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Axiom Genotyping Assay Manual

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1. General Settings Press F1 for more information about data reduction functions and formulas Technique Type Labware Selection Layout Settings First Pass SecondPass Third Pass Fourth Pass Fifth Pass Sixth Pass ep ction Page gt Ia sspe p REDUCTION_A2 a PASE B x DTX880 Abs 280nm Genomic ETI SE eect ed C x_DTx880_Abs_320nm_Genomic Formula B Name of Data Abs 280 Name of Units Notes Apply Formula for Wells with Category v Sample Appendix B Sample Quantitation after Resuspension 122 A x_DTX880_Abs_260nm_Genomic 8 x DTX880 Abs 280nm Genomic C x DTX880 Abs 320nm Genomic Abs 320 Name of Units Notes Apply Formula For Wells with Category Pr x DTX880 Abs 260nm Genomic 8 B x DTX880 Abs 280nm Genomic C x DTX880 Abs 320nm Genomic A C 0 29 120 0 05 Name of Data Concentration Name of Units ug uL Notes 0 29 Pathlength 120 Dilution Factor 0 05 DNA extinction coefficient Apply Formula For Wells with Category p Appendix B Sample Quantitation after Resuspension 123 3 1 A x DTX880 Abs 260nm Genomic
2. B x DTX880 Abs 280nm Genomic C x DTX880 Abs 320nm Genomic Purity Name of Units Notes Apply Formula for Wells with Category F Sende General Settings Press F1 for more information about data reduction Functions and Formulas Technique Type Labware Selection Layout Settings First Pass Second Pass Third Pass Fourth Pass Fifth Pass Sixth Pass Method aia A x DTX880 Abs 260nm Genomic ata Reduction Page REDUCTI Baia ReRUHOn Fng B x DTX880 Abs 280nm Genomic er one F 9 fe C x_DTX880_Abs_320nm_Genomic Formula a C 0 29 690 Name of Data Massirxn Name of Units ug Notes 690 120 x 0 05 x 115 115 volume per reaction of resuspended pellet and Hyb Mix Apply Formula For Wells with Category v Sample Appendix B Sample Quantitation after Resuspension 124 Output Settings Select Export to Microsoft Excel and Show Result Viewer Figure B 15 Output Settings A Create Protocol NewProtocol for 96 array plate Output Settings General Settings Select data output and printer options Technique Type Labware Selection Layout Settings Method Selection Perform after completing Data Reduction Page measurement s pe Export to Microsoft Excel Old Format Version lt 3 2
3. Calculation Project Plate Type Probe Array Type Probe Array Barcode Sample File Name Array Name Default Axiom GW Hu SNP 96 Axiom GW Hu SNP A01 Sample A01 Sample A01 Axiom_GW_Hu_SNP 96 Axiom GW Hu SNP A02 Sample A02 Sample A02 W Hu SNF Axiom GW Hu SNP A03 Sample A03 Sample A04 _ Axiom GW Hu SNP A04 Axiom 2 0 Assay Preparation Before You Start This section provides information on procedures that are performed multiple times during manual target preparation and on steps that are critical to the success of the manual target preparation It is essential that you familiarize yourself with the information in this section prior to running the manual target preparation for Axiom 2 0 Assay A list of all equipment and resources required for the Axiom 2 0 Assay with manual target preparation is in the Axiom 2 0 Assay Manual Workflow Site Prep Guide P N 702991 Differences Between the Axiom Assays The Axiom 2 0 Assay Manual Workflow contains important differences from the original Axiom Genotyping Assay If you are experienced with running the original Axiom Assay it is highly recommended that you familiarize yourself with these changes to prevent errors and ensure the success of the Axiom 2 0 Assay Reagent Kit Changes The new Axiom 2 0 Reagent Kit contains a different Module than the original Axiom Reagent Kit however Modules 2 3 and 4 are identical bet
4. Appendix F GeneTitan Multi Channel Instrument Care 140 Figure F 10 Inserting the Lamp IMPORTANT The lamp bulb faces away from the fan and toward the reflecting mirror Appendix F GeneTitan Multi Channel Instrument Care 141 Resetting the Lamp Counter You must alert the software application that you have replaced the lamp so that the hours of the lamp counter are reset to zero This menu option is only available when the system is not processing any plates 1 On the software application click Tools Reset Counter for Life Remaining Figure F 11 Figure F 11 Inserting the Lamp il AGCC GeneTitan Instrument Control File Help Shutdown System ale x Tools Check Available Disk Space Reset Counter for Lamp Life Remaining Sy atus System Setup Fluidics Status Scan Status Barcode Plate Type Estimated Completion Time Estimated Time Window to Run Next Hyb Wash Scan HT Array Type Same plate type 7 System is available now Hybridization Oven Status Fluidics Status Imaging Device Status Barcode Position 1 Estimated Time Remaining Barcode Barcode Protocol Name Estimated Time Remaining Estimated Time Remaining Lamp Life Remaining 143 hours Barcode Position 2 Estimated Time Remaining m Wash B Tempera
5. Consumables Item Supplier Part Number Mother E Base Device EB M03 Daughter E Base Device Life Technologies EB D03 E Gel 48 4 agarose gels formerlyInvitrogen G8008 04 Tracklt 25 bp DNA Ladder 10488 022 Tracklt Cyan Orange Loading Buffer 10482 028 Table A 3 Gel and reagents required Item Supplier Part Number Adhesive film use one of the following a MicroAmp Clear Adhesive Film Applied Biosystems 4306311 Microseal B Film Bio Rad MSB1001 Pipette Tips Same brand as pipette Appendix A Fragmentation Quality Control Gel Protocol 115 Diluting the Tracklt Cyan Orange Loading Buffer The following recipe is for preparing a 1000 fold dilution of the TrackIt Cyan Orange Loading Buffer To Dilute the Tracklt Cyan Orange Loading Buffer 1 Add 50 uL of TrackIt Cyan Orange Loading Buffer to 49 95 mL nuclease free water Total volume 50 mL Mix well Store at room temperature Fragmentation QC Gel Protocol This protocol is based on running QC gels for 96 samples To Run a Fragmentation QC Gel 1O 199 MC CO UT ee iN Sa Tightly seal the gel QC plate prepared during automated or manual target preparation Vortex the center of the plate for 3 sec and spin at 2000 rpm for 30 sec Power on two E Bases Push the Power Prg button on each to ensure the program is in EG mode not EP mode Place the E Gels onto the base units Remove two combs from each gel Load 20 uL fro
6. Fluidic Diagnostic Messages Table 7 4 Problem messages Problem and Possible Causes About this message HT96CC FLUIDIC DIAGNOSTIC BUFFERX Buffer bottle A B or Rinse a WASHX Wash A or B reservoir in the fluidics station FillUntilSensorState Failure on valve group BUFFERB_TO_WASHA Prime ran out during fill operation BUFFERB_TO_WASHA Recommended actions Plate 550032 345678922xxxxxx 1 1 1 4 Tene 6 16 2009 3 59 24 PM Replenish fluid level in the Rinse or Wash Bottle B to the Fluidic process CleanThenFillWashawithBufferB 1L mark Do not overfill Possible causes For dispense Failure include i Bottle empty or fluid level too low Replenish bottle o Only replenish bottles when prompted by the Ul Bottle cap not secure Check all bottle caps are secure selhs se Clogged fiter Replace fier Replenishing during fluidic processing may cause system malfunction including overflowing inside the Continuing due to time critical nature of the process Fill cannot be guaranteed Underfill was likely system and more problems The only thing to do while a plate is running is to make sure bottle caps are a Replenish fluid level in Wash Bottle A to 2 L Secure the bottle cap a Replace the filter Instructions for filter replacement in the GeneTitan Multi Channel Instrument User s Manual P N 08 0306 If the problem persists call Affymetrix support The typical cause is an unsecure bottle cap HT
7. E Cancel F When drawer 6 opens confirm that the plate stack is securely clamped then press the Confirmation button When processing resumes 1 The plate stack which has finished hybridization is moved from the Hyb oven to drawer 1 temporarily and then moved to the unclamp station after step 2 it remains clamped The plate stack in drawer 6 is moved to the Hyb oven The plate is moved to the unclamped station 4 The plate stack in the unclamp area is unclamped and moved into the fluidics area NOTE At the end of a Hyb Wash Scan run all plate and tray covers and the fixing tray cover should be in the trash Figure 5 36 is an example of how the System Status Workflow window will appear when three Axiom array plates are being processed Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 97 Figure 5 36 Example of the System Status window three Axiom array plates are being processed System Status SystemSetup Barcode Plate Type Location Hyb Status Fluidics Status Scan Status Estimated Completion Ti 5500324059357012609088 550032 Left Position Completed Running Waiting 5 4 2008 11 50 38 AM 550032 plate 20000000 550032 Right Posit Running Waiting Waiting 5 4 2008 11 55 00 AM a 250032 Left Position Running Waiting Waiting 5 4 2008 12 53 02 PM Estimated Time Wi
8. OOOOJDOOO OOOOOOOO OOOQDOOO OOOOOOOO OOOOOOOO0 OOOQO DOOO OOOQO DOOO OOOO DOO NOTE In the procedures vortex twice means to repeat the vortexing step Sample Quantitation This protocol has been optimized using a PicoGreen assay to determine genomic DNA concentrations Other quantitation methods such as UV Absorbance may give different readings Therefore you should correlate readings from other methods to the equivalent PicoGreen determined concentration Please refer to Chapter 2 Genomic DNA Preparation and Requirements on page 11 for more information Chapter 3 Axiom 2 0 Assay Preparation Before You Start 24 About the Reagents and Master Mix Preparation Axiom 2 0 Reagent Kit Components a Caps on the vials are color coded by assay stage Properly store all enzyme reagents especially enzyme containing vials Improper storage methods can profoundly impact activity Lu IMPORTANT The Axiom 2 0 Assay is compatible only with reagents from an Axiom Reagent Kit These reagents are not interchangeable with reagents from other Affymetrix reagent kits such as SNP 6 0 DMET Plus etc The new Axiom 2 0 Reagent Kit contains a different Module 1 than the original Axiom Reagent Kit however Modules 2 3 and 4 are identical between both versions and may be used from either kit for this assay Only use new Module 1 from the Axiom 2 0 Reagent Kit for the Axiom 2 0 Assay Reagents fro
9. gt MIAME Sample Information gt Pedigree Template GeneTitan Array Plate Type Required Axiom GW Hu SNP 96 v Project where to create samples Default v Download 3 Step 2 complete the registration file as follows A Click the Microsoft Excel box on the bottom bar of the monitor to open the Excel spreadsheet B Enter a unique name for each sample Sample File Name and any additional information you would like to include C Do one of the following a If you are ready to load the array plate onto the GeneTitan MC Instrument scan the array plate barcode and proceed to the next step a If you are not ready to load the array plate onto the GeneTitan MC Instrument proceed directly to the next step Figure D 3 Entering sample information into a Batch Registration file f n ld e v GeneTitanArrayPlateRegistration_7 xls Compatibility Mode Microsoft Excel gt fons Insert A Page Layout Formulas 7 Data Review a View Add Ins Acrobat dh Cut Arial jho gt AD A E Wrap Text m Normal Bad 4a Copy 4 pes E j MR il eee Pg Format Painter BF Bene A RR ER EE Merge Center iamen NR CORE ar Table Clipboard E Font EI Alignment Number Styles G23 QX E A B C D E H 1 Sample File Path Project Plate Type Probe Array Type Probe Array Barcode Sample File Name Array Name 2 Default Axiom GW Hu SNP 96 Axiom GW Hu SNP A01 Sample A01 Sample A01 3 Default Axiom GW
10. CAUTION Do not remove the scan tray from its protective black base Leave the scan tray in the base until loaded onto the GeneTitan MC Instrument When handling the scan tray the bottom glass surface of the tray should not be touched Figure 5 26 The Scan Tray with Cover on the Black Base Always leave the scan tray in its protective black base 4 t E Barcoded Scan tray t cover PN 202757 GeneTitan Scan Tray PN 501006 or PN 500860 scan Tay protective base Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 90 Proper Installation of the GeneTitan Tray Consumables It is very important that you install the GeneTitan tray consumables in the proper orientation The barcode faces into the instrument Notch This faces out and left FRONT OF INSTRUMENT FACING YOU Barcode This faces BACK TO THE REAR of the instrument Turn the tray and cover combo so that the barcode faces BACK AND INTO the instrument and the notch faces OUT AND TO THE LEFT Barcode faces in and back Notch faces out and left Affymetrix For Research Use Only faces out NOTE The instrument control software will display a warning if it detects a problem during the fluid dispense operations The filters in the GeneTitan Wash A Wash B and DI Water bottles should be replaced if the software displays such a warning Refer to Appendix F GeneTitan M
11. The consumable is either not the correct consumable not loaded correctly or its barcode is not readable Proceeding with an incorrect or incorrectly loaded consumable can result in a loss of consumables loss of samples and may require a field service engineer to service the instrument Refer to the System Setup Tab or the User Guide provided with the Assay or AGCC for instructions on proper consumable placement Press the flashing blue confirmation button or Press OK GeneTitan will verify the barcode and orientation Press Skip GeneTitan will NOT verify the barcode and orientation se Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 86 Load a Second Axiom Array Plate and Hyb Tray Onto the GeneTitan MC Instrument When You Can Load a Second Array Plate and Hyb Tray Once processing begins you have a specific period of time during which you can load another Axiom array plate and hyb tray This period of time is displayed above the Hyb Oven Status pane Figure 5 23 You cannot load another hyb tray before or after this period of time Lu IMPORTANT You must load the next array plate and hyb tray during the period of time displayed above the Hyb Oven Status You cannot load another hyb tray before or after this period of time You will have to wait until the current process is finished NOTE While the first plate is in the oven you can load another plate if the time spacing requiremen
12. CAUTION Some of the steps in this stage should be performed under a fume hood Duration a Centrifuge and dry plates 1 hour 20 minutes Resuspension and hyb mix preparation 25 min Gel QC and OD 45 min total 2 5 hr Input Required Precipitation plate from Stage 2 Fragmentation and Precipitation on page 38 Equipment Consumables and Reagents Required The equipment and consumables listed in Table 4 11 are required for this stage Table 4 11 Equipment and Consumables Required for Stage 3 Drying Resuspension and QC Quantity Item As required Adhesive seals for 96 well plates 1 Marker fine point permanent 1 each Rainin Pipettes Single channel P20 Single channel P 100 a Multi channel P20 a Multi channel P 200 As needed Pipette tips for pipettes listed above 2 Bio Rad Hard Shell 96 well plate HSP 9631 or any 96 well PCR plate for making the dilutions QC Dilution Plate Gel Samples Plate 1 Bio Rad Hardshell 96 well plate HSP 9631 for Bio Rad PTC 200 TRobot or Bio Rad 0240G thermal cyclers or Bio Rad Hard Shell Full Height 96 Well Semi Skirted PCR Plate P N HSS 9601 for ABI 9700 or ABI 2720 thermal cyclers Hyb Ready Plate 1 OD plate 96 well UV Star 370 uL well 1 Oven set at 37 C 1 Mini microcentrifuge microfuge with microtube rotor Chapter 4 Axiom 2 0 Assay Manual Target Preparation 45 Table 4 11 Equipment and Consumables Required f
13. IMPORTANT Before proceeding to DNA Amplification perform the gDNA preparation described in Chapter 2 Genomic DNA Preparation and Requirements on page 11 Using the manual target preparation protocol a single operator can process three gDNA and array plates a week during a forty hour work week for a total of 288 arrays See Chapter 6 Manual Target Preparation for Processing Three Axiom Array Plates per Week on page 100 for more information Chapter 4 Axiom 2 0 Assay Manual Target Preparation 33 Stage 1 DNA Amplification Duration Lu IMPORTANT Before proceeding to DNA Amplification perform the gDNA preparation described in Chapter 2 Genomic DNA Preparation and Requirements on page 11 NOTE For this protocol the term samples includes the positive control The following sets of steps are necessary to perform DNA amplification 1 Initial Setup for DNA Amplification on page 35 2 Prepare the Denaturation Master Mix on page 36 3 Add Denaturation Master Mix to Samples on page 36 4 Add Neutralization Solution to Samples on page 37 5 Prepare and Add the Amplification Master Mix on page 37 6 Freeze or Proceed on page 38 Lu IMPORTANT Amplification preparation should take place in an a dedicated area such as a biosafety hood with dedicated pipettes tips vortex etc See Amplification Staging Area on page 19 for more information For 96 samples Time to thaw materials 1 hr Hands on ti
14. If the information is correct the application allows you to proceed to the next step If the instrument is unable to read the barcode it will push the tray out and will prompt Figure 5 16 you to load the correct plate with the proper orientation into the instrument Figure 5 15 Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 83 n Click OK to retry and check the loading of the array plate or n Click Skip if the instrument has problems reading the barcode and after verifying that the trays have been placed in the proper orientation IMPORTANT Do not install a 3 plate stack of trays Confirm that you have removed the clear plastic shipping cover as shown in Figure 5 1 on page 68 Clear Tray Shipping Cover To be Discarded Blue Array Plate Protective Base F Select the arrays to scan instructions in Figure 5 17 By default all arrays are selected Status Message in Status Window Please select arrays to scan using array selector Press the Next button to advance to the next step Array Selection Iv Manual Array Selection Default all arrays are selected 1121314 s 6 7 8 9 10 11 12 Single array click one box Multiple arrays o Click one box o Hold down the Ctrl key o Click another box in the same column Group of arrays o Click one box o Hold down the Shift key a Left click and drag the mouse E Will Be Scanned L Will Not Be Scan
15. Multi Channel Pipettes Use 12 channel pipettes when working to add Master Mix or to transfer samples to plates and GeneTitan trays a Use a pipette of appropriate size for the volume of liquid being transferred Change pipette tips after each transfer or addition Chapter 3 Axiom 2 0 Assay Preparation Before You Start 26 GeneTitan MC Instrument Consumables All consumables for the GeneTitan MC Instrument are provided by Affymetrix The following table provides guidance on the consumables that are shipped with the array plate IMPORTANT All GeneTitan trays and tray covers must have barcodes Discard any consumable tray or tray cover without a barcode Table 3 3 Axiom GeneTitan Tray Consumables from the Axiom GeneTitan Consumables Kit P N 901606 Axiom All array plates TE lated The Axiom array plate Genome have the ote Array plate Is shipping package includes Wide or P N 202091 WR ge RE the following Axiom etched on the Consumables Kit a The function of the clear myDesign on the plastic plastic cover for the array Custom plate is to protect the Array Plate array plate during Package transport You can discard this after removing the array plate The array plate must be protected at all times from damage or exposure to dust The array plate must be in the blue array plate protective base at all times The blue array plate protective base in the package must be used to Clear Tray protect the array pla
16. Squeeze and release the trigger slowly 3 times over each section Squeeze for approximately two seconds and release for approximately two seconds Appendix E Deionization Procedure for GeneTitan Trays and Covers 132 4 Place the stain tray cover with the flat surface facing upward on the Kimwipe Aim the anti static gun P N 74 0014 approximately one half inch away from the flat surface and pull the trigger As you pull the trigger move the gun across the cover so that the stream of ionized particles settles on all areas of the cover and dissipates the static electricity Squeeze and release the trigger slowly 3 times over each section Squeeze for approximately two seconds and release for approximately two seconds Place the treated cover or tray on the Kimwipe and lift it up Do one of the following a If the Kimwipe does not adhere to the plastic proceed with the step a Ifthe Kimwipe adheres to the plastic then perform steps 3 and 4 again If it continues to adhere to the plastic then the gun is not working and you should replace it Figure E 3 Removing the Static Charge from Stain Trays and Lids Treat the inside surface of stain trays right and cover left ZEROSTAT RD MILTY zz IMPORTANT Remove static gun cover before use If a Kimwipe sticks to treated surface treat again following this procedure If the Kimwipe still adheres replace the antistatic gun GeneTitan Multi Channe
17. approximately 2 hours Input Required Amplification plate from Stage 1 DNA Amplification on page 33 Chapter 4 Axiom 2 0 Assay Manual Target Preparation Equipment Consumables and Reagents Required Equipment and Consumables The equipment and consumables listed in Table 4 6 are required for this stage Table 4 6 Equipment and Consumables Required for Stage 2 Fragmentation and Precipitation 39 Quantity Item As required Adhesive seals for 96 well plates 1 Freezer set to 20 C Designate a shelf where the precipitation plates can be left undisturbed 1 Cooler chilled to 20 C 1 Ice bucket filled with ice 1 Marker fine point permanent 1 each Rainin Pipettes a Single channel P1000 Single channel P200 a Multi channel P20 Multi channel P200 a Multi channel P1200 As needed Pipette tips for pipettes listed above 1 Pipet aid 1 Plate centrifuge set at room temp 1 Mini microcentrifuge microfuge with microtube rotor 2 3 Ovens see Oven Recommendations on page 22 a One oven set at 37 C One oven set to 65 C 1 15 mL conical tube 1 50 mL conical tube 1 50 mL conical tube holder 4 Solution basin 100 mL sterile multichannel 1 Vortexer Reagents Required Table 4 7 Reagents Required for Stage 2 Fragmentation and Precipitation Reagent Module From the Axiom 2 0 Reagent Kit Axiom Frag Enzyme leave at 20 C u
18. enter your experiment name Click the Eject Plate Carrier icon Load the OD plate onto the DTX 880 Click the Close Plate Carrier icon 8 Click the Run the Selected Protocol icon at the bottom of the window ey Ss ucc ees When the protocol is finished running a list of results is displayed If you used the formula provided in this appendix two XML files are generated Figure B 1 Open the ResultData file with Microsoft Excel to view and assess the OD readings RawData file information is included in the ResultData file Figure B 1 File Edit View Favorites Tools Help B Search E Folders E Address O C Documents and Settings All Users Application DatalMultimodelDetection SoftwarelData Size Type Date Modified 83KB XML Document 9 17 2009 8 41 AM 206KB XML Document 9 17 2009 8 41 AM File and Folder Tasks b Mave the celected itame Appendix B Sample Quantitation after Resuspension 117 Assess the OD Readings If using the formula provided in this appendix the raw data is included in the final Result Data file Figure B 2 is an example of a Result Data file Your OD readings should be similar to those displayed below Figure B 2 Example of Result Data file with acceptable OD readings m quem REDUCTION A1 REDUCTION A2 REDUCTION A3 REDUCTION A4 REDUCTION A5 REDUCTION A6 Abs260 Abs280 Abs320 Purity 0 Concentration Mass rxn ug A1 S1 0 5634 0 3138 0 0493 1 7954 1
19. 12 channel P1200 200 1000 pL a We recommend the use of Rainin pipettes and tips Affymetrix has only verified the use of Rainin 12 channel pipettes in this assay The use of other pipettes such as other brands or 8 channel pipettes may impact the timing of the protocol and may adversely impact the assay Pipette substitution may violate the terms of the Axiom 2 0 Assay and array replacement policy Always use pipettes that have been calibrated a It is essential that you be proficient with the use of single and multi channel pipettes To familiarize yourself with the use of multi channel pipettes we strongly recommend practicing several times before processing actual samples Use water and solution basins to get a feel for aspirating and dispensing solutions to multiple wells simultaneously Single channel Pipettes and Serological Pipettes Use single channel pipettes for preparing Master Mixes and for puncturing bubbles in GeneTitan trays The single channel pipettes will not be used for working with the plates or trays otherwise a Use single channel pipettes for volumes less than or equal to 2 mL For volumes between 1 and 2 mL add the reagent in two portions with a fresh tip for each portion Use serological pipette for volumes gt 2 mL In most cases 25 or 50 mL serological pipettes will not fit into the mouths of the reagents bottles Multiple transfers using 5 or 10 mL serological pipettes will need to be performed
20. 6 399 365 6 420 169 6 551 817 6 610 482 6 733 977 6 955 915 and D430 024 and other U S or foreign patents Products are manufactured and sold under license from OGT under 5 700 637 and 6 054 270 Software products may be covered by one or more of the following patents U S Patent Nos 6 090 555 6 611 767 6 687 692 6 829 376 7 130 458 7 451 047 and other U S or foreign patents Fluidics stations Products may be covered by U S Patent Nos 6 114 122 6 287 850 6 391 623 6 422 249 and other U S or foreign patents Scanners Products may be covered by one or more of the following patents U S Patent Nos 6 141 096 6 262 838 6 294 327 6 403 320 6 407 858 6 597 000 7 406 391 and other U S or foreign patents Hybridization ovens Products may be covered by one or more of the following patents U S Patent Nos 6 050 719 6 386 749 6 705 754 and other U S or foreign patents Copyright Affymetrix Inc All rights reserved Contents Chapter 1 Chapter 2 Chapter 3 About the Axiom 2 0 Assay s s nes 8 Related Documentation xcci cbe rb QAO ROT Pp a 9 Misi CMC L APNETV 9 Overview of the Axiom 2 0 Manual Workflow Assay iilis 9 Running Multiple Plate Workflows 0 0000 ee 10 Safety Warnings and Precautions 0 0 00000 eee 10 Genomic DNA Preparation and Requirements 0 05 11 Sources OF Genormic DNA 3 e onte EM dh PRU ga a Reed
21. Activities Activity Plate ID Approximate Start Times DNA Amplification A 9 30 a m DNA Amplification C 10 30 a m DNA Amplification B 1 30 p m Approximate start time indicates start of thawing of reagents See Stage 1 DNA Amplification on page 33 for more information on the protocol Manual Target Prep Workflow Day 2 The tables below show the steps that need to be performed on the second day Plates A and B are fragmented and precipitated on Day 2 without freezing to preserve a 23 hr amplification incubation Precipitation is carried out at 20 C overnight IMPORTANT Store Plate C at 20 C immediately after the end of the 23 hr Amplification reaction without performing the 65 C Stop Amplification Reaction step Figure 6 3 Manual Target Preparation Workflow Day 2 Activities Day 2 a m l Day 2 p m 8 9 10 11 12 1 2 S 4 5 Plates A B c x Notes Begin thawing required reagents x Freeze Plate C Color Code Fragmentation and precipitation see Stage 2 Fragmentation and Precipitation on page 38 Table 6 7 Manual Target Prep Workflow Day 2 Activities Activity Plate ID Approximate Start Times Fragment and precipitate A 10 00 a m Freeze 20 C 11 00 a m Fragment and precipitate B 2 00 p m Chapter 6 Manual Target Preparation for Processing Three
22. C Rehybridization 126 Storing Hyb Trays for Rehybridization To Store a Hyb Tray 1 When processing on the GeneTitan MC Instrument is complete unload the hyb tray and tightly seal it with an adhesive film Press the four corners and sides of the hyb tray to ensure that there is no open space between the seal and the plate The plate must be well sealed to prevent cross contamination between samples Store the hyb tray at 20 C Rehybridizing an Experiment NOTE It is recommended to perform these steps under a fume hood To Rehybridize an Experiment 1 ee ae ak Remove the plate from the 20 C freezer and spin briefly to collect all of the liquid to the bottom of the plate bring up to 1000 rpm Slowly and carefully remove the seal to prevent cross contamination Using a multichannel pipette transfer the full volume from each well of the hyb tray to the corresponding wells of a new PCR plate Volume per well should be 45 uL To recover all of the remaining material from the hyb tray A Add 50 uL of molecular biology grade water to each well using a multichannel pipette B Pipet up and down 10 times to mix rinsing each corner of the well as you mix C Transfer the full volume from each well to the corresponding wells of the PCR plate Tightly seal the PCR plate with an adhesive film Vortex each corner of the plate at maximum speed Spin briefly again to collect all of the liquid to the bottom of
23. C Variables C Transformation C Concentration C cutoff L validation Technique Type select Absorbance Technique Type Select the desired technique type from the list below Technique Type MO Absorbance mO Luminescence 177 FRET of Fluorescence Intensity Top oe Fluorescence Intensity Bottom m 2 Fluorescence Polarization of Time Resolved Fluorescence Appendix B Sample Quantitation after Resuspension 119 Labware x_Abs_Greiner 96 UV clear std 96 microplate format Select the desired labware type from the list below Type of Labware Name Microplate Format Labware Selection Standard 96 96 Standard 384 384 Standard 1536 1536 x DTX Abs Greiner 384 VIS clear std 384 x Abs Greiner 96 VIS clear std Appendix B Sample Quantitation after Resuspension 120 Layout Settings as appropriate for 96 array plates 1429 Create Protocol NewProtocol 1 General Settings Technique Type Labware Selection Type Samp 3 Filing Flow Replicates Layout Settings gt O Vertical O Constant Number 1 s pisses pees Tees g E Horizontal Incremental Vertical Horizontal Data Reduction Page Output Settings St 52 53 54 55 56 s 58 513 514 515 516 17 518 519 S20 c S25 526 S27 528 529 530 531 532 Method Selection add the three formulas created on the Data Reduction Page to the Group 1 box thod
24. Channel Instrument 95 Figure 5 32 Stain 2 Tray and Stbl Tray Loaded in Drawer 4 Stain 2 Tray and Stbl Tray loaded in drawer 4 Figure 5 33 Stain 1 Tray Loaded in Drawer 5 Stain 1 Tray loaded in drawer 5 3 At the prompt shown in Figure 5 34 click Yes to load another Axiom array plate and hyb tray Figure 5 34 Prompt asking to load another plate Right or left position determined by the position of Axiom array plates already in the GeneTitan MC Instrument WorkFlow Option Do you want to load another plate in the Right Position Please press the Yes button to confirm 4 Follow the prompts and A Setup Option select Setup Another Run then click Next B Scan or manually enter the Axiom array plate barcode then click Next C Select a protocol then click Next Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 96 D When drawer 6 opens 1 Remove the blue cover from the previous Axiom array plate 2 Load anew Axiom array plate and new blue base on the left load a new hyb tray on the right 3 Press the Confirmation button E Click OK when prompted Figure 5 35 Figure 5 35 Confirm Resume Processing Message Confirm Resume Processing xj D This will resume the HybWashScan in the Left Position and S J This will start the another plate with HybWashScan run mode in the Left Position Please press the OK button to confirm
