Infinium HD Gemini Assay Experienced User - Support

Infinium HD Gemini Assay Experienced User - Support
Infinium® HD Assay Gemini, Manual Protocol
Experienced User Card
Day 1
Day 2
Day 3
Wash BeadChip
Quantitate DNA
Fragment AMP3
Hands-on: 30 min/plate
Fluorometer: 5 min/plate
Hands-on: ~30 min
Incubation: 60 min
Hands-on:
~20 min/8 BeadChips
Reagents
Reagents
Reagents
Lambda DNA
PicoGreen dsDNA
1X TE
Output
FRG
PB1
Output
Output
AMP3 Plate
BeadChip
Sample QDNA Plate with
Quantitated DNA
XStain HD
BeadChip
Precip AMP3
Make AMP3
Hands-on:
~20 min/16 samples
Incubation: 20–24 hours
Reagents
0.1N NaOH
MP1
AMM
Hands-on: ~30 min/plate
Incubation: 50 min
Dry: 60 min
Reagents
2-propanol
PA1
Output
AMP3 Plate
Output
AMP3 Plate with
Amplified DNA
Hands-on: ~3 hours
Dry: 60 min
Reagents
RA1
95% Formamide /
1 mM EDTA
PB1
XC1
XC2
XC3
XC4
TEM
STM
ATM
Output
BeadChip
Resuspend AMP3
Hands-on: ~30 min
Incubation: 60 min
Reagents
RA1
Output
AMP3 Plate
Image BeadChip
iScan System Scan Time:
35 min/BeadChip
BeadArray Reader Scan
Time: 45 min/BeadChip
Output
Image and Data Files
Hyb Duo BeadChip
Hands-on:
~30 min/8 BeadChips
Incubation: 16–24 hours
Reagents
PB2
Output
BeadChip
Infinium LIMS
Optional
Pre-Amp
Post-Amp
Cold Storage
Option
Overnight
Incubation
Fill in the lab tracking
form and the sample
sheet as you perform
the assay
Catalog # WG-901-3002
Part # 11311015 Rev. A
Page 1 of 28
Infinium® HD Assay Gemini, Manual Protocol (Pre-AMP)
Experienced User Card
Quant DNA
(Optional)
Estimated Time
Consumables
Preparation
Steps
This process uses the PicoGreen dsDNA quantitation reagent to quantitate
double-stranded DNA samples. You can quantitate up to six plates, each
containing up to 96 samples.
Hands-on time: ~20 minutes per plate, plus 10 minutes to prepare the PicoGreen
Spectrofluorometer read time: ~5 minutes per plate
Item
Quantity
Storage
Supplied By
PicoGreen dsDNA
quantitation reagent
See Instructions
2 to 8°C
User
1X TE
See Instructions
Room
User
temperature
Lambda DNA
See Instructions
2 to 8°C
96-well 0.65 ml microtiter
plate
1 per 96 samples
User
Fluotrac 200 96-well flatbottom plate
1 per Std DNA plate
1 per Sample DNA plate
User
User
[ ]
Thaw PicoGreen to room temperature for 60 minutes in a lightimpermeable container.
[ ]
Hand-label the microtiter plate “Standard DNA.”
[ ]
Hand-label one of the Fluotrac plates “Standard QDNA.” Hand-label the
other Fluotrac plate “Sample QDNA.” In the Sample Sheet, enter the
Sample_Name (optional) and Sample_Plate for each Sample_Well.
Make Standard DNA Plate
[ ] 1. Add stock Lambda DNA to well A1 in the plate labelled “Standard DNA”
and dilute it to 75 ng/μl in a final volume of 233.3 μl. Pipette up and
down several times.
[ ] 2. Add 66.7 μl 1X TEto well B1.
[ ] 3. Add 100 μl 1X TE to wells C, D, E, F, G, and H of column 1.
[ ] 4. Transfer 133.3 μl of Lambda DNA from well A1 into well B1. Pipette up
and down several times.
[ ] 5. Change tips. Transfer 100 μl from well B1 into well C1. Pipette up and
down several times.
[ ] 6. Repeat for wells D1, E1, F1, and G1, changing tips each time. Do not
transfer from well G1 to H1. Well H1 serves as the blank 0 ng/μl
Lambda DNA.
[ ] 7. Cover the Standard DNA plate with an adhesive seal.
Catalog # WG-901-3002
Part # 11311015 Rev. A
Page 3 of 28
Infinium® HD Assay Gemini, Manual Protocol (Pre-AMP)
Experienced User Card
Dilute PicoGreen
[ ] 1. Prepare a 1:200 dilution of PicoGreen into 1X TE, using a sealed 100 ml
or 250 ml Nalgene bottle wrapped in aluminum foil.
Use 115 μl PicoGreen and 23 ml 1X TE for 1 plate, 215 μl PicoGreen and
43 ml 1X TE for 2 plates, and so on up to 6 plates.
[ ] 2. Cap the foil-wrapped bottle and vortex to mix.
Create Standard QDNA Plate with Diluted PicoGreen
[ ] 1. Pour the PicoGreen/1X TE dilution into a clean reagent reservoir.
[ ] 2. Using a multichannel pipette, transfer 195 μl PicoGreen/1X TE dilution
into each well of columns 1 and 2 of the Fluotrac plate labelled
“Standard QDNA“.
