Illumina Infinium HTS Assay, Manual Protocol
Experienced User Card
FOR RESEARCH USE ONLY
ILLUMINA PROPRIETARY
Part # 15045737 Rev. A
October 2013
Page 1 of 24
Illumina Infinium HTS Assay, Manual Protocol
Experienced User Card
Page 2 of 24
Part # 15045737 Rev. A
Illumina Infinium HTS Assay, Manual Protocol
Experienced User Card
Amplify DNA (Pre-Amp)
Move DNA samples into the MSA3 plate. Denature and neutralize samples, and prepare them for amplification. Incubate overnight to amplify.
Estimated Time
Hands-on time:
• 45 minutes for 48 samples
• 60 minutes for 96 samples
Incubation time: 20–24 hours
Consumables
Item
MA1
MA2
MSM
0.1N NaOH
96-well 0.8 ml microplate (MIDI)
WG#-DNA plate with 96 DNA samples (50 ng/μl)
Quantity
1 tube
(per 96 samples)
1 tube
(per 96 samples)
1 tube
(per 96 samples)
15 ml
(per 96 samples)
1 plate
1 plate
Storage
-15°C to -25°C
-15°C to -25°C
-15°C to -25°C
2°C to 8°C
-15°C to -25°C
Supplied By
Illumina
Illumina
Illumina
General lab supplier
General lab supplier
User
Preparation
[_] 1 Preheat the Illumina Hybridization Oven in the post-amp area to 37°C and allow the temperature to equilibrate.
[_] 2 Apply an MSA3 barcode label to a new 0.8 ml microplate (MIDI).
[_] 3 Thaw MA1, MA2, and MSM tubes to room temperature. Gently invert at least 10 times to mix contents.
[_] 4 Thaw DNA samples to room temperature.
Steps to Make MSA3 Plate
[_] 1 If you do not already have a WG#-DNA plate, add DNA into one of the following:
• 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 into the MSA3 plate wells.
Part # 15045737 Rev. A
Page 3 of 24
Illumina Infinium HTS Assay, Manual Protocol
Experienced User Card
[_] 3 Transfer 4 μl of the DNA sample from the WG#-DNA plate to the corresponding wells in the
MSA3 plate.
[_] 4 Record the location of the original DNA sample ID for each well in the MSA3 plate.
[_] 5 Dispense 4 μl 0.1N NaOH into each well of the MSA3 plate that contains MA1 and sample.
[_] 6 Seal the MSA3 plate with the 96-well cap mat.
CAUTION
Orient the cap mat so that A1 on the cap matches A1 on the plate. To prevent evaporation and spills, which could lead to assay variability and cross-contamination, make sure that all 96 caps are securely seated.
[_] 7 Vortex the plate at 1600 rpm for 1 minute.
[_] 8 Centrifuge to 280 × g for 1 minute.
[_] 9 Incubate for 10 minutes at room temperature.
[_] 10 Carefully remove the cap mat.
When you remove a cap mat, set it aside, upside down, in a safe location for use later in the protocol.
[_] 11 Dispense 34 μl MA2 into each well of the MSA3 plate containing sample.
[_] 12 Dispense 38 μl MSM into each well of the MSA3 plate containing sample.
[_] 13 Seal MSA3 plate with cap mat.
When you place the cap mat back on the plate, be sure to match it to its original plate and orient it correctly.
[_] 14 Vortex the sealed MSA3 plate at 1600 rpm for 1 minute.
[_] 15 Pulse centrifuge to 280 × g.
INCUBATION
Incubate in the Illumina Hybridization Oven for 20–24 hours at 37°C.
[_] 16 Proceed to the next step.
Page 4 of 24
Part # 15045737 Rev. A
Illumina Infinium HTS Assay, Manual Protocol
Experienced User Card
Fragment DNA (Post-Amp)
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
FMS
Quantity Storage
1 tube (per 96 samples) -15°C to -25°C
Supplied By
Illumina
Preparation
[_] 1 Preheat the heat block with the MIDI plate insert to 37°C.
[_] 2 Thaw FMS tubes to room temperature.
[_] 3 Gently invert the FMS tubes at least 10 times to mix contents.