25. Click Yes 5 Wait until the status of the array plate TR HEBEI in the WorkFlow window changes from Select Barcode Plate Type Location AbortRequest to Aborted E 550032 1234567800000 x 550032 Left Position 5A and 5B 550032 23456789 00000 X 550032 Right Posit 6 Once aborted retrieve the array plate and other related consumables by Using Setup Option Unload Plates pirat Shep Loading a new array plate Please select Plate s to abort Press Abort button to abort Press Cancel button to cancel Exception If reagents are loading abort the plate using the Cancel button displayed in the reagent load step EL AE Cancel ance Note If the gripper is required to complete the Abort process the plate will Aborting Run x remain in the AbortRequest state until the gripper becomes available Are you sure Teje Work Flow 5A Barcode Plate Type Location Fluidics Status Scan Status 550032 1 23456 78xXxxxxxx ET Left Position Waiting Waiting Work Flow 5B Barcode Plate Type Location Hyb Status Fluidics Status Scan Status 550032 1234567000000 ET Waiting Waiting Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 75 Stage 1 Create and Upload Batch Registration File In the AGCC software you must create and upload a Batch Registration file before you begin St
26. Figure 5 31 on page 94 Status Load the Stain 1 Tray with cover on Left side of Drawer Load the Ligation Tray with cover on Right side of Drawer Press the Confirmation button when done 4 Stain Tray with Stain 2 Stbl Tray Figure 5 32 on page 95 Status Load the Stain 2 Tray with cover on Left side of Drawer Load the Fixing Tray with cover on Right side of Drawer Press the Confirmation button when done Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument Table 5 6 Sequence for Loading the Trays with Reagents Continued 93 Loading Left Right Sequence by Drawer Number 5 Stain Tray with Stain 1 Empty Status Load the Stain 1 Tray with cover on Left side of Drawer Press the Confirmation button when done Figure 5 30 Scan Tray loaded in Drawer 2 Scan tray with cover loaded in drawer 2 Do NOT load the protective black base packaged with the scan tray Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 94 Lu IMPORTANT When you load the plates or trays insert them under the tabs or fingers that may protrude into the stage Confirm that the tray is not resting on these fingers Tab or Finger Figure 5 31 Stain 1 Tray and Ligation Tray Loaded in Drawer 3 Stain 1 Tray and Ligation Tray loaded in drawer 3 Chapter 5 Array Processing with the GeneTitan Multi
27. Manual Target Prep Workflow Day 5 liisssissss ees 107 TOUTES OO IE skaats ca cues app arp ees clo HM wh he ip ara CR dea i 108 GeneTitan Multichannel Instrument 0 0 00000 00 e 108 Miscellaneous Messages 0 0 eee ee ee 109 Failed Messages 2 2 eee eee 110 Fluidic Diagnostic Messages 0 0 eee 110 Wash Scany RESUME xa ius oe CORRER RC Goad Re ER PE RR RR aha 2 113 PAVOSIRIA A RUM e aa a e AT a E E E a a RETRO UTE 113 Appendix A Appendix B Appendix C Appendix D Appendix E Appendix F Contents 6 Fragmentation Quality Control Gel Protocol 00 cee eee 114 Protocol for Running a Fragmentation Quality Control Gel 0 00005 114 Equipment Required nanna anaana eae 114 E Gels and Reagents 2 0 0 0 cee eee eee 114 Consumables sse ec cee Se ae Shay a a EErEerue Rupee rese fuu egy E RR Blan does 114 Diluting the Tracklt Cyan Orange Loading Buffer 0 000002 eee eee 115 Fragmentation QC Gel Protocol nanoa eee ee 115 Sample Quantitation after Resuspension 0200e eee eee 116 Protocol for Sample Quantitation after Resuspension iilii lille 116 Equipment Required 2 0 0 le 116 Quantitate the Diluted Samples no n anaana aaae 116 Assess the OD Readings 0 000 117 Suggested Protocol for OD Quantitation Using the DTX 880 05 118 If Performing Sample Quantitation on a Plate Reader Other than the DTX88
28. Probe Array Barcode Sample File Name Array Name 2 Default Axiom GW Hu SNP 96 Axiom GW Hu SNP A01 Sample A01 Sample A01 3 Default Axiom GW Hu SNP 96 Axiom GW Hu SNP A02 Sample A02 Sample A02 4 Default Axiom GW Hu SNP 96 Axiom GW Hu SNP A03 Sample A03 Sample A03 5 Default Axiom GW Hu SNP 96 Axiom GW Hu SNP A04 Sample A04 Sample A04 Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 76 Stage 2 Hybridization Reagents Required Reagents Required Table 5 1 Reagents Required from the Axiom 2 0 Reagent Kit Reagent Module Axiom Wash Buffer A both bottles 1L Module 3 Room Temperature Axiom Wash Buffer B P N 901472 Axiom Water An Axiom Genome Wide human or non human array plate or an Axiom myDesign Genotyping 96 array plate is required for this step Prior to inserting this plate into the GeneTitan MC Instrument for hybridization the array plate should be brought to room temperature as described on Step 2 on page 52 a A hybridization tray containing denatured samples fromStep 8 on page 53 in Chapter 4 is also required for this step The denatured samples should be transferred to the hyb tray only after the GeneTitan MC Instrument is ready for loading the hyb tray in the Load Trays onto the GeneTitan MC Instrument on page 91 Setup the Instrument To Setup the Instrument 1 Launch AGCC Launcher and select AGCC GeneTitan Control Figure 5 9 The system initializes Af
29. RE EE 40 2 Prepare Fragmentation Master Mix iiiisssssl eee 41 3 Add Fragmentation Master Mix to Wells 0 000 0000 000 cee eee 42 4 Aliquot the Stop Solution to the Fragmentation Plate 0 2 0 0 000 42 5 Prepare and Add Precipitation Master Mix 2 0 0 0 000 eee 43 Stage 3 Drying Resuspension and QC 20 ee eee 44 MUI ON Gack artes a eae seein ses aa mt nei aS Re eet fds th aE a le dy E Seabee ead eas 44 Input Required 2 0 ae eee 44 Equipment Consumables and Reagents Required 0 00000 eee eee eee 44 1 Centrifuge and Dry Pellets and Thaw Reagents 0 000000 e eee eee 46 2 Prepare the Tubes Basins and Trays for Resuspension and Hyb Master Mix Praeparatio cing p ea ng ote S te ee ane evecare ee eats payne A ere ate eed 47 3 Resuspension and Hybridization Master Mix Preparation 0000 47 4 Recommended Perform Quantitation and Fragmentation QC Checks 49 5 Freeze Or Proceed uns accsEE DE UE URN oe ed aun ITE eu s 50 Stage 4 Denaturation and Hybridization liliis 50 Decus ME Er c r T 50 Required Input from Previous Stage 0 0 0 ees 50 Equipment Consumables and Reagents Required llle 50 1 Prepare Hyb Ready Samples Stored at 20 C 0 eee 52 2 Prepare Equipment and Perform Denaturation 00000000 e eee eee 52 3 Prepare Hybridization Tray and Load into GeneTitan MC Instrument 53
30. Selection Available detection and preparation methods are displayed To add detection or preparation methods click and drag the method to the protocol or sel General Settings Technique Type Labware Selection Layout Settings sthod Sel gt x Me OU seeto Single Kinetic Area Scan Wavelength Scan Data Reduction Page Select Method Estimated Time Output Settings Preparation wait A Set Temperature Shake E Eject Load Il Pause Absorbance IVTEXPRESS 260nm 55 x DTX880 Abs 260nm Genomic x DTX880 Abs 280nm Genomic Anm Genomic Appendix B Sample Quantitation after Resuspension 121 Data Reduction Page create the formulas required for scans at 260 280 and 320 This protocol consists of six passes Click Add new Pass to create passes two through six shown in these figures below General Settings Press F1 for more information about data reduction functions and Formulas Technique Type Labware Selection Layout Settings First Pass Second Pass Third Pass Fourth Pass Fifth Pass Sixth Pass Method Selection A x DTX880 Abs 260nm Genomic REDUCT eue ET aunoa Output Settings c x_DTX880_Abs_320nm_Genomic Formula A Name of Data Abs 260 Mame of Units Notes v Sample Apply Formula For Wells with Category
31. Site Prep Guide P N 702991 for vendor information NOTE The ABI 9700 and the ABI 2720 use the half skirted 96 well plates P N HSS 9601 96 well UV Star Plates 370 uL well Thermal Cycler Recommendations The following thermal cyclers are recommended a BIO RAD PTC 200 or a Whatman Biometra TRobot 96 or a BIO RAD DNA Engine Tetrad 2 PTC 0240 or ABI 9700 with gold sliver or aluminum block or ABI 2720 p IMPORTANT Always use the heated lid option when programming protocols Chapter 3 Axiom 2 0 Assay Preparation Before You Start 21 We have verified the performance of this assay using the following thermal cyclers Bio Rad PTC 200 Biometra TRobot 96 ABI 9700 with a gold silver or aluminum block ABI 2720 and the Bio Rad PTC 0240 The performance of this assay has not been verified with other thermal cyclers Use of other thermal cyclers may result in assay failure and may violate the Axiom array and reagent replacement policy The thermocycler needs to be programmed with the Axiom 2 0 Denature protocol 1 95 C 10 min 2 48 C 3 min 3 48 C hold Use the heated lid option when setting up or running the protocol nn WARNING Evaporation during denaturation can negatively impact assay performance Use the recommended thermal cycler consumables and sealing film to eliminate condensation and evaporation For thermal cyclers with variable lid tension such as the Bio Rad PTC 200 or Tetr
32. Stain and Scan steps Wash Scan Resume Use this option if It was necessary to hybridize the array plate in an oven separate from the GeneTitan MC Instrument a Fluidics processing has been interrupted e g a power failure occurs at your facility Scan Use this option a To rescan an entire array plate or specific arrays on a plate that failed to scan for reasons such as bubbles or gridding failure If you have hybridized and performed the fluidics processes off the GeneTitan MC Instrument Unload Plates Use this option to unload plates and trays from the instrument when a Array plate processing is complete a Array plate processing has been aborted Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 74 Aborting a Process If necessary you can abort the processing of one or more array plates Instructions and an example are shown below in Figure 5 7 If the instrument aborts a process you can retrieve the array plate and related consumables as described in Figure 5 7 An instrument initiated abort may occur Due to improper placement of plates a If the UPS detects a long power interruption draining the UPS to 75 power Figure 5 7 Manually Aborting an Array Plate To abort array plate processing HE AGCC GeneTitan Control 96MC 1 Click the Stop button Fie Tools Help 2 Select the array plate that you wantto 4 2 abort E E Stop Email Help 3 Click Abort z 4
33. Useappropriate serological and single channel pipettes to add reagents to the 15 mL tube labeled Lig in the order shown in Table 4 31 Gently flick the Axiom Ligate Enzyme tube 2 3 times then perform a quick spin immediately prior to adding the enzyme to the Master Mix Table 4 31 Ligation Master Mix Preparation Stage 2 Reagent Per Array Master Mix 96 a Ligation Master Mix from Stage 1 a Axiom Probe Mix 1 82 42 uL 9 10 mL 1 16 mL Axiom Probe Mix 2 10 5 uL 1 16 mL a Axiom Ligate Enzyme 174 4 uL 11 59 mL 3 Gently invert 10 times to mix do not vortex 4 Place on ice and protect from direct light e g cover with aluminum foil or ice bucket lid Chapter 4 Axiom 2 0 Assay Manual Target Preparation 62 3 Aliquot Master Mixes and Axiom Hold Buffer into Trays Label the Trays 1 Gather the scan tray and the stain trays and covers from the Axiom GeneTitan Consumables Kit 2 Label two stain trays S1 3 Label the remaining stain trays a 2 a Stbl a Lig When preparing the hybridization and reagent trays to be loaded onto the GeneTitan MC Instrument you will need to mark the front of each tray in a way that identifies its contents Lu IMPORTANT It is critical that you write only on the proper side of the front edge of stain trays as illustrated in Figure 3 3 on page 31 The front edge of the tray is the short side with the lettering A throug
34. Wash A Wash B and Rinse bottles If you run 4 8 plates week then the micro pore filters need to be replaced more frequently Servicing the Outer Enclosure Fan Filters Cleaning Schedule The GeneTitan fan filter cartridge Figure F 1 should be cleaned at least every 90 days of service Note that in some service locations the presence of excessive dust or particulate matter may necessitate cleaning the cartridge more often than 90 days A plugged filter cartridge can cause excessive temperatures within the machine that can cause unwanted evaporation on test media Part details Affymetrix P N 01 0669 Number of filters required per GeneTitan instrument 3 Appendix F GeneTitan Multi Channel Instrument Care 134 Cleaning Procedure 1 Slide the filter cartridge from the fan filter cartridge at the rear of the GeneTitan Instrument 2 Submerse in clean DI water Rinse and agitate gently to dislodge material 3 Remove from water and dry with clean compressed air or towels 4 When the filter cartridge is completely dry to the touch re install the cartridge Appendix F GeneTitan Multi Channel Instrument Care 135 Replacing the Bottle Filters The bottles used in GeneTitan Instrument contain a filter to remove particulates that may exist in the buffers and DI water The filters in the GeneTitan fluidics bottles Wash A Wash B and DI Water need to be replaced when the filters are clogged The software displays
35. Workflow Day 3 Activities Activity Plate ID Approximate Start Times Centrifuge Dry Resuspend QC A B 9 00 a m Thaw Plate C C 12 00 a m Fragment and precipitate C 1 00 p m Denature and Hyb A 4 00 p m Chapter 6 Manual Target Preparation for Processing Three Axiom Array Plates per Week 106 Manual Target Prep Workflow Day 4 Denaturation of Samples Load array plate and hyb tray in the GeneTitan MC Instrument for Plates B and C a Centrifuge dry resuspend and QC Plate C a GeneTitan reagent trays prep and loading for Plate A n WARNING The Hybridization Tray prep should take place under a running fume hood Lu IMPORTANT The GeneTitan reagent trays for array processing cannot be loaded until the array plate has finished hybridization and they should not be prepared more than 1 5 hours before hybridization will finish The GeneTitan reagent trays cannot be prepared ahead of time and stored Figure 6 5 Manual Target Preparation Workflow Day 4 Activities Plates Day 4 a m GT Reagent prep load E Notes A Begin thawing required reagents y Begin warming Axiom array plate to room temperature z Coupled operations on the GeneTitan MC Instrument Load reagent trays for Plate A and hyb tray array plate for Plate C Color Codes Centrifugation Drying Resuspension Hyb Cocktail Prep and QC
36. following steps at room temperature 1 Per Table 4 5 pipette the appropriate amount of Axiom 2 0 Amp Soln into the 50 mL tube labeled Amp MM at room temperature NOTE Use a 10 mL serological pipette to transfer Axiom 2 0 Amp Soln to the tube The bottles have narrow openings and a 25 mL pipette will not fit through the mouth of the bottle Table 4 5 Amplification Master Mix Amp MM Reagent Per Sample pL Master Mix 96 To the 50 mL tube marked Amp MM add Axiom 2 0 Amp Soln 225 uL 26 0 mL Axiom 2 0 Amp Enzyme 5 pL 578 uL Total Volume 230 pL 26 58 mL 2 Remove the Axiom 2 0 Amp Enzyme from the freezer and place in a portable cooler at 20 C A Flick the Axiom 2 0 Amp Enzyme tube three times then spin B Per Table 4 5 on page 37 add the appropriate amount of Axiom 2 0 Amp Enzyme to the tube labeled Amp MM Chapter 4 Axiom 2 0 Assay Manual Target Preparation 38 Vortex the Amplification Master Mix well invert the tube 2 times and then vortex again Slowly pour the Amplification Master Mix to the solution basin labeled Amp MM Carefully remove the seal from the Neutralization plate and discard the seal Using a P1200 12 channel pipette slowly add 230 uL Amplification Master Mix to each well of the Neutralization plate pipetting down the wall of the well there will now be a total volume of 400 uL well Do not mix by pipetting up and down Change tips between e
37. for vendor information Affymetrix GeneChip Hyb Oven 645 NOTE The GeneChip Hybridization Oven 640 is currently not supported with the Axiom 2 0 Assay however if you want to utilize it in the workflow please contact your Field Service Engineer FSE or Affymetrix Technical Support regarding the compatibility of this oven with the Axiom 2 0 Assay u If using an Affymetrix GeneChip Hyb Oven set the rotation speed to 15 RPM to aid in even heat distribution u For either Affymetrix GeneChip Hyb Oven plates are placed in the bottom of the oven To avoid interfering with the rotation apparatus do not stack plates in the oven o Up to 4 plates can fit into a Hyb Oven 645 Multiple ovens are required for manual target preparation The exact number depends upon whether you are running only a single sample plate and array plate through the workflow or if you are trying to run the three plate week manual target preparation workflow a If you are running individual plates you will need two ovens for the workflow a If you are running the three plate week workflow three ovens are recommended See Changing Oven Temperatures for the Three Plate Workflow on page 102 of Chapter 6 Manual Target Preparation for Processing Three Axiom Array Plates per Week for more information Equipment Care and Calibration Lab instrumentation plays an important role in the successful completion of this assay To aid in maintaining consistency across
38. gDNA uL for the Axiom Genome Wide Pan African Array Set only using reduced EDTA TE buffer 4 Seal vortex and spin NOTE Do NOT dilute the Reference Genomic DNA 103 control from the Axiom 2 0 Reagent Kit It is already at a working concentration Chapter 2 Genomic DNA Preparation and Requirements 16 3 Aliquot the Diluted Samples and the Control Next the samples and control are placed in the following deep well plate for target preparation a For the Manual Target Prep protocol use the ABgene 96 Square Well Storage AB 0932 However if you are running the Automated Target prep see the Axiom 2 0 Assay Automated Workflow User Guide P N702963 for deep well plate recommendation Aliquot Diluted Samples and Reference Genomic DNA 103 to the Selected Deep Well Plate 1 20 uL of each diluted gDNA sample this should be the equivalent of 100 to 200 ng of gDNA as required by the Axiom array 2 20 uL of the Reference Genomic DNA 103 control We recommend including at least one positive control on each plate 3 Seal and spin NOTE For samples to be processed on the Axiom Genome Wide Pan African Array Set three identical deep well plates of 100 ng gDNA per well should be made 4 Freeze or Proceed At this point you can Store the sample plate at 20 C or a Proceed to DNA Amplification for Manual Target Prep See Chapter 4 Axiom 2 0 Assay Manual Target Preparation on page 32 NOTE You can l
39. id ene an is processing Please contact your local Alfymetrix representative or FSE to obtain information on procuring new filters Have you replaced the filters elect YES to continue processing using new NO re rre NR aao ls Mo werpig Way apnea each ine Garston ie erci E rece Wil Filter Change Required BUFFERA_TO_WASHA 3 17 2010 950 53 AM Warning TEA Dern e DIO MNA CORN eet OL OI mes EOM QUK beac Dre REO You need 3 fiters GeneTitan instrument for BUFFERA_TO_WASHA Recent fil data shows a trend of increasing fil times Median value for time remaining before timeout is 12 516 sec Most recent average fill time remaining is 6 166 sec Teu DM lace ell Amc TONO DAOS OS SASE DENT eal cae The bottles are depressunzed and fiters can be DANARO Men Gia Baral an B Croce Please contact your local Affymetrix representative or FSE to obtain information on procuring new fiters Have you replaced the filters Select YES to continue processing using new NO to continue One arg tt BE er hi waring mig appeu anch ibis Gave han i leanchedi The filters in the GeneTitan fluidics bottles Wash A Wash B and DI Water need to be replaced when the filters are worn out The software displays warning message boxes for the filter in each reagent bottle when it detects a problem or shows a trend of increased fill times during fluid fill operations If an error is detected as described above then a message box titled Filt
40. loading on the GeneTitan MC Instrument 96 arrays 12 5 hr Total Prep Time includes reagent thawing time and hands on time Chapter 6 Manual Target Preparation for Processing Three Axiom Array Plates per Week 102 Timing Issues for GeneTitan MC Array Processing IMPORTANT Maintaining consistent timing during the set up of the GeneTitan MC Instrument is critical to containing the user interventions of the three plate workflow within a work day Once one process begins late there is little opportunity to catch up until the end of the workflow The hybridization time for the Axiom 2 0 Assay on the GeneTitan MC Instrument is 23 5 to 24 hr Table 6 3 This provides a 30 min window during which you are prompted by the instrument control software to load the reagents required for washing and staining Table 6 3 Time Required for Array Plate Processing on the GeneTitan MC Instrument Steps on the GeneTitan MC Instrument Time Required Hybridization of two plates in one day 23 5 hr each plate a First plate loaded at 9 30 a m Second plate loaded at 5 00 p m Loading reagent trays 15 min Fluidics 5 hr each plate Imaging 96 arrays 7 5 hr Changing Oven Temperatures for the Three Plate Workflow Multiple ovens are required for manual target preparation If you are running the three plate week workflow three ovens are recommended Table 6 4 lists the different temperatures required for each step Though
41. manipulating genomic DNA to avoid contamination with foreign DNA amplified in other reactions and procedures It is recommended that genomic DNA manipulations are performed in a dedicated pre amplification room or area separate from the main laboratory This pre amplification area should have a dedicated set of pipettes and plasticware If no dedicated area is available use of a dedicated bench or a dedicated biosafety hood and dedicated pipettes is suggested If no dedicated bench or biosafety hood is available a set of dedicated pipettes is recommended Ideally this pre amplification area would be separate from the amplification staging area described in Chapter 3 on page 19 however these areas may be combined due to space and equipment limitations Assessing the Quality of Genomic DNA Using 196 Agarose E gels We recommend this quality control step to asses the quality of the gDNA prior to starting the assay Equipment and Reagents Recommended Table 2 1 E Gel and Reagents Required Item Supplier Part Number Mother E Base Device EB M03 Daughter E Base Device Life Technologies EB D03 formerly Invitrogen E Gel 48 196 agarose gels G8008 01 RediLoad 750026 E Gel 96 High Range DNA Marker 12352 019 Guidelines for Preparing the Genomic DNA Plate for Gel Analysis Loading a DNA mass of 10 ng to 20 ng per well is recommended If lower amounts are loaded omission of the loading dye is recomme
42. manually Protocol Name The protocol that GeneTitan MC Instrument will run The list of protocols displayed is based on the first 6 digits of the array plate barcode Only the protocols that are valid for the type of array plate loaded are displayed Workflow Steps This field displays an overview of the user actions required to process an array plate based on the setup option selected Status This field displays the actions that must be performed to prepare or unload the GeneTitan MC Instrument for the setup option that has been selected After each action you are instructed to click the Next button or to press the blinking blue Confirmation button located on the GeneTitan MC Instrument File Tools Help m s a Stop Email Help System Status System Setup Setup Option Hyb Wash Scan M Plate Information gt Barcode 5500324059357012609098 Plate Type Protocol Name Location Workflow Steps Enter Array Plate Barcode Refill glass bottles with buffer Empty trash bin Remove consumable trays and plates Load consumable trays and plates Select arrays to scan Start Processing Status Please selectthe protocol Press the Next button to advance to the next step Cancel Next Select the System Setup tab Figure 5 10 Configure the software as follows A Setup Option Hyb Wash Scan Other options available are
43. on which Axiom array will be used in the downstream protocol All Axiom arrays except the Axiom Genome Wide Pan African Array Set require a total of 200 ng The Axiom Genome Wide Pan African Array Set requires a total of 300 ng or 100 ng per array there are three arrays in the Axiom Genome Wide Pan African Array Set To Prepare gDNA 1 Thaw Samples and Control 2 Quantitate and Dilute gDNA 3 Aliquot the Diluted Samples and the Control 4 Freeze or Proceed 5 Create a Batch Registration File Duration Thirty minutes to an hour for reagents to thaw and half an hour for setup Equipment Consumables and Reagents Required Equipment and Consumables The equipment and consumables listed in Table 2 2 are required for this stage Table 2 2 Equipment and Consumables Required for Genomic DNA Preparation Quantity Item As required Adhesive seals for plates 1 Ice bucket filled with ice 1 each Pipettes Single channel P10 or P20 Optional multi channel P10 or P20 As required Pipette tips 1 Plate deep well ABGene 96 Square Well Storage AB 0932 1 Plate centrifuge 1 Plate spectrophotometer required only if no OD measurements available for samples 1 Vortexer A different deep well plate is used for the Automated Workflow version of the Axiom 2 0 Assay See Chapter 2 of the Axiom 2 0 Assay Automated Workflow User Guide P N 702963 Chapter 2 Genomic DNA Preparation and Requirem
44. only two ovens are strictly required we recommend maintaining separate 37 C ovens for the amplification and fragmentation stages to avoid confusion of plates and to minimize excess opening and closing of oven doors during incubation periods Table 6 5 provides a list of suggested settings for three ovens when performing the three plate week workflow Table 6 4 Oven Temperatures Needed for Each Step of the Workflow Workflow step Oven Temp Amplification 37 C Stopping Amplification 65 C Pre Fragmentation Incubation 37 C Fragmentation Incubation 37 C Drying 37 C Hybridization 48 C Table 6 5 Suggested Settings for Ovens When Performing Three Plate Week Manual Target Prep Workflow Day of Workflow Oven 1 Oven 2 Oven 3 Day 1 37 C N A N A Day 2 37 C 65 C 37 C Day 3 48 C 65 C 37 C Day 4 48 C 65 C 37 C Day 5 N A N A N A Chapter 6 Manual Target Preparation for Processing Three Axiom Array Plates per Week 103 Thawing Frozen Plates of Amplified DNA To Thaw Frozen Plates of Amplified DNA 1 Place the deep well plate in a small water bath For example pour Millipore water into a small tray Place the frozen plate in the water in the tray 2 Leave the plate in the water bath for 50 min until all wells have thawed Spin down at 1000 rpm for 30 sec To avoid cross contamination of wells during vortexing A Remove the seal and blot the top of the plate with a
45. run a set of samples and array plates through the protocol using a minimum of personnel and a forty hour week The timing of steps is critical whether using automated target prep or manual target prep because of the following constraints Incubation after DNA Amplification is 23 hours Hybridization in the GeneTitan Instrument is 23 5 hours Reagent trays for wash stain imaging must be prepared as Hybridization finishes a Limits to when a second hyb tray and array plate can be loaded into the GeneTitan Instrument These limitations require careful timing The details are covered in Chapter 6 Manual Target Preparation for Processing Three Axiom Array Plates per Week on page 100 Safety Warnings and Precautions CAUTION All chemicals should be considered as potentially hazardous We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice Wear suitable protective clothing such as lab coat safety glasses and gloves Care should be taken to avoid contact with skin and eyes In case of contact with skin or eyes wash immediately with water See MSDS Material Safety Data Sheet for specific advice A WARNING The following components contain harmful or toxic ingredients a Axiom Stabilize Soln 8 Gluteraldehyde a Axiom HybSoln 2 100 Formamide Axiom Hyb Buffer lt 55 Tet
46. sample and array plate that are required to perform this workflow Lu IMPORTANT Experienced users and careful timing are critical for the successful execution of this workflow The three plate per week workflow is described in the following sections Overview of the 3 Plate Workflow for Manual Target Preparation a Thawing Frozen Plates of Amplified DNA on page 103 Manual Target Prep and Array Processing on page 103 Detailed instructions for the manual target prep protocol and the array plate processing are given in a Chapter 4 Axiom 2 0 Assay Manual Target Preparation on page 32 a Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument on page 67 Overview of the 3 Plate Workflow for Manual Target Preparation The table below displays the timing and duration of the hands on processing necessary for performing the three plate workflow by one person Figure 6 1 Three Plate per Week Manual Target Prep Workflow Plate Day 1 Day 2 Day 3 Day 4 Day 5 8 9 1011121 2 3 4 5 6 8 9 1011112123 4 5 6 8 9 1011121 2 3 4 5 6 8 9 1011121 2 3 4 5 6 8 9 1011121 2 3 4 5 6 A A Al Z B all E a c z Ni I 1 1 Notes Begin Thawing required reagents for the process y Begin warming Axiom array plate to room temp
47. then restart it If the same error occurs shut down both the application and the computer and then restart If it still occurs shut down the GeneTitan MC Instrument and then restart The log files are produced by different AGCC components The logs provide a record of the tasks performed by different components such as the migration tools and installer These log files provide useful information for troubleshooting problems These files may be requested by your field application specialist FAS field service engineer FSE or the Affymetrix call center AGCC Log Files The following files apply to the GeneTitan Instruments All the AGCC log files from C Command_Console Logs The different log files include Systemlog XML XML file with system information DEC log Text file with information on the use of the Data Exchange Console DECError log Text file with information on errors created while using DEC AGCC LibFilelmporter log Text file with info on use of the Library File Importer with date and time code Other AGCC Files Your FAS and or FSE may request you to send the following files for troubleshooting 1 Library files PARAMS MASTER WORKFLOW SMD MEDIA located in CACommand Console W ibrary excluding the large analysis library files CDF PSI GRC 2 Provide a list of all sub folders and their contents under the library files folder located in CACommand Console W ibrary Please ensure there are no duplicate librar
