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Amplify DNA

This step adds the DNA samples to the plates. The samples are denatured and neutralized to prepare them
for amplification.

Consumables

u

MA1 (2 tubes)

u

MA2 (2 tubes)

u

MSM (2 tubes)

u

0.1N NaOH (15 ml)

u

96-well 0.8 ml microplate (midi) (1 plate)

u

DNA plate with 48 or 96 DNA samples (50 ng/μl) (midi or TCY) (1 plate)

u

Cap mats

Preparation

1

Preheat the Illumina Hybridization Oven in the post-amp area to 37°C and allow the temperature to
equilibrate.

2

Prepare the following consumables:

Item

Storage

Instructions

DNA

-25°C to -15°C

Thaw at room temperature. DNA must be 50 ng/µl, resuspended in TE (10 mM Tris,
1mM EDTA.

MA1

Room
temperature

Thaw at room temperature. Invert 10 times to mix, and then pulse centrifuge to
eliminate bubbles and collect reagent at the bottom of the tube.

MA2

-25°C to -15°C

Thaw at room temperature. Invert 10 times to mix, and then pulse centrifuge to
eliminate bubbles and collect reagent at the bottom of the tube.

MSM

-25°C to -15°C

Thaw at room temperature. Invert 10 times to mix, and then pulse centrifuge to
eliminate bubbles and collect reagent at the bottom of the tube.

3

Apply an MSA1 barcode label to a new midi plate.

Procedure

1

If you do not already have a DNA plate, add DNA into either of the following:

u

Midi plate: 20 µl to each DNA well

u

TCY plate: 10 µl to each DNA well

Apply a barcode label to the new DNA plate.

2

At the robot PC, select MSA1 Tasks | Make MSA1.

3

In the Basic Run Parameters pane, enter the Number of DNA plates.
The robot PC updates the Required Run Items and the bed map to show the correct position of items on
the robot bed.

NOTE

If you are using Illumina LIMS, you cannot change the number of DNA samples on this screen. The Illumina
LIMS software processes the correct number of samples.

Document # 11322427 v03

For Research Use Only. Not for use in diagnostic procedures.

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Infinium HD Super Assay Reference Guide

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Содержание Infinium HD Super Assay

Страница 1: ...Infinium HD Super Assay Reference Guide Document 11322427 v03 August 2019 ILLUMINA PROPRIETARY For Research Use Only Not for use in diagnostic procedures ...

Страница 2: ......

Страница 3: ...ained personnel in order to ensure the proper and safe use of the product s described herein All of the contents of this document must be fully read and understood prior to using such product s FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN MAY RESULT IN DAMAGE TO THE PRODUCT S INJURY TO PERSONS INCLUDING TO USERS OR OTHERS AND DAMAGE TO OTHER PROPERTY AN...

Страница 4: ...ienced User Cards replaced with Infinium HD Super Assay Checklist Manual Protocol document 1000000080218 and Infinium HD Super Assay Checklist Automated Protocol document 1000000080220 which provide current format of experienced user instructions Added list of acronyms Consumables and equipment information moved to Infinium Assay Lab Setup and Procedures Guide document 11322460 Added Tip and Techn...

Страница 5: ...with 550 ml of PB1 Part 11294809 Rev C June 2010 Update Part 11294809 Rev B June 2009 Update Part 11294809 Rev A November 2008 Minor revisions Part 11294809 Rev A May 2008 Initial release Document 11322427 v03 For Research Use Only Not for use in diagnostic procedures v Infinium HD Super Assay Reference Guide ...

Страница 6: ...per Manual Workflow 7 Amplify DNA 9 Incubate DNA 10 Fragment DNA 10 Precipitate DNA 11 Resuspend DNA 13 Hybridize to BeadChip 14 Wash BeadChips 19 Extend and Stain BeadChips 25 Image BeadChip 34 Illumina GenomeStudio 35 Chapter 3 Automated Protocol 37 Introduction 37 Infinium HD Super Automated Workflow 37 Amplify DNA 39 Incubate DNA 41 Fragment DNA 41 Precipitate DNA 42 Resuspend DNA 44 Hybridize...

