My Document - Support
Illumina Infinium LCG Quad Assay, Manual Protocol
Experienced User Card
FOR RESEARCH USE ONLY
ILLUMINA PROPRIETARY
Catalog # WG-901-5002
Part # 15025909 Rev. A
May 2011
Page 1 of 26
Illumina Infinium LCG Quad Assay, Manual Protocol
Experienced User Card
Page 2 of 26
Part # 15025909 Rev. A
Illumina Infinium LCG Quad Assay, Manual Protocol
Experienced User Card
This process creates a AMP5 plate for DNA amplification. The DNA sample is denatured
with 0.1N NaOH and then neutralized with RPM. The last reagent added is AMM
(Amplification Master Mix).
Estimated Time
Hands-on time:
• ~20 minutes per 16 samples
Incubation time: 20–24 hours
Consumables
Item
Quantity
Storage
Supplied By
RPM
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
(per 8–24 samples)
2° to 8°C
User
96-well 0.8 ml microtiter plate
(MIDI)
1 plate
DNA plate with DNA samples
1 plate
User
-15° to -25°C
User
Preparation
} Preheat the Illumina Hybridization Oven in the post-amp area to 37°C and allow the
temperature to equilibrate.
} Apply an AMP5 barcode label to a new MIDI plate.
} Thaw RPM and AMM tubes to room temperature.
} Thaw DNA samples to room temperature.
Steps to Make the AMP5 Plate
[_] 1
If you do not already have a DNA plate, add DNA, normalized to 50 ng/μl into either a:
• MIDI plate: 40 μl to each DNA plate well
• TCY plate: 30 μl to each DNA plate well
[_] 2
Apply a barcode label to the new DNA plate.
[_] 3
Vortex the DNA plate at 1600 rpm for 1 minute.
[_] 4
Pulse centrifuge to 280 xg.
[_] 5
Transfer 8 μl of the DNA sample into each well in the following AMP5 plate columns:
• Column 1 (8 samples)
• Columns 1 and 3 (16 samples)
Part # 15025909 Rev. A
Page 3 of 26
Make the AMP5 Plate (Pre-AMP)
Make the AMP5 Plate (Pre-AMP)
Illumina Infinium LCG Quad Assay, Manual Protocol
Make the AMP5 Plate (Pre-AMP)
Experienced User Card
• Columns 1, 3, 5, 7, 9, and 11 (48 samples)
[_] 6
On the lab tracking form, record the original DNA sample ID for each well in the AMP5
plate.
[_] 7
Dispense 8 μl 0.1N NaOH into each well of the AMP5 plate that contains DNA.
[_] 8
Incubate for 10 minutes at room temperature.
[_] 9
Dispense 135 μl RPM into each well of the AMP5 plate containing sample.
[_] 10 Dispense 150 μl AMM into each well of the AMP5 plate containing sample.
[_] 11 Seal the AMP5 plate with the 96-well cap mat.
[_] 12 Invert the 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.
[_] 15 Proceed to Fragment the AMP5 Plate (Post-AMP).
Page 4 of 26
Part # 15025909 Rev. A
Illumina Infinium LCG Quad Assay, Manual Protocol
Experienced User Card
This process enzymatically fragments the amplified DNA samples. An end-point
fragmentation is used to prevent over-fragmentation.
Estimated Time
Hands-on time: ~30 minutes for 96 samples
Incubation time: 1 hour
Consumables
Item
Quantity
Storage
Supplied By
FRG
1 tube (per 16
samples)
-15° to -25°C
Illumina
Preparation
} Preheat the heat block with the MIDI plate insert to 37°C.
} Thaw FRG tubes to room temperature. Gently invert at least 10 times to mix contents.
} Remove the AMP5 plate from the Illumina Hybridization Oven.
Steps to Fragment the AMP5 Plate
[_] 1
Pulse centrifuge the plate to 280 xg.
[_] 2
Split the sample into 1 additional well, for a total of 2 wells per sample. Each well
should contain 150 μl.
For example, move 150 μl sample from A1 into A2, A3, and A4.
Follow this pattern for rows B–H, columns 1, 3, 5, 7, 9, and 11.
[_] 3
Add 50 μl FRG to each well containing sample.