48. tray Notched corner of the stain tray should face the front Lanek OE Stain ay NEE see Stage 5 Manually Preparing Ligation Staining and Stabilization Reagent Trays for the GeneTitan MC Instrument on page 55 for detailed information Axiom 2 0 Assay Manual Target Preparation Manual target preparation for the Affymetrix Axiom Genome Wide assay enables you to perform target preparation to process 96 samples at a time without the use of automation equipment NOTE Array handling and processing protocols still require the use of a GeneTitan MC Instrument as described in Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument on page 67 Lu IMPORTANT Read all the instructions in Chapter 3 Axiom 2 0 Assay Preparation Before You Start on page 18 before performing manual target preparation A list of all equipment and resources required for the Axiom 2 0 Assay with manual target preparation is in the Axiom 2 0 Assay Manual Workflow Site Prep Guide P N 702991 The protocol for manual target preparation is presented in the following sections a Stage 1 DNA Amplification on page 33 Stage 2 Fragmentation and Precipitation on page 38 Stage 3 Drying Resuspension and QC on page 44 Stage 4 Denaturation and Hybridization on page 50 a Stage 5 Manually Preparing Ligation Staining and Stabilization Reagent Trays for the GeneTitan MC Instrument on page 55 Lu
49. u Label one plate as QC Diln o Label the second plate as Gel Sample 1 each 96 well UV Star 370 uL well plate labeled OD 14 mL Gel Loading dye o 1000 fold dilution of 6x Invitrogen TrackIt Cyan Orange as described in Diluting the TrackIt Cyan Orange Loading Buffer on page 115 14 mL of nuclease free water P N 71786 15 fold dilution of 25bp Invitrogen Ladder P N 10488 022 To Perform the QC Checks carry out the following steps at room temperature 1 NOTE Change tips while transferring samples from the Hyb Ready plate and the QC Dilution plate to avoid cross contamination Pour nuclease free water into the solution basin labeled H20 The water will be used to make the QC Dilution plate and the OD plate Make QC Dilution Plate A B C Add 33 uL nuclease free water to each well of the QC Diln plate Transfer 3 uL of the Hyb Ready sample from each well of the Hyb Ready plate to the corresponding well of the QC Diln plate Change pipette tips after each transfer Seal vortex and spin Make OD Sample plate A B Carefully remove the seal from the QC Dilution plate and discard the seal Transfer 10 uL of each QC Dilution Plate sample to the plate labeled OD This plate is known from here on as the OD plate Change pipette tips after each transfer Add 90 uL nuclease free water to each well of the OD Plate and mix by pipetting up and down Change pipette tips after each addition Final sample mas
50. wells Perform denaturation and transfer samples to a new hybridization tray If using the Biomek FX Target Prep Express run these methods Denature samples Transfer denatured samples to Hyb Tray a If using the Manual Target Prep Workflow a Perform Denaturation and Hybridization see Stage 4 Denaturation and Hybridization on page 50 Lu IMPORTANT Place a new hyb tray on the deck Transfer the hyb tray to the GeneTitan MC Instrument and A Load the hyb tray and a new array plate onto the GeneTitan MC Instrument B When hybridization is complete prepare a new set of reagent trays using either The Biomek FX Target Prep Express Manual Reagent Tray Prep see Stage 5 Manually Preparing Ligation Staining and Stabilization Reagent Trays for the GeneTitan MC Instrument on page 55 C Transfer the reagent trays to the GeneTitan MC Instrument and finish processing the array plate Registering Samples in Affymetrix GeneChip Command Console Creating a GeneTitan Array Plate Registration File A GeneTitan Array Plate Registration file is a Microsoft Excel spreadsheet that includes information on the samples you are processing on a single array plate This information includes the array plate format the array plate barcode and sample file names so that you can track the samples that are loaded onto a particular array plate The version of Microsoft Excel must be 1997 2000 file extension is xls not
51. xlsx To Create a GeneTitan Array Plate Registration File 1 In AGCC Portal open the Samples menu and select GeneTitan Array Plate Registration Figure D 1 Selec File Edit View Favorites Tools Help Q O l A Gl Pom yr O 2 2m 783 Address amp http localhost 3000 AFFyWeb Default aspx 4 E Affymetrix GeneChip Command Console Portal Home Microsoft Internet Explorer B E Search Files By El Array Name x Use HOME DATA SAMPLES ADMINISTRATION HELP Registration Quick Registration Batch Registration Sample Prep Plate Registration GeneTitan Array Plate Registration Add Barcode Version 2 0 Batch Edit Upload 2008 Affymetrix Inc All rights reserved Affymetrix GeneChip NetAltx r Powered by Affymetrix GeneChip compatible asd Genotyping Console are tra registered trademarks of Affymetrix Inc All other trademarks are the y a owners 2 Step 1 Figure D 2 on page 128 A Select the array plate type B Click Download Appendix D Registering Samples in Affymetrix GeneChip Command Console 128 Figure D 2 Selecting the type of array plate to be processed HOME DATA SAMPLES ADMINISTRATION HELP GeneTitan Array Plate Registration 4 Step 1 Create a blank GeneTitan Array Plate registration file with the desired attributes Select the templates with the attributes you wish to use for the sample files H
52. y e S corner should E B face the front E Correct placement of cover on stain tray md us LN t Incorrect placement of cover on stain tray Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 70 Labeling GeneTitan Hybridization and Reagent Trays When preparing the hybridization and reagent trays to be loaded onto the GeneTitan MC Instrument you will need to mark each tray in a way that identifies its contents Lu IMPORTANT It is critical that you write only on the proper locations of the proper sides of hyb and stain trays Do NOT write in any other location as this can interfere with sensors inside the GeneTitan MC Instrument and result in experiment failure To ensure proper placement of lids onto stain trays and trays onto the GeneTitan MC Instrument you can also mark the notched corner of the trays and lids Proper labeling for hyb trays and reagent trays is described in a Labeling for Hyb Trays below a Labeling for Stain Trays on page 71 Lu IMPORTANT Do not confuse hyb trays with stain trays Labeling for Hyb Trays You may label the hyb tray on the front part of the short side of the tray next to the notch at the left as shown in Figure 5 4 The proper section for labeling is closest to the notched corner corresponding to the Al and B1 wells Do NOT label trays on the long side of the tray Notched corner of the hyb tray should f
53. 0 124 oim jordle 72 15 RNC 125 Protocol for Rehybridizing Samples llis 125 Rehybridizatio h user eter eR Md a a feb ahs mae eee ete ed 125 Equipment Consumables and Reagents Required 0 2 00 0000 cee eee 125 Storing Hyb Trays for Rehybridization 0 0 0 0 eee 126 Rehybridizing an Experiment 00 000 nerta eea aE eee ee eee 126 Registering Samples in Affymetrix GeneChip Command Console 127 Creating a GeneTitan Array Plate Registration File 0 0 20 0 0 0 eee 127 Deionization Procedure for GeneTitan Trays and Covers 130 Testing the Anti Static GUNG uem nem pues bae XE IA P EROR ae E 131 Deioriizatiori Procedure as desse keRtfcauieeswbigaewduesd bene PAPE RESI ERE 131 GeneTitan Multi Channel Instrument Care ll l 133 Cleaning and Maintenance n nauau nannaa 133 MORTAIN gorrian paa nn a aE nen teen A ee bfee E E E 9 EE E E EA A R E aac 133 Every SIX MORIS s 2d i holies ER E e gly edo Arak hale E REE e Rt dd 133 Servicing the Outer Enclosure Fan Filters nnana auaa eee a 133 Replacing the Bottle Filters 2 0 0 eee ee 135 Replacing the Xenon Lamp in the GeneTitan MC Instrument 0 137 droubleshooHlid aedes cat tee awe ee PU eM Bee een eee eae ee pa 142 food EE 142 AGCC Log Files for GeneTitan MC Systems 0 000 000 0c es 143 Contents Problems and Solutions 0 0 0 00 eee eee Insufficient Disk Space Notice
54. 0 6366 1223 2034 A2 S2 0 58 0 3195 0 0487 1 8153 10 9924 1264 1276 A3 sa 0 5757 0 3177 0 0494 1 8121 10 889 1252 231 AA S4 0 5792 0 3204 0 0467 1 8077 11 0172 1266 9828 A5 S5 0 5693 0 3136 0 0496 1 8154 10 7524 1236 5276 A6 S6 0 5653 0 315 0 0534 1 7946 10 591 1217 969 AT s7 0 6072 0 3394 0 0488 1 789 11 5531 1328 6069 8 se 0 595 0 329 0 0489 1 8085 11 2986 1299 3414 AQ s9 0 5921 0 3279 0 0498 1 8057 11 22 1290 3 A10 10 0 6149 0 3413 0 0502 1 8016 11 6834 1343 5966 A11 s11 0 6103 0 3377 0 0497 1 8072 11 5986 1333 8414 A12 12 0 5984 0 3309 0 0498 1 8084 11 3503 1305 2897 B1 13 0 5786 0 3229 0 0522 1 7919 10 891 1252 469 B2 S14 0 5757 0 3208 0 0522 1 7946 10 831 1245 569 B3 15 0 5501 0 305 0 0491 1 8036 10 3655 1192 0345 B4 S16 0 5415 0 2987 0 0505 1 8129 10 1586 1168 2414 B5 17 0 5084 0 282 0 0485 1 8028 9 5152 1094 2448 B6 S18 0 5533 0 3061 0 0491 1 8076 10 4317 1199 6483 B7 19 0 5502 0 304 0 052 1 8099 10 3076 1185 3724 B8 20 0 5776 0 3187 0 0498 1 8124 10 92 1255 8 B9 S21 0 5673 0 3136 0 0535 1 809 10 6303 1222 4897 B10 22 0 5602 0 3102 0 0493 1 8059 10 5703 1215 5897 B11 S23 0 5814 0 3206 0 0499 1 8135 10 9966 1264 6034 B12 24 0 583 0 3235 0 0524 1 8022 10 9779 1262 4621 c1 25 0 5424 0 3009 0 0475 1 8026 10 2393 1177 5207 c2 S26 0 5375 0 2973 0 0472 1 8079 10 1441 1166 5759 ca S27 0 5196 0 2868 0 0473 1 8117 9 7717 1123 7483 OD Yield Assessment Guidelines The measurement of the yield of DNA after resuspension of the pellets
55. 2 0 tee ee 7 About the Axiom 2 0 Assay The first Genome Wide Association study GWAS was published in 2005 1 when individuals carrying particular haplotypes of SNP rs380390 were found to have increased risk of developing age related macular degeneration a study performed with the Affymetrix GeneChip Mapping 100K Array Set 2 As of September 2009 there have been over 400 peer reviewed GWAS publications and over 1774 SNPs have been implicated in human disease 3 Initial GWAS studies focused on the common disease common variant hypothesis 1 that held that haplotypes with a minor allele frequency MAF gt 5 would show measurable contribution to human disease research Current research is shifting towards complex disease complex rare variant studies As such these research projects require a broader catalog of human variation such as is being generated by the 1000 Genomes Project http www 1000genomes org This project focuses on identifying alleles with a MAF lt 5 across a broader spectrum of human ethnicities In order to allow our customers to take advantage of this novel and rare content for genome association and candidate gene studies in a cost effective and timely manner Affymetrix is introducing a new genotyping product line the Axiom Genotyping Solution The Axiom Genotyping Solution introduces a new genotyping technology platform that includes novel assay biochemistry array configuration and pr
56. 48 C Log 9 18 24 AM ProductName HTS6CC 9 18 24 AM ProduclVersionS6F 3 0 0 99 9 18 24 AM LastwriteTime 36F 4 29 2009 4 30 30 AM 9 18 24 AM ProductVersion96Scanner 1 6 0 0 9 18 24 AM LastwriteTime 96Scanner 4 29 2008 4 36 13 AM 9 18 24 AM DriveC free space 76 5 GigaBytes 9 18 24 AM DriveD free space 748 4 GigaBytes 9 18 24 AM DriveC free space is less than 96 0 GigaBytes 9 18 24 AM DriveC free space is less than 96 0 GigaBytes 9 18 37 AM MED amp 96S files Copied 26 9 18 37 AM AuditLogDir set to C Command_Console Logs 96F 9 18 37 AM LogFileDir set to C Command_Console Logs S6F 9 18 37 AM Timer started with Interval 1000 msec 9 18 37 AM Homing HTS6F and Scanner 9 18 37 AM Set HybOven temperature to 48 C 9 18 37 AM Set WashB temperature to 25 C 9 19 16 AM Homing HTSBF completed 9 19 16 AM Initializing Scanner 9 19 16 AM Status Scanner drawer not extended or no plate present 9 19 16 AM ScannerOn Option is on 9 20 38 4M Scanner homing completed 9 20 38 AM Checking and removing plate from scanner 9 21 06 AM Status No Plate in Scanner System ready for running 77 Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 78 Figure 5 10 System Setup Tab and the Information Displayed in this Pane System Setup tab Setup Option The various options you can choose for processing Axiom array plates Barcode The array plate barcode Can be scanned or entered
57. 96CC FLUIDIC DIAGNOSTIC x PulseUntilSensorState Failure on group PRIME_RINSE If the failure is detected du ring priming the instrument Plate 550032 345678922xxxxxx i i al paces EM will pause and wait for the problem to be corrected Fluidic process CleanThenFillWashawithRinse Possible causes For dispense failure include Bottle empty or fluid level too low Replenish bottle If the failure is detected during another process and if the Bottle cap not secure Check all bottle caps are secure 3 B P y Clogged fiter Replace filter cause is a clogged filter wait until the end of the run to replace the filter Instructions for filter replacement in the GeneTitan Multi Channel Instrument User s Manual P N 08 0306 Table 7 4 Problem messages Continued Chapter 7 Troubleshooting 111 Problem and Possible Causes When the instrument experiences a loss in Clean Dry Air CDA pressure the software will display the warning message aiclxl 1 20 2010 10 09 45 AM System lost CDA pressure Verify lines are connected and tumed ON System serial number HT96Fluidic O000000 When the pressure is detected again a dialog message confirming the availability of CDA pressure is displayed Wl GeneTitan I labxl 1 20 2010 10 11 00 AM System CDA pressure has been restored The pressure event was detected at 1 20 2010 10 09 45 AM and lasted until 1 20 2010 10 11 00 AM System serial number H
58. AX v Affymetrix ilser Guide Axiom 2 0 Assay Manual Workflow P N 702990 Rev 3 For research use only Not for use in diagnostic procedures Trademarks Affymetrix Axiom Command Console CytoScan DMET GeneAtlas GeneChip GeneChip compatible GeneTitan Genotyping Console myDesign NetAffx OncoScan Powered by Affymetrix Procarta and QuantiGene are trademarks or registered trademarks of Affymetrix Inc All other trademarks are the property of their respective owners Limited License Subject to the Affymetrix terms and conditions that govern your use of Affymetrix products Affymetrix grants you a non exclusive non transferable non sublicensable license to use this Affymetrix product only in accordance with the manual and written instructions provided by Affymetrix You understand and agree that except as expressly set forth in the Affymetrix terms and conditions no right or license to any patent or other intellectual property owned or licensable by Affymetrix is conveyed or implied by this Affymetrix product In particular no right or license is conveyed or implied to use this Affymetrix product in combination with a product not provided licensed or specifically recommended by Affymetrix for such use Patents Arrays Products may be covered by one or more of the following patents U S Patent Nos 5 445 934 5 744 305 5 945 334 6 140 044 6 261 776 6 291 183 6 346 413
59. Alfymetrix representative or FSE to obtain information on procuring new filters Have you replaced the fiers Select YES to continue processing using new fiters NO to continue processing without changing fiers this waming may appear each time GeneTitan is launched o Et iter change Requred eurrera vo wasna E 3 17 2010 9 50 53 AM Warning RES DNA 1 ees me mua ee ONA OON Denke DR MEME I MED DOCENT RCRUM MES You need 3 fiters per GeneTitan instrument Possible fiter for SURFER YO WASHA Recent fil dats shows a trend of increasing fil times Median value for time remaining before timeout is 12 516 sec Most recent average fill time remaining is 6 165 sec Fe MM OCA Ma OM MM HANE DN AC NONO DORMI eM ca ennt The bottles are depressurzed and filters can be now Do not change fiters while GeneTitan is processing Please contact your local Alfymetix representative or FSE to obtain information on procuring new filters Have you replaced the fers Select YES to continue processing NO to continue M anol 6 NT av Chis weg my Nose sach Woe Cu INR Fe Muched o9 The message boxes displayed in Figure F 2 will provide information on fluid dispense errors that were detected by the instrument for any of the bottles or when the instrument detects an increase in the amount of time that is required to perform the fill operations NOTE The reagent bottles are depressurized when this warning message is displaye
60. Axiom Array Plates per Week 105 Manual Target Prep Workflow Day 3 Centrifuge dry resuspend and QC Plates A and B Thaw Plate C see Thawing Frozen Plates of Amplified DNA on page 103 Fragment including the 65 C Stop Amplification Reaction step and precipitate Plate C Perform Denaturation on Plate A Transfer Plate A samples to Hyb Tray A Load Hyb Tray A and array plate into GeneTitan MC Instrument and begin hybridization n WARNING The Hybridization Tray prep should take place under a running fume hood IMPORTANT Amplified plates that are frozen must be thawed and thoroughly mixed by following the procedure under Thawing Frozen Plates of Amplified DNA on page 103 Figure 6 4 Manual Target Preparation Workflow Day 3 Activities Day 3 a m Day 3 p m 8 9 10 11 12 1 2 3 4 5 Plates A B T Notes A Begin thawing required reagents y Begin thawing Plate C Y Begin warming Axiom array plate to room temperature Color Codes ag Fragmentation and Precipitation see Stage 2 Fragmentation and Precipitation on page 38 Centrifugation Drying Resuspension Hyb Cocktail Prep and QC see Stage 3 Drying Resuspension and QC on page 44 Sample Denature load array plate and hyb tray in the GeneTitan MC Instrument see Stage 4 Denaturation and Hybridization on page 50 Table 6 8 Manual Target Prep
61. Axiom Stain 1 B P N 901276 Axiom Stain 2 A Axiom Stain 2 B Axiom Stabilize Diluent Axiom Water Axiom Hold Buffer These solutions are light sensitive Keep tubes out of direct light for a prolonged period of time 1 Prepare the Reagents for Stage 5 To Prepare the Reagents 1 Prepare the reagents from Module 4 1 as described in Table 4 23 Table 4 23 Reagents from Module 4 1 20 C P N 901278 Reagent Temp Out of Treatment Storage before Master Module Mix Axiom Ligate Buffer Thaw at Room Temp 1 Place on bench top at room temp for 30 min Place on ice 2 Examine for precipitate 3 Vortex twice 4 Examine for precipitate If any a Warm bottle with your hands and vortex again for thirty seconds Axiom Ligate Enzyme Keep at 20 C until Just before use Place in 20 C portable ready to use 1 Flick 2 to 3 times to mix cooler 2 Spin 3 Place in 20 C portable cooler until use Axiom Ligate Soln 1 Thaw at Room Temp Vortex and Spin Place on Ice Axiom Probe Mix 1 Thaw at Room Temp Vortex and Spin Place on Ice Axiom Stain Buffer Thaw at Room Temp Vortex and Spin Place on Ice Axiom Stabilize Soln Thaw at Room Temp Vortex and Spin Place on Ice Notes Temperature the reagent is held at immediately after removal from module This bottle can also be thawed in a dish with room temperature Millipore water NOTE The presence of some precipitate i
62. Dh 11 General Requlrerents zer eee etn od qe qubd per od np RR RAD e od qe d 12 Special Requirements 2 2 0 00 00 eh 12 Assessing the Quality of Genomic DNA Using 1 Agarose E gels 12 Genomic DNA Extraction Purification Methods 0 00 000 eee eee 13 Genomic DNA Cleanup isisssssssee hrs 13 Genomic DNA Preparation liissss s 14 B ration TRE 14 Equipment Consumables and Reagents Required 0 000000 iiis 14 1 Thaw Samples and Control 2 ee 15 2 Quantitate and Dilute gDNA ri reee te cents dna Be eed Skeet d eee ees 15 3 Aliquot the Diluted Samples and the Control 0 0 0 0 0000 c cece 16 4 Freeze Or Proceed ss pP d eR exa Bea ete e P Ee 16 5 Create a Batch Registration File 0 2 es 16 Axiom 2 0 Assay Preparation Before You Start 00 18 Differences Between the Axiom Assays liiis 18 Requirements and Recommendations liiisiies eee 19 Room Temperature 0 0 000 hrs 19 Special Requirements 2 2 0 0 0 hh 19 Safety Warnings and Precautions iiiiiis esee 20 Control Recommendations n saaana aaae 20 Plate Requirements and Recommendations o saaa auauna 20 Thermal Cycler Recommendations o a aaa aaaea a 20 Thermal Cycler Consumables 0 0 0 0 0c a 21 Oven Recommendations uuu eed e DR Ue s rots E E E EE E ER 22 Equipment Care and Calibration 0 0 000 es 22 Rrocedures Ax gt ata dus etum
63. Equipment Quantity Rainin Pipettes single channel Rainin Pipettes 12 channel 1 each P200 P200 P1000 Vortexer 1 Table 4 21 Consumables Required for Stage 5 Manually Preparing Ligation Staining and Stabilization Reagent Trays for the GeneTitan MC Instrument Consumable Part Number Quantity Aluminum foil optional As required GeneTitan Consumables Kit Affymetrix 1 kit includes P N 901606 o Scan Tray P N 501006 1 o Stain Tray P N 501025 5 o Covers for trays P N 202757 6 Pipette serological a 5 x 1 10 mL VWR P N 53283 706 1 10 x 1 10 mL VWR P N 53283 708 2 Pipette tips As required for pipettes listed in Table 4 20 Solution basin 100 mL sterile multichannel 5 15 mL conical tube 3 50 mL conical tube 1 Reagents Required Table 4 22 Axiom 2 0 Reagents Required for Stain and Ligation Stage Continued Module Reagent Axiom Ligate Buffer Axiom Ligate Enzyme Module 4 1 20 C Axiom Ligate Soln 1 P N 901278 Axiom Probe Mix 1 Axiom Stain Buffer Axiom Stabilize Soln These solutions are light sensitive Keep tubes out of direct light for a prolonged period of time Chapter 4 Axiom 2 0 Assay Manual Target Preparation 57 Table 4 22 Axiom 2 0 Reagents Required for Stain and Ligation Stage Continued Module Reagent Axiom Ligate Soln 2 Axiom Probe Mix 2 Axiom Wash A Axiom Stain 1 A Module 4 2 2 8 C
64. Figure 2 1 Gel Images Showing Intact gDNA and Degraded gDNA Intact Degraded Samples Samples 10 kb 4 kb 2 kb 0 8 kb 0 4 kb Genomic DNA Extraction Purification Methods Genomic DNA extraction and purification methods that meet the general requirements outlined above should yield successful results Methods that include boiling or strong denaturants are not acceptable because the DNA would be rendered single stranded and can no longer be accurately quantitated using a PicoGreen based assay Genomic DNA Cleanup If a gDNA preparation is suspected to contain inhibitors the following cleanup procedure can be used Add 0 5 volumes of 7 5 M NH4OAc 2 5 volumes of absolute ethanol stored at 20 C to gDNA Vortex and incubate at 20 C for 1 hr Centrifuge at 12 000 x g in a microcentrifuge at room temperature for 20 min Remove supernatant and wash pellet with 8096 ethanol Centrifuge at 12 000 x g at room temperature for 5 min Remove the 80 ethanol and repeat the 80 ethanol wash one more time Resuspend the pellet in reduced EDTA TE Buffer 10 mM Tris HCl pH 8 0 0 1 mM EDTA Se we UNE ES Chapter 2 Genomic DNA Preparation and Requirements 14 Genomic DNA Preparation This step needs to be done before proceeding with the DNA amplification stages The genomic DNA gDNA you will process using the Axiom 2 0 Assay should meet the general requirements listed earlier in this chapter The amount of gDNA depends
65. GeneTitan MC Instrument and result in experiment failure Lu IMPORTANT Do not confuse hyb trays with stain trays Place the hyb tray under the fume hood Using a P200 12 channel pipette set at 105 uL slowly transfer the denatured samples from the Hyb Ready plate into the hyb tray Dispense to the first stop to avoid creating bubbles Change pipette tips after each transfer discard the tip even if it shows some volume left Ensure that there are no air bubbles present in the hyb tray Puncture any air bubbles that you see using a clean pipette tip There is no need to spread the sample around the bottom of the hyb tray wells Sample distribution across the well will occur when the array plate is stacked together with the hyb tray by the GeneTitan MC Instrument Load the array plate and hyb tray into the GeneTitan MC Instrument see Load an Axiom Array Plate and Hyb Tray Onto the GeneTitan MC Instrument on page 81 Chapter 4 Axiom 2 0 Assay Manual Target Preparation 54 Lu IMPORTANT The array plate must be loaded on the left side on its protective blue base as shown in the figure below The clear plastic cover on top of the array plate SHOULD NOT be loaded in the GeneTitan MC Instrument Clear Tray 78 Shipping Cover To be Discarded Blue Array Plate Protective Base Array Plate Load the hyb tray on the right side without any covering The hyb tray should not have any bubbles m IMP
66. Handler User s Manual Beckman Coulter P N 987834 Biomek Software User s Manual Beckman Coulter P N 987835 References 1 Manolio T A and Collins F S The HapMap and Genome Wide Association Studies in Diagnosis and Therapy Annu Rev Medicine 2009 60 443 56 2 Klein RJ Zeiss C Chew EY et al Complement factor H polymorphism in age related macular degeneration Science 2005 308 385 89 3 Hindorff LA Junkins HA Mehta JP and Manolio TA A Catalog of Published Genome Wide Association Studies Available at www genome gov gwastudies Accessed 09 28 2009 Overview of the Axiom 2 0 Manual Workflow Assay Running the Axiom 2 0 Assay requires the following sets of steps 1 Genomic DNA Prep Resulting in samples that meet requirements spelled out in Chapter 2 Genomic DNA Preparation and Requirements on page 11 2 Target Prep of the samples see Chapter 4 Axiom 2 0 Assay Manual Target Preparation on page 32 3 Array Processing done with GeneTitan MC Instrument a GeneTitan Instrument Control software AGCC Portal software See Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument on page 67 A list of the required equipment and supplies for running the Axiom 2 0 Assay manual target preparation can be found in the Axiom 2 0 Assay Manual Workflow Site Prep Guide P N 702991 Chapter 1 About the Axiom 2 0 Assay 10 Running Multiple Plate Workflows Affymetrix provides workflows that allow you to
67. Hu SNP 96 Axiom GW Hu SNP A02 Sample A02 Sample A02 4 Default Axiom GW Hu SNP 96 Axiom GW Hu SNP A03 Sample A03 Sample A03 5 Default Axiom GW Hu SNP 96 Axiom GW Hu SNP A04 Sample A04 Sample A04 4 Save the file as follows A Open File gt Save As B Enter a name for the array plate registration file C Click Save By default the file is saved in the Affymetrix Downloads folder 5 Step 3 when ready to load the array plate onto the GeneTitan MC Instrument A Click the Browse button navigate to the file and click Open B Scan the array plate barcode if not already scanned C Click the Upload button Figure D 4 wait for the information to load then click the Save button located at the bottom of the next page that is displayed If the samples are successfully registered the message in Figure D 5 is displayed Appendix D Registering Samples in Affymetrix GeneChip Command Console 129 Step 3 Upload the GeneTitan Array Plate registration file to create new sample ARR files Enter the path or click Browse to find the GeneTitan Array Plate registration file If a plate barcode is not provided in the excel file being uploaded one MUST be pr barcode field below GeneTitan Array Plate registration file Required C Documents and Settings AFFXUserDesktopuAffymetrix DownloadsiGeneTitanArrayPlateR Browse GeneTitan Array Plate Barcode 5500944077806010110489 HOME DATA SAMPLES AD
68. Kimwipe B Tightly reseal the plate with a fresh seal 5 Vortex the plate for 30 sec to thoroughly mix refer to guidelines described in Seal Vortex and Spin on page 23 6 Spin at 1000 rpm for 30 sec Manual Target Prep and Array Processing Manual Target Prep Workflow Day 1 On this day you start amplification of the three plates each plate must incubate 23 1 hours after amplification begins All amplifications should be set up on Day 1 to allow for a 23 1 hr amplification incubation for each plate and to minimize movement between pre amplification and post amplification areas Begin thawing the amplification reagents particularly the Axiom 2 0 Amp Soln 60 min prior to the start of each reaction IMPORTANT Amplification preparation should take place in an Amplification Staging Room or dedicated area such as biosafety hood with dedicated pipettes tips vortex etc See Amplification Staging Area on page 19 for more information Figure 6 2 Manual Target Preparation Workflow Day 1 Activities Day 1 a m Day 1 p m 8 9 10 1 2 3 4 5 Plate A A B A Cc Notes A Begin thawing reagents and materials for the process Color Code Amplification see Stage 1 DNA Amplification on page 33 Chapter 6 Manual Target Preparation for Processing Three Axiom Array Plates per Week 104 Table 6 6 Manual Target Prep Workflow Day 1
69. MINISTRATION HELP rT eTitan Arrays Plate Sample R Registered GeneTitan Array Plate Samples successfully Deionization Procedure for GeneTitan Trays and Covers Use the following technique to destatic GeneTitan MC Instrument stain trays and lids IMPORTANT Except for the HT array tray and the Hybridization Tray you must deionize all GeneTitan stain trays stain tray covers and scan tray covers using an anti static gun You must do this before you fill the trays with reagents and before you place the covers on the trays Deionization removes the static electricity The presence of static electricity on the underside of the cover can cause the gripper to lift the tray along with the tray cover and can result in an aborted run See Figure E 1 Figure E 2 and Figure E 3 Deionize the inner surface of each tray and lid The surface of the tray with the wells that will hold reagents The surface of the lid that will cover the reagents CAUTION Do not deionize the scan tray or hybridization tray Appendix E Deionization Procedure for GeneTitan Trays and Covers 131 Figure E 2 Stain Tray with Cover Deionize the cover and the tray MM TM Testing the Anti Static Gun Verify that the anti static gun P N 74 0014 Figure E 3 is in working condition You can use the protective cap on the gun to determine if the anti static gun is releasing ions The procedure is as follows Kee
70. ORTANT After the GeneTitan MC Instrument has stacked the array plate and hyb tray the instrument will extend the drawer Manually check the stacking by gently pressing the six latching points to confirm that the two parts are clamped properly and check underneath the arrays to make sure there are no bubbles If bubbles are found gently tap the plate on top and the bubbles should disappear Do NOT tip tilt the array plate hyb tray sandwich while inspecting the bottom for bubbles See Step 3 on page 84 for detailed instructions Hybridization will continue on the GeneTitan MC Instrument for 23 5 24 hours before you can load the Ligation Staining Stabilization reagent trays into the GeneTitan MC Instrument You must wait until the hybridization step on the GeneTitan MC Instrument is approximately 1 5 hours from completion 22 hours after the start of hybridization to begin Stage 5 Manually Preparing Ligation Staining and Stabilization Reagent Trays for the GeneTitan MC Instrument below Long delays between sample denaturation and loading into the GeneTitan MC Instrument for hybridization should be avoided However if denaturation has begun and the GeneTitan MC Instrument is found not to be ready for hybridization then n If the Hyb Ready samples have not been transferred to the hyb tray still in the Hyb Ready plate the Hyb Ready plate should be held at 48 C in the thermocycler until the GeneTitan MC Instrument is ready at which poin
