 µl LTM. Incubate for 10 minutes.
15. 450 µl XC3. Incubate for 1 minute. Repeat once.
16. Incubate for 5 minutes.
17. Immediately remove the Flow-Through Chambers from the Chamber
Rack. Place flat on the lab bench at room temperature (22°C).
Wash and Coat
1. Pour 310 ml PB1 per 8 BeadChips into a wash dish. Cover dish.
2. Place the staining rack inside the wash dish, with the locking arms facing towards you.
Handle the BeadChips only by the edges or the barcode end. Do not let the BeadChips dry out.
3. For each BeadChip: a. Use the dismantling tool to remove the two metal clamps from the Flow-Through Chamber. b. Remove the glass back plate, the spacer, and then the
BeadChip. c. Immediately place each BeadChip into the staining rack that is in the wash dish with the barcode facing away from you. Place half of the BeadChips above the handle and half below. All chips should be completely submerged.
4. Slowly move the staining rack up and down 10 times, breaking the surface of the reagent.
5. Soak for 5 minutes.
Do not leave the BeadChips in the PB1 for more than
30 minutes.
6. Pour 310 ml XC4 into a wash dish. Do not let the XC4 sit for more than 10 minutes.
7. Move the BeadChip staining rack into the XC4 dish, with the barcodes facing away from you.
8. Slowly move the staining rack up and down 10 times, breaking the surface of the reagent.
9. Soak for 5 minutes.
10. Lift the staining rack out of the solution and place it horizontally on a tube rack, BeadChip barcodes facing up.
11. Remove the BeadChips from the staining rack with locking tweezers, working from top to bottom. Place each BeadChip flat on a tube rack to dry. Remove the staining rack handle after removing the first four
BeadChips.
12. Dry the BeadChips in the vacuum desiccator for 50–55 minutes at
508 mm Hg (0.68 bar).
13. Clean the underside of the BeadChip with a ProStat EtOH wipe.
Do not touch the stripes with the wipe or allow EtOH to drip onto the stripes.
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14. Clean and store the glass back plates and Hyb Chamber components.
Good stopping point
Image BC2
Next step
Do one of the following:
•
Proceed to Image BC2 .
•
Store the BeadChips in the Illumina BeadChip Slide Storage Box inside a vacuum desiccator at room temperature (22°C). Image the
BeadChips within 72 hours.
Scan time: 45 minutes per BeadChip
Scan the BeadChips with the Illumina BeadArray™ Reader, which uses a laser to excite the fluorophore of the single-base extension product on the beads.
The scanner records high-resolution images of the light emitted from the fluorophores. The data from these images are analyzed to determine SNP genotypes using Illumina’s genotyping software.
Preparation
Turn on the BeadArray Reader one or two hours before scanning. This allows the lasers to stabilize.
Steps
1. Open the Illumina BeadScan application.
2. Click Scan on the Welcome screen.
3. Select BeadChip from the Docking Fixture menu.
4. In the Settings area, click Edit. The Options dialog box appears.
5. Click Browse to navigate to and select the following directories:
•
Data repository directory containing your data
•
Decode map directory, containing the decode data from the
BeadChip CD
6. To save the image data in compact JPG format, select the
Compressed check box.
7. Click either Save for this Scan or Save for All Scans.
8. Place the BeadChip(s) in the BeadArray Reader tray. Check to ensure that they are seated correctly.
9. Scan each BeadChip barcode. The barcode should appear on the screen in the position corresponding to the tray position.
10. Record the scanner ID and scan date in the lab tracking worksheet.
11. Click to open the Select Scan Settings dialog box.
12. Select a scan method for each BeadChip. Click Select.
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13. Click Scan. Allow 45 minutes for the scanning process.
Tip : If the BeadArray Reader is unable to scan the fiducials, you may need to clean the BeadChips. See the Infinium II Assay Lab Setup
and Procedures Guide (Illumina part # 11207963) for directions.
14. When the Welcome screen reappears, click Open Tray.
15. Remove the BeadChips.
Next step
Do one of the following:
•
Scan the next set of BeadChips.
•
Right click near the Illumina logo on the Welcome screen and select
Exit. Close the BeadArray Reader tray and turn off the machine.
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