Страница 7: ...on In the case of the Infinium II probe design the 3 end of the primer is positioned directly adjacent to the SNP site or if a nonpolymorphic probe directly adjacent to the nonpolymorphic site With the Infinium I probe design the 3 end of the primer overlaps with the SNP site If there is a perfect match extension occurs and signal is generated If there is a mismatch extension does not occur and no...

Страница 8: ...klist Automated Protocol document 1000000080220 Provides a checklist for experienced users of the Infinium HD Super Assay when performing the automated protocol Infinium HD Super Assay Checklist Manual Protocol document 1000000080218 Provides a checklist for experienced users of the Infinium HD Super Assay when performing the manual protocol Infinium Lab Setup and Procedures Guide document 1132246...

Страница 9: ...sed to room temperature air for extended periods of time it is no longer fresh To make best use of RA1 only pour the amount needed for the current step If you plan to perform additional assay steps requiring RA1 the same day leave the remaining thawed reagent in the original closed bottle at room temperature until it is needed Follow the standard RA1 storage procedures described in this guide for ...

Страница 10: ...to 165 C and 2 5 seconds Pipetting u Make sure that pipettes are properly calibrated cleaned and decontaminated u Dispense slowly and carefully to prevent turbulence in the plate wells and flow through chambers u Use a multichannel pipette whenever possible Centrifugation u When centrifuging plates or BeadChips place a balance plate or rack with BeadChips opposite each plate or rack being centrifu...

Страница 11: ...platform before beginning the Hybridize to BeadChip section is optional u Removing the rocker platform is optional for plate incubation in the Amplify DNA Incubate DNA Fragment DNA Precipitate DNA and Resuspend DNA sections Acronyms Acronym Definition EDTA Ethylenediaminetetraacetic acid EtOH Ethanol ATM Anti Stain Two Color Master Mix FMS Fragmentation solution MA1 Multi Sample Amplification 1 Mi...

Страница 12: ...he plate barcode and select Go d Note which step the plate is queued to run and proceed with that step e If the error message indicates the BeadChip did not accession into the system accession it and repeat the verification step f If the BeadChip is not the right type for the batch accession the correct type and repeat the verification step No Illumina LIMS If you are using the automated protocol ...

Страница 13: ...meStudio 35 Introduction This section describes pre and post amplification manual laboratory protocols for the Infinium HD Super Assay Follow the protocols in the order shown Infinium HD Super Manual Workflow The following figure graphically represents the Infinium HD Super Assay manual workflow for 24 BeadChips These protocols describe the procedure for preparing 96 DNA samples Document 11322427 ...

Страница 14: ...Figure 2 Infinium HD Super Manual Workflow Document 11322427 v03 For Research Use Only Not for use in diagnostic procedures 8 Infinium HD Super Assay Reference Guide ...

Страница 15: ...f the tube MA2 25 C to 15 C Thaw at room temperature Invert 10 times to mix and then pulse centrifuge to eliminate bubbles and collect reagent at the bottom of the tube MSM 25 C to 15 C Thaw at room temperature Invert 10 times to mix and then pulse centrifuge to eliminate bubbles and collect reagent at the bottom of the tube 3 Apply an MSA1 barcode label to a new 0 8 ml microplate midi Procedure 1...

Страница 16: ...ol steps in the post amplification area Incubate DNA This step uniformly amplifies the genomic DNA generating a sufficient quantity of each individual DNA sample to be used in the Infinium HD Super Assay 1 Incubate the MSA1 plate in the Illumina Hybridization Oven for 20 24 hours at 37 C Fragment DNA This step enzymatically fragments the DNA An endpoint fragmentation is used to prevent overfragmen...

Страница 17: ... Preparation 1 Do one of the following u If proceeding immediately from Fragment DNA leave the MSA1 plate on the heat block until preparation is complete u If the MSA1 plate was stored at 25 C to 15 C thaw at room temperature pulse centrifuge at 280 g and preheat the heat block to 37 C 2 Prepare the following consumable Item Storage Instructions PM1 2 C to 8 C Thaw to room temperature and invert 1...