71. Output Settings gt oS User Defined Excel Export zs Show Result Viewer r3 Create XML and dat data Files amp o Automatic Print out after measurement Save the protocol If Performing Sample Quantitation on a Plate Reader Other than the DTX880 Your plate reader should be calibrated to ensure accurate readings The total yield in ug per well can be calculated as A C FD V E P Where A the observed OD260 C the observed OD320 an estimate of a blank reading D 120 the net dilution factor when preparing the OD Sample plate as described in the Automated or Manual protocol V 115 the volume of the sample in uL after the resuspension step E 0 05 the extinction coefficient of duplex DNA at 260 nm P the optical path length for the plate type and plate reader used If your plate reader does not record the OD320 the OD260 of a blank solution of water only should be used for the parameter B above The optical path length is dependent on the type of plate and spectrophotometer used Check your manufacturer s recommendations for the path length for your instrument and plate type or for recommendations on how to measure this quantity The SpectraMax Plus384 described as an alternative spectrophotometer in the Axiom 2 0 Assay Manual Workflow Site Prep Guide P N 702991 can employ an automated path length detection system Consult this instrument s manual for more information The resulting yield cal
72. Prepare the Stabilization Master Mix 1 Use appropriate serological and single channel pipettes to add reagents to the 15 mL tube labeled Stbl in the order shown in Table 4 29 Table 4 29 Stabilization Master Mix Reagent Per Array Master Mix 96 To the tube marked Stbl add Axiom Water 93 19 uL 10 3 mL a Axiom Stabilize Diluent a Axiom Stabilize Soln 11 61 mL 2 Vortex the master mix at high speed for 3 sec 3 Place on ice Chapter 4 Axiom 2 0 Assay Manual Target Preparation 61 Prepare Ligation Master Mix The Ligation Master Mix is prepared in two stages Ligation Master Mix Stage 1 To Begin Preparing the Ligation Master Mix 1 Place the 15 mL conical tube marked Lig on ice 2 Use appropriate serological and single channel pipettes to add reagents to the 15 mL tube labeled Lig in the order shown in Table 4 30 Table 4 30 Ligation Master Mix Preparation Stage 1 Reagent Per Array Master Mix 96 To the tube marked Lig add a Axiom Ligate Buffer 66 15 uL 7 3mL a Axiom Ligate Soln 1 13 12 uL 1 45 mL Axiom Ligate Soln 2 3 15 uL 348 uL Sub Total 82 42 pL 9 10 mL 3 Mix well by vortexing the tube for 3 seconds 4 Place the tube marked Lig back on ice Ligation Master Mix Stage 2 To Finish Preparing the Ligation Master Mix 1 Remove the Axiom Ligation Enzyme from the 20 C freezer and place in a cooler chilled to 20 C 2
73. S9 located on the bottom left side of the system has either detected a leak is unpowered or requires adjustment Correct the problem before continuing No fluidic Process will be able to run until the problem is resolved This message reflects the current system state If the leak affects fluidic processing for a specific plate being processed that plate will display a similar message Call Affymetrix Technical Support for Service System serial number HT96Fluidic BETA002 Causes System malfunction a User killing the application using task manager during a fill operation resulting in application exit without stopping flow Solution Contact Affymetrix field support The system cannot be used for any fluidic processing until this is resolved Table 7 4 Problem messages Continued Chapter 7 Troubleshooting 112 Problem and Possible Causes Leak Resolved EI GeneTitan 3 1 2011 11 35 22 AM Fluidic system leak resolved The leak event was detected at 3 1 2011 11 33 41 AM and lasted until 3 1 2011 11 35 22 AM System serial number HT96Fluidic BETAO02 This message is displayed when the leak is resolved meaning the sensor LED is again lit up If the original leak detected message was not acknowledged it will be automatically removed from the GUI and replaced by the following message It will remain displayed until another leak is detected or the user acknowledges it by pressing OK To resolve t
74. Scanning Arrays Total Scanner Status wan Not Be scanned L1 Scan Completed Will Be Scanned KI Scan Failed Scanned System Ready ELLELE 2 The software will display a message that allows you to change your mind Figure F 12 Are you Sure Lamp Life Management Reset Counter for Lamp Life Remaining Are you sure Please make sure 1 You had replaced the lamp before resting the counter for lamp life remaining 2 The lamp is plugged into an active socket and 3 The lamp power switch is in the ON position 4 The scanner is not busy Failure to replace the lamp will cause poor quality data Failure to turn on the lamp will cause Scanner error Ce e 3 Click Yes if you want to reset the counter The software will display a message that confirms that the software has reset the counter Figure F 13 Appendix F GeneTitan Multi Channel Instrument Care 142 Figure F 13 The counter is reset Lamp Life Management x Lamp life remaining has been reset to 500 hours Troubleshooting Log Files This section provides instructions on how to identify and solve simple problems with the GeneTitan MC Instrument If a problem or error occurs that is not listed in this chapter contact a Affymetrix technical support for assistance For software errors that do not involve hardware crashes the most common solution is to shut down the application and
75. Stage 5 Manually Preparing Ligation Staining and Stabilization Reagent Trays for the Genhelittan MC INStrUMeNt dou eee oe des booed ee OR IR eee 55 Equipment Consumables and Reagents Required 0 000000 eee ee eee 55 1 Prepare the Reagents for Stage 5 n anaana naana 57 2 Prepare the Stain Ligation and Stabilization Master Mixes 000 59 3 Aliquot Master Mixes and Axiom Hold Buffer into Trays iliius 62 Chapter 5 Chapter 6 Chapter 7 Contents 5 Array Processing with the GeneTitan Multi Channel Instrument 67 Before Using the GeneTitan MC Instrument 0 00 0000000 eee 67 Proper Tray Alignment and Loading 0 00000 c eee ees 67 Stain Trays and COVES 5 csse enr ehr PPAR bE eee eae DUX T RIPE IEEE 69 E mail and Telephone Notifications from the GeneTitan MC Instrument 71 GeneTitan MC Instrument Lamp issslls e 71 Setup Options for Array Plate Processing 0 2 0 0 0 00 cece 72 Aborting d PIOCeSS 2a caseo e RO Rs d Ib RE a RR AUR S Reit d 74 Stage 1 Create and Upload Batch Registration File 0 0 0 0 0 00 cee eee 75 Stage 2 HybEIdIZatlOnts uud o Fees bu Uwe Pao eo ND e a ele ew A 76 Reagents RegulFed i3 ib bose rehia hated d dod dd ob bidedd a IER M d 76 Setup the Instrument esae ite T Seed edwedueeiteri t b YE E 76 Load an Axiom Array Plate and Hyb Tray Onto the GeneTitan MC Instrument 81 Load a Second Axiom Array Plate
76. T96Fluidic 0000000 4 Possible Causes Please verify that the facility CDA or the portable CDA compressor is in working condition Refer to the GeneTitan MC Site Prep Guide for the portable compressor model that has been validated with the GeneTitan MC instrument Contact your local field application specialist and notify the engineer about the error message Leak Detected Leak checks are performed at application startup and any time a fluidic process priming filling draining etc is performed The leak detection is a hard wired sensor which will shut off fluid flow without software control Leaks are normally confined to the drip pan located inside the system WI LEAK DETECTED Error processing 5500321212121212 while trying to process fluidic macro Fillw ashB Event detected at 9 8 2008 4 55 45 PM possible leak has been detected and valve power is disabled through a hardware interlock Software control of the valve system has been disabled Sensor 9 located on the bottom left side of the system has either detected a leak is unpowered or requires adjustment Correct the problem before continuing Select Retry to continue processing after the problem is resolved or Cancel to abort the process Cancel EI FE22 S9 Leak monitor 3 1 2011 11 33 41 AM possible leak has been detected and valve power is disabled through a hardware interlock Software control of the valve system has been disabled Sensor
77. Titan MC Instrument Chapter 4 Axiom 2 0 Assay Manual Target Preparation 63 Stain 1 Master Mix To Aliquot the Stain 1 Master Mix 1 Pour the S1 Master Mix into the solution basin marked S placed on the bench top at room temperature 2 Using a P200 12 channel pipette with new pipette tips aliquot 105 uL per well to both S7 trays dispense to the first stop only to avoid creating bubbles You do not need to change pipette tips between additions of the Stain 1 Master Mix 3 If Bubbles are present puncture them with a pipette tip Droplets of liquid splashed onto the well dividers place a Kimwipe on top of the tray to blot and remove Figure 4 1 Figure 4 1 Well Dividers in Trays Example of a droplet of liquid that has splashed onto the well divider of a stain tray during reagent aliquoting Ensure no droplets of liquid are on top of the wells dividers Blot with a Kimwipe to remove 4 Place covers on the 7 trays Orient cover correctly on the tray with the notched corners together Figure 4 2 Lu IMPORTANT Leaving liquid on the top of the dividers may cause excessive evaporation or may form a seal that will restrict the removal of the GeneTitan tray cover Figure 4 2 Placing Cover on Stain Tray Notched corner of Stain Tray and Lid Notched corner should face the front 5 Protect the trays from light if not immediately loading onto the GeneTitan MC In
78. a fume hood Duration Hands on 45 minutes including denaturation time a in GeneTitan MC Instrument 23 5 to 24 hours Hyb Time Required Input from Previous Stage Hyb Ready plate Equipment Consumables and Reagents Required The following thermal cyclers are recommended a BIO RAD PTC 200 or a Whatman Biometra TRobot or a BIO RAD DNA Engine Tetrad 2 PTC 0240 or ABI 9700 or ABI 2720 Ei IMPORTANT Always use the heated lid option when programming protocols The thermocycler needs to be programmed with the Axiom 2 0 Denature protocol see Thermal Cycler Recommendations on page 20 Chapter 4 Axiom 2 0 Assay Manual Target Preparation 51 Table 4 15 Equipment Required for Stage 4 Denaturation and Hybridization Equipment Quantity GeneTitan MC Instrument 1 Rainin P200 12 channel Pipette 1 Pipette tips As needed Thermal Cycler Appropriate thermal cycler programmed with the 1 Axiom 2 0 Denature protocol see Thermal Cycler Recommendations on page 20 96 well metal chamber 1 warmed in a 48 C oven The metal chamber coming out of a 48 C oven is warm to the touch Gloves and mitts can be used if it feels too hot Table 4 16 Consumables Required for Stage 4 Denaturation and Hybridization Consumable Vendor and Quantity Part Number One of the following Axiom array plates Affymetrix o One Axiom Genome Wide human or non human 96 array plate i
79. ace the front Label the hyb tray here CAUTION Writing on the wrong side of the hyb tray or on the wrong part of the long side may interfere with the operation of sensors in the GeneTitan MC Instrument Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 71 Labeling for Stain Trays You may label the stain trays on the left side of the front of the tray as shown in Figure 5 5 The correct side is closest to the notched corner corresponding to the Al through C1 wells Figure 5 5 Labeling GeneTitan Stain Tray Stain Tray shown with Lid Do NOT label trays on the long side of the tray Notched corner of the stain tray should face the front tabel th stain tray here E mail and Telephone Notifications from the GeneTitan MC Instrument We strongly recommend that you configure the Affymetrix GeneChip Command Console AGCC software to send you GeneTitan MC notifications It is critical that you know when the instrument requires your attention either for sample handling or troubleshooting Rapid notification can lessen the risk of sample loss Notifications can be sent to e mail addresses and telephones Refer to the AGCC user manual for instructions The types of notifications available will let you know when a process Starts Completes Aborts a Encounters an error GeneTitan MC Instrument Lamp The GeneTitan MC Instrument uses a xenon arc lamp system that i
80. ach addition mon f NOTE After adding the Amplification Master Mix the plate is now known as the Amplification plate G Seal tightly vortex twice and spin the Amplification plate for one minute at 1000 rpm as described in Seal Vortex and Spin on page 23 H Place the sealed amplification plate in an oven set at 37 C and leave undisturbed for 23 1 hr NOTE If using a GeneChip Hybridization Oven place the plate on the bottom of the oven Plates do not rotate Set the rotor for 15 rpm speed See Oven Recommendations on page 22 for more information 6 Freeze or Proceed After the incubation finishes you can either a Proceed to Stage 2 Fragmentation and Precipitation on page 38 a Store the amplification plate at 20 C NOTE If freezing do not perform the stop amplification reaction step before you store the Amplification plate at 20 C The Stop Amplification Reaction step will be performed after thawing the frozen plate as described in 1 Stop Amplification Reaction on page 40 Stage 2 Fragmentation and Precipitation The following sets of steps are necessary to perform fragmentation and precipitation 1 Stop Amplification Reaction on page 40 2 Prepare Fragmentation Master Mix on page 41 3 Add Fragmentation Master Mix to Wells on page 42 4 Aliquot the Stop Solution to the Fragmentation Plate on page 42 5 Prepare and Add Precipitation Master Mix on page 43 Duration Total time
81. ad 0240 or Biometra TRobot please follow the manufacturer s instructions for adjusting lid tension Thermal Cycler Consumables Table 3 1 provides details into the consumables to be used with each thermal cycler Table 3 1 Thermal Cycler Consumables for the Axiom 2 0 Assay Thermal Cycler Model PCR Plate Type Seal Bio Rad PTC 200 Bio Rad Hard Shell Thin Wall 96 Well MicroAmp Clear Adhesive Film from Skirted PCR Plates P N HSP9631 Applied Biosystems P N 4306311 TRobot Bio Rad Hard Shell Thin Wall 96 Well BioRad Arched Auto Sealing Lids with Skirted PCR Plates P N HSP9631 Wide Tabs P N MSL 2032 with BioRad Micro seal P Replacement Pads MSP 1003 ABI 9700 Bio Rad P N HSS 9601 half skirted plate MicroAmp Clear Adhesive Film from Applied Biosystems P N 4306311 ABI 2720 Bio Rad P N HSS 9601 half skirted plate MicroAmp Clear Adhesive Film from Applied Biosystems P N 4306311 Bio Rad Tetrad 2 PTC 0240 Bio Rad Hard Shell Thin Wall 96 Well MicroAmp Clear Adhesive Film from Skirted PCR Plates P N HSP9631 Applied Biosystems P N 4306311 Microseal B film from BioRad P N MSB 1001 may be used in place of MicroAmp Clear Adhesive Film for the BioRad and ABI thermal cyclers Chapter 3 Axiom 2 0 Assay Preparation Before You Start 22 Oven Recommendations The following ovens are recommended ED 53 drying oven by Binder Refer to the Axiom 2 0 Assay Manual Workflow Site Prep Guide P N 702991
82. age 2 Hybridization on page 76 example shown in Figure 5 8 This file contains information critical for a Data file generation during scanning Tracking the experimental results for each sample loaded onto an array plate 1 If you have not already created a batch registration file create one now See Appendix D Registering Samples in Affymetrix Gene Chip Command Console on page 127 for detailed instructions In AGCC select the array plate format 96 samples and open a batch registration file template Scan the array plate barcode into the yellow barcode field Enter a unique name for each sample and any additional information Save the file Upload the file gum E a IMPORTANT It is very important to create and upload a batch registration file with your sample information prior to starting Stage 2 Hybridization on page 76 Figure 5 8 Example of a Batch Registration File for an Array Plate Ca d ME GeneTitanArrayPlateRegistration 7 xls Compatibility Mode Microsoft Excel Home Insert Page Layout Formulas Data Review View Add Ins Acrobat S 4 Cut Arial 10 A wm m Siwrapt E M MI SEE zy Wrap Text z TE 4 Normal Bad 43 Copy aha J Fiy d Pastel A Format Painter PANE Aae EEE E Bi Merge amp Center 8 8 Poe o Clipboard fa Font Tal Alignment Number fa Styles G23 QX f mj A B C D E H 1 Sample File Path Project Plate Type Probe Array Type
83. and Hyb Tray Onto the GeneTitan MC Instrument 86 Status Window Prompts and Actions Required 00 00 ccc eee eee 87 Stage 3 Ligate Wash Stain and Scan 2 eee 89 Equipment Consumables and Reagents Required 00 00000 e cee eee 89 Proper Installation of the GeneTitan Tray Consumables 000 000000 90 Load Trays onto the GeneTitan MC Instrument 0 00000 ccc eee 91 CONTINUING thle Workflow persere oko bd ERR C EIU Ue ed edd 98 Storing Hyb Trays for Rehybridization lille 98 Shutting Down the GeneTitan MC Instrument llslisssle ee 99 Manual Target Preparation for Processing Three Axiom Array Plates Del Week iL cuiua ue S eR qe arde P EROR ER PCR Oa ond d oR Oe 100 Overview of the 3 Plate Workflow for Manual Target Preparation 100 Timing Issues for Manual Target Preparation 0 000 000 liess 101 Timing Issues for GeneTitan MC Array Processing 000 00 eee eee eee eee 102 Changing Oven Temperatures for the Three Plate Workflow 005 102 Thawing Frozen Plates of Amplified DNA 0 0000000 cee 103 Manual Target Prep and Array Processing 0000 cece eee eee eee 103 Manual Target Prep Workflow Day 1 0 00000 eee 103 Manual Target Prep Workflow Day 2 isssssslss ses 104 Manual Target Prep Workflow Day 3 ssssslsss ees 105 Manual Target Prep Workflow Day 4 0 00000 se 106
84. as not yet been started For certain steps you can enter a duration in seconds even if the step requires gt 1 hr to run you must enter the duration in seconds You can set a step for less time than normal but not for longer than the normal duration Aborting a Run Abort can take up to three minutes if a plate is in the Fluidics station Status window Abort Requested changes to Abort Completed Clamped Array Plate Hyb Tray sandwich aborted from the oven or from drawerIN are moved to drawer 1 Proceed as follows o Use the Unload Plates option to remove the aborted plate s o Start another run which will force an unload of the aborted plate s System initiated Power interruption a Plate loaded incorrectly Equipment malfunction The system will abort the processing Follow the instructions displayed in the user interface User initiated Can abort processing of individual array plates If multiple plates are being processed the gripper may continue to process the remaining array plates Fragmentation Quality Control Gel Protocol Protocol for Running a Fragmentation Quality Control Gel Equipment Required Table A 1 Equipment required Item Supplier Part Number Gel imager Your choice Pipette multi or single channel P20 Your choice Plate centrifuge Your choice Vortex E Gels and Reagents Table A 2 E Gel and reagents required Your choice
85. ate gently to avoid disturbing the pellets Do not bump or bang the plate To Centrifuge and Dry the Pellets 1 Turn the oven on and preheat to 37 C If using an Affymetrix GeneChip Hyb Oven set the rotation speed to 15 rpm to distribute heat Begin thawing warming the reagents used in this stage as shown in Table 4 13 on page 46 Remove the Precipitation plate from the 20 C freezer and centrifuge the plate for 40 min at 4 C at 3200 xg 4000 RPM for the Eppendorf 5810R centrifuge with the rotor configuration described in the Axiom 2 0 Assay Automated Workflow Site Prep Guide NOTE WARNING Use rotor buckets with a soft rubber bottom to ensure that the deep well plates do not crack Do not use buckets where the plates sit directly on a metal or hard plastic bottom such as the A 4 62 rotor with a WO 15 plate carrier hard bottom for the Eppendorf 5810R centrifuge Use of hard bottom plate carriers may result in cracked plates loss of sample unbalanced centrifugation damage to the instrument and possible physical injury f NOTE If you are processing two plates at the same time as in the three plate week manual prep workflow you can centrifuge both plates at the same time During the centrifugation time prepare the resuspension and hybridization reagents as shown in Table 4 13 Table 4 13 Reagent Preparation for Resuspension and Hybridization Reagent Treatment Axiom Resuspension Buffer Warm
86. ates on the GeneTitan MC Instrument B C GeneTitan reagent trays preparation and loading aA 5 GeneTitan reagent trays preparation and loading a B C The timing of these steps is critical because of constraints on both the target preparation done on the lab bench and the array processing done using the GeneTitan MC Instrument These constraints are described in more detail in a Timing Issues for Manual Target Preparation on page 101 a Timing Issues for GeneTitan MC Array Processing on page 102 Timing Issues for Manual Target Preparation The GeneTitan reagent trays for array processing cannot be loaded until the array plate has finished hybridization and they should not be prepared more than 1 5 hours before hybridization will finish The GeneTitan reagent trays cannot be prepared ahead of time and stored Table 6 2 Time Required for Manual Target Preparation Manual Preparation Hands on Time Total Prep Incubation Hybridization Required Time Processing Amplification 0 5 hr 1 5 hr 23 1 hr Fragmentation and Precipitation prep 2hr 2hr Overnight Precipitation Re suspension and Hybridization Mix prep 25 min 25 min N A Centrifugation Drying 30 min 1 hr 20 min N A QC gel and OD 45 min 45 min N A Denaturation and hyb tray array plate 25 min 45 23 5 24 hr hybridization loading on the GeneTitan MC Instrument GeneTitan reagent tray preparation and 1 hr 1 5 hr Additional time for processing
87. ble inthe correct loaded improperly ORIENTATION The bar code is missing Details or obscured The consumable is either not the correct consumable not loaded correctly or its barcode is not readable Proceeding with an incorrect or incorrectly loaded consumable can result in a loss of consumables loss of samples and may require a field service engineer to service the instrument Refer to the System Setup Tab or the User Guide provided with the Assay or AGCC for instructions on proper consumable placement Press the flashing blue confirmation button or Press OK The GeneTitan MC Instrument will verify the barcode and orientation Press Skip The GeneTitan MC Instrument will NOT verify the barcode and orientation The barcode entered at registration will be used Table 5 5 Selecting Which Arrays to Scan Status Window Prompt Action Required Reagent and Receptacle Select arrays to scan a Accept the default all arrays selected if appropriate Otherwise select the arrays to be scanned a Click Next then click OK to start processing Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 89 Stage 3 Ligate Wash Stain and Scan Equipment Consumables and Reagents Required Scan Tray with Axiom Hold Buffer Cover the tray by orienting the notched corner of the cover over the notched edge of the tray and leave on the bench top no need to protect from light Figure 5 26
88. cause the GeneTitan MC Instrument to crash and can result in substantial damage to the instrument and loss of samples E Press the Confirmation button re 5 16 Barcode Error Message B Verify Drawer 6 Array Plate Load Warming The system was not able to verify the array plate barcode Please verify that the tray on the left side of the drawer is a blue cover and if applicable an array plate in the corect ORIENTATION The right side of the drawer should contain a hyb tray if applicable in the correct ORIENTATION Details The consumable is either not the correct consumable not loaded correctly or its barcode is not readable Proceeding with an incorrect or incorrectly loaded consumable can result in a loss of consumables loss of samples and may require a field service engineer to service the instrument Refer to the System Setup Tab or the User Guide provided with the Assay or AGCC for instructions on proper consumable placement Press the flashing blue confirmation button or Press OK GeneTitan will verify the barcode and orientation Press Skip GeneTitan will NOT verify the barcode and orientation The barcode entered at registration will be used Cx s When you load the array plate left side of the drawer The internal bar code reader reads the barcode of the array plate and compares it with the barcode and the plate type specified in the Barcode field and Plate Type field on the Setup page
89. ck duration 1 min Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 88 Table 5 3 Emptying the Trash Bin Status Window Prompt Action Required Receptacle Reagent Empty trash bin Open and empty the trash bin Press the Confirmation button to continue NOTE If the trash bin is empty you will not be able to open it Continue the process by pressing the blue confirmation button Table 5 4 Loading the Array Plate and Hyb Tray Barcode Error Messages Status Window Prompt Action Required Reagent Receptacle Load Array Plate Tray on Left Load the array plate with the blue base and the hyb tray in a Hyb tray loaded with Right side of Drawer Loadhyb drawer 6 denatured samples tray without cover on Left a IMPORTANT The blue base must remain in left side HTA in Right side of Drawer even when empty IMPORTANT The trays must be positioned If the trays are placed incorrectly the software will display an error dialog box indicating the barcode could not be read Press the Confirmation button to continue Text version of the error message These messages are Warning The system was not able to verify the array plate barcode displayed if Please verify that the tray on the left side of the drawer is a blue cover and if applicable an array plate in Plate has been the correct ORIENTATION The right side of the drawer should contain a hyb tray if applica
90. culations can be compared against the typical yields shown in column H of Figure B 2 on page 117 and against OD Yield Assessment Guidelines on page 117 Rehybridization Protocol for Rehybridizing Samples Rehybridization The target prep rehybridization protocol is used to help identify potential sources of problem in sample failures You may occasionally encounter sub optimal performance resulting in sample failures with the Axiom Genotyping System Failures that cannot be attributed to instrumentation issues may be due to either sample quality and or reagent and array issues To help isolate the potential cause of the problem you may choose to perform a target prep re hybridization protocol This is a protocol by which customers can re process previously used hybridization cocktails that has been stored at 20 C in combination with a new array The results of this re hybridization can indicate if the original sub optimal performance was due to issues associated with the array array processing the DNA target prep or reagent quality Table C 1 shows conclusions that can be drawn from the results of the re hybridization of Axiom 2 0 Assay hybridization cocktail to the Axiom Genome Wide Arrays Table C 1 Conclusions that can be drawn from the results of the re hybridization of Axiom Genotyping Assay hybridization cocktail to the Axiom Genome Wide Arrays Results of Re hybridization Details Conclusion Pass Significan
91. d It is safe to change the filters in all three fluidic bottles when this message is displayed After changing the filters in all three bottles using the procedure described below please press the Yes button to continue If you choose to ignore the error message press the No button This warning message will be displayed each time AGCC instrument control software is launched You may also experience data quality issues if particulate matter cannot be trapped by the filters if they are clogged Appendix F GeneTitan Multi Channel Instrument Care 136 We recommend that your site keep three spare filters in the event they need to be replaced The procedure for replacing the filters is simple Part details Affymetrix P N 01 0671 Figure F 3 Replacing the Filter Buffer Supply Tube Filter Holder Filter Removing and inspecting the Filter 1 Loosen and remove the cap on the bottle 2 Carefully remove the filter from the end of the filter body 3 Visually inspect the filter If one of the filters appears to have a concentration of dirt or contaminate in it discard it and replace the filter with a new one Replacing the Filter 1 Insert the filter into the end of the filter body 2 Replace the cap onto the bottle and tighten it 3 Repeat for each bottle bu IMPORTANT Replace one filter at a time to ensure the correct connection of the buffer supply tube to its respective bottle The color of the buf
92. d be replaced if the software displays such a warning Refer to Appendix F GeneTitan Multi Channel Instrument Care on page 133 for the message displayed to the user and the procedure for replacing the filters a Scan The array plate is moved to the imaging device in the GeneTitan MC Instrument and each array is scanned o Duration for 96 samples 7 5 hr Hyb Wash If this setup option is selected array plate processing will stop after the array has gone through fluidics processing Use this option if an array plate cannot be scanned on the same GeneTitan MC Instrument as the one used for hybridization and fluidics processing If the Array Plate Cannot Be Scanned Immediately After the Hyb Wash Process is Complete 1 Wrap the array plate in the scan tray with black protective base in aluminum foil to protect from light No lid is required Do not invert the plate stack If inverted the Hold Buffer will spill out of the tray To prevent liquid spillage try to keep the plate level when handling the plates Do not touch the bottom optical surface of the scan tray 2 Store at 4 C 3 Scan the array plate within 3 days or less When Ready to Scan the Array Plate 1 Keeping the plate protected from light bring the plate to room temperature for 20 min 2 Remove the aluminum foil and load onto the GeneTitan MC Instrument Wash Scan Use this option if You wish to bypass the Hybridization step and perform only the Wash
93. d scan trays must be placed in the GeneTitan MC Instrument with the GeneTitan stain tray cover All tray covers must be de ionized to remove static electricity prior to placing the cover on the tray See Appendix E Deionization Procedure for GeneTitan Trays and Covers on page 130 for the anti static procedure GeneTitan Tray 501025 Stain Tray Cover 202757 shown with the Stain Tray cover The GeneTitan stain trays must be placed in the GeneTitan MC Instrument with the GeneTitan stain tray cover It is important to remove the static electricity on the cover and the stain tray prior to loading the tray into the GeneTitan MC Instrument See Appendix E Deionization Procedure for GeneTitan Trays and Covers on page 130 for the anti static procedure GeneTitan 500867 Hyb Tray The GeneTitan hybridization trays are packaged in white pouches with the label HT Hybridization Tray After aliquoting the hyb cocktail into the hybridization tray the tray should be loaded into the GeneTitan Instrument with the barcode facing away from the operator i e barcode should be on the back side Chapter 3 Axiom 2 0 Assay Preparation Before You Start 30 Labeling GeneTitan Hybridization and Reagent Trays When preparing the hybridization and reagent trays to be loaded onto the GeneTitan MC Instrument you will need to mark each tray in a way that identifies its contents Lu IMPORTANT It is crit
94. ddition Proceed immediately to the next step 5 Seal and vortex and do a quick spin at 1000 rpm 6 Leave the Fragmentation plate on the benchtop while you prepare the Precipitation Master Mix 5 Prepare and Add Precipitation Master Mix To Prepare and Add Precipitation Master Mix carry out the following steps at room temperature 1 Prepare Precipitation Master Mix in the 50 mL conical tube labelled Precip MM Table 4 10 Precipitation Master Mix Reagent per sample Master Mix 96 To the 50 mL tube marked Precip MM add Axiom Precip Soln 1 238 uL 26 mL Axiom Precip Soln 2 2 uL 218 uL Total Volume 240 pL 26 22 mL NOTE Use a 5 or 10 mL serological pipette to pipette Axiom Precip Soln 1 The bottle has a narrow opening and a 25 mL serological pipette will not fit through the mouth of the bottle Vortex the Precip MM tube and place on benchtop at room temperature Pour the Precipitation Master Mix into the solution basin labeled Precip MM Carefully remove the seal from the Fragmentation plate and discard the seal Ur S Woo Using a P1200 12 channel pipette add 240 uL Precipitation Master Mix to each sample Rest each pipette tip against the wall of each well while delivering You do not need to mix up and down Change tips after each addition NOTE After adding the Precipitation Master Mix the plate is now known as the Precipitation plate Seal the Precipitation plate and vortex Spin Rem