Part # 15025909 Rev. A
Page 5 of 26
Fragment the AMP5 Plate (Post-AMP)
Fragment the AMP5 Plate (Post-AMP)
Illumina Infinium LCG Quad Assay, Manual Protocol
Fragment the AMP5 Plate (Post-AMP)
Experienced User Card
[_] 4
Seal the AMP5 plate with the 96-well cap mat.
[_] 5
Vortex the plate at 1600 rpm for 1 minute.
[_] 6
Pulse centrifuge the plate to 280 xg.
[_] 7
Place the sealed plate on the 37°C heat block for 1 hour.
[_] 8
Do one of the following:
• Continue to the next step, Precipitate the AMP5 Plate (Post-AMP). Leave plate in 37°C
heat block until setup is complete. Do not leave the plate in the 37°C heat block for
longer than 2 hours.
• If you do not plan to proceed to the next step within the next 4 hours, store the sealed
AMP5 plate at -15° to -25°C for no more than 24 hours.
Page 6 of 26
Part # 15025909 Rev. A
Illumina Infinium LCG Quad Assay, Manual Protocol
Experienced User Card
Add PA1 and 2-propanol to the AMP5 plate to precipitate the DNA samples.
Estimated Time
Hands-on time: ~30 minutes
Incubation and dry time: 2 hours
Consumables
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
temperature
User
Preparation
} If frozen, thaw AMP5 plate to room temperature, then pulse centrifuge the plate to 280
xg.
} Preheat heat block to 37°C.
} Thaw PA1 to room temperature. Gently invert at least 10 times to mix contents.
} Remove the 96-well cap mat.
Steps to Precipitate the AMP5 Plate
[_] 1
Add 100 μl PA1 to each AMP5 plate well containing sample.
[_] 2
Seal the plate with the cap mat.
[_] 3
Vortex the plate at 1600 rpm for 1 minute.
[_] 4
Incubate at 37°C for 5 minutes.
[_] 5
Pulse centrifuge to 280 xg.
NOTE
Set centrifuge to 4°C in preparation for the next centrifuge step.
[_] 6
Add 300 μl 100% 2-propanol to each well containing sample.
[_] 7
Carefully seal the AMP5 plate with a new, dry cap mat, taking care not to shake the plate
in any way until the cap mat is fully seated.
[_] 8
Invert the plate at least 10 times to mix contents thoroughly.
[_] 9
Incubate at 4°C for 30 minutes.
[_] 10 Centrifuge to 3,000 xg at 4°C for 20 minutes. Immediately remove the AMP5 plate from
centrifuge.
[_] 11 Remove the cap mat and discard it.
Part # 15025909 Rev. A
Page 7 of 26
Precipitate the AMP5 Plate (Post-AMP)
Precipitate the AMP5 Plate (Post-AMP)
Illumina Infinium LCG Quad Assay, Manual Protocol
Precipitate the AMP5 Plate (Post-AMP)
Experienced User Card
[_] 12 Over an absorbent pad, decant the supernatant by quickly inverting the AMP5 plate.
Drain liquid onto the absorbent pad and then smack the plate down, avoiding the liquid
that was just drained onto the pad.
[_] 13 Tap firmly several times for 1 minute or until all wells are devoid of liquid.
[_] 14 Leave the uncovered, inverted plate on the tube rack for 1 hour at room temperature to
air dry the pellet.
At this point, blue pellets should be present at the bottoms of the wells.
[_] 15 Do one of the following:
• Continue to the next step, Resuspend the AMP5 Plate (Post-AMP).
• If you do not plan to proceed to the next step immediately, seal the AMP5 plate with
a new cap mat and store it at -15° to -25°C for no more than 24 hours.
Page 8 of 26
Part # 15025909 Rev. A
Illumina Infinium LCG Quad Assay, Manual Protocol
Experienced User Card
Add RA1 to the AMP5 plate to resuspend the precipitated DNA samples.
Estimated Time
Hands-on time: ~30 minutes
Incubation time: 1 hour
Consumables
Item
Quantity
Storage
Supplied By
RA1
Bottle (9 ml per 48
samples)
-15° to -25°C
Illumina
NOTE
Pour out only the recommended volume of RA1 needed for the suggested
number of samples listed in the consumables table. Additional RA1 is used later in
the XStain LCG BeadChip step.