Страница 18: ... Drain liquid onto the absorbent pad and then smack the plate down on a dry area of the pad 17 Keeping the plate inverted firmly tap until all wells are free of liquid 1 minute Do not allow supernatant to pour in to other wells 18 Place the uncovered inverted plate on the tube rack for 1 hour at room temperature to air dry the pellets 19 Make sure that a blue pellet is still present in the bottom ...

Страница 19: ...unused contents in accordance with the governmental safety standards for your region Preparation 1 If the MSA1 plate was stored at 25 C to 15 C thaw to room temperature then remove cap mat 2 Prepare the following consumable Item Storage Instructions RA1 25 C to 15 C Warm to room temperature in a 20 C to 25 C water bath Mix to dissolve any remaining crystals 3 Preheat the Illumina Hybridization Ove...

Страница 20: ...en thaw the MSA1 plate at room temperature and then pulse centrifuge at 280 g 2 Preheat the heat block to 95 C 3 Preheat the Illumina Hybridization Oven to 48 C Procedure Denature DNA 1 Place the MSA1 plate on the preheated heat block for 20 minutes to denature the DNA 2 Cool the MSA1 plate on the benchtop at room temperature for 30 minutes 3 Pulse centrifuge at 280 g Assemble the Hybridization Ch...

Страница 21: ...dges 3 Place the gasket into the hybridization chamber u Match the wider edge of the hybridization chamber gasket to the barcode ridge side of the hybridization chamber u Press down on the edges of the gasket to make sure it is properly seated 4 Make sure that the gaskets are properly placed and seated Document 11322427 v03 For Research Use Only Not for use in diagnostic procedures 15 Infinium HD ...

Страница 22: ...s on the benchtop at room temperature until the BeadChips are loaded with DNA 1 hour Load BeadChip 1 Pulse centrifuge the MSA1 plate at 280 g 2 Remove the BeadChips from all packaging Hold BeadChips by the ends away from the sample inlets 3 Place each BeadChip into an insert so that the barcode ends align Document 11322427 v03 For Research Use Only Not for use in diagnostic procedures 16 Infinium ...

Страница 23: ...nsert the pipette into the sample inlet before dispensing u Load A1 H1 as shown in the following graphic 6 Wait for the DNA to disperse over the entire surface 7 Inspect the loading port for excess liquid Document 11322427 v03 For Research Use Only Not for use in diagnostic procedures 17 Infinium HD Super Assay Reference Guide ...

Страница 24: ...over the ridges indicated on the chamber u Keep the inserts steady and level 2 Place the back of the lid onto the chamber and then slowly lower the front to avoid dislodging the inserts 3 Close all four clamps so that the lid is secure and sits evenly on the base without any gaps Close the clamps in the following order top left bottom right top right bottom left 4 Place the chamber into the prehea...

Страница 25: ...le upright on the lab bench overnight u Add the 100 EtOH and place the XC4 on its side on a rocker to resuspend until the BeadChips are ready for coating 3 Optional Store at 2 C to 8 C and use up to six times over a period of 2 weeks Wash BeadChips This step prepares the BeadChips for the staining process Consumables u 70 EtOH as needed u 95 formamide 1 mM EDTA 15 ml for up to 8 BeadChips 17 ml fo...

Страница 26: ...hamber from the hybridization oven Allow to cool for 30 minutes before opening 2 Prepare the following items u Fill two wash dishes with 200 ml PB1 each and label them Wash 1 and Wash 2 u Using a graduated cylinder fill the Multi Sample BeadChip Alignment Fixture with 150 ml PB1 3 Remove the following Te Flow flow through chamber components from storage u Black frames u Spacers separated for ease ...

Страница 27: ... hybridization chamber inserts from the hybridization chambers 3 Remove the BeadChips from the hybridization inserts 4 Remove the cover seals from the BeadChips Using powder free gloved hands hold the BeadChip securely and by the edges in one hand Remove the entire seal in a single continuous motion Do not touch exposed arrays 5 Immediately and carefully slide each BeadChip into the wash rack in W...

