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Cytoscan HD Assay Manual

1. 40 Affymetrix CytoScan Assay User Manual Table 5 17 Equipment and Consumables Required for Stage 7 Labeling Quantity Item 1 8 12 well strip tubes 0 2 mL As required 8 12 tube strip caps 1 Vortexer 4 GeneMate 96 Well PCR Tube Storage Rack IMPORTANT Use only the thermal cyclers tubes 96 well plates and adhesive film and listed under Thermal Cyclers 96 Well Plate and Adhesive Seals on page 8 Reagents Required The following reagents are required for this stage Table 5 18 Reagents Required for Stage 7 Labeling Reagent 30 mM DNA Labeling Reagent TdT 5X TdT Buffer About Stage 8 Target Hybridization TM During this stage each sample is hybridized onto a CytoScan Array You will 1 Prepare a Hybridization Master Mix and add it to each sample 2 Denature the samples on a thermal cycler 3 Load each sample onto a CytoScan Array 4 Place the arrays into a hybridization oven at 50 C for 16 to 18 hr For the detailed protocol see Stage 8 Target Hybridization on page 91 Location and Duration Post PCR Area Hands on time 45 minutes Hybridization time 16 to 18 hr Equipment and Consumables Required The following equipment and consumables are required for this stage Chapter 5 Assay Overview 41 IMPORTANT Hybridization only in the GeneChip Hybridization Oven 645 is recommended for this assay While prep
2. KG Affymetrix User Manual CytoScan Assay For research use only Not for use in diagnostic procedures Trademarks OAffymetrix Inc All rights reserved 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 Cartridge 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 6 399 365 6 42
3. or equivalent Sterile barrier pipette tips and non barrier pipette tips Tygon Tubing 0 04 inner diameter Cole Parmer H 06418 04 Tough Spots Label Dots 3 8 USA Scientific 9185 0000 100 Affymetrix CytoScan Assay User Manual Reagents Required The following reagents are required for washing and staining arrays These reagents are recommendations and have been tested and evaluated by Affymetrix scientists Table 7 2 Reagents Required for Washing and Staining Arrays Reagent Stain Buffer 1 Stain Buffer 2 Affymetrix GeneChip Array Holding Buffer Affymetrix GeneChip Wash A Affymetrix GeneChip Wash B Fluidics Station and Scanner Control Software You will use the Affymetrix GeneChip Command Console AGCC Version 3 2 2 or higher to operate the fluidics station and the scanner For more information on the AGCC application refer to the Affymetrix GeneChip Command Console User s Guide Prime the Fluidics Station Priming ensures the lines ofthe fluidics station are filled with the appropriate buffers and the fluidics station is ready to run fluidics station protocols Priming should be done When the fluidics station is first started When wash solutions are changed Before washing if a shutdown has been performed a If the LCD window instructs the user to prime The Fluidics Station 450 is used to wash and stain the arrays it is o
4. Cancel 6 7 After you click OK click the Resume icon If any arrays in the carousel are to be rescanned select the check box Allow rescans Shutting Down the Fluidics Station To shut down the Fluidics Station 1 Gently lift up the cartridge lever to engage close the washblock After removing an array from the holder the LCD window displays the message ENGAGE WASHBLOCK The instrument automatically performs a Cleanout procedure The LCD window indicates the progress of this procedure When REMOVE VIALS is displayed in the LCD remove the vials The REMOVE VIALS message indicates the cleanout procedure is complete If no other processing is to be performed place the wash lines into a bottle filled with deionized water Using AGCC choose the Shutdown 450 protocol for all modules Run the protocol for all modules The Shutdown protocol is critical to instrument reliability Refer to the instrument User s Guide for more information When the protocol is complete turn the instrument off Empty the waste bottle 106 Affymetrix CytoScan Assay User Manual IMPORTANT To maintain the cleanliness of the fluidics station and obtain the highest quality image and data possible a weekly bleach protocol is highly recommended see Chapter 9 Fluidics Station Care and Maintenance on page 119 Troubleshooting General Assay Performance Recommendations As with any assay using PCR the Cyto
5. The Affymetrix CytoScan Assay is designed for processing 8 24 samples including controls Instructions are provided for processing 8 16 or 24 samples in parallel The protocol is presented in the following stages a Preparing the Genomic DNA a Stage I Restriction Enzyme Digestion on page 48 Stage 2 Ligation on page 53 Stage 3 PCR on page 58 a Stage 4 PCR Product Purification on page 68 Stage 5 Quantitation on page 75 Stage 6 Fragmentation on page 79 a Stage 7 Labeling on page 87 a Stage 8 Target Hybridization on page 91 Preparing the Genomic DNA This protocol has been optimized using UV absorbance to determine genomic DNA concentrations Other quantitation methods such as PicoGreen may give different readings Set Up the Work Area To set up the work area 1 Place a double cooling block on ice Figure 6 1 2 Place a 96 well plate in the upper half of the cooling block 44 Affymetrix CytoScan Assay User Manual Figure 6 1 Diluting genomic DNA samples to 50 ng uL Genomic DNA samples Low EDTA TE Buffer NOTE The illustrations in this user manual depict the setup recommended for eight samples six genomic DNA samples plus one positive control and one negative control If running less than eight samples follow the same plate layout If running more than eight samples refer to or for more information Dilute the Genomic DNA To dilute the genomic DNA 1
6. 140 Thermal Cycler Programs ves nA E x vada wenes 143 CytOScan Digest aeres ido or he Ed oka wR Obed ei ea he BEES 143 CytOScamligate ie eris bb REIP d ees Bowe Pee bb ES ng 143 CytoScan PCR aue ka he unika iga pei bee DA pi bd Puis 143 For the GeneAmp PCR System 9700 0 143 vi Affymetrix CytoScan Assay User Manual Appendix F CytoScan Fragment 1 0 0 es 144 CytoScarilabel 5 144 CytoscaMm HYD aac ake a eter antt desee d pue ios D A AaS e eres 144 Reagents Equipment and Consumables 145 About this Appendix 0 0 0000 000 ccc es 145 Affymetrix Equipment Required 00 000 eee 145 Affymetrix Software Required 0 000000 145 Affymetrix Arrays Required 0 0 0 000000 eee 146 Affymetrix Reagents Required 0 0000 eee 147 Reagents Required from Other Suppliers 000002000 148 Optional Affymetrix Equipment 2 0 0 0 00002 148 Equipment Required from Other Suppliers 0005 148 Pre PCR Clean Area Equipment Required 005 148 Post PCR Area Equipment Required llli cee eee 150 Consumables Required from Other Suppliers 0 152 Supplier Contact List 0 IIR 153 Introduction Topics in this chapter include a About the Affymetrix CytoScan Solution a About This Manual on page 2 About the Affymetrix CytoScan Solution E IMPORTANT The CytoScan Assay protocol is optimized for