[ ] 3. Add 2 μl of each stock Lambda DNA dilution from the Standard DNA
plate to columns 1 and 2 of the Standard QDNA Fluotrac plate.
[ ] 4. Immediately cover the plate with an adhesive aluminum seal.
Prepare Sample QDNA Plate with PicoGreen and DNA
[ ] 1. Using a multichannel pipette, transfer 195 μl PicoGreen/1X TE dilution
into each well of columns 1 and 2 of the plate labelled “Sample QDNA“.
[ ] 2. Add 2 μl of DNA sample to all 96 wells of the Sample QDNA plate.
[ ] 3. Immediately cover the plate with an adhesive aluminum seal.
Read QDNA Plate
[ ] 1. Turn on the spectrofluorometer. At the PC, open the SoftMax Pro
program.
[ ] 2. Load the Illumina QDNA.ppr file from the installation CD that came with
your system.
[ ] 3. Select Assays | Nucleic Acids | Illumina QDNA.
[ ] 4. Place the Standard QDNA Fluotrac Plate into the spectrofluorometer.
[ ] 5. Click the blue arrow next to Lambda Standard then click Read.
[ ] 6. When the software finishes reading the data, remove the plate from the
drawer.
[ ] 7. Click the blue arrow next to Standard Curve to view the standard curve
graph.
[ ] 8. Place the first Sample QDNA plate in the spectrofluorometer.
[ ] 9. Click the blue arrow next to QDNA#1 then click Read.
[ ] 10. When the software finishes reading the plate, remove the plate from the
drawer.
[ ] 11. Repeat steps 8 through 10 to quantitate all Sample QDNA plates.
[ ] 12. Once all plates have been read, click File | Save to save the output data
file (*.pda).
[ ] 13. When you have saved the *.pda file, click File | Import/Export | Export
and export the file as a *.txt file.
[ ] 14. Do one of the following:
Catalog # WG-901-3002
Part # 11311015 Rev. A
•
Proceed to Make AMP3.
•
Store the quantitated DNA at 2 to 8ºC for up to one month.
Page 4 of 28
Infinium® HD Assay Gemini, Manual Protocol (Pre-AMP)
Experienced User Card
Make AMP3
Estimated Time
Consumables
Move DNA samples into AMP3 plate. Denature and neutralize samples,
and prepare them for amplification. Incubate overnight to amplify.
Hands-on time: ~20 minutes per 16 samples
Incubation time: ~20–24 hours
Item
Quantity
Storage
Supplied By
MP1
1 tube per
16 samples
-15 to -25°C
Illumina
AMM
1 tube per
16 samples
-15 to -25°C
Illumina
0.1N NaOH
15 ml for 16–48
samples
2 to 8°C
User
96-well 0.8 ml microtiter plate 1 plate per
(MIDI)
48 samples
WG#-DNA plate with up to
96 DNA samples (50 ng/μl)
Preparation
Steps
1 plate for up to 48
samples
User
-15 to -25°C
User
[ ]
Preheat the Illumina Hybridization Oven in the post-amp area to 37°C.
[ ]
Apply an AMP3 barcode label to a new MIDI plate.
[ ]
Thaw MP1 and AMM tubes to room temperature. Gently invert to mix,
then pulse centrifuge to 280 xg.
[ ]
Thaw DNA samples to room temperature.
[ ] 1. If you do not already have a WG#-DNA plate, create one by adding
DNA, normalized to 50 ng/μl, into either a:
•
MIDI plate: 40 μl to each WG#-DNA plate well
•
TCY plate: 30 μl to each WG#-DNA plate well
Apply a barcode label to the new WG#-DNA plate.
[ ] 2. Vortex the WG#-DNA plate at 1600 rpm (actual vortex speed) for
1 minute.
[ ] 3. Centrifuge to 280 xg for 1 minute.
[ ] 4. Transfer 8 μl DNA sample, into each well in the following AMP3 plate
columns:
•
Column 1 (8 samples)
•
Columns 1 and 3 (16 samples)
•
Columns 1, 3, 5, 7, 9, and 11 (48 samples)
[ ] 5. Dispense 8 μl 0.1N NaOH into each well that contains DNA.
[ ] 6. Incubate for 10 minutes at room temperature.
[ ] 7. Dispense 135 μl MP1 into each well containing sample.
Catalog # WG-901-3002
Part # 11311015 Rev. A
Page 5 of 28
Infinium® HD Assay Gemini, Manual Protocol (Pre-AMP)
Experienced User Card
[ ] 8. Dispense 150 μl AMM into each well containing sample.
[ ] 9. Seal the plate with a cap mat.
[ ] 10. Invert the sealed plate at least 10 times to mix contents.
[ ] 11. Pulse centrifuge to 280 xg.
[ ] 12. Incubate in the Illumina Hybridization Oven for 20–24 hours at 37°C.
[ ] 13. Proceed immediately to Fragment AMP3.
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.
Catalog # WG-901-3002
Part # 11311015 Rev. A
Page 6 of 28
Infinium® HD Assay Gemini, Manual Protocol (Post-AMP)
Experienced User Card
Fragment
AMP3
Estimated Time
Consumables
Preparation
Steps
Enzymatically fragment DNA, using end-point fragmentation to avoid
over-fragmentation.