Pulse centrifuge to 280 × g.
[_] 4 Remove the MSA3 plate from the Illumina Hybridization Oven.
Steps to Fragment the MSA3 Plate
[_] 1 Pulse centrifuge the plate to 50 × g.
[_] 2 Carefully remove the cap mat.
[_] 3 Add 25 μl FMS to each well containing sample.
[_] 4 Seal the MSA3 plate with the 96-well cap mat.
[_] 5 Vortex the plate at 1600 rpm for 1 minute.
[_] 6 Centrifuge the plate to 50 × g at 22°C for 1 minute.
[_] 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 DNA (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 immediately, store the sealed WG#-DNA plate at -15°C to -25°C.
SAFE STOPPING POINT
Now is a good stopping point in the process.
Part # 15045737 Rev. A
Page 5 of 24
Illumina Infinium HTS Assay, Manual Protocol
Experienced User Card
Page 6 of 24
Part # 15045737 Rev. A
Illumina Infinium HTS Assay, Manual Protocol
Experienced User Card
Precipitate DNA (Post-Amp)
Add PM1 and 2-propanol to the MSA3 plate to precipitate the DNA samples.
Estimated Time
Hands-on time: ~30 minutes for 96 samples
Incubation and dry time: 2 hours
Consumables
Item
PM1
100% 2-propanol
Quantity
1 tube (per 96 samples)
30 ml (per 96 samples)
Storage
2°C to 8°C
Room temperature
Supplied By
Illumina
General lab supplier
Preparation
[_] 1 If frozen, thaw MSA3 plate to room temperature, then pulse centrifuge the plate to 50 × g.
[_] 2 Preheat heat block to 37°C.
[_] 3 Thaw PM1 to room temperature. Centrifuge to 280 × g for 1 minute.
[_] 4 Remove the 96-well cap mat.
Steps to Precipitate the MSA3 Plate
[_] 1 Add 50 μl PM1 to each MSA3 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 × g for 1 minute.
NOTE
Set centrifuge to 4°C in preparation for the next centrifuge step.
[_] 6 Carefully remove the cap mat and discard it.
[_] 7 Add 155 μl 100% 2-propanol to each well containing sample.
[_] 8 Carefully seal the MSA3 plate with a new, dry cap mat, taking care not to shake the plate in any way until the cap mat is fully seated.
[_] 9 Invert the plate at least 10 times to mix contents thoroughly.
[_] 10 Incubate at 4°C for 30 minutes.
[_] 11 Centrifuge to 3,000 × g at 4°C for 20 minutes. Immediately remove the MSA3 plate from centrifuge.
[_] 12 Remove the cap mat and discard it.
Part # 15045737 Rev. A
Page 7 of 24
Illumina Infinium HTS Assay, Manual Protocol
Experienced User Card
[_] 13 Quickly invert the MSA3 plate and drain the liquid onto an absorbent pad to decant the supernatant. Then smack the plate down on a dry area of the pad, avoiding the liquid that was drained onto the pad.
[_] 14 Tap firmly several times for 1 minute or until all wells are devoid of liquid.
[_] 15 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.
[_] 16 Do one of the following:
• Continue to the next step, Resuspend DNA (Post-Amp).
• If you do not plan to proceed to the next step immediately, seal the MSA3 plate with a new cap mat and store it at -15°C to -25°C.
SAFE STOPPING POINT
Now is a good stopping point in the process.
Page 8 of 24
Part # 15045737 Rev. A
Illumina Infinium HTS Assay, Manual Protocol
Experienced User Card
Resuspend DNA (Post-Amp)
Add RA1 to the MSA3 plate to resuspend the precipitated DNA samples.
Estimated Time
Hands-on time: ~30 minutes for 96 samples
Incubation time: 1 hour
Consumables
Item
RA1
Quantity
7 ml per 96 samples
Storage
-15°C to -25°C
Supplied By
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
BeadChip step.
Preparation
[_] 1 If you stored the MSA3 plate at -15°C to -25°C, thaw it to room temperature. Remove the cap mat and discard it.
[_] 2 Preheat the Illumina Hybridization Oven to 48°C.