7. 3 Aliquot the diluted genomic DNA samples and controls into the sample processing plate For the detailed protocol see Preparing the Genomic DNA on page 43 Location and Duration Pre PCR Clean Area Hands on time dependent upon number of samples to be processed Using Controls We recommend including one positive and one negative control with every set of samples processed For the positive control use the Genomic DNA included in the CytoScan Assay Kit For the negative control use the Low EDTA TE Buffer included in the CytoScan Assay Kit Equipment and Consumables Required The equipment and consumables listed in Table 5 1 are required for this stage Table 5 1 Equipment and Consumables Required for Preparing the Genomic DNA Quantity Item As required Adhesive seals for 96 well plates 1 Cooling chamber double block chilled to 4 C placed on ice do not freeze 1 Ice bucket filled with ice 1 Marker fine point permanent 1 Mini microcentrifuge microfuge 1 Pipette single channel P20 1 Pipette single channel P100 or P200 1 Pipette 12 channel 2 20 uL Chapter 5 Assay Overview 27 Table 5 1 Equipment and Consumables Required for Preparing the Genomic DNA Continued Quantity Item 1 Pipette 12 channel 20 200 HL As needed Pipette tips for pipettes listed above 2 Plate Bio Rad 96 well unskirted As needed Tubes Eppendorf Safe Lock Tubes 1 5 mL Natur
8. Bright hybridization artifact s obscure gridding oligo locations on the array Try to manually align the grid See the Affymetrix GeneChip Command Console User Manual P N 702569 for instructions If manual grid alignment fails to produce a CEL file repeat the experiment 116 Affymetrix CytoScan Assay User Manual Data QC Failures Likely Cause Solution Low or failing SNPQC a Cross contamination between samples within a plate Contaminated reagents equipment or input DNA Over or under fragmentation of the PCR product Hybridization oven out of calibration or oven model is not compatible with this assay a Repeat assay using a control sample of known integrity such as Ref103 Review and follow best practices o Ensure a tight plate seal at every step a Use fresh filter tips at each pipetting step a Use caution when pooling PCR product a If the problem persists use fresh reagents and fresh input DNA a Decontaminate the pre PCR room and equipment if necessary Process only 6 8 arrays at a time When processing arrays for washing it is important to work quickly as delays in this step will impact data quality Perform all steps after removal of arrays from the oven to the time the washing begins with minimal delays See above a Ensure that only the GeneChip Hybridization Oven 645 is used for this assay Have the oven serviced Elevated or faili
9. 2 TBE precast or house made 1X TBE Buffer Ethidium Bromide Solution 5X RapidRun Loading Dye Plates 96 well reaction 34 Affymetrix CytoScan Assay User Manual About Stage 4 PCR Product Purification During this stage you will purify the PCR products as follows 1 Pool the PCR reactions 2 Add Purification Beads to each pooled reaction and incubate the mix 3 Wash DNA bound beads with Purification Wash Buffer 4 Add Elution Buffer to elute the DNA For the detailed protocol see Stage 4 PCR Product Purification on page 68 Location and Duration a Post PCR Area Hands on time 2 hr DNA binding to magnetic bead 15 to 20 minutes EtOH wash approximately 10 to 20 minutes Elution 15 to 30 minutes Total time for this stage approximately 2 0 to 3 0 hr Equipment and Consumables Required The following equipment and materials are required to perform this stage Table 5 10 Equipment and Consumables Required for Stage 4 PCR Product Purification Quantity Item 1 Adhesive seals for 96 well plates 1 Microcentrifuge Eppendorf 5424 with rotor for 24 tubes 2 0 mL 1 Magnetic stand 1 Marker fine point permanent 1 Microtube Foam Insert for vortexing 2 0 mL tubes 1 Pipette single channel P20 1 Pipette single channel P200 1 Pipette single channel P1000 1 Pipette 12 channel 2 20 HL 1 Pipette 12 channel 20 200 uL 1 Pipette 12 channel
10. Biotech 1 Tough Spots 3 8 USA Scientific 9185 0000 C1 Tube Safe Lock Tube 1 5 mL Amber Eppendorf 022363221 1 Tube Safe Lock Tube 1 5 mL Blue Eppendorf 022363247 1 Tube Safe Lock Tube 1 5 mL Natural Eppendorf 022363204 1 Tube centrifuge 50 mL VWR 93000 036 1 Tube centrifuge 15 mL VWR 21008 103 O Tube strips 8 well 0 2 mL VWR 20170 004 Supplier Contact List Table F 12 Supplier Contact List Appendix F Reagents Equipment and Consumables 153 Supplier Web Site Address Affymetrix www affymetrix com Agilent Technologies www genomics agilent com Applied Biosystems www appliedbiosystems com Bio Rad www bio rad com Bio Smith www biosmith com Clontech www clontech com Diversified Biotech www divbio com E amp K Scientific www eandkscientific com Eppendorf www eppendorf com ESCO www escoglobal com Fisher Scientific www fishersci com Life Technologies www lifetechnologies com ISC BioExpress www bioexpress com Lonza www lonza com Molecular Devices www moleculardevices com Molecular Probes www molecularprobes com NanoDrop www nanodrop com Neptune Scientific www neptunescientific com New England Biolabs www neb com Pierce Biotechnology part of Thermo Fisher Scientific www piercenet com Promega www promega com Rainin www rainin com 154 Affymetrix Cy
11. Thaw the genomic DNA gDNA as follows A Place on the bench top at room temperature until thawed B Once thawed place in the cooling block on ice 6 7 Chapter 6 CytoScan Assay Protocol 45 Vortex the gDNA samples at high speed for 3 seconds Spin down for 1 minute then place back in the cooling block If sample concentration is unknown take an OD measurement of each sample now Consult your spectrophotometer handbook for more information on how to determine the sample concentration Lu IMPORTANT To avoid contaminating samples with PCR product take only an aliquot of each sample not stock to the plate spectrophotometer or NanoDrop Based on OD measurements dilute each sample in a separate well of the 96 well plate to 50 ng hL using Low EDTA TE buffer Lu IMPORTANT Do NOT dilute the Genomic DNA provided in the CytoScan Reagent Kit it is already at a working concentration Seal the plate vortex at high speed for 3 seconds then spin down for 1 minute Place back on the cooling block Aliquoting the Prepared Genomic DNA and Controls Set Up the Work Area To set up the work area i 2 Mark a 96 well plate as shown in Figure 6 2 The digestion and ligation reactions will be performed in this plate Place the plate on the lower half of the cooling block Figure 6 3 on page 47 Figure 6 2 Marking a 96 well plate for digestion and ligation I OOOOOO00k OOO00606