Hands-on time: ~30 minutes per 48 samples
Incubation time: 1 hour
Item
Quantity
Storage
FRG
1 tube per 16 samples -15 to -25°C
Supplied By
Illumina
[ ]
Preheat the heat block with the MIDI plate insert to 37°C.
[ ]
Thaw the FRG tube to room temperature. Invert several times to mix
contents. Pulse centrifuge to 280 xg for 1 minute.
[ ] 1. Centrifuge the plate to 50 xg for 1 minute.
[ ] 2. Thoroughly pipette-mix all wells containing sample to evenly distribute
precipitate.
[ ] 3. Split the sample into 1 additional well, for a total of 2 wells per sample.
Each well should contain 150 μl.
48
41 to
40
33 to
Samp
les
Samp
les
32
25 to
24
Samp
les
17 to
Samp
les
Samp
les
Samp
les
9 to
1 to
8
16
For example, move 150 μl sample from A1 into A2.
A
B
WG1123456789-AMP3
C
D
E
F
AMP3 Plate
G
H
1
2
3
4
5
6
7
8
9 10 11 12
[ ] 4. Dispense 50 μl FRG to each well containing sample.
[ ] 5. Seal the AMP3 plate with the cap mat.
[ ] 6. Vortex the AMP3 plate at 1600 rpm for 1 minute.
[ ] 7. Centrifuge the plate to 50 xg for 1 minute at 22°C.
[ ] 8. Incubate the sealed plate on the 37°C heat block for 1 hour.
[ ] 9. Do one of the following:
Catalog # WG-901-3002
Part # 11311015 Rev. A
Page 7 of 28
Infinium® HD Assay Gemini, Manual Protocol (Post-AMP)
Experienced User Card
Catalog # WG-901-3002
Part # 11311015 Rev. A
•
Proceed to Precip AMP3. Leave the plate in 37°C heat block until
setup is complete.
•
Store the sealed AMP3 plate at -15 to -25°C if you do not plan to
proceed to the next step immediately.
Page 8 of 28
Infinium® HD Assay Gemini, Manual Protocol (Post-AMP)
Experienced User Card
Precip AMP3
Estimated Time
Consumables
Preparation
Steps
Precipitate the DNA sample using 2-propanol and PA1.
Hands-on time: ~30 minutes per 48 samples
Incubation time: 2 hours
Item
Quantity
Storage
Supplied By
PA1
1 tube per
16 samples
2 to 8°C
Illumina
100% 2-propanol
12 ml per 16 samples Room
40 ml per 48 samples temperature
User
[ ]
If frozen, thaw AMP3 plate to room temperature. Pulse centrifuge to
50 xg.
[ ]
Thaw PA1 to room temperature. Centrifuge to 280 xg for 1 minute.
[ ]
Preheat the heat block to 37°C, if it is not already.
[ ]
Turn on the heat sealer.
[ ]
Set the centrifuge to 4°C.
[ ] 1. Dispense 100 μl PA1 to each well containing sample.
[ ] 2. Seal the plate with the cap mat.
[ ] 3. Vortex the plate at 1600 rpm for 1 minute.
[ ] 4. Centrifuge to 50 xg at 22°C for 1 minute.
[ ] 5. Incubate at 37°C for 5 minutes.
[ ] 6. Add 300 μl 100% 2-propanol to each well containing sample.
[ ] 7. Seal the plate with a new, dry cap mat.
[ ] 8. Invert at least 10 times to mix contents.
[ ] 9. Incubate at 4°C for 30 minutes.
[ ] 10. Place the sealed AMP3 plate in the centrifuge opposite another plate of
equal weight.
[ ] 11. Centrifuge to 3000 xg at 4°C for 20 minutes.
Perform the next steps immediately, to avoid dislodging the blue pellet.
If any delay occurs, repeat the 20-minute centrifugation.
[ ] 12. Remove the cap mat.
[ ] 13. Decant the supernatant by quickly inverting the AMP3 plate and
smacking it down onto an absorbent pad.
[ ] 14. Tap the plate firmly on the pad several times over a period of 1 minute or
until all wells are completely devoid of liquid.
Catalog # WG-901-3002
Part # 11311015 Rev. A
Page 9 of 28
Infinium® HD Assay Gemini, Manual Protocol (Post-AMP)
Experienced User Card
[ ] 15. Place the inverted, uncovered plate on a tube rack for 1 hour at room
temperature to air dry the pellet.
[ ] 16. Do one of the following:
Catalog # WG-901-3002
Part # 11311015 Rev. A
•
Proceed to Resuspend AMP3.
•
Heat-seal the AMP3 plate and store it at -15 to -25°C for up to 24
hours or -80°C for long-term storage.
Page 10 of 28
Infinium® HD Assay Gemini, Manual Protocol (Post-AMP)
Experienced User Card
Resuspend
AMP3
Estimated Time
Consumables
Preparation
Steps
Resuspend the precipitated DNA using RA1.
Hands-on time: ~30 minutes per 48 samples
Incubation time: 1 hour
Item
Quantity
Storage
Supplied By
RA1
9 ml per 48 samples
-15 to -25°C
Illumina
[ ]
Gradually warm the RA1 reagent to room temperature. Gently mix to
dissolve crystals.
[ ]
If you stored the AMP3 plate at -15 to -25°C, thaw it to room
temperature.