[_] 3 Turn on the heat sealer to preheat. Allow 20 minutes.
[_] 4 Thaw RA1 to room temperature. Invert several times to re-dissolve the solution.
Steps to Resuspend the MSA3 Plate
[_] 1 Add 23 μl RA1 to each well of the MSA3 plate containing a DNA pellet.
Reserve any leftover reagent for XStain BeadChip.
[_] 2 Apply a foil heat seal to the MSA3 plate by firmly and evenly holding the heat sealer sealing block down for 5 seconds.
[_] 3 Place the sealed plate in the Illumina Hybridization Oven and incubate for 1 hour at 48°C.
[_] 4 Vortex the plate at 1800 rpm for 1 minute.
[_] 5 Pulse centrifuge to 280 × g.
[_] 6 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 MSA3 plate at room temperature for up to 1 hour.
• If you do not plan to proceed to the next step immediately, store the sealed MSA3 plate at -15°C to -25°C for no more than 24 hours. For more than 24 hours, store at 80°C. Store
RA1 at -15°C to -25°C.
SAFE STOPPING POINT
Now is a good stopping point in the process.
Part # 15045737 Rev. A
Page 9 of 24
Illumina Infinium HTS Assay, Manual Protocol
Experienced User Card
Page 10 of 24
Part # 15045737 Rev. A
Illumina Infinium HTS Assay, Manual Protocol
Experienced User Card
Hybridize to BeadChip (Post-Amp)
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:
• 24x1 HTS BeadChip: ~16 minutes for 4 BeadChips (96 samples)
Incubation time: 16–24 hours
Consumables
Item
PB2
BeadChips
Hyb Chambers
Hyb Chamber gaskets
Hyb Chamber inserts
1
4
4
1
Quantity
(per 96 Samples)
1 tube
Storage
Room temperature
Supplied By
Illumina
Illumina
Illumina
Illumina
Illumina
Preparation
[_] 1 If frozen, thaw MSA3 plate to room temperature, and then pulse centrifuge the MSA3 plate to 280 × g.
[_] 2 Preheat the heat block to 95°C.
[_] 3 Preheat the Illumina Hybridization Oven to 48°C and set the rocker speed to 5.
Assemble the Hybridization Chambers
[_] 1 Place the resuspended MSA3 plate on the heat block to denature the samples at 95°C for
20 minutes.
[_] 2 Remove the BeadChips from 2°C to 8°C storage, leaving the BeadChips in their plastic bags and mylar packages until you are ready to begin hybridization.
[_] 3 During the 20 minute incubation, prepare the Hyb Chambers.
[_] 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. It is not necessary to lock down the lid.
Part # 15045737 Rev. A
Page 11 of 24
Illumina Infinium HTS Assay, Manual Protocol
Experienced User Card
[_] 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.
NOTE
You can also prepare the Hyb Chambers later, during the 30 minute cool down.
[_] 4 After the 20 minute incubation, remove the MSA3 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 MSA3 plate to 280 × g. Remove the foil seal.
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.
For the 24x1 BeadChip
[_] 1 Using a single-channel precision pipette or an adjustable spacer multi-channel pipette, dispense 14 μl of each DNA sample onto the appropriate BeadChip section.
NOTE
The spacing of the BeadChip is not compatible with a standard multi-channel pipette. Use a singlechannel pipette or an adjustable spacer multi-channel pipette to load samples.
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.
[_] 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.
OVERNIGHT INCUBATION
Incubate at 48°C for at least 16 hours but no more than 24 hours.
Resuspend XC4 Reagent for XStain BeadChip
[_] 1 Add 330 ml 100% EtOH to the XC4 bottle.
Page 12 of 24
Part # 15045737 Rev. A
Illumina Infinium HTS Assay, Manual Protocol
Experienced User Card
[_] 2 Shake vigorously for 15 seconds.
[_] 3 Leave the bottle upright on the lab bench overnight.
[_] 4 Shake the XC4 bottle vigorously to ensure complete resuspension. If any coating is visible, vortex at 1625 rpm until it is in complete suspension. After it is resuspended, use XC4 at room temperature.