Preparation
} If you stored the AMP5 plate at -15° to -25°C, thaw it to room temperature. Remove the
cap mat and discard it.
} Preheat the Illumina Hybridization Oven to 48°C.
} Turn on the heat sealer to preheat. Allow 20 minutes.
} Thaw RA1 to room temperature. Invert several times to re-dissolve the solution.
Steps to Resuspend the AMP5 Plate
[_] 1
Add 46 μl RA1 to each well of the AMP5 plate containing a DNA pellet.
Reserve any leftover reagent for Hyb Multi BeadChip and XStain LCG BeadChip.
[_] 2
Apply a foil heat seal to the AMP5 plate by firmly and evenly holding the heat sealer
sealing block down for 3 seconds.
[_] 3
Immediately remove the AMP5 plate from the heat sealer and forcefully roll the rubber
plate sealer over the plate until you can see all 96 well indentations through the foil.
Repeat application of the heat sealer if all 96 wells are not defined.
[_] 4
Place the sealed plate in the Illumina Hybridization Oven and incubate for 1 hour at
48°C.
[_] 5
Vortex the plate at 1800 rpm for 1 minute.
[_] 6
Pulse centrifuge to 280 xg.
[_] 7
Do one of the following:
• Continue to the next step, Hybridize Multi BeadChip (Post-AMP). If you plan to do so
immediately, it is safe to leave the AMP5 plate at room temperature for up to 1 hour.
Part # 15025909 Rev. A
Page 9 of 26
Resuspend the AMP5 Plate (Post-AMP)
Resuspend the AMP5 Plate (Post-AMP)
Illumina Infinium LCG Quad Assay, Manual Protocol
Resuspend the AMP5 Plate (Post-AMP)
Experienced User Card
• If you do not plan to proceed to the next step immediately, store the sealed AMP5
plate at -15° to -25°C for no more than 24 hours. Store RA1 at -15° to -25°C.
Page 10 of 26
Part # 15025909 Rev. A
Illumina Infinium LCG Quad Assay, Manual Protocol
Experienced User Card
Dispense the fragmented, resuspended DNA samples onto BeadChips. Incubate the
BeadChips in the Illumina Hybridization Oven to hybridize the samples onto the BeadChips.
Estimated Time
Hands-on time:
• 4x1 LCG BeadChip: ~40 minutes for 12 BeadChips (48 samples)
Incubation time: 16–24 hours
Consumables
Item
Quantity
Storage
Supplied By
PB2
2 tubes (per 16
samples)
Room
temperature
Illumina
BeadChips
4 (per 16 samples)
Illumina
Hyb Chambers
1 (per 16 samples)
Illumina
Hyb Chamber gaskets
1 (per 16 samples)
Illumina
Hyb Chamber inserts
4 (per 16 samples)
Illumina
EtOH
330 ml
User
Preparation
} Preheat the heat block to 95°C.
} Preheat the Illumina Hybridization Oven to 48°C.
Assemble the Hybridization Chambers
[_] 1
Place the resuspended AMP5 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, leaving the BeadChips in their ziplock
bags and mylar packages until you are ready to begin hybridization.
[_] 3 During the 20-minute incubation, prepare the Hyb Chamber(s).
[_] a Place the BeadChip Hyb Chamber gaskets into the BeadChip Hyb Chambers.
[_] b Dispense 400 μl PB2 into the humidifying buffer reservoirs in the Hyb Chambers.
[_] c After you fill the Hyb Chamber reservoirs with PB2, place the lid on the Hyb
Chamber right away to prevent evaporation. The lid does not need to be locked
down.
[_] d Leave the closed Hyb Chambers on the bench at room temperature until the
BeadChips are loaded with DNA sample. Load BeadChips into the Hyb Chamber
within one hour.
Part # 15025909 Rev. A
Page 11 of 26
Hybridize Multi BeadChip (Post-AMP)
Hybridize Multi BeadChip (Post-AMP)
Illumina Infinium LCG Quad Assay, Manual Protocol
Hybridize Multi BeadChip (Post-AMP)
Experienced User Card
NOTE
You can also prepare the Hyb Chambers later, during the 30-minute cool down.
[_] 4
After the 20-minute incubation, remove the AMP5 plate from the heat block and place it
on the benchtop at room temperature for 30 minutes.