95. described under Setup Options for Array Plate Processing on page 72 Click Next NOTE If there is not enough disk space a message is displayed Delete or move dat files to another location to free up enough disk space for the data that will be generated by eight Axiom array plates 96 Axiom array plate requires 80 GB Plate Information Barcode Scan or manually enter the Axiom array plate barcode and click Next Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 79 The first six characters of the barcode identify the type of plate being loaded the protocol GeneTitan MC Instrument will use to process the plate and the imaging device parameters required for this type of plate 550094 lt barcode gt Affymetrix 96 array plate Figure 5 11 Barcode Error Message It this error message is displayed a Ensure that the library files for the type of array plate you are Cannot find the protocol file For the entered barcode Please make sure the protocol files are properly installed and or using are correctly installed eae ae me ae Try manually entering the array plate barcode a Library files must be installed prior to launching the GeneTitan MC Instrument If a library file must be installed exit the GeneTitan Instrument install libraries and relaunch Array Registration Protocol Name Select the protocol name and click Next The system reads the first 6 di
96. e and Hyb Tray Onto the GeneTitan MC Instrument on page 81 Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 81 Load an Axiom Array Plate and Hyb Tray Onto the GeneTitan MC Instrument The System Layout pane indicates the position of the various trays in each drawer during a GeneTitan MC Instrument run at maximum throughput This pane does not change as plates are loaded or removed Figure 5 13 System Layout Location of Plates Inside the GeneTitan MC Instrument Drawer Numbers System Layout Used Hyb Tray Used Hyb Tray 1 Scan Tray Scan Tray 2 Stain 1 Tray Ligation Tray 3 Stain 2 Tray Stabilizing Tray 4 NOTE Earlier versions of the software may show as Fix Tray rather than Stain 1 Tray 5 Stabilizing Tray Array Plate Hyb Tray I Trash Bin To Load an Axiom Array Plate and Hyb Tray onto GeneTitan MC Instrument 1 When drawer 6 opens load the array plate and hyb tray as follows A Examine the wells of the hyb tray for bubbles puncture any bubbles with a pipette tip bu IMPORTANT Removing bubbles at this step greatly reduces the chance of bubbles under the arrays when the hyb tray and the Axiom array plates are clamped Bubbles under an array can result in black spots on the array image B Load the hyb tray without the cover on the right side of the drawer Figure 5 15 on page 82 The array p
97. e laboratories at Affymetrix for DNA that meets the requirements below Blood Semen Nasal swab a Hair bulbs a Ear punch tissue x NOTE DNA derived from Formalin Fixed Paraffin Embedded FFPE blocks should not be used with this assay Chapter 2 Genomic DNA Preparation and Requirements 12 General Requirements a Starting DNA must be double stranded for the purpose of accurate concentration determination a DNA must be of high purity DNA should be free of DNA polymerase inhibitors Examples of inhibitors include high concentrations of heme from blood and high concentrations of chelating agents 1 e EDTA The gDNA extraction purification method should render DNA that is generally salt free because high concentrations of particular salts can also inhibit enzyme reactions DNA purity is indicated by OD260 OD2s0 and OD260 OD soratios The OD260 OD280 ratio should be between 1 8 and 2 0 and the OD260 ODz230 ratio should be greater than 1 5 We recommend that DNA samples that do not meet these criteria be cleaned up as described under Genomic DNA Cleanup on page 13 DNA must not be degraded The approximate average size of gDNA may be assessed on a 1 agarose gel using an appropriate size standard control Approximately 90 of the DNA must be greater than 10 Kb in size Control DNA can be run on the same gel for side by side comparison Special Requirements Pre Amplification Area Precautions are required when
98. e repeated B Do NOT stop to measure volumes proceed without delay Complete the 10 minute incubation on the benchtop at room temperature While completing the incubation at room temperature pour the Neutralization Soln into the solution basin as described in Step 1 on page 37 After incubation immediately add the Neutralization Soln as described in 4 Add Neutralization Solution to Samples on page 37 4 Add Neutralization Solution to Samples To Add the Neutralization Master Mix to Your Samples carry out the following steps at room temperature 1 2 3 mom D Pour the Axiom 2 0 Neutral Soln into the solution basin marked N Soln Carefully remove the seal from the Denaturation plate and discard the seal Using a P200 12 channel pipette pipetting down the wall of each well add 130 uL of Axiom 2 0 Neutral Soln to each sample total volume 170 uL well Change tips between each addition The plate is now known as the Neutralization Plate Seal vortex and spin the Neutralization plate Visually examine the volume in each well should be 170 n L well and A Keep a record of any wells that visually appear to have a particularly low or high volume these samples may need to be repeated B Do NOT stop to measure volumes Proceed immediately to 5 Prepare and Add the Amplification Master Mix on page 37 5 Prepare and Add the Amplification Master Mix To Prepare and Add the Amplification Master Mix carry out the
99. eady Clamping Points on Axiom Array Plate and Hyb Tray Array Plate EL Hyb Tray Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 85 A Remove the plate stack and gently press the two plates together at each clamping point Listen for a clicking sound which indicates that the plates are now clamped No clicking sound indicates the plates are already clamped See Figure 5 21 for an example of a array plate hybridization tray sandwich Figure 5 21 Array Plate Hyb Sandwich B Inspect the bottom of the plate stack for bubbles under the arrays do NOT invert the plates C If bubbles are present gently tap the plate until the bubbles move out from under the arrays do NOT unclamp the plate stack D Return the plate stack to the drawer and press the Confirmation button to proceed The message in Figure 5 22 may be displayed again if plate orientation is incorrect or if the hyb tray barcode cannot be read Click OK to proceed Figure 5 22 Verification Message Gl verify Drawer 6 Load 15 x Warning The system was not able to verify the GeneTitan Consumable Tray using the barcode on the Tray Please verify that the tray on the left side of the drawer is a blue cover and if applicable an array plate in the correct ORIENTATION The right side of the drawer should contain a hyb tray if applicable in the correct ORIENTATION Details
100. eave the gDNA sample plate at room temperature if proceeding immediately to DNA Amplification 5 Create a Batch Registration File Ls IMPORTANT It is very important to create and upload a GeneTitan Array Plate Registration file with your sample information prior to loading the array plate and hyb tray in the GeneTitan Instrument We recommend that you create but not upload this file at the same time you prepare your plate of genomic DNA When your samples are ready for hybridization you will scan the array plate barcode and upload the file to Affymetrix GeneChip Command Console AGCC GeneTitan Array Plate Registration files contain information that is critical for a Data file generation during imaging Tracking the experimental results for each sample loaded onto an array plate Detailed instructions for creating this file are located in Appendix D Registering Samples in Affymetrix GeneChip Command Console on page 127 See also Figure 2 2 for a screen shot showing an example of a batch registration file 1 Open AGCC Portal Samples and select A GeneTitan Array Plate Registration B The array plate format C Click Download 2 Enter a unique name for each sample and any additional information Save the file Chapter 2 Genomic DNA Preparation and Requirements 17 The array plate barcode will not be scanned until you are ready to load the array plate and samples onto the GeneTitan MC Instrument for processing
101. efore setting up hybridization on the GeneTitan MC Instrument A Leave the array plate in the pouch at room temperature for a minimum of 25 minutes before opening and loading on the GeneTitan MC Instrument to allow the plate to come to room temperature B At the end of the array warm up time open the pouch and scan the array plate barcode into the Batch Registration file see Stage 1 Create and Upload Batch Registration File on page 75 Make sure the thermal cycler is powered on and the Axiom 2 0 Denature program with the heated lid option has been selected WARNING Do not remove the array plate from the protective base or touch the surface of any arrays Open the lid of the thermal cycler and place the sealed Hyb Ready plate on the thermal cycler Check the integrity of the seal as evaporation during denaturation can negatively impact assay performance Close the lid For thermal cyclers with variable lid tension such as the Bio Rad PTC 200 or the BIO RAD DNA Engine Tetrad 2 PTC 0240 follow manufacturer s instructions for adjusting lid tension Start the Axiom 2 0 Denature program described on Thermal Cycler Recommendations on page 20 While the program is running A Prepare the reagents from Module 3 as described in Table 4 18 Table 4 18 Reagents from Module 3 P N 901472 Temp Out of Module Treatment Reagent Axiom Wash Buffer A Axiom Wash Buffer B Axiom Water Notes Temp Ou
102. en to the 65 C oven and incubate for 20 minutes If working with a thawed plate place the thawed Amplification plate in the 65 C oven and incubate for 20 minutes 2 Prepare reagents as shown in Table 4 8 at the start of the 65 C incubation of the amplification plate NOTE Leave the Axiom Frag Enzyme at 20 C until ready to use Table 4 8 Reagent Preparation for Fragmentation and Precipitation Reagent Treatment Axiom 10X Frag Buffer Thaw vortex and keep on ice Axiom Frag Diluent Thaw vortex spin and keep on ice Axiom Frag Enzyme Flick tube 3X spin and keep in 20 C cooler until ready to use Axiom Frag Rxn Stop Thaw vortex and keep at room temperature Axiom Precip Soln 1 Thaw vortex and keep at room temperature Axiom Precip Soln 2 Thaw vortex spin and keep at room temperature Isopropanol Keep at room temperature Chapter 4 Axiom 2 0 Assay Manual Target Preparation 41 3 Optional Remove samples for quantifying amplification yield by the PicoGreen Assay A Carefully remove the seal from the Amplification plate and discard the seal B Transfer 4 uL samples from each well to a 96 well PCR plate such as a Bio Rad Hard Shell 96 well plate HSP 9631 and set aside for later quantitation e g using the Quant iT PicoGreen dsDNA Kit from Life Technologies C Reseal the Amplification plate and continue with the Stop Amplification Step Transfer the Amplification plate f
103. ents 15 Reagents The reagents listed in Table 2 3 are required for this stage Table 2 3 Reagents Required for Genomic DNA Preparation Reagent Supplier Part Number From the Axiom 2 0 Reagent Kit a Axiom Reference Genomic DNA 103 use as a positive control Located in Module 1 20 C User supplied Reduced EDTA TE Buffer 10 mM Tris HCl pH 8 0 0 1 mM EDTA Affymetrix 75793 1 Thaw Samples and Control Thaw the components listed below to room temperature gDNA samples Axiom Reference Genomic DNA 103 from the Axiom 2 0 Reagent Kit To Thaw either a Place items on benchtop for one hour Thaw in a water bath A Fill a small plastic dish with Millipore water Do not overfill as the level of the water should not overflow when the sample tubes or plates are placed in the bath B Thaw the sealed sample plate and Reference sample for a half hour C Wipe offthe sample plate after removing and before removing the lid to minimize the chances that the water will enter the well and cause contamination or reaction failure 2 Quantitate and Dilute gDNA To Quantitate and Dilute the gDNA 1 Gently vortex 50 maximum and spin the gDNA and Reference Genomic DNA 103 2 Recommendation quantitate each sample e g using the Quant iT PicoGreen dsDNA Kit 3 Dilute each sample to a concentration of either 10 ng for all Axiom arrays except the Axiom Genome Wide Pan African Array Set or 5 ng
104. enzyme are now thawed and kept at room temperature a There are new recipes for Denaturation Master Mix and Amplification Master Mix for Axiom 2 0 Assay The Neutral Soln no longer requires dilution before use a Amplification master mix is now made and added to the plate at room temperature The new incubation time for the Denaturation Plate is 10 minutes The new oven temperature for Amplification Plate incubation is 37 C Chapter 3 Axiom 2 0 Assay Preparation Before You Start 19 Fragmentation and Precipitation Stage Changes There is a new recipe for Fragmentation Master Mix preparation for Axiom 2 0 Assay Regents for Precipitation Master Mix preparation but not Fragmentation Master Mix preparation are now handled at room temperature Drying Resuspension and QC Stage Changes Reagents for Hybridization Master Mix preparation are now handled at room temperature a There is a new recipe for Hybridization Master Mix for Axiom 2 0 Assay Changes to the QC steps are o The observed OD260 yields from in process QC are higher It is recommended to consider troubleshooting if median yield of a plate is lt 1000 ug o QC gel samples are diluted more Denaturation and Hybridization Stage Changes The new Axiom 2 0 Denature thermal cycler protocol for denaturation of the hyb ready samples is 95 C for 10min 48 C for 3 min and 48 C hold Requirements and Recommendations This section describes r
105. equirements and recommendations for facilities and equipment needed to perform the Axiom 2 0 Assay with manual target preparation Room Temperature When referred to in the Axiom 2 0 Assay room temperature is 18 to 25 C Special Requirements Amplification Staging Area Precautions are required when setting up amplification reactions to avoid contamination with foreign DNA amplified in other reactions and procedures It is recommended that amplification reaction set up is performed in a dedicated amplification staging area separate from the main laboratory This amplification staging area should have a dedicated set of pipettes and plasticware If no dedicated amplification staging area is available use of a dedicated bench or a dedicated biosafety hood and dedicated pipettes is suggested If no dedicated bench or biosafety hood is available a set of dedicated pipettes is recommended Fume Hood At certain steps in the protocol we recommend the use of adequate local or general ventilation to keep airborne concentrations low A fume hood is suggested as a way to achieve the desired concentration Thus a fume hood is strongly recommended for several steps of this assay Chapter 3 Axiom 2 0 Assay Preparation Before You Start 20 Safety Warnings and Precautions CAUTION All chemicals should be considered as potentially hazardous We therefore recommend that this product is handled only by those persons who have been trained i
106. er Change Required is displayed along with the information on the specific dispense operation You should change all three filters when a warning is displayed for any one of the three filters Refer to the section Replacing the Filter on page 136 in Appendix F Chapter 7 Troubleshooting 113 Wash Scan Resume If a run is aborted during fluidics processing the instrument will place the aborted array plate into the scan tray To restart this process remove the Axiom array plate from the scan tray and place it in its protective blue base The step at which the run was aborted can be identified by Viewing the System Status window if you are aborting the last plate through the fluidics system a Initiating the resume process 1 System Setup tab Select Wash Scan Resume 2 Follow the prompts to unload and reload all drawers The trays will be loaded It is up to you to determine whether or not to load fresh reagents or reuse the trays already in the GeneTitan Multichannel Instrument Base your decision upon the step where the problem occurred To Help Ensure that the Samples are Processed Correctly We Recommend that You 1 Load new stain trays with fresh reagents 2 Load a new scan tray We do not recommend the use of trays without reagents or holding buffers for steps that appear to have already executed Resume Step You must select the step at which you wish to resume plate processing You can select any step that h
107. erature x Day 211 AM Freeze Plate C y Day3 Noon start plate C thawing z Day4 5 00 PM Coupled operations on GeneTitan MC Instrument Load reagents for plate A and hyb tray and array plate for Plate C Color Code Amplification Fragmentation and Precipitation Centrifugation Drying Resuspension Hybridization Mix Prep and QC Sample Denature load array plate and hyb tray into the GeneTitan MC Instrument GeneTitan MC Instrument reagent trays prep and loading The three plates are referred to as Plates A B and C in the manual target prep and in the GeneTitan Array Processing Chapter 6 Manual Target Preparation for Processing Three Axiom Array Plates per Week 101 In order to process three plates during a 40 hour week the steps must be performed in the order and with the timing described in this chapter Table 6 1 Daily Steps for Manual Target Prep Workflow Day Activities Plates 1 Amplify 3 plates of genomic DNA A B and C 2 Fragment and precipitate two plates amplified on Day 1 a A B Freeze one plate of amplified DNA for fragmentation later in the week aC 3 a Fragment and precipitate one plate aC a Centrifuge dry resuspend and QC two plates precipitated on Day 2 a A B a Denature and begin hybridization for one plate on the GeneTitan MC Instrument A 4 a Centrifuge dry resuspend and QC plates precipitated on Day 3 aC a Denature and begin Hybridization for two pl
108. erile multichannel Chapter 4 Axiom 2 0 Assay Manual Target Preparation 35 Reagents Required Table 4 2 Reagents Required for Stage 1 DNA Amplification Axiom 2 0 Reagent Kit Module Axiom 2 0 Denat Soln 10X Axiom 2 0 Neutral Soln Module 1 20 C P N 901711 Axiom 2 0 Amp Soln Axiom Water Axiom 2 0 Amp Enzyme 1 Initial Setup for DNA Amplification To Perform the Initial Setup 1 Set an incubator oven temperature at 37 C 2 Set the centrifuge temp to room temperature 3 Prepare reagents as shown in Table 4 3 NOTE Leave the Axiom Amp Enzyme at 20 C until ready to use Table 4 3 Initial Preparation of Reagents for Amplification Reagent Treatment Axiom 2 0 Denat Soln 10X Thaw vortex spin and keep at room temperature Axiom 2 0 Neutral Soln Thaw see Note below vortex and keep at room temperature Axiom 2 0 Amp Soln Thaw see Note below vortex and keep at room temperature Axiom Water Thaw see Note below vortex and keep at room temperature Axiom 2 0 Amp Enzyme Flick tube 3X spin and keep in 20 C cooler until ready to use NOTE Allow 1 hour for Axiom 2 0 Amp Soln to thaw on the benchtop at room temperature If the solution is not completely thawed after 1 hour vortex briefly and return to the benchtop to complete thawing The bottles can also be thawed in a dish with Millipore water The Axiom 2 0 Amp Soln must be t
109. fer supply tubing matches the bottle color code Appendix F GeneTitan Multi Channel Instrument Care 137 Replacing the Xenon Lamp in the GeneTitan MC Instrument This section applies to your site only if you have the GeneTitan Multi Channel MC instrument After the normal life expectancy of the lamp has expired the software application will alert you to the requirement to replace the lamp This procedure is simple but you must follow good health and safety precautions Affymetrix Part Number 01 0740 Lu IMPORTANT Please DO NOT try to replace the lamp when a plate is being processed either in the fluidics or scanner system Lamp Life Imaging Device Status Notices The Imaging Status pane displays lamp life and Imaging Device status notices for the GeneTitan MC In normal operation the pane displays the hours of life left in the lamp Figure F 4 Lamp Life above tolerance Imaging Device Status Barcode Estimated Time Remaining Lamp Life Remaining 166 hours It displays a red or yellow notice when the lamp life is getting short Figure F 5 Lamp Life above tolerance Imaging Device Status Barcode Estimated Time Remaining It also displays a red notice when the Imaging Device is offline Figure F 6 Imaging Device Offline Imaging Device Status Barcode Estimated Time Remaining NOTE The 300 Watt Xenon lamp in the GeneTitan MC instrument is war
110. function Plate in Fluidics use Wash Scan Resume to resume the fluidics process Do not manually or through the AGCC transfer utility move any data associated with the current plate that is being processed scanned Hybridization aborted a System initiated abort a User initiated abort System initiated abort Power loss Array plate and hyb tray are still clamped Contact your local field service engineer with information on the workstation model The plate stack is moved to drawer 1 Remove the plate stack and finish hybridization offline a Return the hybridized array plate to the GeneTitan Multichannel Instrument and finish processing using the Wash Scan process FAILED messages See Failed Messages on page 110 FLUIDIC DIAGNOSTIC messages See Fluidic Diagnostic Messages on page 110 Fluidics aborted System initiated abort a User initiated abort System initiated abort Power loss User initiated abort a Incorrect protocol selected Follow the recommendations and instructions under Wash Scan Resume on page 113 Chapter 7 Troubleshooting 109 Miscellaneous Messages Table 7 2 Miscellaneous messages and recommended actions Message and Recommended Action Recommendation click Yes Homing recovery of gripped item If you click No nothing will occur Homing will not complete and you not be able to use the system The item held by the gripper will be moved to ei
111. g immediately to the next stage 2 Prepare the Tubes Basins and Trays for Resuspension and Hyb Master Mix Preparation To Prepare the Tubes Basins and Trays Used in the Procedure 1 Label the 15 mL tube as indicated in the table below Label Tube Size Temperature Contents a Hyb MM 15 mL Room Temperature in Fume Hood Hybridization Master Mix 2 Label solution basins as indicated in the table below Label Temperature Contents a Resus Room Temperature Axiom Resusp Buffer a Hyb MM Room Temperature in Fume Hood Hybridization Master Mix 3 If performing the recommended QC checks label solution basins as indicated in the table below Label Temperature Contents a NF H2O Leave basin at room temperature Nuclease Free Water a Loading Dye Leave basin at room temperature Diluted Loading dye 3 Resuspension and Hybridization Master Mix Preparation NOTE If a plate was stored at 20 C after drying the pellets it is recommended to allow the plate to sit at room temperature for 1 5 hour before carrying out resuspension NOTE Make sure the Axiom Resusp Buffer has equilibrated to room temperature before adding to dry pellets in Step 3 below To Resuspend the Pellets carry out the following steps at room temperature 1 Pour Axiom Resusp Buffer in the solution basin labeled Resus NOTE If you are processing two plates at the same time as in the three plate week manual pre
112. gits of the array plate barcode to determine which protocols can be run for the type of array plate that has been loaded Only valid protocols are displayed 550094 protocol for Affymetrix 96 array plate barcodes 4 Complete the remaining workflow steps as follows A Refill bottles with buffer Figure 5 12 on page 80 1 Fill these bottles a Wash A fill with Axiom Wash Buffer A keep at 2 L full Wash B fill with Axiom Wash Buffer B Use all 600 mL of Wash B from the reagent kit per Axiom plate Fill to 1L mark when processing two plates on the same day a Rinse fill with Axiom Water keep at 1 L full IMPORTANT a Always ensure that the GeneTitan bottles containing Wash A and Rinse are above the 50 mark when setting up the system to process an Axiom HT array plate All 600 mL of the Wash buffer B from the Axiom reagent kit should be emptied into the GeneTitan Wash B bottle when setting up the system to process a plate This ensures that the GeneTitan Wash B bottle is filled to more than the requisite 3596 of Wash B bottle volume Also do not overfill the bottles Fill Wash Buffer B and Water bottles to the 1 L mark only Wash A keep at 2 L We strongly recommend refilling these bottles every time you are prompted to do so If the volume in any of these bottles becomes too low during a run a message is displayed see Chapter 7 Troubleshooting on page 108 However even if you fill the bottle at this time the instrumen
113. glass from damage The black scan tray protective base is distinct from the blue array plate protective base and must not be used with the array plate Remove and discard the protective base from the scan tray before loading into the GeneTitan MultiChannel Instrument Chapter 3 Axiom 2 0 Assay Preparation Before You Start 28 Table 3 3 Axiom GeneTitan Tray Consumables from the Axiom GeneTitan Consumables Kit P N 901606 Continued Item Scan Tray with cover GeneTitan Stain Trays Part Number Labware Image Information Scan Tray The GeneTitan scan tray 501006 must be loaded with the Cover 202757 scan tray cover into the GeneTitan MC Instrument a Do not load the scan tray with the protective base 501025 The GeneTitan stain trays are packaged in ziplock bags to keep them free of dust The GeneTitan stain trays are barcoded and tray have separator walls that are flush with the frame of the stain tray as shown by the yellow line Chapter 3 Axiom 2 0 Assay Preparation Before You Start 29 Table 3 3 Axiom GeneTitan Tray Consumables from the Axiom GeneTitan Consumables Kit P N 901606 Continued Item Part Number Labware Image Information GeneTitan 202757 Scan and Stain Tray cover a The GeneTitan scan and stain tray covers are provided to prevent any evaporation of the stains in stain trays and the array holding buffer in the scan tray All Stain an
114. h H Do NOT write on any other side as this can interfere with sensors inside of the GeneTitan MC Instrument and result in experiment failure To ensure proper placement of lids onto stain trays and trays onto the GeneTitan MC Instrument you can also mark the notched corner of the trays and lids Lu IMPORTANT Do not confuse hyb trays with stain trays Deionize Trays and Covers Deionize the inside of each tray and cover now Return the trays and covers to the bench top after deionizing See Appendix E Deionization Procedure for GeneTitan Trays and Covers on page 130 for the recommended technique About Aliquoting Reagents to Trays IMPORTANT Always aliquot reagents to the bottom of the tray Avoid touching the sides or the top of the wells with the pipette tips Droplets close to or on the top of the well dividers may cause the lid to stick to the tray during GeneTitan MC Instrument processing For all trays pipette into trays on the bench top Ifthe trays are not being used immediately protect them from light by covering with foil or placing in a cabinet Lu IMPORTANT Remember to deionize the stain trays and the covers before aliquotting master mixes When aliquoting ligation staining and stabilization reagents to the trays it is not necessary to spread the reagent to each corner of the well The reagent will spread evenly when the array plate is inserted into the reagent tray during processing with the Gene
115. h the Clear Cover and Protective Base lt A Barcoded Scan tray cover PN 202757 GeneTitan Scan Tray PN 501006 or PN 500860 Scan Tray protective base Notched corners of tray and cover line up Notched corners of cover tray and base Figure 4 4 Loading the Scan Tray with Axiom Hold Buffer Leave the scan tray in its protective black base while loading with Axiom Hold Buffer sano fa weds pue Weis muen 095 MACAN Cover Protective Black Base 66 Array Processing with the GeneTitan Multi Channel Instrument The Axiom 2 0 Assay is designed for processing 96 samples at a time on Axiom Genome Wide and Custom myDesign Array Plates The protocol is performed in two sets of steps Target Preparation performed on the lab bench without advanced automation See Chapter 4 Axiom 2 0 Assay Manual Target Preparation on page 32 a Array processing performed on the GeneTitan Multi Channel MC Instrument This chapter includes instructions for Part 2 Array Processing These instructions are presented as follows a Before Using the GeneTitan MC Instrument on page 67 a Stage 1 Create and Upload Batch Registration File on page 75 Stage 2 Hybridization on page 76 Stage 3 Ligate Wash Stain and Scan on page 89 Before Using the GeneTitan MC Instrument Proper Tray Alignment and Loading Proper alignment and loading of plates covers and trays is critical when usi
116. he GeneTitan Multi Channel Instrument 92 Table 5 6 Sequence for Loading the Trays with Reagents Note If the software is unable to verify the barcode on the scan tray and the scan tray cover the software will display the following error message E Verily Drawer 2 Left Side Array Plate Load joj xil Warning The system was not able to verify the array plate barcode Please verify that the tray on the left side of the drawer is a SCAN tray with an ARRAY PLATE in the correct ORIENTATION Details The consumable is either not the correct consumable not loaded correctly or its barcode is not readable Proceeding with an incorrect or incorrectly loaded consumable can result in a loss of consumables loss of samples and may require a field service engineer to service the instrument Refer to the System Setup Tab or the User Guide provided with the Assay or AGCC for instructions on proper consumable placement Press the flashing blue confirmation button or Press OK GeneTitan will verify the barcode and orientation Press Skip GeneTitan will NOT verify the barcode and orientation The barcode entered at registration will be used Eos Ew 2 Scan Tray with cover do not load the protective black base left side of drawer as indicated in Status window Figure 5 30 on page 93 Status Load the Scan Tray with cover on Left side of Drawer Press the Confirmation button when done 3 Stain Tray with Stain 1 Ligation Tray