Part # 15045737 Rev. A
Page 13 of 24
Illumina Infinium HTS Assay, Manual Protocol
Experienced User Card
Page 14 of 24
Part # 15045737 Rev. A
Illumina Infinium HTS Assay, Manual Protocol
Experienced User Card
Wash BeadChip (Post-Amp)
Prepare the BeadChips for the staining process.
Estimated Time
• 20 minutes for 4 BeadChips
• 30 minutes for 8 BeadChips
Consumables
Item
PB1
Quantity
550 ml for 1 to 8
BeadChips
700 ml for 9 to 16
BeadChips
850 ml for 17 to 24
BeadChips
1 (per 8 BeadChips) Multi-sample BeadChip alignment fixture
Te-Flow LCG flow-through chambers, with black frames,
LCG spacers, LCG glass back plates, and clamps
Wash dish
Wash rack
1 (per BeadChip)
2 (up to 8 BeadChips)
1 (up to 8 BeadChips)
Storage
Room temperature
Supplied By
Illumina
Illumina
Illumina
Illumina
Illumina
Preparation
[_] 1 Remove each Hyb Chamber from the Illumina Hybridization Oven. Let cool on the benchtop for 30 minutes before opening.
[_] 2 While the Hyb Chamber is cooling:
[_] a Fill two wash dishes with PB1 (200 ml per wash dish). Label each dish "PB1".
[_] b Fill the Multi-Sample BeadChip Alignment Fixture with 150 ml PB1.
[_] c Separate the clear plastic spacers from the white backs.
[_] d 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.
Part # 15045737 Rev. A
Page 15 of 24
Illumina Infinium HTS Assay, Manual Protocol
Experienced User Card
NOTE
To make sure that no solution splatters on you, Illumina recommends removing the cover seal over an absorbent cloth or paper towels, preferably in a hood.
[_] a Using powder-free gloved hands, hold the BeadChip securely and by the edges in one hand. Avoid contact with the sample inlets. Make sure that the barcode is facing up and closest to you, and that the top side of the BeadChip is angled slightly away from you.
[_] b 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.
NOTE
You can use the two 200 ml PB1 wash dishes for up to 24 BeadChips. However, use 150 ml of fresh
PB1 for every 8 BeadChips in the Multi-Sample BeadChip Alignment Fixture.
[_] 7 After 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 8 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 Return to this procedure and follow the steps described above to wash the next set of 8
BeadChips.
[_] c Repeat for each remaining set of 8 BeadChips.
Assemble Flow-Through Chambers
NOTE
Confirm that you are using the correct Infinium LCG 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 24
Part # 15045737 Rev. A
Illumina Infinium HTS Assay, Manual Protocol
Experienced User Card
Figure 1 Correct LCG Back Plates and Spacers
[_] 1 If you have not done so, fill the Multi-sample BeadChip Alignment Fixture with 150 ml PB1.
If you plan to process more than 4 BeadChips, this 150 ml of PB1 can be reused for an additional set of 4 BeadChips. Use 150 ml of fresh PB1 for every additional set of 8
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 fits 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 is 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 DiH
2
O and scrub them with a small cleaning brush, ensuring that no PB2 remains in the Hyb Chamber reservoir.
[_] 10 Proceed to the next step.
Part # 15045737 Rev. A
Page 17 of 24
Illumina Infinium HTS Assay, Manual Protocol
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 flowthrough chambers in the chamber rack until all necessary steps are completed.