12. large 9L 16 x 13in 41 x 33cm LabScientific RECB 1202 1 96 well tube storage racks GeneMate R 7909 2 O MicroAmp Adhesive Film Applicator Applied 4333183 Biosystems 1 Magnetic stand select one of the following MagnaRack Life Technologies C 15000 DynaMag 2 Magnet Life Technologies 123 21D PureProteome Magnetic Stand Millipore LSKMAGSOS 1 Microcentrifuge 5415D or R Eppendorf 022621408 1 Microcentrifuge Standard Rotor F 45 24 11 24 bores Eppendorf 22636502 1 Microfuge for tubes and strip tubes Any vendor 1 Microtube Foam Insert Scientific 504 0234 00 Industries 1 6 Platform Head for the Microtube Foam Insert Scientific 146 6005 00 Industries 1 Pipette single channel 2 20 uL Rainin L 20 Pipette single channel 20 200 HL Rainin L 200 Pipette single channel 100 1000 uL Rainin L 1000 Appendix F Reagents Equipment and Consumables 151 Table F 10 Post PCR Area Equipment Required Continued Item Vendor Part Number 1 Pipette 12 channel 2 20 HL Rainin L12 20 Pipette 12 channel 20 200 HL Rainin L12 200 1 Pipette 12 channel 100 1200 uL Rainin L12 1200 1 Plate centrifuge multipurpose Eppendorf 5804R or 5810R Refrigerator 4 C 6 cu ft Any vendor Spectrophotometer select one of the following SpectraMax Plate Spectrophotometer Molecular Devices Spectramax Plus384 a NanoDrop NanoDrop ND 1000 1 GeneAmp PCR System 9700 gol
13. the dilution followed by OD reading being sure to vortex the eluted DNA and the OD plate thoroughly at each step Verify instrument and pipette calibration and settings during operation Verify the formula used to calculate the yields from a given O D OD 260 280 ratio is not between 1 8 and 2 0 PCR product may not have been adequately washed An error may have been made while taking the O D readings Ensure that proper volume of absolute ethanol is added to the Purification Wash Buffer and follow the procedure provided in Chapter 6 on page 68 Retake the O D following the instructions provided in Chapter 6 on page 75 OD 320 measurement is gt 0 1 Purification beads may have been carried over into purified samples Scratches or dust particles on the OD plate Air bubbles are present in the diluted DNA within the OD plate Spin down the sample for 5 minutes Place on the MagnaRack and pipette out the eluate Retake the OD measurement Ensure that the bottom surface of the OD plate is clean and scratch free Vortex the OD plate spin it down again following the guidelines provided in Chapter 6 on page 75 and retake the OD Chapter 8 Troubleshooting 113 Fragmentation QC Step Gel or Bioanalyzer Likely Cause Solution Over fragmentation Majority of fragmented sample appears 50 bp on a 496 agarose gel Excess Fragmentation Reagent was added during pr
14. 0 000 0 00 0000000200 64 Check the PCR Reaction by Running a Gel 5 65 What TO Do Next os eed o decade E RET ERR PEE dps 67 Stage 4 PCR Product Purification cisco sees llle 68 Prepare Purification Wash Buffer 0 0000 000 ccc ee eee 68 Pool the PCR Products essen snecta nieret irani niir dep Ted 68 Purify the Pooled PCR Products 0 69 What TODO Next sca muc oleae adda A oe cheats 74 Stage 5 QuantitatlOn Lx saec eto hata chad ale ood ane dd 75 Important Information About This Stage 00005 75 Prepare the Reagents Equipment and Consumables 75 Procedure if Using a Microplate Spectrophotometer 76 Procedure if Using a NanoDrop isslilssisssl sess 77 Assess the Yield 2444 54 nisada Pewee owes eee ded akc MER E 78 What lo Do Next 22 s lt 29 enterrat be Sede Sed bigee Phe oes 78 Stage 6 Fragmentation 0 0 0 0 bisnes cee ene 79 Important Information About This Stage 00005 79 Prepare the Reagents Equipment and Consumables 79 What TOo4DO Next lt s iste RR ree ps eter detiene deste 84 Check the Fragmentation Reaction by Running a Gel 85 Stage 7 Labeling og eeu REI o e oe obe ee ge gy woe 87 Prepare the Reagents Equipment and Consumables 87 Prepare the Labeling Master Mix llle 89 What To Do Next ss Rs 90 iv Affymetrix CytoScan Assay User Manual Chapter 7 Chapte
15. 3 1 The Post PCR Room has airborne contamination with PCR product and template After entering the Post PCR Room do not re enter the Pre PCR Clean Room without first showering and changing into freshly laundered clothes Activities that take place in this room include Preparation of non amplified genomic DNA Digestion and ligation reactions a Preparation of PCR reactions Figure 3 1 Pre PCR Clean Room Equipment Shown Vortexer Microfuge Pipettes on stand Ice bucket Thermal cycler Plate centrifuge Freezer db 9o IN OY odo Be UND Refrigerator To help prevent sample contamination All ofthe reagents and master stocks required for the steps performed in the Pre PCR Clean Room should be stored in this room under the appropriate conditions Chapter 3 Laboratory Setup and Recommendations 13 a All ofthe equipment required for the steps performed in this room should be dedicated Do not move any equipment including ice buckets and pipettes between the Pre and the Post PCR Rooms Always wear a fresh gown booties and gloves to prevent PCR carryover and to minimize the risk of trace levels of contaminants being brought into the room Post PCR Room Activities that take place in this room include PCR amplification PCR product purification and quantitation PCR product fragmentation and labeling a Sample hybridization onto arrays Washing and staining of arrays a Sca
16. Can be held overnight Volume 100 pL Specify Maximum mode Check the PCR Reaction by Running a Gel To ensure consistent results run a 3 uL aliquot from each PCR reaction on a gel WARNING Use good laboratory practices and always wear the appropriate personal protective equipment when handling ethidium bromide Run the Gels When the CytoScan PCR program is finished 1 2 3 4 Remove the plate from the thermal cycler Make sure the plate ls sealed tightly and spin down at 2000 rpm for 1 minute Place in the cooling block on ice Label a fresh 8 to 12 well strip tube as shown in Figure 6 11 This is referred to as the ge strip tube Aliquot 5 uL of Affymetrix Nuclease Free water with 2 uL of 5X RapidRun Loading Dye to each well of the strip tube to be used 66 Affymetrix CytoScan Assay User Manual 6 Unseal the PCR plate and discard the seal 7 Transfer 3 uL of PCR product from each well of row A only of the plate to the corresponding wells of the strip tube Figure 6 11 Transfer aliquots of each PCR product to the gel plate PCR Plate P4 AO Transfer 3 uL from only row A of the PCR plate to the corresponding wells of the gel strip tube ee ecees SO ee Cee O60 6 es ee OOOOOOOOO Gel Strip Tube 8 Seal the PCR plate tightly with a new seal and store it at 20 C if not proceeding to the purification step 9 Sealthe gel stri
17. Cycler Program CytoScan Ligate Program Temperature Time 16 C 3 hours 70 C 20 minutes 4 C Hold What To Do Next Do one of the following a If following the recommended workflow Figure 5 1 on page 24 proceed immediately to Stage 3 PCR on page 56 Samples can be stored in a cooling block on ice for up to 60 minutes The sample plate can also be left in the thermal cycler at 4 C hold over night If not proceeding directly to the next step ensure the plate is sealed tightly and then store the plate at 20 C 58 Affymetrix CytoScan Assay User Manual Stage 3 PCR About Controls To assess the presence of contamination always include one PCR negative control with every set of samples run Lu IMPORTANT It is crucial to dilute the ligated DNA with chilled Affymetrix Nuclease Free water prior to PCR Dilute the Ligated Samples To dilute the samples 1 wo PWM e Place the Affymetrix Nuclease Free water on ice 20 minutes prior to use Place a double cooling block on ice Figure 6 8 Place a reagent reservoir on the upper half of the cooling block on ice Pour chilled Affymetrix Nuclease Free water into the reagent reservoir When the CytoScan Ligate program is finished take the plate out Make sure the plate is sealed tightly and spin down at 2000 rpm for 1 minute Place the plate in the lower half of the cooling block on ice Unseal the ligated sample plate