[_] 5
After the 30-minute cool down, pulse centrifuge the AMP5 plate to 280 xg. Remove the
foil seal.
[_] 6
Combine the two separate wells back into the original well. For example, move 46 μl
sample from the A2 well back into A1.
Figure 1 Consolidating Sample Back Into Original Sample Well
Load BeadChip
[_] 1
Just before loading DNA samples, remove all BeadChips from their ziplock bags and
mylar packages.
When handling the BeadChip, avoid contacting the beadstripe area and sample inlets.
[_] 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.
[_] 3
Using a single-channel precision pipette, dispense 70 μl of each DNA sample onto the
appropriate BeadChip section, according to the chart shown below.
Make sure that the pipette tip is in the sample inlet prior to dispensing.
Page 12 of 26
Part # 15025909 Rev. A
Illumina Infinium LCG Quad Assay, Manual Protocol
Experienced User Card
Hybridize Multi BeadChip (Post-AMP)
[_] 4
On the lab tracking form, record the BeadChip barcode for each group of samples.
[_] 5
Visually inspect all sections of the BeadChip to ensure the DNA sample covers all areas
of each bead stripe.
[_] 6
Record any sections that are not completely covered on the lab tracking form.
Set up Multi BeadChip for Hybridization
[_] 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 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 so that the lid is secure and even on
the base (no gaps).
NOTE
Keep the Hyb Chamber steady and level when moving it or transferring it to the
Illumina Hybridization Oven.
Part # 15025909 Rev. A
Page 13 of 26
Illumina Infinium LCG Quad Assay, Manual Protocol
Hybridize Multi BeadChip (Post-AMP)
Experienced User Card
[_] 4
Place the Hyb Chamber in the 48°C Illumina Hybridization Oven so that the clamps of
the Hyb Chamber face the left and right side of the oven and the Illumina logo on top of
the Hyb Chamber is facing you.
[_] 5
Incubate at 48°C for at least 16 hours but no more than 24 hours.
[_] 6
Cover the residual sample in the AMP5 plate with a foil seal.
You can store the plate indefinitely at -80°C.
[_] 7
Proceed to Wash the BeadChip (Post-AMP) after the overnight incubation.
Resuspend XC4 Reagent for XStain LCG BeadChip
[_] 1
Add 330 ml 100% EtOH to the XC4 bottle.
Each XC4 bottle (350 ml) has enough solution to process up to 24 BeadChips.
[_] 2
Shake the XC4 bottle vigorously to ensure complete resuspension.
Once resuspended, use XC4 at room temperature.
You can store it at 2° to 8°C for 2 weeks if unused.
Page 14 of 26
Part # 15025909 Rev. A
Illumina Infinium LCG Quad Assay, Manual Protocol
Experienced User Card
Prepare the BeadChips for the staining process.
Estimated Time
• 20 minutes for 4 BeadChips
• 30 minutes for 8 BeadChips
Consumables
Item
Quantity
Storage
Supplied By
PB1
550 ml (up to 8
BeadChips)
Room
temperature
Illumina
Multi-Sample BeadChip
Alignment Fixture
1 (per 8 BeadChips)
Illumina
Te-Flow LCG Flow-Through
Chambers (with Black Frames,
LCG Spacers, LCG Glass Back
Plates, and Clamps)
1 (per BeadChip)
Illumina
Wash Dish
2 (up to 8 BeadChips)
Illumina
Wash Rack
1 (up to 8 BeadChips)
Illumina
Preparation
} Remove each Hyb Chamber from the Illumina Hybridization Oven. Let cool on the
benchtop for 25 minutes prior to opening.
} While the Hyb Chamber is cooling:
• Fill two wash dishes with PB1 (200 ml per wash dish). Label each dish "PB1".
• Fill the Multi-Sample BeadChip Alignment Fixture with 150 ml PB1.
• Separate the clear plastic spacers from the white backs.
• Clean the glass back plates if necessary.
Steps to Wash BeadChip
[_] 1
Attach the wire handle to the rack and submerge the wash rack in the wash dish
containing 200 ml PB1.
[_] 2
Remove the Hyb Chamber inserts from the Hyb Chambers.
[_] 3
Remove BeadChips from the Hyb Chamber inserts one at a time.
[_] 4
Remove the cover seal from each BeadChip.