Page 18 of 24
Part # 15045737 Rev. A
Illumina Infinium HTS Assay, Manual Protocol
Experienced User Card
Extend and Stain (XStain) BeadChip (Post-Amp)
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: 55 minutes
Consumables
Item
RA1
Storage
-15°C to -25°C
Supplied By
Illumina
LX1
LX2
EML
XC3
SML (Make sure that all SML tubes indicate the same stain temperature on the label)
ATM
Quantity
10 ml for 1–8
BeadChips
20 ml for 9–16
BeadChips
30 ml for 17–24
BeadChips
2 tubes (per 8
BeadChips)
2 tubes (per 8
BeadChips)
2 tubes (per 8
BeadChips)
50 ml for 1–8
BeadChips
100 ml for 9–16
BeadChips
150 ml for 17–24
BeadChips
2 tubes (per 8
BeadChips)
-15°C to -25°C
-15°C to -25°C
-15°C to -25°C
Room temperature
-15°C to -25°C
-15°C to -25°C
Illumina
Illumina
Illumina
Illumina
Illumina
Illumina
PB1
2 tubes (per 8
BeadChips)
310 ml for 1–8
BeadChips
285 ml for 9–24
BeadChips
Room temperature
Illumina
Part # 15045737 Rev. A
Page 19 of 24
Illumina Infinium HTS Assay, Manual Protocol
Experienced User Card
Item
XC4
Alconox Powder Detergent
EtOH
95% formamide/1 mM EDTA
Quantity
310 ml for 1–8
BeadChips
285 ml for 9–24
BeadChips
As needed
As needed
15 ml for 1–8
BeadChips
17 ml for 9–16
BeadChips
25 ml for 17–24
BeadChips
Storage
Room temperature
Room temperature
Room temperature
-15°C to -25°C
Supplied By
Illumina
General lab supplier
General lab supplier
General lab supplier
Preparation
[_] 11 Place all reagent tubes in a rack in the order you plan to use them. If frozen, allow them to thaw to room temperature, and then gently invert the reagent tubes at least 10 times to mix contents.
[_] 12 Make sure the water circulator is filled to the appropriate level.
[_] 13 Turn on the water circulator. Set it to a temperature that brings the Chamber Rack to 44°C at equilibrium.
[_] 14 Remove bubbles trapped in the Chamber Rack.
[_] 15 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. Finally, move to the last set of 8
BeadChips before you start the incubation time.
Steps marked with an asterisk (*) indicate when to 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:
Page 20 of 24
Part # 15045737 Rev. A
Illumina Infinium HTS Assay, Manual Protocol
Experienced User Card
[_] a 150 μl RA1. Incubate for 30 seconds. Repeat 5 times.
[_] 1 [_] 2 [_] 3 [_] 4 [_] 5 [_] 6
[_] b 225 μl LX1. Repeat one time*. Incubate for 10 minutes.
[_] 1 [_] 2
[_] c 225 μl LX2. Repeat one time*. 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.
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. Finally, move to the last set of 8
BeadChips before you start the incubation time.
Steps marked with an asterisk (*) indicate when to 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 minutes.
[_] 1 [_] 2 [_] 3
[_] c 250 μl ATM. Incubate for 10 minutes.
[_] d 250 μl XC3. Incubate for 1 minute. Repeat twice*. Wait 5 minutes.
[_] 1 [_] 2 [_] 3
[_] e 250 μl SML. Incubate for 10 minutes.
[_] f 250 μl XC3. Incubate for 1 minute. Repeat twice*. Wait 5 minutes.
[_] 1 [_] 2 [_] 3
[_] g 250 μl ATM. Incubate for 10 minutes.
[_] h 250 μl XC3. Incubate for 1 minute. Repeat twice*. Wait 5 minutes.
[_] 1 [_] 2 [_] 3
[_] i 250 μl SML. Incubate for 10 minutes.
[_] j 250 μl XC3. Incubate for 1 minute. Repeat twice*. Wait 5 minutes.
[_] 1 [_] 2 [_] 3
[_] 3 Immediately remove the flow-through chambers from the chamber rack and place horizontally on a lab bench at room temperature.
Part # 15045737 Rev. A
Page 21 of 24
Illumina Infinium HTS Assay, Manual Protocol
Experienced User Card
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.
[_] 6 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 Make sure 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.
Page 22 of 24
Part # 15045737 Rev. A
Illumina Infinium HTS Assay, Manual Protocol
Experienced User Card
Image BeadChip (Post-Amp)
Follow the instructions in the iScan System User Guide or HiScan System User Guide to scan your
BeadChips. Use the Infinium LCG scan setting for your BeadChip.
Part # 15045737 Rev. A
Page 23 of 24
Illumina Infinium HTS Assay, Manual Protocol
Experienced User Card
Page 24 of 24
Part # 15045737 Rev. A