18. Fragmentation 0000000 eee 36 About Stage 7 Labeling 0 0 000000 ccc ee eee 39 About Stage 8 Target Hybridization 20 000 000 cece 40 CytoScan Assay Protocol 232 cr rk o ha n 43 Preparing the Genomic DNA 02 sess 43 Aliquoting the Prepared Genomic DNA and Controls 45 What TO DOINGXt asiten toe ce ce torte Eto re e noctua 47 Stage 1 Restriction Enzyme Digestion 4 48 Prepare the Reagents Equipment and Consumables 48 Prepare the Digestion Master Mix lisse 50 Add Digestion Master Mix to Samples iliis iles 51 Load Samples onto the Thermal Cycler n n nannan aana 52 Contents iii What TODO Next Lue scere ue ipii Boe cod ael Baker eon Send 52 Stage 2 Ligation llle ees 53 Prepare the Reagents Consumables and Other Components 53 Prepare the Ligation Master Mix 0 55 Add Ligation Master Mixto Reactions llle 56 Load the Samples Onto the Thermal Cycler iliius 57 What TODO Next o ene tuc ERREUR eU we de ENS 57 Stage 3 PCR sees edad eS Hdepa e doe edd am ea eee 58 Abo t Cohttols 42d sia niasa eee te date Raed E 58 Dilute the Ligated Samples 00 0000 00 cece ee eee 58 Transfer Diluted Ligated Samples to the PCR Plate o oo nnan aaaaaaa 60 Prepare the PCR Master Mix nnana nananana 0000 cee eee eee 63 Add PCR Master Mix to Each Sample 000000 0000 ee 64 Load PCR Plate onto a Thermal Cycler 0
19. Set the run time to 21 minutes Push the Power Prg button again it will change from red to green When the run time is reached the system will automatically shut off the dye should be near the end of the lane The gel is then ready for imaging Figure D 1 Gel Image of the PCR Product from Ref103 Genomic DNA on 2 E Gel 140 Affymetrix CytoScan Assay User Manual Fragmented Product on 4 E Gel Diluting the Tracklt Cyan Orange Loading Buffer The following instructions prepare a 1000 fold dilution of the TrackIt Cyan Orange Loading Buffer 1 2 Add 50 uL of TrackIt Cyan Orange Loading Buffer to 49 95 mL Nuclease Free water total volume is 50 mL Mix well and store at room temperature Diluting Fragmented Product Dilutions can be prepared in strip tubes or 96 well plates 1 After the Fragmentation step is complete aliquot 4 uL and make 1 8 dilution by adding 28 uL water Add 8 uL of the above diluted fragmented product to 12 uL of the 1 1000 fold diluted Loading Buffer to give a total volume of 20 uL Briefly vortex and spin down the diluted samples before loading onto the E Gel Running the E Gel 1 2 3 Turn on the power for the E Base red light Push the Power Prg button to make sure the program is set to EG mode not EP Remove the comb s from the E Gel and wipe away any buffer that comes out of the gel or is on the surface Insert the 48 well 4 Agarose E Gel into
20. all supported racks This is an example of pellet formation in the MagnaRack Avoid contact with the bead pellet when pipetting off the supernatant Add Purification Wash Buffer IMPORTANT Ensure that absolute ethanol has been added to the Purification Wash Buffer bottle 1 Using a P1000 pipette add 1 mL of Purification Wash Buffer to each tube 72 Affymetrix CytoScan Assay User Manual For gt 8 samples pour the wash buffer into a reservoir Add 1 mL of Purification Wash Buffer to each tube using a multi channel pipette 3 samples at a time Ensure that the pipette tips are arranged to enable this 2 Cap the tubes and load them into the foam tube adaptor Figure 6 17 Fully insert tubes into the foam to ensure they are secure Space seven sample tubes and a balance tube adequately to balance Figure 6 17 3 Vortex at maximum setting for 2 minutes The bead pellet may not be completely resuspended that is OK 4 Centrifuge the tubes for 3 minutes at maximum speed position tubes with cap hinges facing out 16 100 rcf 5 Place the tubes on the magnetic stand until all of the pellets move to the magnet Figure 6 17 Resuspended Bead Clump and Vortexer with Foam Tube Adaptor 6 Usea P1000 pipette to pipet off the supernatant without disturbing the bead pellet and discard For gt 8 samples use a multi channel pipette to remove the supernatant from 3 samples at a time Ensure
21. carousel positions 1 4 at room temperature If the arrays are not at room temperature do not select this option The scanner will wait 10 minutes before scanning begins to allow the arrays to reach room temperature Only one scan per array is required Pixel resolution and wavelength are preset and cannot be changed warnine The door is locked while the instrument is scanning Do not attempt to open the door manually Adding Arrays During an Autoloader Run To add arrays while an AutoLoader run is in progress 1 Click the Add Chips icon Add Chips The GeneChip Scanner message appears Figure 7 2 GeneChip Scanner Message GeneChip Scanner Warming Adding chips without completing the scan will cause the chip currently being scanned to be rescanned Add Now Add after Scan Cancel 2 Click Add after Scan Lu IMPORTANT Do not use the Add Now feature Use only the Add after Scan feature when working with CytoScan Arrays 5 Chapter 7 Washing Staining and Scanning Arrays 105 When the status on the scanner reads Autoloader Door Unlocked open the scanner and add the arrays Close the scanner When the following message is displayed click OK Figure 7 3 GeneChip Scanner Message GeneChip Scanner Load your samples in the autoloader then click OK or press Enter JY Arrays in carousel positions 1 4 at room temperature Allow rescans
22. loss of enzyme activity o Store the enzymes in a cooler placed in a 20 C freezer to preserve activity When taking out enzymes for reaction setup always use a cooler chilled to 20 C u Take care when pipetting enzymes stored in glycerol which is viscous Do not store enzymes at 80 C u Because Fragmentation Reagent activity can decline over time after dilution on ice add it to the samples as quickly as possible Preparing the Ligation Master Mix with 2096 to 2596 overage and the PCR Master Mix with 15 overage ensures consistency in reagent preparation by minimizing pipetting errors and reducing handling time of temperature sensitive reagents The success of this assay depends on the accurate pipetting and subsequent thorough mixing of small volumes of reagents 108 Affymetrix CytoScan Assay User Manual The PCR reaction for this assay has been validated using the specified thermal cyclers We highly recommend that your PCR thermal cyclers be calibrated regularly Take care when programming your thermal cycler and use the recommended 96 well plate It is essential to run gels to monitor both the PCR and the fragmentation reactions For the PCR reaction individual PCR products are run on a gel Product bands should be visible in the 150 to 2000 bp size range See Check the PCR Reaction by Running a Gel on page 65 for more information See Appendix D on page 137 for E Gel information Following fragmentation run your