117. his issue complete the following tasks Verify all internal and external tubing is connected and clean a Verify wash reservoirs are clean Verify all bottle caps are secure and that no bottle cap is crimping a supply line a Verify vacuum is working properly a Do not refill bottles or empty waste except when prompted to by the GeneTitan application Contact your facility group to ensure CDA is supplied to your GeneTitan system Contact Affymetrix Field Service to have the sensor adjusted or replaced if the problem persists even after correcting for the usual causes outlined above Filter Error Messages EI Filter Change Required BUFFERA_TO_WASHA 3 9 2010 5 21 00 PM Warming ordern elg ee re eee EI You need 3 fiters instrument foe BUFFERA_TO_WASHA is listed below 1 27 2010 4 29 16 PM dispense time remaining before timeout was too short 1 75 sec 1 27 2010 4 41 45 PM dispense time remaining before timeout was too short 1 407 sec This warming will stop appearing when fiters have been replaced or 5 acceptable dispenses have been recorded The on 5 cheroree erit fee BUFFER TOA WASHA were 1 27 2010 ene PM dispense time remaining 1275 sec 1 27 2010 4 41 45 PM dispense time remaning 12 407 sl 1 27 2010 4 26 28 PM dispense time remaning 1596 1 27 2010 4 23 16 PM dispense time remaning 1 1 27 2010 4 41 45 PM dispense time remaining eh Fister Go EN oes ce e PR UR I AN PANE The unies e i Stas ei nag ees o
118. horoughly mixed before use 4 Thaw Samples in gDNA Plate A Bring your gDNA samples to room temperature on the bench top B Vortex and spin C Leave at room temperature Lu IMPORTANT a gDNA samples must be brought to room temperature before proceeding with denaturation gDNA samples must be 20 pL volume of each gDNA at a concentration of 5 ng pL or 10 ng pL depending on the array type in an ABgene 96 square well storage plate 2 2 mL see Genomic DNA Preparation on page 14 Chapter 4 Axiom 2 0 Assay Manual Target Preparation 36 5 Label the 15 mL and 50 mL conical tubes as indicated in the table below Label Tube Size Temperature Contents a DMM 15 mL leave tube at room temperature Denaturation Master Mix a Amp MM 50 mL leave tube at room temperature Amplification Master Mix 6 Label three solution basins as indicated in the table below Label Temperature Contents a DMM Leave basin at room temperature Denaturation Master Mix a N Soln Leave basin at room temperature Neutralization Solution a Amp MM Leave basin at room temperature Amplification Master Mix 2 Prepare the Denaturation Master Mix To Prepare the Denaturation Master Mix carry out the following steps at room temperature 1 Per Table 4 4 on page 36 dilute the appropriate volume of Axiom 2 0 Denat Soln 10X using the Axiom Water Table 4 4 Preparing Denaturation Master Mix D MM Reagent pe
119. ical that you write only on the proper locations of the proper sides of hyb and stain trays Do NOT write in any other location as this can interfere with sensors inside the GeneTitan MC Instrument and result in experiment failure To ensure proper placement of lids onto stain trays and trays onto the GeneTitan MC Instrument you can also mark the notched corner of the trays and lids Proper labeling for hyb trays and reagent trays is described in a Labeling for Hyb Trays below a Labeling for Stain Trays on page 31 Labeling for Hyb Trays You may label the hyb tray on the front part of the short side of the tray next to the notch at the left as shown in Figure 3 2 The proper section for labeling is closest to the notched corner corresponding to the Al and B1 wells Do NOT label trays on the long side of the tray Notched corner of the hyb tray Label the hyb tray here should face the front Writing on the wrong side of the hyb tray or on the wrong part of the long side may interfere with the operation of sensors in the GeneTitan MC Instrument Chapter 3 Axiom 2 0 Assay Preparation Before You Start 31 Labeling for Stain Trays You may label the stain trays on the left side of the front of the tray as shown in Figure 3 3 The correct side is closest to the notched corner corresponding to the A1 through C1 wells Figure 3 3 Labeling GeneTitan Stain Tray Do NOT label trays on the long side of the
120. ication 33 drying resuspension and QC 44 duration 33 equipment and perform denaturation 52 fragmentation and precipitation 38 GeneTitan MC consumables 26 hybridization tray and load into GeneTitan MC 53 ligation staining and stabilization reagent trays 55 master mixes and Axiom hold buffer 62 Plate requirements and recommendations 20 stain ligation and stabilization master mixes 59 thermal cycler recommendations 20 timing issues 101 Messages 109 Multiple plate workflows 10 Neutralization master mix 37 0 OD quantitation 118 OD readings 117 OD yield assessment guidelines 117 Oven temperatures for 3 plate workflow 102 Index 145 P Pipettes and pipetting 25 Plate reader other than the DTX880 124 Precautions 10 Problems and solutions 143 Procedure deionization 130 procedures 23 Protocol rehybridization 125 Q QC control gel protocol 114 Quantitation 116 Quantitation and fragmentation QC checks 49 References 9 Registering samples 127 Registration file 127 Rehybridization 125 storing hyb trays 126 Rehybridizing an experiment 126 Related documentation 9 Resuspension and hybridization master mix preparation 47 S Safety warnings 10 Safety warnings and precautions Manual target preparation 20 Sample quantitation 116 Scan 73 Seal vortex and spin 23 Setup options GeneTitan MC Instrument 72 Shutting down the GeneTitan MC Instrument 99 Stain trays and covers 69 Storing Hyb Trays for reh
121. ing washing and imaging Cel files generated by the GeneTitan Multi Channel Instrument are processed using the Axiom Genotyping Algorithm version 1 Axiom GT1 available through Affymetrix Power Tools or Genotyping Console v4 1 In summary the Axiom Genotyping Solution is a product line that provides catalog arrays that a Are optimized for high genetic coverage of their population in question a Provide highly automated reproducible results suitable for GWAS Chapter 1 About the Axiom 2 0 Assay 9 Related Documentation a Axiom 2 0 Assay Manual Workflow Site Prep Guide P N 702991 Axiom 2 0 Manual Target Prep Protocol QRC P N 702989 a Axiom Genotyping Solution Analysis Guide P N 702961 a Axiom 2 0 gDNA Sample Prep Protocol QRC P N 702987 Axiom gDNA Sample Prep for Genome Wide BOS 1 Array Plate QRC P N 702975 GeneTitan MC Protocol for Axiom 2 0 Array Plate Processing QRC P N 702988 GeneTitan Multichannel Instrument User s Manual P N 08 0306 a GeneTitan Multichannel Instrument Site Preparation Guide P N 08 0305 a Affymetrix GeneChip Command Console Software User Manual P N 702569 a Affymetrix Genotyping Console 4 1 User Manual P N 702982 Axiom 2 0 Assay Automated Workflow a Axiom 2 0 Assay Automated Workflow User Guide P N 702963 a Axiom 2 0 Assay Automated Workflow Site Prep Guide P N 702984 Axiom 2 0 Assay Automated Target Prep Protocol QRC P N 702962 Biomek Liquid
122. is an important QC checkpoint in the Axiom 2 0 Assay If the median yield for the plate is 1000 ug DNA per sample a Pause the protocol a Assess each of the steps performed to that point to determine the possible source of the low yields This DNA yield corresponds to an A260 value of approximately 0 49 and an A260 A320 value of approximately 0 42 Appendix B Sample Quantitation after Resuspension 118 Suggested Protocol for OD Quantitation Using the DTX 880 The formula suggested below requires six passes The settings and formula are shown below Protocol Type Analysis Figure B 3 Protocol Type S Select Protocol Type New Protocol Template Protocols Select Protocol Type Protocol Type Analysis Analysis applications allow measurement data to be transformed and analyzed using formulas variables and parameters configured in the protocol General Settings enter a name for the protocol Figure B 4 General Settings ig Create Protocol NewProtocol for 96 array plate General Settings General Settings P Please enter a name and notes for this protocol Technique Type Labware Selection Layout Settings Protocol name NewProtocol For 96 array plate Method Selection Date Created Monday September 28 2009 Data Reduction Page Date Edited Monday September 28 2009 Output Sattngs Date last run Monday September 28 2009 Notes Run Notes Analysis Options
123. itan MC Instrument array processing 67 cleaning and maintenance 133 continuing the workflow 98 create and upload batch registration file 75 hybridization 76 ligate wash stain and scan 89 load an array plate and hyb tray 81 load trays 91 proper installation of the GeneTitan tray consumables 90 setup options 72 setup the instrument 76 shutting down 99 status window prompts and actions required 87 tray alignment and loading 67 troubleshooting 108 142 Xenon lamp 137 GeneTitan MC lamp 71 Genomic DNA 11 GWAS 8 Human genomic DNA 11 Hyb ready samples stored at 20 C 52 Hyb tray loading 81 Hyb Trays storing for rehybridization 98 Hybridization GeneTitan MC Instrument 76 Hyb Wash 73 l Insufficient disk space notice 143 L Labeling GeneTitan hybridization and reagent trays 70 Labeling GeneTitan hybridization and reagent trays 30 Lambda LS Xenon Arc Lamp replacing the lamp 137 Lamp Lambda LS Xenon Arc Lamp 137 Lamp counter resetting 141 Lamp life Imaging Device status notices 137 Ligate Wash Stain and Scan 89 Load an array plate and hyb tray 81 Load trays on to GeneTitan MC Instrument 91 Loading a second array plate and hyb tray 86 Log files 142 GeneTitan MC Fluidics 143 GeneTitan MC Imaging Device 143 Maintenance 133 Manual target prep 3 plate 103 Manual target Preparation 3 array pates per week 100 Manual target preparation 23 32 control recommendations 20 denaturation and hybridization 50 DNA amplif
124. ition at all times Setup Options for Array Plate Processing The processes setup options available for processing array plates are shown in Figure 5 6 A brief description of each option is given below Figure 5 6 Setup Options for Processing Array Plates a AGCC GeneTitan Instrument Control File Tools Help Stop Email Help System Status System Setup Setup Option m Plate Information Barcode Wash Scan Resume Scan Unload Plates Plate Type Protocol Name Location Hyb Wash Scan This setup option enables you to hybridize wash ligate stain fix and scan an array plate on the GeneTitan MC Instrument Lu IMPORTANT When running a multi plate workflow you must pay careful attention to the software prompts that tell you which side of the drawer to place or remove a plate tray Hyb the array plate is moved to the hybridization oven inside the instrument Each denatured sample in the hyb tray is hybridized to an array on the array plate o Duration for 96 samples 23 5 hr a Wash samples on arrays are ligated washed stained and fixed o Duration for 96 samples 5 hr Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 73 NOTE The instrument control software will display a warning if it detects a problem during the fluid dispense operations The filters in the GeneTitan Wash A Wash B and DI Water bottles shoul
125. l Instrument Care This chapter provides instructions on caring for and maintaining the instrument and on troubleshooting if problems arise Always run a Shutdown protocol when the instrument will be off or unused overnight or longer This will prevent salt crystals from forming within the Fluidics system Always use deionized water to prevent contamination of the lines Change buffers with freshly prepared buffer at each system startup The GeneTitan MC Instrument should be positioned on a sturdy level bench away from extremes in temperature and away from moving air Lu IMPORTANT Before performing maintenance turn off power to the instrument to avoid injury in case of an electrical malfunction Cleaning and Maintenance Monthly The GeneTitan family of instruments require little in the way of customer maintenance The instruments must be kept clean and free of dust Dust buildup can degrade performance Wipe the exterior surfaces clean using a mild dish detergent solution in water Do not use ammonia based cleaners or organic solvents such as alcohol or acetone to clean the system because they may damage the exterior surfaces The following tasks should be performed regularly to ensure the Imaging Device remains in working order Wipe down the outer surface of the Imaging Device with a dry cloth Every Six Months Replace the cooling fan air filters at the rear of the instrument Replace the Micropore filters in the
126. late must be loaded on its protective blue base as shown in Figure 5 15 on page 82 below The clear plastic cover on top of the array plate SHOULD NOT be loaded in the GeneTitan MC Instrument See Figure 5 1 on page 68 for more details on the correct way of loading the array plate C Remove the array plate and protective blue base from its package To avoid dust or other damage leave the array plate packaged until ready to load onto the GeneTitan MC Instrument Figure 5 14 Figure 5 14 Array Plate Packaging Clear Tray 748 Shipping Cover To be Discarded Blue Array Plate Protective Base Array Plate Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 82 Figure 5 15 Array Plate with Protective Blue Base and the Hyb Tray Properly Loaded into Drawer 6 Array Plate with Protective Blue Base Hyb Tray D Load the array plate with the protective blue base on the left side of the drawer Figure 5 15 Lu CAUTION The notched corner of each plate cover and tray must be aligned When loading onto the GeneTitan MC Instrument the notched edge plates covers and trays must be aligned as indicated by the Tray Alignment guide in the drawer Figure 5 15 on page 82 The error message shown in may be displayed Plate barcodes must face the internal barcode reader back of the drawer Improper tray positioning can
127. lume 57 uL 7 48 mL Add the reagents from Table 4 9 to the Frg MM tube in the order shown using appropriate single channel and serological pipettes Chapter 4 Axiom 2 0 Assay Manual Target Preparation 42 Just before the end of the 45 minute 37 C incubation flick the Axiom Frag Enzyme tube 2 to 3 times and spin Add the Axiom Frag Enzyme to the Fragmentation Master Mix at the end of the 45 minute 37 C incubation NOTE Leave the Axiom Frag Enzyme at 20 C until ready to use Vortex twice and place on ice Slowly pour the Fragmentation Master Mix in the solution basin labelled Frg MM placed at room temperature 3 Add Fragmentation Master Mix to Wells Ka IMPORTANT Work quickly to perform this set of steps to minimize the time that the Fragmentation plate is out of the 37 C oven Carefully remove the Amplification plate from the 37 C oven and place on the bench top at room temperature Do not place the Amplification plate on ice Carefully remove the seal from the Amplification plate and discard the seal Pipetting directly into the liquid of each well use a P200 12 channel pipette to add 57 uL of Fragmentation Master Mix to each reaction Change tips after each addition After adding the Fragmentation Master Mix to the plate the plate is now known as the Fragmentation plate Seal the Fragmentation plate and vortex twice Start the timer for 30 min Spin the Fragmentation plate in the pla
128. m Other Suppliers a Use only fresh reagents from the recommended vendors to help eliminate changes in pH or the salt concentration of buffers a Consult the appropriate MSDS for reagent storage and handling requirements Master Mix Preparation a Carefully follow each master mix recipe Use pipettes that have been calibrated to 5 a If you run out of master mix during any of these procedures a volume error has been made or the pipettes are not accurate We recommend that you stop and repeat the experiment NOTE The volumes of Master Mixes prepared are designed to provide consistent handling of reagents and consistent assay results The percent overage of different master mixes may differ depending upon the reagent volumes involved When Using Reagents at the Lab Bench Properly chill essential equipment such as reagent coolers before use a Ensure that enzymes are kept at 20 C until needed When removed from the freezer immediately place in a cooler that has been chilled to 20 C Chapter 3 Axiom 2 0 Assay Preparation Before You Start 25 Pipettes and Pipetting To efficiently process samples a Use a pipette of appropriate size for the volume of liquid being transferred Table 3 2 Table 3 2 Recommended Pipette Sizes Pipette Size Recommended Volume Range Single channel P20 12 channel P20 1 20 uL P50 optional 20 50 uL Single channel P200 12 channel P200 20 200 uL Single channel P1000
129. m each well of the gel QC plate onto the gels Load 15 uL of diluted TrackIt 25 bp ladder into the marker wells M Load 20 uL nuclease free water into any unused wells 10 Run the gels for 22 min 11 Take a gel image Fragmentation QC gel images should look similar to the gel shown in Figure A 1 Figure A 1 Example of a Typical Fragmentation QC E gel Fragments should fall between 25 bp and 125 bp 25 bp ladder 25 bp ladder 125 bp 25bp 125 bp 25 bp Sample Quantitation after Resuspension Protocol for Sample Quantitation after Resuspension Equipment Required The following equipment is required for this protocol Table B 1 Equipment required for sample quantitation after resuspension Quantity Item 1 DTX 880 Multimode Detector with Genomic Filter Slide Quantitate the Diluted Samples During target prep two plates of diluted samples are prepared one for OD quantitation and one for a QC gel to check the fragmentation reaction For OD quantitation readings should be taken at wavelengths of 260 280 and 320 nm See Suggested Protocol for OD Quantitation Using the DTX 880 on page 118 for more information To Quantitate the Diluted Samples Prepared for OD Quantitation Launch the Multimode Analysis Software When the Protocol Selection List is displayed select the appropriate protocol Right click the protocol and select Run the selected protocol In the Result Name field
130. me approximately 0 5 hr Incubation at 37 C 23 1 hr a Total time required approximately 24 5 hr Input Required gDNA Sample Plate with 20 uL of each gDNA diluted to a concentration of 5 ng uL or 10 ng L as required according to the Axiom array that will be used in an ABgene 96 square well storage plate 2 2 mL See Genomic DNA Preparation on page 14 for more information Chapter 4 Axiom 2 0 Assay Manual Target Preparation 34 Equipment Consumables and Reagents Required Equipment and Consumables The equipment and consumables listed in Table 4 1 are required for this stage Table 4 1 Equipment and Consumables Required for Stage 1 DNA Amplification Quantity Item As required Adhesive seals for 96 well plate Applied Biosystems MicroAmp Clear adhesive film 1 Cooler chilled to 20 C 1 50 mL tube holder 1 15 mL tube holder 1 Marker fine point permanent 1 Mini microcentrifuge microfuge with microtube rotor 1 each Rainin Pipettes Single channel P200 a Single channel P1000 a Multi channel P20 Multi channel P200 Multi channel P1200 As needed Pipette tips As needed Pipette serological 5b x 1 10 mL VWR P N 53283 706 10 x 1 10 mL VWR P N 53283 708 1 Pipet aid 1 Plate centrifuge at room temperature 1 Oven set at 37 C 1 50 mL conical tube 1 15 mL conical tube 1 Vortexer 1 Timer 3 Solution basin 100 mL st
131. n laboratory techniques and that it is used in accordance with the principles of good laboratory practice Wear suitable protective clothing such as lab coat safety glasses and gloves Care should be taken to avoid contact with skin and eyes nn WARNING The following components contain harmful or toxic ingredients a Axiom Stabilize Soln 8 Gluteraldehyde Axiom HybSoln 2 100 Formamide a Axiom Hyb Buffer lt 55 Tetramethylammonium Chloride As such we recommend the use of a fume hood when using these products during the Manual Target Preparation protocol In all cases customers should use adequate local and general ventilation in order to minimize airborne concentrations Copies ofthe Material Safety Data Sheets for the kit components are available on the Affymetrix website at www affymetrix com Control Recommendations A negative control is not required for this assay We recommend including one positive control with every set of samples processed A positive control Axiom Reference Genomic DNA 103 is included in the Affymetrix Axiom 2 0 Reagent Kit Plate Requirements and Recommendations The following types of plates are required for performing manual target preparation Refer to the Axiom 2 0 Assay Manual Workflow Site Prep Guide P N 702991 for vendor information ABgene 96 Square Well Storage Plate 2 2 mL Bio Rad Hard Shell 96 well plate P N HSP 9631 Refer to the Axiom 2 0 Assay Manual Workflow
132. n Axiom Ligate Buffer is OK and will not adversely impact assay performance Follow the instructions above to resuspend any precipitate before use Chapter 4 Axiom 2 0 Assay Manual Target Preparation 58 2 Prepare the reagents from Module 4 2 as described in Table 4 24 Table 4 24 Reagents from Module 4 2 2 8 C P N 901276 Reagent Temp Out of Treatment Storage before Master Mix Module Axiom Ligate Soln 2 Thaw at Room Vortex and Spin Store at Room Temp Temp do not place on ice Axiom Probe Mix 2 Place on Ice Vortex and Spin Place on ice Axiom Wash A Leave on bench 1 Vortex twice Place on bench top at room temp 2 Place on Bench for 30 min 3 Look for precipitate 4 Vortex again if necessary Axiom Stain 1 A Place on ice Flick 2 to 3 times to mix then spin Place on ice Axiom Stain 1 B Place on ice Flick 2 to 3 times to mix then spin Place on ice Axiom Stain 2 A Place on ice Flick 2 to 3 times to mix then spin Place on ice Axiom Stain 2 B Place on ice Flick 2 to 3 times to mix then spin Place on ice Axiom Stabilize Diluent Place on ice Place on ice 1 Vortex and Spin 2 Look for precipitate If any a Warm tube to room temperature and vortex again Axiom Water Place on ice Place on ice Axiom Hold Buffer Room Temp Place at Room Temp away from light Notes Temp Out of Module temperature reagent is held at immediately after removal from module These solutions are light
133. n a various P Ns 1 protective base or o One Axiom myDesign Genotyping 96 array plate in a protective base a Hyb Tray P N 500867 1 The Consumables for the GeneTitan MC Instrument are packaged separately from the Axiom array plates The consumables are available in the Axiom GeneTitan Consumables Kit PIN 901606 The hyb tray is available in the Axiom GeneTitan Consumables Kit Table 4 17 Reagents Required from the Axiom 2 0 Reagent Kit Reagent Module Axiom Wash Buffer A both bottles 1L Module 3 Room Temperature Axiom Wash Buffer B P N 901472 Axiom Water Chapter 4 Axiom 2 0 Assay Manual Target Preparation 52 1 Prepare Hyb Ready Samples Stored at 20 C To Prepare Hyb Ready Samples That Were Stored at 20 C 1 Warm up the Hyb Ready plate at room temperature for 5 minutes It is not necessary to equilibrate the plate for longer duration Make sure the Hyb Ready plate is sealed well If the plate is not sealed well A Spin the plate and carefully remove the old seal B If there is condensation on the top of the plate blot dry gently with a Kimwipe C Use a fresh seal and tightly reseal the plate Vortex the Hyb Ready plate briefly then spin at 1000 rpm for 30 seconds Place the Hyb Ready plate at room temperature 2 Prepare Equipment and Perform Denaturation 1 2 Preheat the 96 well metal chamber in a 48 C oven Warm up the array plate on the bench top b
134. n lost of data If you see this notice you will need to free up sufficient disk space before imaging starts Index Numerics 3 array pates per week 100 3 plate manual target prep 103 3 plate workflow oven temps 102 A Aborting a process 74 Aborting a run 113 AGCC files 142 AGCC log files 142 AGCC Log Files for GeneTitan MC Systems 143 Amplification master mix 37 Amplification staging room 12 19 Amplified DNA thawing 103 Anti static gun testing 131 Array plate loading 81 Array processing GeneTitan MC Instrument 67 Axiom 2 0 Assay 9 Axiom 2 0 Assay 8 Batch registration file 16 Bottle filters replacing 135 Cc Care 133 GeneTitan MC Instrument 133 Centrifuge and dry pellets and thaw reagents 46 Cleaning 133 Cleaning and maintenance 133 Command Console 127 Consumables Manual target preparation 34 39 44 50 rehybridization 125 Cyan Orange Loading Buffer 115 D Deionization 130 Deionization procedure 131 Denaturation master mix 36 Documentation 9 DTX 880 118 Duration 14 Manual target preparation 33 44 50 E mail and telephone notifications 71 Equipment Consumables and Reagents Required 14 F Failed messages 110 Fan filters cleaning schedule 133 servicing 133 Fluidic diagnostic messages 110 Fragmentation QC gel protocol 114 Fume hood 19 G gDNA 15 Gel protocol 114 GeneChip Command Console 127 GeneTitan MC array processing timing issues 102 GeneT
135. n this prompt to resume reagent loading is displayed to the user there is no need to wait for the estimated time to count down to zero Follow the prompts displayed to continue with staining ligation fixing and scanning 1 Follow the prompts in the Status window A Wash Bottles A and B and the Rinse Bottle refill as necessary the system will prime itself again Waste bottle empty if necessary Wash bottle A 2 L Wash Bottle B and Rinse Bottle fill to 1 L mark only B Empty the trash bin C Remove consumable trays and plates as instructed except for the blue base Leave the blue array plate base in drawer 6 even though the base is empty 2 Load consumable trays and plates as follows A Follow the prompts in the Status window load sequence and prompts in Table 5 6 B Once loaded examine each cover for droplets of liquid C If any liquid is present remove the tray clean the cover and top of the tray with Kimwipes and reload the tray CAUTION a Orient trays as indicated by the guide inside the drawer Improper orientation may cause the run to fail a Remove the protective black base from the scan tray immediately prior to loading Figure 5 30 on page 93 a Examine each cover for droplets of liquid after loading Liquid on the cover can result in capillary phenomenon As a result the tray may stick to the cover and be lifted out of place inside the instrument Chapter 5 Array Processing with t
136. n was started 2 C Affymetrix GeneChipHTScanControIMC RunLog collect all dated directories and contents since the GeneTitan application was started Problems and Solutions This section provides instructions on how to identify and solve problems with the unit If problems arise with the instruments use the following tables to locate the description that matches the problem If you cannot find a solution call Affymetrix technical support for assistance For software errors that do not involve hardware crashes the most common solution is to shut down the application and then restart it If the same error occurs shut down both the application and the computer and then restart If it still occurs shut down the entire unit and then restart Insufficient Disk Space Notice If there is not enough memory on the computer s drives to save the data from an array plate a notice appears when You first initialize the software and instrument a You select arrays for imaging Figure F 14 Insufficient Disk Space Notice Insufficient Disk Space Error Barcode DriveID SpaceReqGB FreeSpaceGB lateState ScannerState PI 5500321234567890123456 C 12 311 Hyb Waiting DriveID FreeSpaceGB FreeSpaceRemainingGB Status e 2 Insufficient disk space Please free up sufficient disk space before scanning starts You can check for sufficient disk space with the the menu command under Tools Check Available Disk Space Failure to do so will result i
137. nd click and the drawer is fully seated a Check that the array plate barcode has been entered Array Registration k correctly Ensure that the library files required for the type of array Cannot find the protocol file For the entered barcode lat 7 h b installed d installed Please make sure the protocol files are properly installed and or plate you are using have been installed and are installe Re register array plate by scanning the barcode or in the correct di rectory Type in the barcode Restart the GeneTitan MC instrument control software after library files have been installed Chapter 7 Troubleshooting 110 Failed Messages Table 7 3 Problem and Possible Causes z If this message is displayed FAILED PRIME a during a water wash step array processing has been compromised a during cleanup array processing is OK but cleanup will Retr Cancel not be complete Always ensure that the GeneTitan bottles containing Rinse bottle fluid level too low or bottle empty Wash A and Rinse are above the 50 mark when setting up the system to process an Axiom HT array plate All 600 mL of the Wash buffer B from the Axiom reagent kit should be emptied into the GeneTitan Wash B bottle when setting up the system to process a plate This ensures that the GeneTitan Wash B bottle is filled to more than the requisite 35 of Wash B bottle volume Rinse Failed on plate 550032 laureenxxxxxxxx
138. nded in order to improve visualization Loading 25 ng gDNA per well can improve the image a Add 3 uL of 0 1X of RediLoad dye to each sample Bring each sample to a total volume of 20 uL using H20 for example if the volume of genomic DNA is 5 uL add 3 uL of RediLoad and bring to 20 uL total by adding 12 uL of H20 Seal vortex and spin Chapter 2 Genomic DNA Preparation and Requirements 13 To Run the E Gel Power on for E Base red light Push the Power Prg button to make sure the program is at EG mode not EP Insert the two 48 well 1 Agarose E Gels into the slots Remove 2 combs Load 20 uL from the above plate onto two 48 well 1 agarose E Gels Load 15 uL of diluted High Range DNA Marker 1 3 dilution or 0 34 X from stock into all marker wells as needed I 1 WNS 7 Fill all empty wells with water 8 Adjust the run time to 27 min 9 Push the Power Prg button again it will change from red to green When run time is reached the ladder band reaches the end ofthe lane the system will automatically shut off The gel is then ready for imaging Figure 2 1 shows gel images of intact gDNA that is suitable for use in the Axiom 2 0 Assay and degraded gDNA samples Customers whose gDNA is degraded similar to the image in Figure 2 1 should perform a test experiment to investigate the performance of their samples in the Axiom Genotyping Assay prior to beginning any large scale genotyping projects
139. ndow to Run Next Hyb Wash Scan HT Array Type E ame plate type z System not available processing 2 plates Hybridization Oven Status Fluidics Status TEER 50032 plate IO Barcode 550032405935701 2609098 Position 1 EN 00 58 02 Protocol Name 550032 protocol Estimated Time Remaining 00 03 26 Seah Barcode 550032 plate2900000099 Wash B Temperature Estimated Time Remaining Q0 O0 OO Current Target Heater is OFF ven Temperature jg D TL Target 48 C 11 32 06 AM Unclamp completed 550032405935701 2609098 in Unclamp Hta 11 32 06 AM Step completed 11 32 06 AM UnclampStationPresentPlate 11 32 06 AM 550032 plateDooOOOOO wait for FluidicSystem Gripper 11 32 11 AM UnclampStationPrepareF orPlatel Insertion 11 32 38 AM WashBLid open 11 41 55 AM Hyb 0 59 min 00 00 30 00 00 31 00 00 00 00 33 00 00 30 00 00 3 00 00 00 00 33 00 00 30 00 00 33 FIXING 00 00 10 00 00 10 Workflow indicates the number of plates being processed and where they are in the instrument In this example three Axiom array plates are being processed 2 in the Hyb Oven and 1 in Fluidics Estimated Completion Time is for the current process Estimated Time Remaining for fluidics is adjusted as necessary Adjustments can be due to process interruptions such as a dra