[ ]
Preheat the Illumina Hybridization Oven to 48°C.
[ ]
Turn on the heat sealer.
[ ] 1. Add 46 μl RA1 to each well of the AMP3 plate containing a DNA pellet.
[ ] 2. Heat-seal the AMP3 plate with a foil seal.
[ ] 3. Incubate it in the Illumina Hybridization Oven for 1 hour at 48°C.
[ ] 4. Vortex the plate at 1800 rpm for 1 minute.
[ ] 5. Pulse centrifuge to 280 xg.
If you stored the pellets at -15 to -25°C for more than 72 hours after the
Precip AMP3 process, you may need to repeat steps 3 to 5 until the pellets are completely resuspended.
[ ] 6. Do one of the following:
Catalog # WG-901-3002
Part # 11311015 Rev. A
•
Proceed to Hyb Duo BeadChip. If you plan to do so immediately, it is
safe to leave the RA1 at room temperature.
•
Store the sealed AMP3 plate and the RA1 at -15 to -25°C (-80°C if
storing for more than 24 hours).
Page 11 of 28
Infinium® HD Assay Gemini, Manual Protocol (Post-AMP)
Experienced User Card
Hyb Duo
BeadChip
Estimated Time
Dispense the fragmented, resuspended DNA samples onto
BeadChips. Incubate the BeadChips in the Illumina Hybridization Oven
to hybridize the samples onto the BeadChips.
Hands-on time:
• ~30 minutes for 8 BeadChips (16 samples)
•
~45 minutes for 16 BeadChips (32 samples)
•
~1 hour for 24 BeadChips (48 samples)
Incubation time: 16–24 hours
Consumables
Preparation
Steps
Item
Quantity
(per 16 samples)
Storage
Supplied By
PB2
2 tubes
Room
temperature
Illumina
BeadChips
8
Illumina
Hyb Chambers
2
Illumina
Hyb Chamber gaskets
2
Illumina
Hyb Chamber inserts
8
Illumina
[ ]
Preheat the heat block to 95°C.
[ ]
Preheat the Illumina Hybridization Oven to 48°C and set the rocker
speed to 5.
[ ]
If you plan to perform the XStain process tomorrow, begin thawing the
XC4 reagent. For instructions, see Resuspend XC4 Reagent for XStain
HD BeadChip.
Prepare Hyb Chamber(s)
[ ] 1. Place the Hyb Chamber gaskets into the Hyb Chambers.
[ ] 2. Dispense 400 μl PB2 to each of the 8 humidifying buffer reservoirs in
each Hyb Chamber.
[ ] 3. Place the Hyb Chamber inserts into the Hyb Chambers.
[ ] 4. Secure the lid of each Hyb Chamber. Keep on bench at room
temperature until ready to load BeadChips.
Hyb Duo BeadChip
[ ] 1. Place the resuspended AMP3 plate on the heat block to denature the
samples at 95°C for 20 minutes.
[ ] 2. Remove the BeadChips from 2 to 8°C storage but do not unpackage.
[ ] 3. After the 20-minute incubation, pulse centrifuge the AMP3 plate to
280 xg.
Catalog # WG-901-3002
Part # 11311015 Rev. A
Page 13 of 28
Infinium® HD Assay Gemini, Manual Protocol (Post-AMP)
Experienced User Card
48
41 to
Samp
les
33 to
Samp
les
Samp
les
Samp
les
25 to
40
32
24
17 to
9 to
Samp
les
Samp
les
1 to
8
16
[ ] 4. Combine the two separate wells back into the original well. For example,
move 46 μl sample from the A2 well back into A1.
A
B
WG1123456789-AMP3
C
D
E
F
AMP3 Plate
G
H
1
2
3
4
5
6
7
8
9 10 11 12
Load BeadChip
[ ] 1. Just before loading DNA samples, remove all BeadChips from their
packages.
[ ] 2. Place each BeadChip in a Hyb Chamber insert, orienting the barcode
end so that it matches the barcode symbol on the Hyb Chamber insert.
Make sure you place each BeadChip into the Hyb Chamber insert prior
to loading the DNA sample.
[ ] 3. Dispense 84 μl of each DNA sample into the appropriate BeadChip inlet
port according to the lab tracking form.
[ ] a. Load samples in the A1 and B1 wells of the AMP3 plate into the
first BeadChip.
[ ] b. Load samples in C1 and D1 into the second BeadChip.
[ ] c. Load samples in E1 and F1 into the third BeadChip.
[ ] d. Load samples in G1 and H1 into the fourth BeadChip.
Repeat the same pattern to transfer sample from column 3 to BeadChips
5–8, and from column 5 to BeadChips 9–12.
Catalog # WG-901-3002
Part # 11311015 Rev. A
Page 14 of 28
Infinium® HD Assay Gemini, Manual Protocol (Post-AMP)
Experienced User Card
WG1123456789-AMP3
AMP3 Plate
12
11
10
9
8
7
6
5
4
3
2
1
A B C D E F G H
A1
B1
1123456789
Two-Sample
BeadChips
1, 5, 9, 13,17, 21
C1
D1
1123456789
Two-Sample
BeadChips
2,6,10,14,18, 22
E1
F1
1123456789
Two-Sample
BeadChips
3, 7,11,15,19,23
G1
H1
1123456789
Two-Sample
BeadChips
4, 8,12 ,16, 20 ,2 4
[ ] 4. Visually inspect all sections of the BeadChips to ensure the DNA sample
covers all of each bead stripe. Record any sections that are not
completely covered.