23. manufacturer s recommendation a Retrain personnel on pre lab best practices a Repeat the assay using fresh reagents and sample Chapter 8 Troubleshooting 111 Purification Yield QC Step Likely Cause Solution Low eluate volume lt 47ul Insufficient volume due to pipetting error or pipet out of calibration Check pipette calibration Make sure 52 uL of elution buffer is added to the beads for elution and the tubes are centrifuged before placing on the magnet Low yields the average purification yield of 7 or more samples is 3 0 ug ul or individual yield is 2 5 ug ul Loss of sample prior to purification If the yield is not adequate repeat the assay Possible problems with input genomic DNA a Use the recommended collection and purification procedures to avoid carryover of inhibitors such as heme EDTA etc a Starting amount of 250 ng genomic DNA should be used Confirm the concentration using a calibrated spectrophotometer Confirm that the genomic DNA sample meets the quality and integrity guidelines provided in Chapter 4 Purification Wash Buffer was prepared incorrectly Verify that the correct volume of absolute ethanol was added to the Purification Wash Buffer before use Inadequate mixing of Purification Beads and PCR reactions during binding Take care to completely mix the PCR reactions and the Purification Beads during sample binding I
24. microfuge 1 Pipette single channel P20 1 Pipette single channel P200 1 Pipette 12 channel 2 20 HL 1 Pipette 12 channel 20 200 uL As needed Pipette tips for pipettes listed above 1 Plate 96 well if using NanoDrop 1 UV Plate 96 well 370ul UV Star if using microplate spectrophotometer 1 Spectrophotometer microplate or NanoDrop 1 Reagent reservoir 25 mL Reagents Required The following reagents are required for this stage Table 5 13 Reagents Required for Stage 5 Quantitation Reagent Affymetrix Nuclease Free Water About Stage 6 Fragmentation During this stage the purified samples are fragmented using the Fragmentation Reagent You will 1 2 3 4 Prepare a Fragmentation Master Mix Quickly add the mix to each sample Place the samples onto a thermal cycler and run the CytoScan Fragment program Check each reaction on a gel Chapter 5 Assay Overview 37 For the detailed protocol see Stage 6 Fragmentation on page 79 Location and Duration a Post PCR Area Hands on time 30 minutes CytoScan Fragment thermal cycler program time 1 hr Equipment and Consumables Required The following equipment and consumables are required for this stage Table 5 14 Equipment and Consumables Required for Stage 6 Fragmentation Quantity Item As required Adhesive seals for 96 well plates 1 Refrigerated plate cen
25. place on the cooling block on ice Allow the following reagents to thaw at room temperature Immediately place on the cooling block on ice when reagents are thawed 10x Nsp I Buffer 100x BSA Lu IMPORTANT Leave the Nsp enzyme at 20 C until ready to use Prepare the 10X Nsp I Buffer and 100X BSA as follows A Vortex 3 times 1 second each time B Pulse spin for 3 seconds C Place in the cooling block on ice Place the Affymetrix Nuclease Free water on ice 50 Affymetrix CytoScan Assay User Manual Prepare the Digestion Master Mix Keeping all reagents tubes and the cooling block on ice prepare the Nsp I Digest Master Mix as follows 1 w 4 5 6 7 To the 1 5 mL Eppendorf tube labeled Dig add the appropriate volumes of the following reagents see Table 6 1 a Chilled Affymetrix Nuclease Free water 10X Nsp I Buffer a 100X BSA Place the master mix in the cooling block Remove the Nsp I enzyme from the freezer and immediately place in a cooler chilled to 20 C Vortex at high speed for 1 second Pulse spin the enzyme for 3 seconds Keep it in the 20 C cooler Immediately add the enzyme to the master mix Return the enzyme to the 20 C cooler Table 6 1 Digestion Master Mix 8 Vortex the master mix at high speed 3 times 1 second each time 9 Pulse spin for 3 seconds 10 Place in the cooling block Reagent 1 Sample 8 Sa
26. pooled Chapter 6 CytoScan Assay Protocol 69 Figure 6 13 Pooll the PCR Products Pool PCR products for each sample in a 1 5 mL Eppendorf tube Do not pool the negative control Discard P WwW OOOOOOQOO OJOGO OOOOOOQOQO Purify the Pooled PCR Products Add Purification Beads and Incubate To add the Purification Beads and incubate 1 Thoroughly mix the Purification Beads stock by shaking and inverting the bottle Examine the bottom of the bottle and ensure that the solution appears homogenous 2 Open the tube caps slowly to ensure that the PCR sample does not spill out IMPORTANT The bead solution is viscous Pipet slowly to ensure that you aspirate and dispense 720 pL 3 Add 720 uL of Purification Beads to each pooled sample a For lt 8 samples Use a single channel P1000 pipette to add 720 ul of beads directly from the bottle to the sample Change tips between pipetting steps 70 Affymetrix CytoScan Assay User Manual For 16 and 24 sample workflows 720 ul of beads may be added directly from the bottle to the sample using a single channel P1000 pipette as described above Alternatively the beads can be added with a multi channel P1000 pipette as follows a 16 samples Aliquot 15 ml of beads into a reagent reservoir 24 samples Aliquot 21 ml of beads into a reagent reservoir Using a multi channel P1000 pipette add 720 uL of Purification Beads to each poole
27. samples on a gel Successful fragmentation is confirmed by the presence of a majority of the distribution between 25 to 125 bp See Check the Fragmentation Reaction by Running a Gel on page 85 for more information Alternatively the fragmented samples can be analyzed using the Agilent 2100 Bioanalyzer See Appendix D on page 137 for E Gel information Always run positive and negative controls in parallel with each group of samples The absence of bands on your PCR gel for the negative control confirms no previously amplified PCR product has contaminated your samples Use Genomic DNA from the CytoScan Reagent Kit as a positive control These controls are effective troubleshooting tools that will help you confirm the successful completion of each stage of the assay Oligonucleotide controls are included in the reagent kit These controls are added to the target samples prior to hybridization and act to confirm successful hybridization washing staining and scanning of the array Regularly calibrate all single channel and multi channel pipettes Check that your spectrophotometer or Nanodrop is accurately calibrated and be sure the OD measurement is within the linear range of the instrument as per the manufacturer s recommendations Hybridization oven temperature is critical to the performance of the assay Use the GeneChip Hybridization Oven 645 only Hybridization ovens should be serviced at least once a year to ensure that they are opera