140. ned imi Not Available 2 Click Next then click OK to begin processing the samples Figure 5 18 The array plate is placed on top of the hyb tray and clamped now referred to as the plate stack Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 84 Start Processing x Click OK to confirm that you wish to proceed with hybridization Ue eel ey yell ashe DE The plate stack is in the left position the left side of the drawer The software starts the process for clamping the array plate to the hybridization tray Press OK on the dialog shown in Figure 5 19 and wait for the drawer to open before retrieving the array plate and hybridization tray combo for inspection The sandwich of the array plate and hybridization tray needs to be manually inspected before the array processing can begin Once clamping is complete the dialog shown in Figure 5 20 on page 84 will be displayed If you do not press OK in Figure 5 19 the dialog box will go away without intervention and Figure 5 20 on page 84 will be displayed El Clamping in progress 550094 test030SXOOOX is being clamped Please remain present to complete the post clamp verification step After the array plate and sample are clamped you will be prompted to verify clamping 3 When drawer 6 opens and the prompt in Figure 5 20 is displayed erify Clamping Ble x Verify plate clamping is secure Press Ok or the blue Confirmation button when r
141. ng the GeneTitan MC Instrument Each plate cover and tray has one notched corner The notched corner of plates trays covers and bases must be in vertical alignment with each other and placed in position A1 per the Tray Alignment guide inside each GeneTitan MC drawer Figure 5 1 on page 68 and Figure 5 2 on page 69 Lu IMPORTANT When running a multi plate workflow you must pay careful attention to the software prompts that tell you which side of the drawer to place or remove a plate tray E TIP Mark the notched corner of each plate cover and tray with permanent marker to help ensure proper alignment and loading onto the GeneTitan MC Instrument CAUTION Take care not to damage the consumables or bend the blue cover posts or scan tray posts NOTE The instrument control software will display a warning if it detects a problem during the fluid dispense operations The filters in the GeneTitan Wash A Wash B and DI Water bottles should be replaced if the software displays such a warning Refer to Appendix F GeneTitan Multi Channel Instrument Care on page 133 for the message displayed to the user and the procedure for replacing the filters Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 68 Figure 5 1 Proper Alignment and Loading of Plates Covers and Trays in the GeneTitan MC Instrument Clear Tray 8 Shipping Cover To be Discarded IMPORTANT Remove the
142. nstrument as soon as possible and should not be stored To prepare the reagent trays for the GeneTitan MC Instrument 1 Prepare the Reagents for Stage 5 on page 57 2 Prepare the Stain Ligation and Stabilization Master Mixes on page 59 3 Aliquot Master Mixes and Axiom Hold Buffer into Trays on page 62 The following instructions are for manually preparing the reagents and trays required to process Axiom array plates on the GeneTitan MC Instrument The reagents and trays required are as follows Table 4 19 Reagent Trays Required for the Axiom 2 0 Assay on the GeneTitan MC Instrument Type of Tray Number of Trays Tray Designation Master Mix Reagent Stain Tray with cover 2 S1 Stain 1 Master Mix Stain Tray with cover 1 2 Stain 2 Master Mix Stain Tray with cover 1 Stbl Stabilization Master Mix Stain Tray with cover 1 Lig Ligation Master Mix Scan Tray 1 Scan Tray Hold Buffer Equipment Consumables and Reagents Required Table 4 20 Equipment Required for Stage 5 Manually Preparing Ligation Staining and Stabilization Reagent Trays for the GeneTitan MC Instrument Equipment Quantity GeneTitan MC Instrument 1 Ice bucket with ice 1 Microcentrifuge 1 Pipetaid 1 Chapter 4 Axiom 2 0 Assay Manual Target Preparation 56 Table 4 20 Equipment Required for Stage 5 Manually Preparing Ligation Staining and Stabilization Reagent Trays for the GeneTitan MC Instrument Continued
143. ntil ready to use Axiom 10X Frag Buffer Axiom Precip Soln 2 Module 2 1 20 C P N 901528 Chapter 4 Axiom 2 0 Assay Manual Target Preparation 40 Table 4 7 Reagents Required for Stage 2 Fragmentation and Precipitation Continued Reagent Module Axiom Frag Diluent Module 2 2 2 8 C Axiom Frag Rxn Stop P N 901529 Axiom Precip Soln 1 User supplied Refer to the Axiom 2 0 Assay Manual Workflow Site Prep Guide P N 702991 Isopropanol 2 Propanol 99 5 96 samples 65 mL per array plate 1 Stop Amplification Reaction If you are running one plate per week you will need two ovens to perform this step One oven set at 37 C Use an oven that can sustain a constant temperature of 37 C and has a temperature accuracy of t C One oven set at 65 C If you are running the three plate per week manual target preparation workflow three ovens are recommended See Chapter 6 Manual Target Preparation for Processing Three Axiom Array Plates per Week on page 100 for more information NOTE If the plate has been frozen and stored it must be thawed using the instructions in Thawing Frozen Plates of Amplified DNA on page 103 Allow an hour to thaw To Stop the Amplification Reaction 1 Place the Amplification plate in the 65 C oven If proceeding directly from the end of Stage 1 DNA Amplification on page 38 transfer the Amplification plate from the 37 C ov
144. ocessing and automated target preparation This solution has applications in human disease research and basic and applied agriculture research For human disease research applications Affymetrix conducted an empirical screen of genomic content from dbSNP http www ncbi nlm nih gove projects SNP The screen included markers from HapMap and the 1000 Genomes Project as well as other sources using HapMap phase 3 samples and or the original 270 HapMap samples All of this information has gone into creating a proprietary Affymetrix database of validated markers that can be interrogated using the Axiom 2 0 Assay There are several arrays available for use with the Axiom 2 0 Assay which leverage the content of this proprietary Affymetrix database For a complete list of supporting products please visit www affymetrix com The Axiom 2 0 Assay interrogates biallelic SNPs and simple indels human only in a single fully automated assay workflow Starting with genomic DNA the samples are processed by performing either an automatic or manual target prep protocol followed by automated processing of the array plates in the GeneTitan MC Instrument Target prep uses methods including DNA amplification fragmentation purification and resuspension of the target in hybridization cocktail a The hyb ready targets are then transferred to the Affymetrix GeneTitan Multi Channel MC Instrument for automated hands free processing including hybridization stain
145. om Hold Buffer Prepare Stain 1 Master Mix To Prepare the Stain 1 Master Mix 1 Use appropriate serological and single channel pipettes to add reagents to the 50 mL tube labeled S7 in the order shown in Table 4 27 This recipe will provide enough for both S reagent trays Table 4 27 Stain 1 Master Mix Reagent Per Array Master Mix 96 To the tube marked S1 add Axiom Wash A 201 6 uL 22 2 mL Axiom Stain Buffer 4 2 uL 463 uL Axiom Stain 1 A 2 1 uL 231 uL a Axiom Stain 1 B 2 1 uL 231 uL Total 210 pL 23 13 mL 105 pL x 2 Chapter 4 Axiom 2 0 Assay Manual Target Preparation 60 2 Gently invert the tube 10 times to mix 3 Place on ice and protect from direct light e g cover with aluminum foil or ice bucket lid Prepare Stain 2 Master Mix To Prepare the Stain 2 Master Mix 1 Use appropriate serological and single channel pipettes to add reagents to the 15 mL tube labeled S2 in the order shown in Table 4 28 Table 4 28 Stain 2 Master Mix Reagent Per Array Master Mix 96 To the tube marked S2 add Axiom Wash A 100 8 uL 11 1 mL Axiom Stain Buffer 2 1 uL 231 uL a Axiom Stain 2 A 1 05 uL 115 6 uL a Axiom Stain 2 B 1 05 uL 115 6 uL Total 105 pL 11 56 mL 2 Gently invert the 2 MM tube 10 times to mix 3 Place on ice and protect from direct light e g cover with aluminum foil or ice bucket lid Prepare Stabilization Master Mix To
146. or Codes GT Reagent prep load GeneTitan reagent trays prep and load see Stage 5 Manually Preparing Ligation Staining and Stabilization Reagent Trays for the GeneTitan MC Instrument on page 55 Table 6 10 Manual Target Prep Workflow Day 5 Activities Activity Plate ID Approximate Start Times GeneTitan reagent tray prep and loading B 8 00 a m GeneTitan reagent tray prep and loading C 3 30 p m Troubleshooting GeneTitan Multichannel Instrument Refer to the GeneTitan Multi Channel Instrument User s Manual P N 08 0306 for further troubleshooting information Table 7 1 GeneTitan Multichannel Instrument troubleshooting guidelines for the Axiom 2 0 Assay Problem Possible Causes Possible Actions Plate trapped in GeneTitan Multichannel Instrument a Plate or plate with lid not properly loaded in drawer a Cut edge of lid and plate not aligned a Gripper failed to retrieve plate a System requires adjustment Restart the GeneTitan Multichannel Instrument 2 Run the setup option Unload Plates 3 If the plate remains trapped in the instrument call Affymetrix support Computer frozen Too many processes running a Attempting to transfer data while an array plate is being scanned imaged Restart the computer and unload all of the plates a Plates in Hyb station finish hybridization off line Plate in Scanner rescan using Scan Only
147. or Stage 3 Drying Resuspension and QC Continued Quantity Item 1 Fume Hood 1 Plate centrifuge set at 4 C 1 15 mL conical tube 1 10 mL Serological Pipette 1 Pipet aid 1 Shaker either Titer Plate Shakers 4PL 120V a Jitterbug 1 Vortexer As needed Solution basin 100 mL sterile multichannel Reagents Required Table 4 12 Reagents Required for Stage 3 Drying Resuspension and QC Reagent Module From the Axiom 2 0 Reagent Kit Axiom Hyb Buffer Module 2 1 20 C P N 901528 Axiom Hyb Soln 1 Axiom Resusp Buffer Module 2 2 2 8 C P N 901529 Axiom Hyb Soln 2 Other Reagents Required for QC steps optional Tracklt Gel Loading Buffer 14 mL of 1000 fold dilution see Appendix A Fragmentation Quality Control Gel Protocol on page 114 for dilution instructions Gel Sample Plate 15 fold dilution of 25bp Invitrogen Ladder P N 10488 022 Nuclease free water ultrapure MB Grade 14 mL P N 71786 for OD and Dilution Plate preparation Gels and Related Materials Required At the end of this stage verifying the fragmentation reaction is highly recommended See Appendix A Fragmentation Quality Control Gel Protocol on page 114 for the required gel and related materials Chapter 4 Axiom 2 0 Assay Manual Target Preparation 46 1 Centrifuge and Dry Pellets and Thaw Reagents CAUTION During this step handle the Precipitation pl
148. osition and P This will start another plate with HybWashScan run mode in the Right Position Please press the OK button to confirm Select the System Status tab to view Axiom array plates status in the WorkFlow window Figure 5 25 Figure 5 25 Example of the WorkFlow Window When Two Plates are Loaded and are in The Hybridization Oven System Setup Work Flow Plate Type Hyb Status Fluidics Status Scan Status Estimated Completion Time Waiting Waiting 5 4 2009 11 20 42 AM Waiting Waiting 5 4 2009 10 25 36 AM Left and Right positions the position of the scan tray in drawer 2 left or right side of the drawer Status Window Prompts and Actions Required Table 5 2 Refilling Buffer Bottles and Emptying the Waste Bottle Status Window Prompt Action Required Receptacle Reagent Buffer bottles have been depressurized Please refill buffer into the bottles Empty the waste bottle a Replenish the fluid in Wash Bottles A and B and the Rinse bottle a Empty the Waste Bottle a Press the Confirmation button to continue a Wash Bottle A fill with Axiom Wash Buffer A up to 2 L a Wash Bottle B fill with Axiom Wash Buffer B to the 1 L mark a Rinse fill with Axiom Water to the 1 L mark Do not overfill these bottles Every time you are prompted to refill the buffer bottles the system runs a fluidics che
149. ove the Precipitation plate from the centrifuge and place on benchtop at room temperature Pour isopropanol into the solution basin labeled ZSO 0 9x o Carefully remove the seal from the Precipitation plate and discard the seal 10 Using a P1200 12 channel pipette add 600 uL isopropanol to each sample and mix well by pipetting up and down within the solution to ensure mixing The solution should look homogenous in the tips after pipetting 5 7 times If not repeat mixing a few more times until the solution looks mixed Do not vortex the plate after isopropanol addition to avoid cross contamination of the samples Change the tips after each addition 11 Blot the top of the plate with Kimwipe and seal tightly with a Microamp seal 12 Carefully transfer the Precipitation plate into the 20 C freezer and incubate overnight 16 24 hrs EH TIP It is recommended to designate a shelf in a 20 C freezer where the plates can be left undisturbed Chapter 4 Axiom 2 0 Assay Manual Target Preparation 44 Stage 3 Drying Resuspension and QC This stage requires the following sets of steps 1 Centrifuge and Dry Pellets and Thaw Reagents on page 46 2 Prepare the Tubes Basins and Trays for Resuspension and Hyb Master Mix Preparation on page 47 3 Resuspension and Hybridization Master Mix Preparation on page 47 4 Recommended Perform Quantitation and Fragmentation QC Checks on page 49 5 Freeze or Proceed on page 50
150. p the cap on the gun and press the trigger and release it Observe the discharge through the viewing slot on the cap of the anti static gun There is a visible light observed in the viewing window on the cap when charged ions are discharged If you cannot see the light the gun may be un usable and you should replace it Each anti static gun is capable of 50 000 trigger operations which is sufficient for approximately 200 250 runs on the GeneTitan MC Instrument Lu IMPORTANT Make sure you remove the cap from the gun when you deionize a tray or cover Deionization Procedure The following process provides guidance on how to use the anti static gun on the stain and scan tray covers only See Figure E 3 A WARNING The deionization steps 4 and 5 will damage the HT arrays on the plate Before using the anti static gun ensure that the HT array plates remain in their protective pouch and placed away from the deionization area You must place the scan tray and hybridization tray away from the area where you are performing deionization Treat the plate or lid as if it were divided into 6 sections and deionize as follows 2 Place a Kimwipe on the benchtop Place the stain tray on a table top Use the anti static gun to aim at the center of each of the six sections on a 96 well cover or tray and pull the trigger Ensure that a stream of ionized particles settles on all wells of the stain tray or cover to dissipate the static electricity
151. p workflow you can add resuspension buffer to both plates at the same time and then place them both in the shaker 2 If the Precipitation plate has a seal on it carefully remove the seal from the Precipitation plate and discard the seal 3 Using a P200 12 channel pipette transfer 35 uL Axiom Resusp Buffer to each well of the Precipitation plate with a dry pellet Avoid touching pellets with tip Change pipette tips after each addition After adding Resuspension buffer the plate is known as the Resuspension plate 4 Sealthe Resuspension plate Chapter 4 Axiom 2 0 Assay Manual Target Preparation 48 Put the sealed Resuspension plate on one of the following shakers a Titer Plate Shakers 4PL at speed 9 for 10 min a Jitterbug at speed 7 for 10 min CAUTION It is recommended that the remainder of the steps in this stage be preformed under a fume hood While the Resuspension plate is shaking prepare the Hybridization Master Mix in the Hyb MM 15 mL tube A Add the reagents in Table 4 14 to the Hyb MM tube in the order shown using serological and single channel pipettes as needed Table 4 14 Hybridization Master Mix Reagent per Sample Master Mix 96 To the 15 mL tube labeled Hyb MM add Axiom Hyb Buffer 70 5 pL Axiom Hyb Soln 1 0 5 uL 10 11 12 13 14 15 Axiom Hyb Soln 2 Total Volume 9 uL B Vortex twice to mix Inspect the Resuspension pla
152. plastic protective shipping tray cover Blue Array Plate Protective Base Array Plate Notched corner of array plate aligned with notched corner of blue base Tip Mark the notched corner of each plate cover and tray with permanent marker to help ensure proper alignment and loading Plates and trays must be seated in this groove The notched corner of all plates bases and Covers and must be seated in this corner of the drawer per the Tray Alignment guide Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 69 Figure 5 2 Array Plate with Protective Blue Base and the Hyb Tray Aligned and Properly Loaded into Drawer 6 Array Plate with Protective Blue Base Hyb Tray Lu IMPORTANT When you install the consumables ensure that the fingers are retracted Do not lay the consumables on top of the drawer fingers this indicates that the instrument is not functioning correctly Please notify your Field Service Engineer if the fingers do not retract automatically You should place the trays into the instrument drawers when a drawer is fully extended by the instrument The fingers are retracted when the drawer is open and are extended when the drawer is closed in order to restrain the consumable Stain Trays and Covers Lu IMPORTANT Always place the flat side of the cover against the stain tray Figure 5 3 Placement of Covers on Trays U SS d Notched
153. r sample Master Mix 96 To the 15 mL tube marked D MM add Axiom 2 0 Denat Soln 10X 2 uL 400 uL Axiom Water 18 uL 3 6 mL Total Volume 2 Vortex and leave at room temperature 3 Add Denaturation Master Mix to Samples To Add the Denaturation Master Mix to Your Samples carry out the following steps at room temperature 1 Spin down the Sample plate Remember Samples must be at room temperature for this step Pour the Denaturation Master Mix into the solution basin marked D MM 3 Carefully remove the seal from the Sample plate and discard the seal Using a P20 12 channel pipette and pipetting directly into the liquid of each well add 20 uL of Denaturation Master Mix to each sample of the plate total volume 40 uL well Do not mix by pipetting up and down Change tips between each addition This plate is now known as the Denaturation plate 5 Seal and vortex the Denaturation plate Start the timer for 10 minute incubation 6 Do a quick spin on the Denaturation plate in a room temperature centrifuge by bringing centrifuge speed to 1000 rpm takes minute NOTE The quick spin time is included in the 10 minute incubation 7 eo so Chapter 4 Axiom 2 0 Assay Manual Target Preparation 37 Visually examine the volume in each well should be 40 pL well and A Keep a record of any wells that visually appear to have a particularly low or high volume these samples may need to b
154. rName AFFXUser 3 10 08 42 AM ExecutablePath C Program Files Affymetrix Command Consoles 10 08 42 AM ProductName HTS6CC 10 08 42 AM ProductVersionSbF 3 0 0 1214 10 08 42 AM LastwiiteTime 96F 9 10 2009 6 12 00 PM 10 08 42 AM ProductVersion96S canner 3 0 0 1214 10 08 42 AM LastWriteTime S6Scanner 9 10 2009 6 11 56 PM 10 08 44 AM MED amp 365 files Copied 30 10 08 44 AM AuditLogDir set to C Command_Console Logs 96F 10 08 44 AM LogFileDir set to C Command_Console Logs 96F 10 08 44 AM Timer started with Interval 1000 msec 10 08 44 AM Homing HTSBF and Scanner 10 08 44 AM Set HybOven temperature to 48 C 10 08 44 AM Set WashB temperature to 39 C Select the System Setup tab 2 Load an Axiom array plate and hyb tray in the same manner that you loaded the previous plate and tray A Scan or manually enter the Axiom array plate barcode then click Next Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 87 B Load the Axiom array plates with the blue base and the hyb tray without the cover then press the Confirmation button C Select the arrays to scan then click Next D Ensure that the plates are clamped securely when prompted then press the Confirmation button E Click OK when prompted to resume plate processing Figure 5 24 Figure 5 24 Confirm Resume Processing Prompt Confirm Resume Processing This will resume the HybWashScan in the Left P
155. ramethylammonium Chloride As such we recommend the use of a fume hood when using these products during the Manual Target Preparation protocol In all cases customers should use adequate local and general ventilation in order to minimize airborne concentrations For research use only Not recommended or intended for diagnosis of disease in humans or animals Do not use internally or externally in humans or animals Copies of the Material Safety Data Sheets for the kit components are available on the Affymetrix website at www affymetrix com Genomic DNA Preparation and Requirements The general requirements for genomic DNA gDNA sources and extraction methods are described in this chapter The success of this assay requires uniform amplification of the genome starting with relatively intact gDNA To achieve this the gDNA must be of high quality and must be free of contaminants that may affect the enzymatic reactions to be performed For this protocol you will use the Axiom 2 0 Reagent Kit The kit contains a tube labeled Genomic DNA This DNA meets the requirements outlined below and is included for use as a control The size and purity of sample gDNA can be compared with those of the control DNA to assess sample quality The control DNA should also be used routinely as an experimental positive control and for troubleshooting purposes Assay performance may vary for gDNA samples that do not meet the general requirements described below Ho
156. ranted for 500 hours The instructions to replace the lamp are available on the following page After changing the lamp it is necessary to reset the lamp life clock manually a WARNING You must turn off the lamp using the power switch in the rear of the unit and remove the power cord Allow the lamp to cool before attempting to replace the lamp Appendix F GeneTitan Multi Channel Instrument Care 138 Removing the Xenon Lamp 1 Unscrew the four retaining bolts They should be finger tight Figure F 7 Figure F 7 Unscrewing the Bolts Unscrew these four bolts HIGH VOLTAGR DISCONNECT POWER BEFORE REMOVING LAMP ACCESS PANEL Caution Risk DN Shock 2 Place each hand on each side of the blue plastic flange and lift out the lamp in a vertical motion Figure F 8 You must use both hands to remove the lamp successfully Apply equal pressure on each side of the lamp and gently lift Figure F 8 Lifting out the lamp Appendix F GeneTitan Multi Channel Instrument Care 139 Replacing the Lamp CAUTION Ensure that you install the lamp in the correct orientation 1 Hold the lamp by the blue plastic flanges Ensure that the lamp bulb faces inward toward the reflecting mirror Figure F 9 and vertically insert the lamp Figure F 10 2 Replace the warning cover and hand tighten the bolts Figure F 7 Figure F 9 The Reflecting mirror Reflecting Mirror
157. rd or Ae ees He oes TR eee eae 23 Seal Vortex aNd SPIN sis 8 lt 22 2204 4 354646 Rene t pia iq RES Lid bh ede 23 Sample Quantitation 0 eee eee 23 About the Reagents and Master Mix Preparation 0 00 0002 cee eee eee 24 Pipettes and Pipetting 0 0 00 eee eee 25 Contents 4 GeneTitan MC Instrument Consumables 0 0 00 00 000 cee eee 26 Labeling GeneTitan Hybridization and Reagent Trays 0 00 000 cee eee 30 Chapter 4 Axiom 2 0 Assay Manual Target Preparation 00 00 eee ee 32 Stage 1 DNA Amplification llis 33 DIU e Won PRAETERITO a A R re e e A a ee a E e a a aa S EI e ETA aoa a 33 INOUTIREGUINCGS dree eat i E EE NAA E e E GLa E eae a E E EAE 33 Equipment Consumables and Reagents Required 0 2 0 2 0000000 eee 34 1 Initial Setup for DNA Amplification 0 00 00 00 cee 35 2 Prepare the Denaturation Master Mix ilslssslssi ene 36 3 Add Denaturation Master Mix to Samples nananana anaa 000 eee eee 36 4 Add Neutralization Solution to Samples illii 37 5 Prepare and Add the Amplification Master Mix liliis 37 6 Freeze or Proceed 4 4 sexed cidr tetta UR Idee icis dui oP ERT SSS Bea dd 38 Stage 2 Fragmentation and Precipitation n saaa asuaan eee 38 IPIE CIO RECEPIT 38 Input Required lsissssssele s 38 Equipment Consumables and Reagents Required liiis eee eee 39 1 Stop Amplification Reaction liississeseee ian DEERE
158. rom the 65 C oven to the 37 C oven and incubate for 45 minutes 5 Set the plate centrifuge to room temperature TIP Keep a labeled balance plate of equal weight ready to minimize any time delay before spinning the Fragmentation plate during later steps 6 Labelthe 15 mL and 50 mL conical tubes as indicated in the table below Label Tube Size Temperature Contents a Frg MM 15 mL Place tube on ice Fragmentation Master Mix a Precip MM 50 mL Place tube at room temperature Precipitation Master Mix 7 Label solution basins as indicated in the table below Label Temperature Contents a Frg MM Leave basin at room temperature Fragmentation Master Mix Stop Leave basin at room temperature Frag Rxn Stop Precip MM Leave basin at room temperature Precipitation Master Mix a SO Leave basin at room temperature Isopropanol 2 Prepare Fragmentation Master Mix To Prepare the Fragmentation Master Mix 1 Start making the Fragmentation Master Mix when there is still five minutes to the finish of the 37 C incubation using the values in the table below Transfer the Axiom Frag Enzyme to a 20 C portable cooler until ready to use Table 4 9 Axiom Fragmentation Master Mix Reagent per sample Master Mix 96 To the 15 mL tube marked Frg MM add Axiom 10X Frag Buffer 45 7 uL 6 0 mL Axiom Frag Diluent 10 3 uL 1 35 mL Axiom Frag Enzyme 1 0 uL 131 0 uL Total Vo
159. rs on page 130 Place the cover as shown in Figure 4 4 on page 66 to prevent dust or static from accumulating on the bottom of the cover Use a 12 channel P200 pipette with new pipette tips to aliquot 150 uL to each well of a scan tray dispense to the first stop and avoid touching the bottom of the tray You do not need to change pipette tips between additions of the Hold buffer If droplets of liquid splashed onto the well dividers place a Kimwipe on top of the tray to blot and remove Cover the tray by orienting the notched corner of the scan tray cover over the notched edge of the tray and the flat side of the cover against the scan tray Figure 4 3 Lu IMPORTANT If your scan tray came with a cover that does not match the picture of the scan tray cover or if your scan tray cover does not have a barcode then please contact your Field Application Specialist for a replacement scan tray cover All tray covers must have a machine readable barcode IMPORTANT The Hold buffer requires 150 pL per well CAUTION Do not remove the scan tray from its protective black base until loading onto the GeneTitan MC instrument To avoid scratching do not touch the bottom of the tray with pipette tips Dispense hold buffer to the first stop only See Stage 3 Ligate Wash Stain and Scan on page 89 for instructions on loading the reagent trays Chapter 4 Axiom 2 0 Assay Manual Target Preparation Figure 4 3 Scan Tray wit
160. s dilution is 120 fold See Appendix B Sample Quantitation after Resuspension on page 116 for more information on performing the Sample Quantitation Make Gel Samples A B C Add 120 uL gel loading dye to each well of the Gel Sample Plate Transfer 3 uL of each QC Dilution Plate sample to the Gel Sample Plate Change pipette tips after each transfer Seal vortex and spin the plate Run gel as described in Appendix A Fragmentation Quality Control Gel Protocol on page 114 After the QC checks the QC dilution plate OD plate and remaining gel samples can be discarded once satisfactory results from the gel and OD 260 readings have been obtained Chapter 4 Axiom 2 0 Assay Manual Target Preparation 50 5 Freeze or Proceed At this point you can a Proceed to Stage 4 Denaturation and Hybridization below or Store the Hyb Ready samples at 20 C Stage 4 Denaturation and Hybridization You will proceed to Stage 4 in one of two ways Directly from Stage 3 without interruption With Hyb Ready samples that were stored at 20 C after Stage 3 To Perform Stage 4 If the Hyb Ready plate was stored at 20 C go to 1 Prepare Hyb Ready Samples Stored at 20 C on page 52 If you are proceeding directly from the end of Stage 4 Denaturation and Hybridization on page 50 go to 2 Prepare Equipment and Perform Denaturation on page 52 CAUTION Parts of this stage should be performed under
161. s warranted for 500 hours to provide illumination for imaging the array at two wavelengths The xenon lamp has a limited lifetime and needs to be replaced at regular intervals The GeneTitan Instrument Control software provides a timer that indicates the remaining useful life of the bulb and notifies you when it requires replacement It is important to adhere to the warnings specified in the GeneTitan MC Instrument user guide Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 72 Refer to the GeneTitan MC Instrument User Guide P N 08 0308 or Appendix F GeneTitan Multi Channel Instrument Care on page 133 of this user guide for details on replacing the lamp Refer to the GeneTitan MC Instrument User Guide P N 08 0308 for the Lambda LS and Smart controller system The Lamp and the controller should NEVER be switched ON or OFF manually The GeneTitan MC Instrument control software manages the lamp activity and will switch the lamp ON and OFF as required It takes 10 minutes to warm up the lamp In idle mode the lamp will remain ON for 2 hours before it is automatically switched OFF and if there are no more plates being transferred from the fluidics to the imaging station This is by design and intended behavior Please do not try to save the lamp life by turning OFF the switch on the lamp NOTE The power switch on the shutter box should be ON at all times The OPEN CLOSE switch on the shutter box should be at AUTO pos
162. samples and operators all equipment must be regularly calibrated and well maintained including All pipettes thermal cyclers and ovens Plate spectrophotometer Chapter 3 Axiom 2 0 Assay Preparation Before You Start 23 Procedures This section covers procedures you may need to do repeatedly during the workflow or which are critical to the performance of the assay Seal Vortex and Spin Unless otherwise noted when the protocol instructs you to seal vortex and spin Seal plates we recommend using MicroAmp Clear Adhesive Films to seal your plates IMPORTANT Always ensure that your plates are tightly sealed A tight seal will prevent sample loss and cross well contamination particularly when plates are being vortexed Spin when instructed to perform a brief spin down of plates or reagent vials follow these guidelines unless otherwise instructed o Plates Spin at room temperature Start the centrifuge allow it to reach 1000 rpm and spin for 1 min n Reagent Vials 3 sec Vortex reagents 3 times sec each time Vortex plates o For deep well plates such as ABgene 2 2 mL square well storage plates vortex 5 seconds in each sector for a total of 5 sectors Figure 3 1 o For PCR plates such as Bio Rad Hard Shell or semi skirted plates vortex 2 seconds in each sector for a total of 5 sectors Figure 3 1 Figure 3 1 Vortexing Plates 7 VU O OO OO OO