NOTE
To ensure no solution splatters on you, Illumina recommends removing the
cover seal over an absorbent cloth or paper towels, preferably in a hood.
Part # 15025909 Rev. A
Page 15 of 26
Wash the BeadChip (Post-AMP)
Wash the BeadChip (Post-AMP)
Illumina Infinium LCG Quad Assay, Manual Protocol
Wash the BeadChip (Post-AMP)
Experienced User Card
[_] a
[_] b
Using powder-free gloved hands, hold the BeadChip securely and by the edges in
one hand. 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.
Remove the entire seal in a single, continuous motion. 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 touch the
exposed arrays.
[_] 5
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.
[_] 6
Repeat steps 4 through 5 until all BeadChips (a maximum of 8) are transferred to the
submerged wash rack.
[_] 7
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.
[_] 8
Move the wash rack to the other wash dish containing clean PB1. Make sure the
BeadChips are completely submerged.
[_] 9
Move the wash rack up and down for 1 minute, breaking the surface of the PB1 with
gentle, slow agitation.
[_] 10 When you remove the BeadChips from the wash rack, inspect them for remaining
residue.
[_] 11 If you are processing more than 8 BeadChips
[_] a Assemble the Flow-Through Chambers for the first eight BeadChips, as described in
the next section, and place them on the lab bench in a horizontal position.
NOTE
Keep the Flow-Through Chambers in a horizontal position on the lab bench until
all assembled Flow-Through Chambers are ready to be loaded into the Chamber
Rack. Do not place the Flow-Through Chambers in the Chamber Rack until all
BeadChips are prepared in Flow-Through Chambers.
[_] b
[_] c
Return to this procedure and follow the steps described above to wash the next set of
eight BeadChips.
Repeat for each remaining set of eight BeadChips.
Assemble Flow-Through Chambers
NOTE
Confirm you are using the correct Infinium LCG Quad Assay glass back plates
and spacers before assembling the Flow-Through Chambers. Refer to the
following image for the correct Flow-Through Chamber components.
Page 16 of 26
Part # 15025909 Rev. A
Illumina Infinium LCG Quad Assay, Manual Protocol
Experienced User Card
If you have not done so, fill the Multi-sample BeadChip Alignment Fixture with 150 ml
PB1.
If more than four BeadChips will be processed, this 150 ml of PB1 can be reused for an
additional set of four BeadChips. You must use 150 ml of fresh PB1 for every additional
set of eight BeadChips.
[_] 2
For each BeadChip to be processed, place a black frame into the Multi-Sample BeadChip
Alignment Fixture pre-filled with PB1.
[_] 3
Place each BeadChip to be processed into a black frame, aligning its barcode with the
ridges stamped onto the Alignment Fixture.
NOTE
Inspect the surface of each BeadChip for residue left by the seal. Use a pipette tip
to remove any residue under buffer and be careful not to scratch the bead area.
[_] 4
Place a clear LCG spacer onto the top of each BeadChip. Use the Alignment Fixture
grooves to guide the spacers into proper position.
NOTE
Be sure to use the clear plastic spacers, not the white ones.
[_] 5
Place the Alignment Bar onto the Alignment Fixture.
The groove in the Alignment Bar should fit over the tab on the Alignment Fixture.
[_] 6
Place a clean LCG 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.
[_] 7 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
approximately 5 mm from the top edge.
[_] c Place the second metal clamp around the Flow-Through Chamber at the barcode end,
approximately 5 mm from the reagent reservoir.
[_] 8
Using scissors, trim the ends of the clear plastic spacers from the Flow-Through Chamber
assembly. Slip scissors up over the barcode to trim the other end.
[_] 9
Immediately wash the Hyb Chamber reservoirs with DiH2 O and scrub them with a small
cleaning brush, ensuring that no PB2 remains in the Hyb Chamber reservoir.
Part # 15025909 Rev. A
Page 17 of 26
Wash the BeadChip (Post-AMP)
[_] 1
Illumina Infinium LCG Quad Assay, Manual Protocol
Wash the BeadChip (Post-AMP)
Experienced User Card
CAUTION
Place all assembled Flow-Through Chambers on the lab bench in a horizontal
position while you perform the preparation steps for XStain BeadChip. Do not
place the Flow-Through Chambers in the Chamber Rack until all necessary steps
are completed.