28. that the pipette tips are arranged to enable this 7 Spin the tubes for 30 seconds at maximum speed hinges facing out 16 100 rcf 8 Place the tubes back on the magnetic stand Lu IMPORTANT While pipetting out be careful not to disturb or break off any of the bead pellet 9 Using a single channel P20 pipette remove the remaining drops of the Purification Wash Buffer from the bottom of each tube one sample at a time 10 Remove the tubes OFF the magnetic stand and allow the remaining Purification wash Buffer to evaporate by leaving the tubes uncapped at room temperature for 10 minutes Chapter 6 CytoScan Assay Protocol 73 Add Elution Buffer To add Elution Buffer to each sample Lu IMPORTANT To ensure better resuspension of the beads add Elution Buffer directly onto the beads 1 Using a P200 pipette add 52 uL of Elution Buffer to each tube directly onto the beads 2 Cap the tubes and load them into the foam tube adaptor Make sure the tubes are balanced 3 Vortex at maximum setting for 10 minutes Vortexing will resuspend the purification beads 4 Examine each tube to ensure that the beads are resuspended in a homogeneous slurry If the beads are not fully resuspended flick the tube to dislodge the pellet and vortex an additional 2 minutes Re examine 5 Centrifuge the tubes for 3 minutes at maximum speed position tubes with cap hinges facing out 16 100 rcf For centrifuging be sure t
29. the tubes to ensure the accurate transfer of 10 uL to each sample Lu IMPORTANT Add the master mix to the samples as quickly as possible Purified PCR product 45 uL Fragmentation Master Mix 10 uL Total 55 HL Seal the plate tightly with a new seal IMPORTANT Always carry the sample plate to the centrifuge or the thermal cycler on the cooling block in the ice box Vortex at high speed for 1 second in all corners and in the center according to the guidelines in Seal Vortex and Spin on page 5 Bring the sample plate to the centrifuge on the cooling block in the ice box Spin the plate in the pre chilled centrifuge at 2000 rpm for 1 minute Quickly remove the plate from the centrifuge and place in the cooling block in the ice box Carry the sample plate on the cooling block in the ice box and immediately load the Fragmentation plate onto the thermal cycler with preheated lid Run the CytoScan Fragment program Table 6 8 84 Affymetrix CytoScan Assay User Manual Table 6 8 CytoScan Fragment Thermal Cycler Program CytoScan Fragment Program Temperature Time 37 C 35 minutes 95 C 15 minutes 4 C Hold 9 Remove and discard any remaining Fragmentation Master Mix Never re use Fragmentation Master Mix 10 At this point the plate centrifuge may be turned back to room temperature What To Do Next Check the fragmentation reaction by running gels as described under Che
30. times 6 Repeat this process until all samples are loaded onto arrays and are placed in the hybridization oven All samples should be loaded within 30 minutes 7 Allow the arrays to rotate at 50 C 60 rpm for 16 to 18 hr IMPORTANT Allow the arrays to rotate in the hybridization oven for 16 to 18 hr at 50 C and 60 rpm This temperature is optimized for this product and should be stringently followed Washing Staining and Scanning Arrays This chapter describes how to wash stain and scan the Affymetrix CytoScan Arrays The instruments that you will use include the a Fluidics Station 450 to wash and stain the arrays a GeneChip Scanner 3000 7G to scan the arrays Once the arrays are scanned the array image dat file is ready for analysis Equipment and Consumables Required The following equipment and consumables are required for washing staining and scanning arrays Table 7 1 Equipment and Consumables Required for Washing Staining and Scanning Arrays Item Vendor Part Number GeneChip Scanner 3000 7G Affymetrix GeneChip Fluidics Station 450 Affymetrix The instrument control application Affymetrix GeneChip Affymetrix Command Console v 3 2 2 or higher Tube Safe Lock Tube 1 5 m Amber Eppendorf 022363221 Tube Safe Lock Tube 1 5 mL Blue Eppendorf 022363247 Tube Safe Lock Tube 1 5 mL Natural Eppendorf 022363352 Pipets P 2 P 20 P 200 P 1000 Rainin Pipetman
31. tube strip on the upper half of the cooling block Figure 6 6 Cut an adhesive seal into strips wide enough to seal 8 or 12 strip tubes Thaw the Reagents and Digested Samples To thaw the reagents and digested samples Lu IMPORTANT Leave the T4 DNA Ligase at 20 C until ready to use Allow the following reagents to thaw at room temperature Immediately place on the cooling block on ice when reagents are thawed 50 uM Adaptor Nsp I a 10X T4 DNA Ligase Buffer requires approximately 20 minutes to thaw If the digested samples were frozen allow them to thaw at room temperature Immediately spin down the plate at 2000 rpm for 1 minute and place on the cooling block on ice 54 Affymetrix CytoScan Assay User Manual Strip tubes to aliquot Ligation Master Mix 50 uM Adaptor Nsp 7123 45 6 a 10X T4 DNA Ligase Buffer Ligation Master Mix tube Prepare the Digested Samples and Reagents 1 Prepare the digested samples as follows A Spin down at 2000 rpm for 1 minute B Place in the lower half of the cooling block on ice 2 To prepare the reagents IMPORTANT Vortex the buffer as long as necessary before use to ensure any precipitate is re suspended and the buffer is clear A Vortex the 10X T4 Ligase Buffer and the 50 uM Adaptor Nsp I at high speed 3 times 1 second each time B Pulse spin for 3 seconds C Place in the cooling block Chapter 6 CytoScan Assay Prot
32. unless otherwise instructed Plates n Spin at room temperature except for the fragmentation step During the fragmentation step spin the plates at 4 C in a refrigerated centrifuge u Start the centrifuge allow it to reach 2000 rpm and spin at that speed for 1 minute Reagent Vials 3 seconds using bench top mini centrifuge Enzyme Vials 3 seconds using bench top mini centrifuge Fragmentation Step Cool the plate centrifuge to 4 C at least 15 to 20 minutes prior to proceeding with the fragmentation step a Pre chill the reagents empty tube for master mix and empty strip tube before starting the fragmentation step a Leave the Fragmentation Reagent at 20 C until ready to use All reagent additions in this step must be performed on ice Always carry the sample plate to the centrifuge or the thermal cycler on the cooling block on ice Running Gels a Run gels at 5 V cm for 45 minutes or until the dye front reaches at least 75 of distance down the gel Be sure to add ethidium bromide to the gel running buffer in the gel box Add two drops of ethidium bromide per 1L of 1X TBE 8 Affymetrix CytoScan Assay User Manual Hybridization a Load only 6 to 8 arrays at a time Remove the seal from the hybridization plate for only 6 8 samples at a time a Preheat the hybridization oven to 50 C at least one hour prior to use Washing Arrays It is important to work quickly when processing arrays for washing