163. see Stage 3 Drying Resuspension and QC on page 44 Sample Denature load array plate and hyb tray in the GeneTitan MC Instrument see Stage 4 Denaturation and Hybridization on page 50 AETH GeneTitan reagent trays prep and load see Stage 5 Manually Preparing Ligation Staining and Stabilization e a e ey le Reagent Trays for the GeneTitan MC Instrument on page 55 Table 6 9 Manual Target Prep Workflow Day 4 Activities Activity Plate ID Approximate Start Times Denature and Hyb B 8 45 a m Centrifugation Drying Resuspension QC C 9 30 a m GeneTitan reagent prep and loading A 3 30 p m Denature and Hyb C 4 15 p m Chapter 6 Manual Target Preparation for Processing Three Axiom Array Plates per Week 107 Manual Target Prep Workflow Day 5 GeneTitan reagents prep and loading for Plates B and C Lu IMPORTANT The GeneTitan reagent trays for array processing cannot be loaded until the array plate has finished hybridization and they should not be prepared more than 1 5 hours before hybridization will finish The GeneTitan reagent trays cannot be prepared ahead of time and stored Figure 6 6 Manual Target Preparation Workflow Day 5 Activities Day 5 a m Day 5 p m 8 B 10 11 12 1 2 3 4 5 Plate A B GT Reagent A prep load C GT Reagent A prep load Notes A Begin thawing required reagents Col
164. sensitive Keep tubes out of direct light for a prolonged period of time N A not applicable in this case NOTE Occasionally crystals are observed in Axiom Wash A and Axiom Stabilize Diluent upon removal from 2 8 C storage Before using these solutions the crystals should be dissolved by warming the solutions to room temperature and then vortexing Chapter 4 Axiom 2 0 Assay Manual Target Preparation 59 2 Prepare the Stain Ligation and Stabilization Master Mixes Label the Tubes and Solution Basins To Label the Tubes 1 Mark the side of each tube with one of designations shown in Table 4 25 Table 4 25 Labeling Master Mix Tubes for Stain Ligation and Stabilization Reagents Conical Tube Number of Tubes Tube Designation Contents Place Tube 50 mL 1 S1 a Stain 1 Master Mix On ice 15mL 1 S2 a Stain 2 Master Mix On ice 15 mL 1 Stbl Stabilization Master On ice Mix 15 mL 1 Lig a Ligation Master Mix On ice NOTE Use a 5 mL or 10 mL serological pipette to transfer Axiom Wash A Axiom Water and Axiom Ligate Buffer These bottles have narrow openings and a 25 mL serological pipette will not fit 2 Mark the side of each solution basin with one of the designations shown in Table 4 26 Table 4 26 Labeling Solution Basins Basin Designation Contents S1 a Stain 1 Master Mix S2 a Stain 2 Master Mix Stbl a Stabilization Master Mix Lig a Ligation Master Mix Hold a Axi
165. strument Chapter 4 Axiom 2 0 Assay Manual Target Preparation 64 Stain 2 Master Mix To Aliquot the Stain 2 Master Mix 1 4 5 Pour the Stain 2 Master Mix into the solution basin marked 2 placed on the bench top at room temperature Using a P200 12 channel pipette with new pipette tips aliquot 105 uL per well to the S2 tray dispense to the first stop You do not need to change pipette tips between additions of the Stain 2 Master Mix If Bubbles are present puncture them with a pipette tip Droplets of liquid splashed onto the well dividers place a Kimwipe on top of the tray to blot and remove Place a cover on the 52 tray Orient the cover correctly on the tray with the notched corners together Figure 4 2 Protect the tray from light if not immediately loading onto the GeneTitan MC Stabilization Master Mix To Aliquot the Stabilization Master Mix 1 Pour the Stabilization Master Mix into the solution basin marked Stbl placed on the bench top at room temperature Using a 12 channel P200 pipette with new pipette tips aliquot 105 uL per well to the Stbl tray dispense to the first stop You do not need to change pipette tips between additions of the Stabilization Master Mix If Bubbles are present puncture them with a pipette tip a Droplets of liquid splashed onto the well dividers blot the top of the tray with a Kimwipe Place a cover on the tray Orient cover correctly on
166. t improvement over original hybridization Re hybridization of hyb cocktail results in data that passes performance metrics and is significantly better than original hybridization Problems in original hyb were not due to target prep Possibly due to the array array processing and or reagents used for array processing Fail No improvement over original hybridization Re hybridization of hyb cocktail results in data that is not significantly better than the original hybridization Inconclusive Target Prep may be suspect however cannot exclude problems with array and reagents or array processing issues Affymetrix has not established an upper limit for how long the hybridization tray can be stored at 20 C prior to re hybridization We have been successful in re hybridizing plates that have been stored up to 11 days but individual results may vary as success depends on the quality of the original target prep and amount of target available for re hybridization Equipment Consumables and Reagents Required Equipment and Consumables Required Multi channel pipette and tips P200 PCR plate BIO RAD P N HSP9631 or o PCR Plate HSS 9601 plate for use with manual target prep a Axiom Genome Wide or Custom myDesign Array Plate Kit 96 array Axiom GeneTitan Consumables Kit Reagents Modules 3 and 4 of the Axiom 2 0 Reagent Kit Water nuclease free ultrapure molecular biology grade Appendix
167. t is met This is to ensure that the second plate does not have to wait for system resources in its workflow The time spacing is roughly equal to the longer of the wash stain or scan time of the first plate Figure 5 23 Loading a Second Hyb Tray and Hybridization Oven Status Information Work Flow Barcode Plate Type Location Hyb Stat RP IF EKIBREIIEDEEN 550032 Left Position NoAction Additional plates cannot be loaded before or after this period of time while the instrument is operating Estimated Time Window to Run Next Hyb Wash Scan In this figure the system is currently available S E This pane displays the period of time during which Samik aribawa another array plate and hyb tray can be loaded Hybridization Oven Status HT Array Type TRU chad lt lt Position of plate stack in the hybridization oven Only 1 Gn essent dus plate being processed in this figure As such position 2 is blank E Barcode Position 1 left side of oven osition 2 Estimated Time Remaining Position 2 right side of oven Oven Temperature Curent 48 1G j Green indicates the current oven temperature is within Target 48 C the target temperature range cn Yellow indicates oven temperature outside of target temperature range 10 08 42 AM HTSBCC started at 10 1 2009 10 08 34 AM 10 08 42 amp M MachineName D254RJF1 10 08 42 AM OSVersion Microsoft Windows NT 5 1 2600 Service Pack 3 10 08 42 AM Use
168. t may not be able to successfully complete the step that was in progress Wash B if you intend to load two array plates on the same day fill the Wash B bottle to the 1L mark use both bottles from the Axiom 2 0 Reagent Kit 2 Empty the waste bottle 3 Press the Confirmation button on the GeneTitan MC Instrument to continue A fluidics check is run 1 min Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 80 Figure 5 12 Example of the Remaining Workflow Steps Workflow Steps Ent Workflow Step EE Empty trash bin Remove consumable trays and plates Load consumable trays and plates Select arrays to scan Start Processing Specific instructions for gt Buffer bottles have been depressurized each workflow step Please refill buffer into the battles Empty the waste bottle Press the Confirmation button when done This is followed by a Fluidics check 1 Minute Cancel B Empty trash bin 1 Open the trash bin and empty If already empty the trash bin remains locked and the Status pane reads Trash bin is empty 2 Press the Confirmation button to continue C Remove consumable trays and plates 1 Remove used trays and plates when drawers open If no consumables to remove the Status window reads Drawers are empty 2 Press the Confirmation button to continue D Continue to Load an Axiom Array Plat
169. t of Module temperature the reagent is held at immediately after removal from module N A not applicable in this case Room Temp Invert 2 3X for mixing before filling GT bottle Room Temp Invert 2 3X for mixing before filling GT bottle Room Temp B Set up the GeneTitan MC Instrument see Setup the Instrument on page 76 Chapter 4 Axiom 2 0 Assay Manual Target Preparation 53 C Upload the Batch Registration File see Stage 1 Create and Upload Batch Registration File on page 75 3 Prepare Hybridization Tray and Load into GeneTitan MC Instrument CAUTION It is recommended to perform the next set of steps under a fume hood 1 ur de After the Axiom 2 0 Denature program has completed remove the Hyb Ready plate from the thermocycler and place into a 96 well metal chamber that has been pre warmed in an oven at 48 C Move the metal chamber containing the denatured Hyb Ready plate to a fume hood Remove Microamp seal from Hyb Ready plate and discard Remove the hyb tray from Axiom Array GeneTitan Consumables Kit from packaging Label the hyb tray See the note below and Figure 3 2 on page 30 for more information Lu IMPORTANT It is critical that you write only on the proper location of the hyb tray on the edge in front of wells A1 and B1 as illustrated in Figure 3 2 on page 30 Do NOT write on any other side as this can interfere with sensors inside of the
170. t you should begin at Step 1 of 3 Prepare Hybridization Tray and Load into GeneTitan MC Instrument on page 53 n If the samples have already been transferred to the hyb tray the hyb tray should be sealed with plate sealing film and placed in an oven at 48 C until the GeneTitan MC Instrument is ready Be sure to remove the plate sealing film before loading into the GeneTitan MC Instrument Chapter 4 Axiom 2 0 Assay Manual Target Preparation 55 Stage 5 Manually Preparing Ligation Staining and Stabilization Reagent Trays for the GeneTitan MC Instrument This stage needs to be done when hybridization in the GeneTitan MC Instrument is near completion 1 5 hours before completion so the reagent trays can be loaded for the GeneTitan MC array processing steps Total time for this step 1 5 hours including reagent preparation hands on time and GeneTitan MC Instrument loading Lu IMPORTANT The reagent trays prepared in this step Stage 5 Manually Preparing Ligation Staining and Stabilization Reagent Trays for the GeneTitan MC Instrument are for the continued processing of an Axiom array plate that has completed the hybridization stage is ready for transfer to the fluidics area The reagent trays for the fluidics stage on the GeneTitan MC Instrument should not be prepared in advance Do not prepare these plates if there is no array plate ready for the fluidics stage Once prepared these plates must be loaded onto the i
171. te Shipping Cover from damage To be Discarded Chapter 3 Axiom 2 0 Assay Preparation Before You Start 27 Table 3 3 Axiom GeneTitan Tray Consumables from the Axiom GeneTitan Consumables Kit P N 901606 Continued Item Part Number Labware Image Information ET Barcoded Scan tray cover PN 202757 GeneTitan Scan Tray PN 501006 or PN 500860 San Ty protective base The HT scan tray shipping package includes the following a The HT scan tray includes a scan tray cover The tray cover should be used to cover the scan tray before placing the tray in the GeneTitan MC Instrument The Tray cover should be de ionized before use See the section Appendix E Deionization Procedure for GeneTitan Trays and Covers on page 130 for the anti static procedure The scan tray must be protected at all times from damage or exposure to dust The scan tray must be in the black plate cover at all times The black scan tray protective base in the package is used to protect the scan tray glass from damage The black scan tray protective base is distinct from the blue array plate protective base and must not be used with the array plate Remove the protective base from the scan tray before loading the scan tray with the scan tray cover Black Scan P N 202096 Tray protective base The black scan tray protective base in the package is used to protect the scan tray
172. te centrifuge at room temperature by bringing the centrifuge to 1000 rpm and stopping it Lu IMPORTANT Keep your timer in a safe place It is helpful to note down the actual time when the incubation began in case the timer stops accidentally Quickly transfer plate to 37 C oven and incubate for 30 min P CAUTION Be watchful for the end of the thirty minute incubation period Fragmentation is an exact 30 minute incubation step Longer and shorter incubation times may lead to poor performance of the assay Prepare the Stop solution a few minutes before the end of the 30 minute incubation period as described in 4 Aliquot the Stop Solution to the Fragmentation Plate below 4 Aliquot the Stop Solution to the Fragmentation Plate To Add the Stop Solution carry out the following steps at room temperature 1 A few minutes before the end of the 30 minute incubation period pour the Axiom Frag Rxn Stop solution in the solution basin labelled Stop Remove the Fragmentation plate from the oven and place on the bench top at room temperature At the end of the 30 minute fragmentation incubation period carefully remove the seal from the Fragmentation plate and discard the seal Chapter 4 Axiom 2 0 Assay Manual Target Preparation 43 4 Using a P20 12 channel pipette end the fragmentation reaction by adding 19 uL of Stop Solution to each reaction Pipette directly into the liquid of each well Change tips after each a
173. te from the bottom If the pellets are not dissolved repeat Step 5 Quickly spin at 1000 rpm Label a Bio Rad Hard Shell 96 well plate HSP 9631 as Hyb Ready Sample ID and keep covered When using the ABI 9700 or ABI 2720 thermal cyclers you should label a Bio Rad Full Height 96 Well Semi Skirted PCR Plate P N HSS 9601 as Hyb Ready Sample ID and keep covered Set a P200 12 channel pipette to 45 uL this is slightly higher than the volume of sample in each well Using the P200 pipette transfer the entire contents of each well of the Resuspension plate to the labeled Hyb Ready plate Change pipette tips after each transfer Pour the Hyb Master Mix to the solution basin labelled Hyb MM placed Using a P200 12 channel pipette add 80 uL of the Hyb Master Mix to each well of the Hyb Ready plate Change tips after each addition Seal vortex twice and spin Prepare the dilutions for the QC steps as described in the next section Chapter 4 Axiom 2 0 Assay Manual Target Preparation 49 4 Recommended Perform Quantitation and Fragmentation QC Checks Before proceeding to Stage 4 Denaturation and Hybridization we highly recommend that you perform quantitation and fragmentation quality control checks The QC checks requires 2 each 100 ml solution basin u Label one basin as H20 u Label the second basin as Loading Dye 2 each Bio Rad Hard Shell 96 well plate HSP 9631 or any 96 well PCR plate for making the dilutions
174. ter initialization the System Status tab is selected and the status of the Hybridization Oven is displayed at the bottom of the Log window The status should read lt Time of day gt System Ready NOTE The instrument control software will display a warning if it detects a problem during the fluid dispense operations The filters in the GeneTitan Wash A Wash B and DI Water bottles should be replaced if the software displays such a warning Refer to Appendix F GeneTitan Multi Channel Instrument Care on page 133 for the message displayed to the user and the procedure for replacing the filters Lu IMPORTANT Please do not close the scanner application by right clicking on it and choosing the Close option This will cause the scanner application to exit abnormally and cause undue delay in processing the next plate The correct way to close the application is described in Shutting Down the GeneTitan MC Instrument on page 99 Command Console aj AGCC Portal r AGCC Viewer Kh GeneTitan Library File Installer Email Configuration E ditor e Data Uploader Resources ASCH Affymetrix com ASC Support at Affymetrix com ASC NetAtts System ready Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument l Hybridization Oven Status Barcode Position 1 Estimated Time Remaining Barcode Position 2 Estimated Time Remaining Oven Temperature Target
175. the tray with the notched corners together Figure 4 2 Ligation Master Mix To Aliquot the Ligation Master Mix 1 Pour the Ligation Master Mix into the solution basin marked Lig placed on the bench top at room temperature Using a 12 channel P200 pipette with new pipette tips aliquot 105 uL per well to the Lig tray dispense to the first stop You do not need to change pipette tips between additions of the Ligation Master Mix If Bubbles are present puncture them with a pipette tip Droplets of liquid splashed onto the well dividers place a Kimwipe on top of the tray to blot and remove Place a cover on the tray Orient cover correctly on the tray with the notched corners together Figure 4 2 Protect the tray from light if not immediately loading onto the GeneTitan MC Chapter 4 Axiom 2 0 Assay Manual Target Preparation 65 Axiom Hold Buffer To Aliquot the Axiom Hold Buffer to the Scan Tray 1 Ensure that the Axiom Hold Buffer has equilibrated to room temperature Vortex and then pour the Axiom Hold Buffer into the solution basin marked Hold placed on the bench top at room temperature Remove the scan tray from its pouch Remove the scan tray cover but leave the scan tray on its protective black base Prepare the barcoded scan tray cover P N 202757 that came with the scan tray by completing the deionization procedure described in Appendix E Deionization Procedure for GeneTitan Trays and Cove
176. ther a Drawer 2 plates and trays a Trash Bin covers Indicates that an item is in the gripper and normal startup of the The drawer names will reflect the location left or Right GeneTitan Multichannel Instrument is not possible The item must be and the drawer number 1 through 6 removed from the instrument before you can begin processing array Examples plates Drawer2L_Hta_DOWN Scan Tray on left side of drawer 2 HtaHyb Clamped Hyb Tray and Array Plate Drawer n L R Hta DOWN where n is the drawer number and L or R to indicate the left or right side The Hta second term indicates the item held An example is drawer1R HtaHyb DOWN indicating it is an array plate with a Hyb Tray or Drawer2L ScanHta Pk DOWN indicating it is an array plate with a scan tray Recover gripped item 550032 laureenxxxxxxxx to location HtaIn Hta DOWN DRAWER NOT RETRACTED ERROR Drawer 1 is not fully closed 6 16 2009 3 38 49 PM The drawers are in an unexpected state not fully retracted You must ensure all drawers are in their Fully retracted state before continuing Manual adjustment is required Open the drawer cover and push the drawer in Fully Press Retry to continue or Cancel to abort the plate Plate 550032 34567892Z2xxxxxx The drawer listed in the message is not fully closed Manually push the drawer back into the instrument until it is fully closed There are two stop positions with audible clicks push until you hear the seco
177. to room temperature 1 hour Axiom Hybridization Buffer Vortex and keep at room temperature Axiom Hybridization Solution 1 Thaw vortex spin and keep at room temperature Axiom Hybridization Solution 2 Vortex spin and keep at room temperature Following centrifugation empty the liquid from the Precipitation plate as follows A Carefully remove the seal from the Precipitation plate and discard the seal B Invert the plate over a waste container and allow the liquid to drain C While still inverted gently press the plate on a pile of Kimwipes on a bench and leave it for 5 min Turn the plate top side up and place in an oven for 20 min at 37 C to dry NOTE If using an Affymetrix 645 oven turn off the rotor during the 20 min drying time If you are proceeding directly to 3 Resuspension and Hybridization Master Mix Preparation on page 47 you can prepare the Hybridization Master Mix at this time Step 6 on page 48 You should also prepare the consumables detailed in 2 Prepare the Tubes Basins and Trays for Resuspension and Hyb Master Mix Preparation on page 47 Chapter 4 Axiom 2 0 Assay Manual Target Preparation 47 8 After 20 min remove the plate from the oven and either a Proceed directly to 3 Resuspension and Hybridization Master Mix Preparation on page 47 even if some droplets of liquid remain Leave the Precipitation plate at room temperature or a Tightly seal the plate and store at 20 C if not proceedin
178. ture 39 C Current Target M Oven Temperature Current Target 48 C Log 11 08 40 AM OSVersion Microsoft Windows NT 5 1 2600 Service Pack 3 11 08 40 AM UserName AFFXUser 11 09 40 AM ExecutablePath C Program Files Affymetrix Command Consoles 11 08 40 AM ProductName HTS6CC 11 03 40 AM ProductVersion36F 3 0 0 1212 11 08 40 AM LastwiriteTime S6F 8 29 2009 10 10 38 PM 11 09 40 AM ProductVersion36Scanner 3 0 0 1212 11 08 40 AM LastwiriteTime 96Scanner 8 29 2009 10 16 10 PM 11 03 41 AM MED amp 965 files Copied 45 11 08 41 AM AuditLogDir set to C Command_Console Logs 96F 11 09 41 AM LogFileDir set to C Command_Console Logs 96F 11 03 41 AM Timer started with Interval 1000 msec 11 08 41 AM Set WashB temperature to 39 C 11 10 13 AM Homing HTS6F completed 11 10 13 AM Initializing Scanner 11 10 13 AM Status Scanner drawer not extended or no plate present 11 10 13 AM ScannerOn Option is on 11 11 13 AM Scanner homing completed 11 11 13 AM Checking and removing plate from scanner 11 11 42 AM Status No Plate in Scanner System ready for running 11 11 45 AM System Ready 11 11 45 AM System Ready 11 11 45 AM Lamp Life Remaining is 143 hours Protocol Log Step Task Time Fluidics Status Plate Status Log z o m im o o e L1 Will Be Scanned Ll Now
179. ulti Channel Instrument Care on page 133 for the message displayed to the user and the procedure for replacing the filters Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 91 Load Trays onto the GeneTitan MC Instrument To Load Trays onto the GeneTitan MC Instrument When hybridization of an Axiom array plate has finished a message Figure 5 29 will alert you to resume the workflow setup Press OK and the software takes you directly back to the System Setup tab Figure 5 29 The Resume Workflow Setup Message ll Resume Workflow Setup zl x 3 30 2010 9 42 24 AM Array Plate S5009POOOOOOOOOOOOOOKK Press OK for reagent loading to continue using the System Setup tab This prompt to continue into reagent load Figure 5 29 occurs when the hyb is complete Estimated Time Remaining displayed under Hybridization Oven Status may display a time remaining of 0 to 30 minutes when the prompt occurs The GeneTitan MC Instrument will allow reagent load to take place after either a the estimated time counts down to zero or a the actual real world hyb time as indicated by the computer clock indicates the hyb is complete NOTE The time estimate displayed on some systems may lag due to high CPU utilization The GeneTitan MC Instrument allows the workflow to synchronize with the system clock to compensate for this situation during the final half hour of the hyb time estimate Whe
180. warning message boxes for the filter in each reagent bottle when it detects a problem or shows a trend of increased fill times during fluid fill operations If an error is detected as described above then a message box titled Filter Change Required is displayed Figure F 2 along with the information on the specific dispense operation You should change all three filters when a warning is displayed for any one of the three filters Figure F 2 Filter change required messages il Filter Change Required BUFFERA_TO_WASHA Rei xl 3 9 2010 5 21 00 PM Waming E EHI E ee PON NEUE OO EEE bow pcena wv ov an Dole You need 3 fitters per GeneTRan Problem dispense fot BUFFERA TO WASHA is Ested below 1 27 2010 4 29 16 PM dispense time remaining before timeout was too short 1 75 sec 1 27 2010 4 41 45 PM dispense time remaining before timeout was too short 1 407 sec This weening will stop appearing when have been replaced or 5 acceptable dispenses have been recorded The lest S alepense erties for BUFFER TO WASHA were 1 27 2010 44148 PM depente tme temaning 12407 ee time n 1 27 2010 4 26 28 PM dispense time remaning 1956 si d 1 27 2010 4 29 16 PM dispense time remaning 1 75 1 27 2010 4 41 45 PM dispense time remaining 140 fel Anay plates processed with dity fiters in buffer ot rinse bottles may exhibit quality issues ae changed now Do not change fiters while GeneTitan is processing plates Please contact your local
181. ween both versions and may be used from either kit for this assay Only use new Module from the Axiom 2 0 Reagent Kit for the Axiom 2 0 Assay Amount of gDNA Required The Axiom Genome Wide Pan African Array Set requires 100 ng of gDNA per array This array is packaged as a three array set so for each gDNA sample a total of 300 ng is required 100 ng array x 3 arrays in the Axiom Genome Wide Pan African Array Set 300 ng gDNA of each sample The Axiom Genome Wide BOS CEU ASI CHB and EUR still require 200 ng gDNA per sample Manual Method and Equipment Consumable Changes New Vortex guideline For deep well plates such as ABgene 2 2 mL square well storage plates the vortexing time per sector has been increased to 5 seconds Refer to Seal Vortex and Spin on page 23 Only 3 incubator oven temperatures are now required during the assay 37 C 65 C and 48 C Refer to Table 6 5 on page 102 Manual Assay Workflow Changes There are many changes in Axiom 2 0 manual assay workflow including how the reagents are handled out of modules various master mixes preparation incubation times and incubation temperature changes The Amplification stage of the assay has changed most These changes are indicated for all the five stages but step by step instructions are detailed in each section of Chapter 4 Axiom 2 0 Assay Manual Target Preparation on page 32 Amplification Stage Changes All reagents of Module 1 except Amp
182. wer being opened Step currently executing in Fluidics Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 98 Continuing the Workflow Once a plate has gone through the fluidics stage of the process it is moved to the imaging device When the scanning process begins the window shown in Figure 5 37 is displayed This window must remain open while Axiom array plates are being scanned CAUTION a The Scan Control window must remain open while Axiom array plates are being scanned Closing this window will halt the scanning process You can minimize this window if necessary without creating any interference to the imaging a Do not manually or through the AGCC transfer utility move any data associated with the current plate that is being processed scanned Transferring data will dramatically slow scanning and may cause the computer to freeze Figure 5 37 Scan Control Window This window must remain open while scanning is in progress If you close this window scanning will stop and delay sample processing Piate Properties AFFYMETRIX cy Current Status Scanning subarrays Estimated Time Remaining O1h 17m Storing Hyb Trays for Rehybridization Hyb trays can be stored after processing for the purpose of rehybridization Store hyb trays as directed below See also Appendix C Rehybridization on page 125 for more information To Store a H
183. wever the reliability of any given result should be assessed in the context of overall experimental design and goals The genomic DNA requirements and preparation are described in the following sections Sources of Genomic DNA a General Requirements on page 12 Genomic DNA Extraction Purification Methods on page 13 a Genomic DNA Cleanup on page 13 Genomic DNA Preparation on page 14 Sources of Genomic DNA The following sources of human gDNA have been successfully tested in the laboratories at Affymetrix for DNA that meets the above requirements Blood Saliva Cell line a WGA pre amplified DNA Genomic DNA amplified with the REPLI g Kit a whole genome amplification kit QIAGEN P N 150025 has been tested successfully with the Axiom 2 0 Genome Wide Human Reagent Kit Assay The REPLI g Kit was used to amplify 20 ng genomic DNA and the resulting yields were quantitated by a PicoGreen assay The amplified products either 100 or 200 ng amplified DNA as required according to the Axiom array type were used without purification as the input DNA sample in the subsequent Axiom 2 0 Assay steps The stability of this amplified product to storage and repeated cycles of freeze thaw have not been evaluated by Affymetrix Success with other types of samples will depend on quality degree of degradation level of purity etc and quantity of gDNA extracted The following sources of bovine gDNA have been successfully tested in th
184. y files as these can cause problems 3 AGCC system configuration file located at C Command Console Configuration Calvin System config Pending job order files located in C Command_Console Jobs 5 Other AGCC related information such as A The number of files under CXCommand Console Data including sub directory B If the system is a networked system or a standalone system C Other applications installed on the system such as antivirus application MS Office Internet Explorer versions Appendix F GeneTitan Multi Channel Instrument Care 143 AGCC Log Files for GeneTitan MC Systems Log files for the GeneTitan MC Instrument control processes are placed in subdirectories of the Command Console Logs folder Affymetrix may need the following files for troubleshooting GeneTitan MC Fluidics 1 C Command_Console Logs 96F A subdirectories named by date e g Log7 29 2009 1 Collect all dated directories and contents since the GeneTitan application was started not just the date of the event some logging goes into files from the date the application started so this can be critical for us 2 Absolutely required are all the log directories from the date the run was started to the date of the event 2 C Command_Console Logs 96F FluidicErrorLog all files in this directory GeneTitan MC Imaging Device 1 C Affymetrix GeneChipHTScanControlIMC Log collect all dated directories and contents since the GeneTitan applicatio
185. yb Tray with Processed Samples 1 After completion of the GeneTitan MC Instrument run unload the hyb tray and tightly seal it with an adhesive film The plate must be well sealed to prevent cross contamination between samples 2 Press the four corners and sides of the tray to ensure that there is no space between the seal and plate Store the hyb tray at 20 C Chapter 5 Array Processing with the GeneTitan Multi Channel Instrument 99 Shutting Down the GeneTitan MC Instrument This procedure assumes that all of the Axiom array plates loaded onto the GeneTitan MC Instrument have been processed nn WARNING Do not attempt to shut down the GeneTitan MC Instrument while array plates are being processed To Shutdown the GeneTitan MC Instrument 1 On the System Setup page open the Setup Options drop down menu and select Unload Plates Unload all of the consumables as prompted Power off the GeneTitan MC Instrument by opening Tools gt Shutdown from the menu ML Exit the AGCC software if it does not close automatically NOTE If the instrument is processing an array plate the software will not allow you to shut down the system 6 Manual Target Preparation for Processing Three Axiom Array Plates per Week When using the manual target prep protocol one person can process up to three Axiom Genome Wide 96 array plates in one forty hour work week This chapter describes the timing of the steps for each
186. ybridization 98 T Timing issues GeneTitan MC Instrument 102 Manual target Prep 101 Trays 47 alignment and loading into GeneTitan MC Instrument 67 Troubleshooting 108 GeneTitan MC Instrument 108 U Unload Plates 73 WwW Wash Scan Resume 113 Index 146 Wash Scan 73 Wash Scan Resume 73 Workflow 98

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