Страница 28: ...d inspect them for remaining residue If you see residue submerge the BeadChip in PB1 and carefully use a pipette tip to remove the remaining residue Assemble Flow Through Chambers 1 Confirm that you are using the correct Infinium glass back plates and spacers before proceeding 2 Fill the BeadChip alignment fixture with 150 ml PB1 for up to 8 BeadChips 3 For each BeadChip place one black frame into...

Страница 29: ...a substitute for the clear spacers 6 Place the alignment bar onto the alignment fixture Fit the groove on the alignment bar over the tab on the alignment fixture 7 Place a clean glass back plate on top of each clear spacer Position the plate reservoir at the barcode end of the BeadChip facing inward to create a reservoir against the BeadChip surface Document 11322427 v03 For Research Use Only Not ...

Страница 30: ...rough chamber at the barcode end 5 mm from the bottom of the reagent reservoir A One stripe is visible between the first clamp and the alignment bar B Glass back plate pressed against alignment bar C Stripes are not visible between the second clamp and the barcode 9 Remove the assembled flow through chamber from the alignment fixture 10 Starting at the nonbarcode end trim the spacers from each end...

Страница 31: ...nucleotides to extend primers hybridized to the sample and stains the primers After the flow through chambers are disassembled the BeadChips are coated for protection Consumables u 70 EtOH as needed u 95 formamide 1 mM EDTA 15 ml for up to 8 BeadChips 17 ml for 16 25 ml for 24 u Alconox Powder Detergent as needed u ATM 2 tubes per 8 BeadChips u PB1 310 ml for up to 8 BeadChips 285 ml for 24 u RA1 ...

Страница 32: ...eadChip firmly and help prevent damage WARNING This protocol uses an aliphatic amide that is a probable reproductive toxin Personal injury can occur through inhalation ingestion skin contact and eye contact For more information consult the material data safety sheet for this assay at www illumina com sds Dispose of containers and any unused contents in accordance with the governmental safety stand...

Страница 33: ...ber rack temperature 4 Remove bubbles trapped in the chamber rack a Separate the heat exchanger from the reagent pan b Lift the heat exchanger upright and away from you with the tubing at the bottom and turn 90 counter clockwise c Return the heat exchanger to a horizontal position d Repeat steps b and c 3 times for a total of 4 rotations or until all bubbles are removed e Using Kimwipes dampened w...

Страница 34: ...ck CAUTION To avoid assay failure complete this procedure without interruption 2 Make sure that each flow through chamber is properly seated on the rack to allow adequate heat exchange between the rack and the chamber 3 Without allowing pipette tips to touch BeadChip surfaces fill the reservoir of each flow through chamber as follows a 150 µl RA1 Incubate for 30 seconds Repeat 5 times b 450 µl XC1...

Страница 35: ...µl STM Incubate for 10 minutes h 450 µl XC3 Incubate for 1 minute Repeat 1 time i Wait 5 minutes j 250 µl ATM Incubate for 10 minutes k 450 µl XC3 Incubate for 1 minute Repeat 1 time l Wait 5 minutes m 250 µl STM Incubate for 10 minutes n 450 µl XC3 Incubate for 1 minute Repeat 1 time o Wait 5 minutes 3 Immediately remove the flow through chambers from the chamber rack and place in reserved alignm...

Страница 36: ...be rack that fits the internal dimensions of vacuum desiccator for removal of the BeadChips Allow one rack per eight BeadChips Kimwipes are not needed under this tube rack 6 Set up two top loading wash dishes labeled PB1 and XC4 7 To indicate fill volume of each wash dish a Add 310 ml water b Mark the water level on the side c Empty the water Indicating fill volume before adding reagents allows re...