Set Up Duo BeadChip for Hyb
[ ] 1. Load the Hyb Chamber inserts containing BeadChips into the Illumina
Hyb Chamber. Position the barcode end over the ridges indicated on the
Hyb Chamber.
[ ] 2. Place the back side of the lid onto the Hyb Chamber and then slowly
bring down the front end to avoid dislodging the Hyb Chamber inserts.
[ ] 3. Close the clamps on both sides of the Hyb Chamber.
[ ] 4. Place the Hyb Chamber into the 48°C Illumina Hybridization Oven so
that the clamps of the Hyb Chamber face the left and right side of the
oven. The Illumina logo on top of the Hyb Chamber should be facing
you.
Catalog # WG-901-3002
Part # 11311015 Rev. A
Page 15 of 28
Infinium® HD Assay Gemini, Manual Protocol (Post-AMP)
Experienced User Card
[ ] 5. If you are loading multiple Hyb Chambers, stack them on top of each
other. You can stack up to 3 Hyb Chambers, for a total of 6 in the Hyb
Oven.
[ ] 6. Start the rocker (optional).
[ ] 7. Incubate at 48°C for at least 16 hours but no more than 24 hours.
Resuspend XC4 Reagent for XStain HD BeadChip
[ ] 1. Add 330 ml 100% EtOH to the XC4 bottle.
[ ] 2. Shake vigorously for 15 seconds.
[ ] 3. Leave the bottle upright on the lab bench overnight.
[ ] 4. Shake again to ensure that the pellet is completely resuspended. If any
coating is visible, vortex at 1625 rpm until it is in complete suspension.
[ ] 5. Proceed to Wash BeadChip.
Catalog # WG-901-3002
Part # 11311015 Rev. A
Page 16 of 28
Infinium® HD Assay Gemini, Manual Protocol (Post-AMP)
Experienced User Card
Wash BeadChip
Estimated Time
Prepare the BeadChips for the staining process.
Hands-on time:
• 20 minutes per 8 BeadChips (16 samples)
•
30 minutes per 16 BeadChips (32 samples)
•
50 minutes per 24 BeadChips (48 samples)
Consumables
Preparation
Steps
Item
Quantity
(per 8 BeadChips)
Storage
Supplied By
PB1
550 ml
Room
temperature
Illumina
Multi-Sample BeadChip
Alignment Fixture
1
Illumina
Te-Flow Flow-Through
1 per BeadChip
Chambers (with black frames,
spacers, glass back plates,
and clamps)
Illumina
Wash Dish
8 BeadChips: 2 dishes
24 BeadChips: 6 dishes
Illumina
Wash Rack
8 BeadChips: 1 rack
24 BeadChips: 3 racks
Illumina
[ ]
Fill 2 wash dishes with PB1 (200 ml per wash dish). Label each dish
“PB1”.
[ ]
Fill the BeadChip Alignment Fixture with 150 ml PB1.
[ ]
Separate the clear plastic spacers from the white backs.
[ ]
Clean the glass back plates according to the directions in the Infinium
Assay Lab Setup and Procedures Guide.
Wash BeadChip
[ ] 1. Remove each Hyb Chamber from the Illumina Hybridization Oven.
[ ] 2. Attach the wire handle to the rack and submerge the wash rack in the
first wash dish containing 200 ml PB1.
[ ] 3. Remove the Hyb Chamber inserts from the Hyb Chambers.
[ ] 4. Remove BeadChips from the Hyb Chamber inserts one at a time.
Catalog # WG-901-3002
Part # 11311015 Rev. A
Page 17 of 28
Infinium® HD Assay Gemini, Manual Protocol (Post-AMP)
Experienced User Card
[ ] 5. Remove the IntelliHyb seal from each BeadChip as follows:
[ ] a. Using powder-free gloved hands, hold the BeadChip in one
hand with your thumb and forefinger on the long edges of the
BeadChip. The BeadChip may also be held with the thumb and
forefinger on the short edges of the BeadChip. In either case
avoid contact with the sample inlets. The barcode should be
facing up and be closest to you, and the top side of the
BeadChip should be angled slightly away from you.
[ ] b. Remove the entire seal in a single, rapid motion by pulling it off
in a diagonal direction. Start with a corner on the barcode end
and pull with a continuous upward motion away from you and
towards the opposite corner on the top side of the BeadChip.
Do not stop and start the pulling action. Do not touch the
exposed active areas.
[ ] 6. Immediately and carefully slide each BeadChip into the wash rack one at
a time, making sure that the BeadChip is completely submerged in the
PB1.
[ ] 7. Repeat steps 5 and 6 until all BeadChips are transferred to the
submerged wash rack. The wash rack holds up to 8 BeadChips.
[ ] 8. Once all BeadChips are in the wash rack, move the wash rack up and
down for 1 minute, breaking the surface of the PB1 with gentle, slow
agitation.
[ ] 9. Move the wash rack to the other wash dish containing PB1. Make sure
the BeadChips are completely submerged.
[ ] 10. Move the wash rack up and down for 1 minute, breaking the surface of
the PB1 with gentle, slow agitation.