33. you are transferring to on the PCR plate Example When transferring samples from row A of the Digest Ligate plate to PCR plate o Cap all wells in row B through row H on the Digest Ligate plate o Cap all wells in row E through row H on PCR Plate 128 Affymetrix CytoScan Assay User Manual PCR to Purification Figure A 2 16 Reaction Workflow PCR to Purification 1 5 mL tubes 1 per reaction Gel check for PCR product 2000 bp Em S500 bp Ss 300 bp OOOO 1 DI AMV 5 8 89 0 2 2 gt GG CO GG 3G GI eO G Gedesguedeoec GOGOOOG gt 150 bp Do not pool negative control GGG COO OC o H 19 3l Add purification beads to each tube and incubate in the tube rack Appendix A Guidelines for Processing 16 Samples 129 Purification Continued to Fragmentation and Labeling Figure A 3 16 Reaction Workflow Purification to Labeling Transfer eluted sample to the appropriate well of a fresh 96 well plate Fragment Label Plate roy Q DP u o gt OOOOOOQ0Qft OOOOOOQ0Rw OOOOOO009 OOOOOOO00 OOOOOOO00 OOOOOO00 OOoooooee OOCOOOOO8 OOODOOOGOGO OOODOGOGOGOGO OOOOOOOQ OOOOOOOQ X Quantitate label and hyb samples onto arrays After incubation centrifuge and pl
34. 0 169 6 551 817 6 733 977 7 629 164 7 790 389 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 Fluidics Stations Products may be protected by one or more of the following patents 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 protected by one or more of the following patents U S Patent Nos 5 578 832 5 631 734 5 834 758 5 936 324 5 981 956 6 025 601 6 141 096 6 171 793 6 185 030 6 201 639 6 207 960 6 218 803 6 225 625 6 252 236 6 335 824 6 403 320 6 407 858 6 472 671 6 490 533 6 650 411 6 643 015 6 813 567 7 682 782 7 689 022 and other U S or foreign patents Autoloaders Products may be protected by one or more of the following patents U S Patent Nos 6 511 277 6 604 902 6 705 754 7 108 472 and other U S or foreign patents Hybridization Oven Rotational Mixer Products may be protected 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 Reagents For DNA Labeling Reagent DLR Products may be protected by one or more of the following patents U S Patent Nos 6 864 059 6 965 020 7 423 143 Cartridge Array Software Products may be protected by one or more of the following patents U S Patent Nos 5 733 729 5 795 716 5 974 164 6 066 454 6 090 555 6 185 561 6 188 783 6 22
35. 00 46 Affymetrix CytoScan Assay User Manual Aliquot the gDNA and Controls NOTE 5 uL of the 50 ng pL working stock is equivalent to 250 ng genomic DNA per well To aliquot the prepared genomic DNA and controls 1 Thaw the Control Genomic DNA from the CytoScan Reagent Kit as follows A Place on the bench top at room temperature until thawed B Once thawed place in the cooling block on ice Vortex the genomic DNA for 3 seconds then quickly spin down for 1 minute Transfer a 5 uL aliquot of the first sample to well Al of the digest ligate plate Figure 6 3 Transfer 5 uL aliquots of each remaining gDNA sample in the same manner 5 For the controls aliquot 5 uL of A Genomic DNA from CytoScan Reagent Kit to well A7 B Low EDTA TE buffer to well A8 6 Tightly seal the digest ligate plate with a new seal then spin down for 1 minute at 2000 rpm Chapter 6 CytoScan Assay Protocol 47 Figure 6 3 Setup for aliquoting diluted gDNA and controls to a 96 well plate labeled for digestion ligation Transfer 5 uL aliquots of each diluted gDNA to the digest ligate plate for digestion reactions positive control 5 uL Genomic DNA from the CytoScan Reagent Kit negative control 5 uL Low EDTA TE Buffer Diluted gDNA 000000 samples h at 50 ng uL v 9 9 f f concentration 9 29 4 Y lel 666664 e2eea8hi d rrr FH 96 well plat
36. 100 1000 HL As needed Pipette tips for pipettes listed above Chapter 5 Assay Overview 35 Table 5 10 Equipment and Consumables Required for Stage 4 PCR Product Purification Quantity Item 1 Plate Bio Rad 96 well One per 96 well Plate holder plate 1 Optional Tube 50 mL conical One per sample Tube Eppendorf Safe Lock Tube 1 5 mL Natural minus neg control 1 Tube holder 1 Vortexer with foam tube adaptor attached 1 Reagent reservoir 25 mL IMPORTANT Use only the magnetic racks listed in Table F 10 Post PCR Area Equipment Required on page 150 Reagents Required The following reagents are required for this stage Table 5 11 Reagents Required for Stage 4 PCR Product Purification Reagent Purification Wash Buffer Elution Buffer Purification Beads Absolute Ethanol About Stage 5 Quantitation During this stage you will quantitate each sample For the detailed protocol see Stage 5 Quantitation on page 75 Location and Duration a Post PCR Room Hands on time 30 minutes 36 Affymetrix CytoScan Assay User Manual Equipment and Consumables Required The following equipment and consumables are required for this stage Table 5 12 Equipment and Consumables Required for Stage 5 Quantitation Quantity Item As required Adhesive seals for 96 well plates 1 Marker fine point permanent 1 Mini centrifuge
37. 3 127 6 228 593 6 229 911 6 242 180 6 308 170 6 361 937 6 420 108 6 484 183 6 505 125 6510 391 6 532 462 6 546 340 6 687 692 6 607 887 7 062 092 7 451 047 7 634 363 7 674 587 and other U S or foreign patents Copyright 2011 2012 Affymetrix Inc All rights reserved Contents Chapter 1 Chapter 2 Chapter 3 Contents i Introduction Soe ose asad heath ota Goa ems dhe Sew ace ae ond 1 About the Affymetrix CytoScan Solution 0 2 llle 1 Abo t This Manual 222 6 ben4edeekkbE RU ee dess9ePPRER e Gee REX 2 Best Practices PMCID 3 CONOIS seisean gaea ee eee CALE cc i TETE EA AAR hda a EE EEE EA 3 Equipment and Calibration anaana eae 3 PUD SUING CREER m 4 Reagent Handling and Storage 9 4 When Using Reagents at the Lab Bench 9 5 Master Mix Preparation 0 0000 cee tee 5 Laboratory Workflow isses eb RE eS E ed ee be 5 Seal Vortex and Spin liiis es 5 Handling the Plate Seal liliis 6 Sealirig Strip TUbDes i sut eee Eo doc CR OG a ada 6 IE 6 JU 7 Fragmentation Step sirere daa a be PCR E 7 RUNNING Gels ied ed Garay RR Sa ew dd dcos NE ed 7 su rr 8 Washing Antay tiet aetate ome ta boca rA ee Ae ate Pate 8 Preparing the Work Area for Each Stage 0 0 0 0 anaana eee 8 Thermal Cyclers 96 Well Plate and Adhesive Seals 000 8 Program Your Thermal Cyclers liliis 9 Hybridization Oven i iiississlssees e 10 Laboratory Setup and Re
38. 3 2 2 or higher If not please update your version of AGCC to v 3 2 2 or latest available 2 Under the Samples tab select Batch Registration The Batch Registration window opens Figure 6 26 Figure 6 26 Barch Registration Window ortal Batch Create Sample Files Windows Internet Explore Aided by Affymetrix localhost wv x Uu P Fle Edt View Favortes Took Mep x O Mchfee ji He evertes ij Connecting SR gt O mR Pages seye Tose C Search Files By Aray Name Use for wildcard El Advanced Search e HOME DATA SAMPLES ADMINISTRATION HELP Batch Sample Registration Create and Upload Batch Registration File gt Confirm gt Finish Step 1 Create a blank batch registration file with the desired attributes Select the templates with the attributes you wish to use for the sample files amp gt MIAME Sample Information gt Pedigree Template For use with Excel or compatible application Create a spreadsheet for 0 Range from 0 500 samples optional project set to ba optional probe array type set to pale wien Semple defaults You can change the project and probe array type when editing the locument Download Step 2 Enter the values for the Sample ARR files in the batch registration file Enter values for the attributes using Excel or a text editing program The first row the heading row of the spreadsheet defines which fields to collect Each additional row belo