Page 18 of 26
Part # 15025909 Rev. A
Illumina Infinium LCG Quad Assay, Manual Protocol
Experienced User Card
In this process, you use RA1 reagent to wash away unhybridized and non-specifically
hybridized DNA sample. LX1 and LX2 are added to condition the BeadChip surface for the
extension reaction. Dispense EML reagent into the Flow-Through Chambers to extend the
primers hybridized to DNA on the BeadChip. This reaction incorporates labeled nucleotides
into the extended primers. 95% formamide/1 mM EDTA is added to remove the hybridized
DNA. After neutralization using the XC3 reagent, the labeled extended primers undergo a
multi-layer staining process on the Chamber Rack. Next, you disassemble the Flow-Through
Chambers and wash the BeadChips in the PB1 reagent, coat them with XC4, and then dry
them.
Estimated Time
Hands-on time: ~2 hours and 45 minutes for 8 BeadChips
Dry time: 1 hour
Part # 15025909 Rev. A
Page 19 of 26
Single-Base Extension and Stain LCG BeadChip (Post-AMP)
Single-Base Extension and Stain LCG BeadChip (Post-AMP)
Illumina Infinium LCG Quad Assay, Manual Protocol
Single-Base Extension and Stain LCG BeadChip (Post-AMP)
Experienced User Card
Consumables
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
LX1
2 tubes (per 8
BeadChips)
-15° to -25°C
Illumina
LX2
2 tubes (per 8
BeadChips)
-15° to -25°C
Illumina
EML
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 17-24
BeadChips
Room
temperature
Illumina
SML (Make sure that all SML
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
Room
temperature
Illumina
Alconox Powder Detergent
as needed
Room
temperature
User
EtOH
as needed
Room
temperature
User
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
Page 20 of 26
Part # 15025909 Rev. A
Illumina Infinium LCG Quad Assay, Manual Protocol
Experienced User Card
} 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 then gently invert the reagent tubes at least 10
times to mix contents.
} 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. All
locations should be at 44°C ± 0.5°C.
Single-Base Extension
CAUTION
The remaining steps must be performed without interruption.
NOTE
If you are processing more than 8 BeadChips complete the reagent dispensing
step for each reagent for the first set of 8 BeadChips, then continue the same
reagent dispensing steps for the second set of 8 BeadChips. Then move to the last
set of 8 BeadChips before you start the incubation time.
Steps marked with an asterisk (*) indicate when you should follow this reagent
dispensing method.
[_] 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.
[_] 1 [_] 2 [_] 3 [_] 4 [_] 5 [_] 6
[_] b 225 μl LX1. Repeat once*. Incubate for 10 minutes.
[_] 1 [_] 2
[_] c 225 μl LX2. Repeat once*. Incubate for 10 minutes.
[_] 1 [_] 2
[_] d 300 μl EML. Incubate for 15 minutes.
[_] e 250 μl 95% formamide/1 mM EDTA. Incubate for 1 minute. Repeat twice.
[_] 1 [_] 2 [_] 3
[_] f
Incubate 5 minutes.
[_] g Begin ramping the Chamber Rack temperature to the temperature indicated on the
SML tube.
[_] h 250 μl XC3. Incubate for 1 minute. Repeat twice*.
[_] 1 [_] 2 [_] 3
[_] 3
Wait until the Chamber Rack reaches the correct temperature.
Part # 15025909 Rev. A
Page 21 of 26
Single-Base Extension and Stain LCG BeadChip (Post-AMP)
Preparation
Illumina Infinium LCG Quad Assay, Manual Protocol
Single-Base Extension and Stain LCG BeadChip (Post-AMP)
Experienced User Card
Stain BeadChip
NOTE
If you are processing more than 8 BeadChips complete the reagent dispensing
step for each reagent for the first set of 8 BeadChips, then continue the same
reagent dispensing steps for the second set of 8 BeadChips. Then move to the last
set of 8 BeadChips before you start the incubation time.
Steps marked with an asterisk (*) indicate when you should follow this reagent
dispensing method.
[_] 1
If you plan to image the BeadChip immediately after the staining process, turn on the
scanner now to allow the lasers to stabilize.