Страница 37: ...adChip only by the barcode end or edges 15 Repeat steps 11 14 to disassemble each flow through chamber one at a time 16 Place the BeadChips into the submerged staining rack Make sure that the BeadChip barcodes face away from you and the locking arms face toward you CAUTION Submerge each BeadChip as quickly as possible to prevent drying 17 If necessary to seat a BeadChip briefly lift the staining r...

Страница 38: ... 10 minutes 23 Transfer the staining rack from the PB1 wash dish to the XC4 wash dish 24 Slowly lift the staining rack up and down 10 times breaking the XC4 surface If the tops of the BeadChips touch gently wiggle the staining rack to separate the slides 25 Soak for 5 minutes 26 Remove the staining rack in one quick motion and place it onto the prepared tube rack Document 11322427 v03 For Research...

Страница 39: ...h the handle away from you unlocking the handle c Pull up the handle and remove 29 For each BeadChip working top to bottom a Holding the staining rack handle if present use self locking tweezers to grip the BeadChip by the barcode end b Place the BeadChip onto a tube rack with the barcode facing up and toward you Do not place on the bottom rack or allow BeadChips to rest on the tube rack edge or t...

Страница 40: ... instructions see the documentation included with the desiccator 35 Return the desiccator to storage Store with the red valve plug in the three way valve of the desiccator to prevent dust and lint from accumulating in the valve port 36 Touch the edges of the BeadChips do not touch arrays to make sure etched barcoded sides are dry 37 Clean the back of each BeadChip using a Kimwipe sprayed with 70 E...

Страница 41: ... idat files collected from your Illumina scanning instrument For feature descriptions and instructions on using the GenomeStudio platform to visualize and analyze genotyping data see the GenomeStudio Framework User Guide and the GenomeStudio User Guide or online help Document 11322427 v03 For Research Use Only Not for use in diagnostic procedures 35 Infinium HD Super Assay Reference Guide ...

Страница 42: ...Document 11322427 v03 For Research Use Only Not for use in diagnostic procedures 36 Infinium HD Super Assay Reference Guide ...

Страница 43: ...ocols for the Infinium HD Super Assay both with and without using the IlluminaIllumina Laboratory Information Management System to track barcodes and other project information Follow the protocols in the order shown For information on using Illumina LIMS see the comprehensive User Guide Infinium HD Super Automated Workflow The following diagram illustrates the Infinium HD Super Assay automated wor...

Страница 44: ...Figure 3 Infinium HD Super Automated Workflow Document 11322427 v03 For Research Use Only Not for use in diagnostic procedures 38 Infinium HD Super Assay Reference Guide ...

Страница 45: ...imes to mix and then pulse centrifuge to eliminate bubbles and collect reagent at the bottom of the tube MSM 25 C to 15 C Thaw at room temperature Invert 10 times to mix and then pulse centrifuge to eliminate bubbles and collect reagent at the bottom of the tube 3 Apply an MSA1 barcode label to a new midi plate Procedure 1 If you do not already have a DNA plate add DNA into either of the following...

Страница 46: ...elect Run 8 When prompted enter the barcode of each DNA plate The robot bed map is updated with the DNA plate locations 9 Place the DNA plates on the robot bed according to the bed map and select OK The robot begins when the plates are in place 10 When the robot has completed the run vortex the sealed MSA1 plate at 1600 rpm for 1 minute 11 Centrifuge at 280 g at 22 C for 1 minute 12 Remove the cap...

Страница 47: ...nsumables u FMS 2 tubes u Cap mats Preparation 1 Preheat the heat block with the midi plate insert at 37 C 2 Prepare the following consumable Item Storage Instructions FMS 25 C to 15 C Thaw to room temperature Invert 10 times to mix 3 Preheat the heat block with the midi plate insert at 37 C 4 Remove the MSA1 plate from the Illumina Hybridization Oven 5 If resuspending the MSA1 plate today remove ...

Страница 48: ...eat block for 1 hour If you are continuing leave the plate in the 37 C heat block until you have completed preparation for the next step Do not leave the plate on the heat block for longer than 2 hours SAFE STOPPING POINT If you are stopping seal the plate s and store at 25 C to 15 C Precipitate DNA This step uses 100 2 propanol and PM1 to precipitate the DNA Consumables u 100 2 propanol u PM1 2 t...