[ ] 11. If you are processing more than 8 BeadChips:
[ ] a. Complete the steps in the next section, Assemble Flow-Through
Chambers, for the first eight BeadChips.
[ ] b. Place the assembled Flow-Through Chambers of the first eight
BeadChips on the lab bench in a horizontal position.
[ ] c. Repeat steps 3 through 11 from this section for any additional
BeadChips. Use new PB1 for each set of eight BeadChips.
Catalog # WG-901-3002
Part # 11311015 Rev. A
Page 18 of 28
Infinium® HD Assay Gemini, Manual Protocol (Post-AMP)
Experienced User Card
Assemble Flow-Through Chambers
[ ] 1. If you have not done so yet, fill the BeadChip Alignment Fixture with 150
ml PB1.
[ ] 2. For each BeadChip to be processed, place a black frame into the MultiSample BeadChip Alignment Fixture.
[ ] 3. Place each BeadChip to be processed into a black frame, aligning its
barcode with the ridges stamped onto the Alignment Fixture. Each
BeadChip should be fully immersed in PB1.
[ ] 4. Place a clear spacer onto the top of each BeadChip. Use the Alignment
Fixture grooves to guide the spacers into proper position.
[ ] 5. Place the Alignment Bar onto the Alignment Fixture.
[ ] 6. Use a laboratory air gun to quickly remove any accumulated dust from
the glass back plates just before placing them onto the BeadChips.
[ ] 7. Place a clean glass back plate on top of the clear spacer covering each
BeadChip. The plate reservoir should be at the barcode end of the
BeadChip, facing inward to create a reservoir against the BeadChip
surface.
[ ] 8. Attach the metal clamps to the Flow-Through Chambers as follows:
[ ] a. Gently push the glass back plate up against the Alignment Bar
with one finger.
[ ] b. Place the first metal clamp around the Flow-Through Chamber
so that the clamp is about 5 millimeters from the top edge.
[ ] c. Place the second metal clamp around the Flow-Through
Chamber at the barcode end, about 5 millimeters from the
reagent reservoir.
[ ] 9. Using scissors, trim the ends of the clear plastic spacers from the FlowThrough Chamber assembly. Slip scissors up over the barcode to trim the
other end.
[ ] 10. Immediately wash the Hyb Chamber reservoirs with dH2O and scrub
them with a small cleaning brush, ensuring that no PB2 remains.
[ ] 11. Proceed to XStain HD BeadChip.
Catalog # WG-901-3002
Part # 11311015 Rev. A
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Infinium® HD Assay Gemini, Manual Protocol (Post-AMP)
Experienced User Card
XStain HD
BeadChip
Estimated Time
Consumables
Catalog # WG-901-3002
Part # 11311015 Rev. A
Wash unhybridized and non-specifically hybridized DNA sample from
the BeadChips. Add labeled nucleotides to extend the primers
hybridized to the DNA. Stain the primers, disassemble the FlowThrough Chambers, and coat the BeadChips for protection.
Hands-on time: ~3 hours for 8–24 BeadChips
Dry time: 55 minutes
Item
Quantity
Storage
Supplied By
RA1
10 ml for 1–8
BeadChips
20 ml for 9–16
BeadChips
30 ml for 17–24
BeadChips
-15 to -25°C
Illumina
XC1
2 tubes
(per 8 BeadChips)
-15 to -25°C
Illumina
XC2
2 tubes
(per 8 BeadChips)
-15 to -25°C
Illumina
TEM
2 tubes
(per 8 BeadChips)
-15 to -25°C
Illumina
XC3
50 ml for 1–8
BeadChips
100 ml for 9–16
BeadChips
150 ml for 24
BeadChips
Room
temperature
Illumina
STM (Make sure that all STM
tubes indicate the same stain
temperature on the label)
2 tubes
(per 8 BeadChips)
-15 to -25°C
Illumina
ATM
2 tubes
(per 8 BeadChips)
-15 to -25°C
Illumina
PB1
310 ml for 1–8
BeadChips
285 ml for 9– 24
BeadChips
Room
temperature
Illumina
XC4
310 ml for 1–8
BeadChips
285 ml for 9– 24
BeadChips
-15 to -25°C
Illumina
Alconox Powder Detergent
as needed
Room
temperature
User
EtOH
as needed
Room
temperature
User
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Infinium® HD Assay Gemini, Manual Protocol (Post-AMP)
Experienced User Card
Preparation
Steps
Item
Quantity
Storage
Supplied By
95% formamide/1 mM EDTA
15 ml for 1–8
BeadChips
17 ml for 9–16
BeadChips
25 ml for 17–24
BeadChips
-15 to -25°C
User
[ ]
Gradually warm the RA1 reagent to room temperature. Gently mix to
dissolve crystals.
[ ]
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.
[ ]
Remove bubbles trapped in the Chamber Rack.
[ ]
Test several locations on the Chamber Rack, using the Illumina
Temperature Probe, to ensure that it is uniformly 44°C.
[ ]
Place all reagent tubes in a rack in the order in which they will be used. If
frozen, allow them to thaw to room temperature and centrifuge to
3000 xg for 3 minutes.
Single-Base Extension
[ ] 1. When the Chamber Rack reaches 44°C, quickly place each Flow-Through
Chamber assembly into the Chamber Rack.