[_] 2 Into the reservoir of each Flow-Through Chamber, dispense:
[_] a 250 μl SML. Incubate for 10 minutes.
[_] b 250 μl XC3. Incubate for 1 minute. Repeat twice*. Wait 5
[_] 1 [_] 2 [_] 3
[_] c 250 μl ATM. Incubate for 10 minutes.
[_] d 250 μl XC3. Incubate for 1 minute. Repeat twice*. Wait 5
[_] 1 [_] 2 [_] 3
[_] e 250 μl SML. Incubate for 10 minutes.
[_] f
250 μl XC3. Incubate for 1 minute. Repeat twice*. Wait 5
[_] 1 [_] 2 [_] 3
[_] g 250 μl ATM. Incubate for 10 minutes.
[_] h 250 μl XC3. Incubate for 1 minute. Repeat twice*. Wait 5
[_] 1 [_] 2 [_] 3
[_] i
250 μl SML. Incubate for 10 minutes.
[_] j
250 μl XC3. Incubate for 1 minute. Repeat twice*. Wait 5
[_] 1 [_] 2 [_] 3
[_] 3
minutes.
minutes.
minutes.
minutes.
minutes.
Immediately remove the Flow-Through Chambers from the Chamber Rack and place
horizontally on a lab bench at room temperature.
Wash and Coat 8 BeadChips
[_] 1
Pour 310 ml PB1 per 8 BeadChips into a wash dish.
[_] 2
Place the staining rack inside the wash dish.
[_] 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. All chips should be completely submerged.
[_] 4
Slowly move the staining rack up and down 10 times, breaking the surface of the reagent.
[_] 5
Allow the BeadChips to soak for an additional 5 minutes.
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Part # 15025909 Rev. A
Illumina Infinium LCG Quad Assay, Manual Protocol
Experienced User Card
Shake the XC4 bottle vigorously to ensure complete resuspension. If necessary, vortex
until completely dissolved.
[_] 7
Pour 310 ml XC4 into a wash dish.
CAUTION
Do not let the XC4 sit for longer than 10 minutes.
[_] 8
Move the BeadChip staining rack into the XC4 dish.
[_] 9
Slowly move the staining rack up and down 10 times, breaking the surface of the reagent.
[_] 10 Allow the BeadChips to soak for an additional 5 minutes.
[_] 11 Lift the staining rack out of the solution and place it on a tube rack with the staining rack
and BeadChips horizontal, barcodes facing up.
[_] 12 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 rack handle if it
facilitates removal of the BeadChips.
[_] 13 Dry the BeadChips in the vacuum desiccator for 50–55 minutes at 675 mm Hg (0.9 bar).
[_] 14 Ensure that the XC4 coating is dry before continuing to the next step.
[_] 15 Clean the underside of each BeadChip with a ProStat EtOH wipe or Kimwipe soaked in
EtOH.
CAUTION
Do not touch the stripes with the wipe or allow EtOH to drip onto the stripes.
[_] 16 Clean and store the glass back plates and Hyb Chamber components.
[_] 17 Do one of the following:
• Proceed to Image BeadChip (Post-AMP).
• Store the BeadChips in the Illumina BeadChip Slide Storage Box at room temperature.
Image the BeadChips within 72 hours.
Part # 15025909 Rev. A
Page 23 of 26
Single-Base Extension and Stain LCG BeadChip (Post-AMP)
[_] 6
Illumina Infinium LCG Quad Assay, Manual Protocol
Single-Base Extension and Stain LCG BeadChip (Post-AMP)
Experienced User Card
Page 24 of 26
Part # 15025909 Rev. A
Illumina Infinium LCG Quad Assay, Manual Protocol
Experienced User Card
Follow the instructions in the iScan System User Guide or HiScanSQ System User Guide to scan
your BeadChips. Use the appropriate scan setting for your BeadChip, as outlined in the
following table:
Table 1 Scan Settings for Infinium LCG Quad
BeadChip
Scan Setting Name
HumanOmni5-4
Infinium LCG
Part # 15025909 Rev. A
Page 25 of 26
Image BeadChip (Post-AMP)
Image BeadChip (Post-AMP)
Illumina Infinium LCG Quad Assay, Manual Protocol
Image BeadChip (Post-AMP)
Experienced User Card
Page 26 of 26
Part # 15025909 Rev. A
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