Страница 49: ... the sealed plate at 280 g 3 Remove the cap mat and place the MSA1 plate on the robot bed according to the bed map 4 Place a half reservoir in the reservoir frame according to the robot bed map and add PM1 as follows u For 48 samples add 1 tube PM1 u For 96 samples add 2 tubes PM1 A PM1 in Half Reservoir B 2 Propanol in Full Reservoir C MSA1 Plate 5 At the robot PC select Run 6 When prompted remov...

Страница 50: ... in the bottom of each sample well 22 Remove and discard the cap mat 23 Hold the plate over an absorbent pad and do as follows a Quickly invert to decant the supernatant b Drain liquid onto the absorbent pad and then smack the plate down Avoid the liquid drained onto the pad 24 Keeping the plate inverted firmly tap until all wells are free of liquid 1 minute Do not allow supernatant to pour into o...

Страница 51: ... to 15 C thaw at room temperature and then remove the cap mats 2 Prepare the following consumable Item Storage Instructions RA1 25 C to 15 C Thaw at room temperature and invert to mix 3 Preheat the Illumina Hybridization Oven to 48 C 4 Preheat the heat sealer for at least 20 minutes before use Procedure 1 At the robot PC select MSA1 Tasks Resuspend MSA1 2 Place the MSA1 plate on the robot bed acco...

Страница 52: ...1 will be used the next day seal it and store it overnight at 4 C Hybridize to BeadChip This step dispenses the fragmented resuspended DNA onto BeadChips Incubation then hybridizes each DNA sample to a section of the BeadChip Consumables u 1 aqueous Alconox solution u 100 EtOH u DI H2O u PB2 3 tubes 1 tube per 4 BeadChips u XC4 Preparation 1 If frozen thaw the MSA1 plate at room temperature and th...

Страница 53: ...de on page 1 Procedure Denature DNA 1 Place the MSA1 plate on the preheated heat block for 20 minutes to denature the DNA 2 Cool the MSA1 plate on the benchtop at room temperature for 30 minutes 3 Pulse centrifuge at 280 g Document 11322427 v03 For Research Use Only Not for use in diagnostic procedures 47 Infinium HD Super Assay Reference Guide ...

Страница 54: ... hybridization chamber u Match the wider edge of the hybridization chamber gasket to the barcode ridge side of the hybridization chamber u Press down on the edges of the gasket to make sure it is properly seated 2 Add 400 µl PB2 into each of the eight humidifying buffer reservoirs in the hybridization chamber 3 Place the hybridization chamber insert into the hybridization chamber Position the barc...

Страница 55: ...ve all BeadChips from packaging 2 Place BeadChips into the robot BeadChip alignment fixtures Align the barcode end with the ridges stamped into the robot BeadChip alignment fixture 3 Stack the robot BeadChip alignment fixtures and carry them to the robot 4 Place the robot BeadChip alignment fixtures onto the robot deck according to the deck map in Figure 8 5 Pulse centrifuge the MSA1 plate at 280 ...

Страница 56: ... tip alignment guide on top of each robot BeadChip alignment fixture a Make sure that the Guide E barcode is upside down and facing away from you b Push both the tip guide and alignment fixture to the upper left corner in its section of the robot bed 3 At the robot PC select OK u The robot scans the barcode on the robot tip alignment guide to confirm that the correct tip guide is being used u The ...

Страница 57: ...room temperature when you load the BeadChips Do not place the hybridization chamber in the Illumina Hybridization Oven when loading the BeadChips 2 Open each hybridization chamber and then carefully place each BeadChip in a hybridization chamber insert Orient the barcode end so that it matches the barcode symbol on the Hyb Chamber Insert 3 Make sure that hybridization chamber inserts are seated pr...