For 4 BeadChips, place the Flow-Through Chambers in every other position, starting at 1, in the first row of the Chamber Rack. For larger numbers of BeadChips, fill all positions in the first row, then the second and
third.
[ ] 2. Into the reservoir of each Flow-Through Chamber, dispense:
[ ] [ ] [ ] [ ] [ ] [ ] a. 150 μl RA1. Incubate for 30 seconds. Repeat 5 times.
[ ] b. 450 μl XC1. Incubate for 10 minutes.
[ ] c. 450 μl XC2. Incubate for 10 minutes.
[ ] d. 200 μl TEM. Incubate for 15 minutes.
[ ] [ ] e. 450 μl 95% formamide/1 mM EDTA. Incubate for 1 minute.
Repeat once.
[ ] f. Incubate 5 minutes.
[ ] g. Begin ramping the Chamber Rack temperature to the
temperature indicated on the STM tube, or to 37°C if none is
shown.
[ ] [ ] h. 450 μl XC3. Incubate for 1 minute. Repeat once.
[ ] 3. 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 iScan or BeadArray Reader now.
[ ] 2. Into the reservoir of each Flow-Through Chamber, dispense:
[ ] a. 250 μl STM and incubate for 10 minutes.
[ ] [ ] b. 450 μl XC3 and incubate for 1 minute. Repeat once, and then
wait 5 minutes.
Catalog # WG-901-3002
Part # 11311015 Rev. A
Page 22 of 28
Infinium® HD Assay Gemini, Manual Protocol (Post-AMP)
Experienced User Card
[ ] c. 250 μl ATM and incubate for 10 minutes.
[ ] [ ] d. 450 μl XC3 and incubate for 1 minute. Repeat once, and then
wait 5 minutes.
[ ] e. 250 μl STM and incubate for 10 minutes.
[ ] [ ] f. 450 μl XC3 and incubate for 1 minute. Repeat once, and then
wait 5 minutes.
[ ] g. 250 μl ATM and incubate for 10 minutes.
[ ] [ ] h. 450 μl XC3 and incubate for 1 minute. Repeat once, and then
wait 5 minutes.
[ ] i. 250 μl STM and incubate for 10 minutes.
[ ] [ ] j. 450 μl XC3 and incubate for 1 minute. Repeat once, and then
wait 5 minutes.
[ ] 3. Immediately remove the Flow-Through Chambers from the Chamber
Rack and place horizontally on a lab bench at room temperature.
Wash and Coat
[ ] 1. Dispense PB1 into a wash dish as follows, and then cover the dish:
•
For 8 Beadchips, dispense 310 ml
•
For 16 Beadchips, dispense 300 ml
•
For 24 BeadChips, dispense 285 ml
[ ] 2. Place the staining rack inside the wash dish. The locking arms and tab
should face towards you.
CAUTION
Handle the BeadChips only by the edges or the barcode
end. Do not let the BeadChips dry out.
[ ] 3. For each BeadChip:
•
Use the dismantling tool to remove the two metal clamps from the
Flow-Through Chamber.
•
Remove the glass back plate, the spacer, and then the BeadChip.
•
Immediately place each BeadChip into the staining rack that is in the
PB1 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.
CAUTION
Do not leave the BeadChips in the PB1 for more than
30 minutes.
[ ] 6. Dispense XC4 into a wash dish as follows:
Catalog # WG-901-3002
Part # 11311015 Rev. A
•
For 8 Beadchips, dispense 310 ml
•
For 16 Beadchips, dispense 300 ml
•
For 24 BeadChips, dispense 285 ml
Page 23 of 28
Infinium® HD Assay Gemini, Manual Protocol (Post-AMP)
Experienced User Card
Do not let it sit for more than 10 minutes.
[ ] 7. Move the BeadChip staining rack into the XC4 dish. The barcodes should
face away from you and the locking arms towards 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, with the BeadChip barcodes facing up.
[ ] 11. Remove the BeadChips from the staining rack with locking tweezers,
working from top to bottom. Place each BeadChip on a tube rack to dry.
Remove the staining rack handle before removing the BeadChips below
it.
[ ] 12. Dry the BeadChips in the vacuum desiccator for 50–55 minutes at
508 mm Hg (0.68 bar). Each desiccator can hold 8 BeadChips.
[ ] 13. Clean the underside of the BeadChip with a ProStat EtOH wipe.
[ ] 14. Clean and store the glass back plates and Hyb Chamber components.
[ ] 15. Do one of the following:
Catalog # WG-901-3002
Part # 11311015 Rev. A
•
Proceed to Image BeadChip on iScan System or Image BeadChip on
BeadArray Reader.
•
Store the BeadChips in the Illumina BeadChip Slide Storage Box
inside a vacuum desiccator at room temperature. Image the
BeadChips within 72 hours.
Page 24 of 28
Infinium® HD Assay Gemini, Manual Protocol (Post-AMP)
Experienced User Card
Image
BeadChip on
iScan System
Estimated Time
Preparation
Steps
The iScan Reader uses a laser to excite the fluor of the single-base
extension product on the beads of the BeadChip sections. Light
emissions from these fluors are then recorded in high-resolution images
of the BeadChip sections. Data from these images are analyzed using
Illumina’s GenomeStudio Genotyping Module.