Страница 58: ...clamps of the hybridization chamber facing the front and back of the oven If you are stacking multiple hybridization chambers in the Illumina Hybridization Oven fit the feet of each hybridization chamber into the matching indents on the lid of the hybridization chamber below it You can stack up to 3 hybridization chambers in two rows for a maximum of 6 total hybridization chambers in the Illumina ...

Страница 59: ...minates before next use Wash BeadChips This step prepares the BeadChips for the staining process Consumables u 95 Formamide 1 mM EDTA u ATM u TEM u PB1 u ATM u STM u XC1 u XC2 u XC3 u XC4 About Reagents u Decant only the reagent volume needed for each step Some reagents are needed later in the protocol u Excepting PB1 all reagents are prepared in this step for use in a subsequent step WARNING This...

Страница 60: ...C to 15 C Thaw at room temperature Invert 10 times to mix TEM 25 C to 15 C Thaw at room temperature Invert 10 times to mix PB1 Room temperature Thaw at room temperature Invert 10 times to mix RA1 25 C to 15 C Shake vigorously to resuspend If necessary vortex until dissolved STM 25 C to 15 C Thaw at room temperature Invert 10 times to mix XC1 25 C to 15 C Thaw at room temperature Invert 10 times to...

Страница 61: ...e hand Remove the entire seal in a single continuous motion Do not touch exposed arrays 5 Immediately and carefully slide each BeadChip into the wash rack in Wash 1 making sure that the BeadChip is submerged in PB1 a maximum of 8 BeadChips 6 Repeat steps 4 5 until all BeadChips are transferred to the submerged wash rack in Wash 1 Document 11322427 v03 For Research Use Only Not for use in diagnosti...

Страница 62: ...ette tip to remove the remaining residue Assemble Flow Through Chambers 1 Confirm that you are using the correct Infinium glass back plates and spacers before proceeding 2 Fill the BeadChip alignment fixture with 150 ml PB1 for up to 8 BeadChips 3 For each BeadChip place one black frame into the BeadChip alignment fixture For example if you are processing four BeadChips place four black frames int...

Страница 63: ...eadChip facing inward to create a reservoir against the BeadChip surface A Reservoir at barcode end of glass back plate B Glass back plate in position 8 Secure each flow through chamber assembly with metal clamps as follows a Using one finger gently push the glass back plate against the alignment bar b Place a metal clamp around the flow through chamber 5 mm from the top edge c Place a second meta...

Страница 64: ...il ready to load onto chamber rack in the Extend and Stain BeadChips step u Do not place on absorbent paper u Do not place in the chamber rack until instructed to do so 12 Wash the hybridization chamber reservoirs with DI H2O Immediate and thorough washing ensures complete removal of PB1 from the wells Extend and Stain BeadChips This step washes unhybridized and nonspecifically hybridized DNA samp...

Страница 65: ...esh u RA1 might form visible precipitate or crystals Before each use hold in front of a light and inspect Invert several times to redissolve the solution as needed u The XC4 coat is slippery and makes the BeadChips difficult to hold Self locking tweezers grip the BeadChip firmly and help prevent damage WARNING This protocol uses an aliphatic amide that is a probable reproductive toxin Personal inj...

Страница 66: ... the appropriate level See the VWR Operator Manual VWR part 110 229 2 Turn on the water circulator Set it to a temperature that brings the Chamber Rack to 44 C at equilibrium This temperature can vary depending on facility ambient conditions Document 11322427 v03 For Research Use Only Not for use in diagnostic procedures 60 Infinium HD Super Assay Reference Guide ...

Страница 67: ...ampened with laboratory grade water clean all surfaces between the heat exchanger and reagent pan Discard Kimwipes with formamide waste f Place the Te Flow back on the reagent pan Using the two guide pins in the reagent pan make sure that the Te Flow is flush 5 Using the Illumina temperature probe test at least three locations on the chamber rack a For accurate measurements make sure that the temp...

Страница 68: ...w the lasers to stabilize 3 Place reservoirs on the robot deck according to the deck map and add reagents to reservoirs as follows Reagent BeadChips Volume 95 formamide 1 mM EDTA 1 8 15 ml 9 16 17 ml 17 24 25 ml RA1 1 8 10 ml 9 16 20 ml 17 24 30 ml XC3 1 8 50 ml 9 16 100 ml 17 24 150 ml 4 Invert the XC1 XC2 TEM STM and ATM tubes to mix Remove the caps and place on the robot deck according to the d...

Страница 69: ...TION Start the robot immediately to prevent BeadChips from drying 9 At the robot PC select OK 10 When the robot finishes remove the flow through chambers from the chamber rack and place them horizontally on the lab bench at room temperature Wash and Coat BeadChips 1 Gather the following equipment u Kimwipes large u Staining rack u Self locking tweezers u Tube rack u Vacuum desiccator u Vacuum hose...

Страница 70: ... are not needed under this tube rack 6 Set up two top loading wash dishes labeled PB1 and XC4 7 To indicate fill volume of each wash dish a Add 310 ml water b Mark the water level on the side c Empty the water Indicating fill volume before adding reagents allows reagents to be added directly from the bottles minimizing contamination A Labeled and filled wash dishes B Staining rack 8 Add 310 ml PB1...

Страница 71: ...adChip only by the barcode end or edges 15 Repeat steps 11 14 to disassemble each flow through chamber one at a time 16 Place the BeadChips into the submerged staining rack Make sure that the BeadChip barcodes face away from you and the locking arms face toward you CAUTION Submerge each BeadChip as quickly as possible to prevent drying 17 If necessary to seat a BeadChip briefly lift the staining r...

Страница 72: ... 10 minutes 23 Transfer the staining rack from the PB1 wash dish to the XC4 wash dish 24 Slowly lift the staining rack up and down 10 times breaking the XC4 surface If the tops of the BeadChips touch gently wiggle the staining rack to separate the slides 25 Soak for 5 minutes 26 Remove the staining rack in one quick motion and place it onto the prepared tube rack Document 11322427 v03 For Research...

Страница 73: ...h the handle away from you unlocking the handle c Pull up the handle and remove 29 For each BeadChip working top to bottom a Holding the staining rack handle if present use self locking tweezers to grip the BeadChip by the barcode end b Place the BeadChip onto a tube rack with the barcode facing up and toward you Do not place on the bottom rack or allow BeadChips to rest on the tube rack edge or t...

Страница 74: ... instructions see the documentation included with the desiccator 35 Return the desiccator to storage Store with the red valve plug in the three way valve of the desiccator to prevent dust and lint from accumulating in the valve port 36 Touch the edges of the BeadChips do not touch arrays to make sure etched barcoded sides are dry 37 Clean the back of each BeadChip using a Kimwipe sprayed with 70 E...

Страница 75: ... idat files collected from your Illumina scanning instrument For feature descriptions and instructions on using the GenomeStudio platform to visualize and analyze genotyping data see the GenomeStudio Framework User Guide and the GenomeStudio User Guide or online help Document 11322427 v03 For Research Use Only Not for use in diagnostic procedures 69 Infinium HD Super Assay Reference Guide ...

Страница 76: ... 800960230 Ireland 353 1800936608 353 016950506 Italy 39 800985513 39 236003759 Japan 0800 111 5011 Netherlands 31 8000222493 31 207132960 New Zealand 0800 451 650 Norway 47 800 16836 47 21939693 Singapore 1 800 579 2745 South Korea 82 80 234 5300 Spain 34 911899417 34 800300143 Sweden 46 850619671 46 200883979 Switzerland 41 565800000 41 800200442 Taiwan 00806651752 United Kingdom 44 8000126019 4...

Страница 77: ...Document 11322427 v03 For Research Use Only Not for use in diagnostic procedures 71 Infinium HD Super Assay Reference Guide ...

Страница 78: ......

Страница 79: ...alifornia 92122 U S A 1 800 809 ILMN 4566 1 858 202 4566 outside North America techsupport illumina com www illumina com For Research Use Only Not for use in diagnostic procedures 2019 Illumina Inc All rights reserved Document 11322427 v03 ...

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