Scanning: 35 minutes per BeadChip
[ ]
On the lab tracking form, record the following for each BeadChip:
•
Scanner ID
•
Scan date
[ ] 1. Turn on the iScan Reader, boot up the iScan PC, and start the
GenomeScan application.
Starting Up the iScan System
[ ] 1. Turn on the iScan Reader and the attached PC.
[ ] 2. Let the iScan Reader warm up for at least 5 minutes.
[ ] 3. For each BeadChip, copy the mini-CD provided with the BeadChip into
the Decode folder. The folder name should be the BeadChip barcode
(for example, 4264011131).
[ ] 4. Double-click the GenomeScan icon
on the desktop.
[ ] 5. Set the LIMS dropdown list to None and enter your Windows user name.
[ ] 6. Click Start.
Loading BeadChips and Starting the Scan
[ ] 1. Load the BeadChips into their carrier and place the carrier into the iScan
Reader tray. Click Next.
[ ] 2. Click any BeadChip section to remove it from the scan. The section will
no longer be highlighted blue.
[ ] 3. If you want to remove an entire BeadChip from the scan, delete the
barcode from the Setup window.
[ ] 4. To begin scanning the BeadChips, click Scan.
[ ] 5. At the end of the scan, a Review window appears. If any stripes fail to
scan successfully, click Rescan to automatically rescan all failed areas.
[ ] 6. When you finish reviewing the data, click Done to return to the Start
window.
Catalog # WG-901-3002
Part # 11311015 Rev. A
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Infinium® HD Assay Gemini, Manual Protocol (Post-AMP)
Experienced User Card
Image
BeadChip on
BeadArray
Reader
Estimated Time
Preparation
Initializing the
BeadArray
Reader (Daily)
The Illumina BeadArray Reader uses a laser to excite the fluor of the
hybridized single-stranded product on the beads of the BeadChip
sections. Light emissions from these fluors are then recorded in highresolution images of the BeadChip sections. Data from these images
are analyzed using Illumina’s GenomeStudio Genotyping Module.
1–2 hours warmup for the BeadArray Reader (first use of the day only)
45 minutes to scan each BeadChip using BeadScan 3.2 FastScan settings
[ ]
If this is the first time the BeadArray Reader is being used today, follow
the steps described in this section.
[ ]
On the lab tracking form, record the following for each BeadChip:
•
Scanner ID
•
Scan date
If this is the first time the scanner is being used today, follow these steps.
[ ] 1. Locate the power switch on the lower-left side of the BeadArray Reader
back panel and turn it to the ON position.
[ ] 2. Wait for the ready indicator to stop flashing.
[ ] 3. Open the BeadScan software.
[ ] 4. Log in and click Scan.
Imaging
BeadChip
When the BeadArray Reader is initialized, follow these steps to perform the
scanning process.
[ ] 1. From the Docking Fixture listbox, select BeadChip.
[ ] 2. Check the Data Repository path and the Decode Map path in the
Settings area.
[ ] 3. Copy the decode map (*.dmap) files for each BeadChip from the
BeadChip CD to the Decode Map path directory.
[ ] 4. For each BeadChip:
[ ] a. Place the BeadChip into the BeadArray Reader tray.
[ ] b. If either the Sentrix Type or Scan Settings are not correct, click
Browse (...) to open the Select Scan Settings dialog box.
[ ] c. Select the appropriate scan method and click Select.
[ ] 5. Make sure that the BeadChips are properly seated in the BeadArray
Reader tray.
[ ] 6. Click Scan.
Scanning Process
BeadScan begins the BeadArray Reader Tilt and Align processes.
Once the Tilt and Align processes are complete, the Scan process
begins. Hover over any of the green dots in the closeup image to see the
relative intensity and the XY position. The red value should be at or close
to zero, because this is a one-color assay.
As the BeadArray Reader scans, the front panel blue Scanning indicator lights
flash in sequence.
Catalog # WG-901-3002
Part # 11311015 Rev. A
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Infinium® HD Assay Gemini, Manual Protocol (Post-AMP)
Experienced User Card
When the BeadArray Reader finishes scanning, a green message screen appears
if the scan was successful, or a red message if it completed with any warnings.
If Scan is Successful
[ ] 1. Click OK on the Scan Completed message to view the next screen.
[ ] 2. Click Done in the Review pane.
[ ] 3. When the application returns to the Welcome screen, click Open Tray.
The BeadArray Reader tray, loaded with the scanned BeadChips, will
eject.
[ ] 4. Remove the BeadChips from the tray.
[ ] 5. Do one of the following:
[ ]
If you have more BeadChips to scan, repeat the scanning process.
[ ]
If this is the last use of the day:
[ ] a. Wipe the BeadArray Reader tray with a lint-free, absorbent
towel. Pay particular attention to the tray edges where reagent
may have wicked out.
[ ] b. Close the tray.
[ ] c. Turn the power switch at the back of the scanner to the OFF
position.
[ ] d. Shut down the BeadArray Reader BeadScan software. To exit,
right-click near the Illumina logo and click Exit.
If Scan is not Successful
Re-scan the array. For more information, refer to the Illumina BeadArray Reader
User Guide.
If the scanner was unable to locate the alignment fiducials (focus points), you may
need to clean the edges of the BeadChip before re-scanning.
Catalog # WG-901-3002
Part # 11311015 Rev. A
Page 28 of 28
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