39. 8 Affymetrix CytoScan Assay User Manual Blank the NanoDrop with water 5 Take 2 uL of the diluted sample and A Measure the OD of each sample at 260 280 and 320 nm OD280 and OD320 are used as controls B Calculate the undiluted concentration for each sample as follows Undiluted sample concentration in ug uL Nanodrop Concentration in ng uL x 10 1000 Assess the Yield Acceptable DNA Yield The average purification yield for 7 or more samples should be 2 3 0 ug l If the average yield is lt 3 0 ug nL please consult the troubleshooting section We do not recommend further processing of samples with yields lt 2 5 ug uL The following OD ranges are based on the use of a conventional UV spectrophotometer plate reader and assume a path length of 1 cm The OD260 OD280 ratio should be between 1 8 and 2 0 Do not proceed if this metric falls outside of this range The OD320 measurement should be very close to zero lt 0 1 If your OD readings are not within the acceptable range refer to Chapter 8 Troubleshooting on page 107 What To Do Next Do one of the following a Proceed immediately to Stage 6 Fragmentation on page 79 a If not proceeding immediately to the next step seal the plate of purified samples and store at 20 C Chapter 6 CytoScan Assay Protocol 79 Stage 6 Fragmentation Important Information About This Stage The degree of fragmentation is critical Perform this stage ca
40. Buffer Part 3 Hyb Buffer Part 4 Oligo Control Reagent 0100 Chapter 6 CytoScan Assay Protocol 95 Prepare the reagents as follows 1 Vortex each reagent at high speed 3 times 1 second each time 2 Pulse spin for 3 seconds then place in the cooling block as shown in Figure 6 28 Reagent reservoir for Hyb Master Mix Hyb Master Mix Hyb Buffer Part 1 1203 yay sveys y Hyb Buffer Part 2 Hyb Buffer Part 3 Hyb Buffer Part 4 Oligo Control Reagent 96 Affymetrix CytoScan Assay User Manual Prepare the Hybridization Master Mix 1 Tothe 15 mL Hyb Master Mix centrifuge tube on ice add the appropriate volume of each reagent in the order shown in Table 6 11 Lu IMPORTANT Some of the Hyb Buffer components are viscous carefully pipette and dispense when preparing the master mix 2 Mix well by vortexing the master mix at high speed 3 times 3 seconds each time until the mixture is homogeneous Table 6 11 Hybridization Master Mix Reagent 1 Sample 8 Samples 16 Samples 24 Samples 20 Overage 20 Overage 20 Overage 20 Overage Hyb Buffer Part 1 165 0 uL 1584 0 uL 3168 0 uL 4752 0 HL Hyb Buffer Part 2 15 0 uL 144 0 uL 288 0 uL 432 0 uL Hyb Buffer Part 3 7 0 uL 67 2 uL 134 4 uL 201 6 uL Hyb Buffer Part 4 1 0 uL 9 6 uL 19 2 uL 28 8 uL Oligo Control Reagent 0100 2 0 HL 19 2 HL 38 4 HL 57 6 HL Total 190 pL 1824 uL 3648 HL 5472 uL Lu IMPORTANT Mak
41. Delays during this step will impact data quality To optimize this step we suggest the following a 30 minutes before hybridization is complete prime the fluidics stations with the correct wash buffers Start the Fluidics Protocol and follow the directions on the LCD panel of the fluidics station Load Stain 1 Stain 2 and the Array Holding buffer in their respective positions on the fluidics station Eject the wash block to avoid sensor time out Process only 6 8 arrays at a time Minimize delays when performing all steps after the arrays are removed from the oven up to the time when washing begins Preparing the Work Area for Each Stage Many of the stages in the CytoScan Assay must be performed rapidly and on ice to carefully control enzyme activity and temperature transitions Therefore we recommend that you set up all of the equipment consumables and reagents except for the enzymes prior to beginning each stage Thermal Cyclers 96 Well Plate and Adhesive Seals The CytoScan Assay has been optimized using the following thermal cyclers 96 well plate and adhesive films Lu IMPORTANT Use only the 96 well plate and adhesive seals listed in Table 2 1 and only the thermal cyclers listed in Table 2 2 Using other plates and seals that are incompatible with these thermal cyclers can result in loss of sample or poor results Chapter 2 Best Practices 9 Table 2 1 96 Well Plate and Adhesive Seals Optimized F
42. Drops USB 75816 Reagents Required from Other Suppliers Table F 7 Reagents Required from Other Suppliers v item Vendor Part Number O TITANIUM DNA Amplification Kit 300 rxn Clontech 639240 O TITANIUM DNA Amplification Kit 400 rxn Clontech 639243 1 Absolute Ethanol Sigma Aldrich 459844 1 Bleach 6 1596 Sodium Hypochlorite VWR 21899 504 or equivalent Optional Affymetrix Equipment Table F 8 Optional Affymetrix Equipment v item Part Number C1 GeneChip System 3000Dx v 2 with Data Transfer Server 00 0349 Equipment Required from Other Suppliers Pre PCR Clean Area Equipment Required TM When performing the pre PCR stages of the CytoScan Assay great care should be taken to avoid sample contamination with PCR products If the assay is to be run ina single room we strongly recommend that the pre PCR stages be performed in a laminar flow or PCR cabinet Appendix F Reagents Equipment and Consumables 149 Table F 9 Pre PCR Clean Area Equipment Required v tem Vendor Part Number C Recommended if protocol is to be performed in one Laminar Cabinet ESCO SVE 6A room only or equivalent Laminar Flow Cabinet 6 ft ESCO SVE 6A PCR Cabinet PCR Cabinet C B S Scientific P 048 02 or equivalent 1 Benchtop Cooler 20 C Agilent 401349 Technologies 1 Biocooler aluminum block 96 well Bio Smi
43. OOOOO OOOOOO0O0Q OOOOOOO0Q OOOOOOOQ 52 Affymetrix CytoScan Assay User Manual Load Samples onto the Thermal Cycler 1 Vortex the plate at high speed for 1 second in all corners and in the center according to the guidelines in Seal Vortex and Spin on page 5 then spin down at 2000 rpm for minute Ensure that the lid of thermal cycler is preheated Load the plate onto the thermal cycler and run the CytoScan Digest program Table 6 2 Table 6 2 CytoScan Digest Program CytoScan Digest Program Temperature Time 37 C 2 hours 65 C 20 minutes 4 C Hold 4 Return any remaining reagents to the freezer 5 When the program is finished remove the plate Make sure the plate is sealed tightly and spin down at 2000 rpm for 1 minute What To Do Next Do one of the following Place the plate in a cooling block on ice and proceed immediately to Stage 2 Ligation on page 53 If not proceeding directly to Ligation make sure the plate is sealed tightly and store the plate at 20 C Chapter 6 CytoScan Assay Protocol 53 Stage 2 Ligation Prepare the Reagents Consumables and Other Components Turn On the Thermal Cycler Power on the thermal cycler to preheat the lid Leave the block at room temperature Set Up the Work Area 1 2 3 4 Place a double cooling block on ice Figure 6 6 Label a 1 5 mL Eppendorf tube as Lig and place in the cooling block Place an 8
44. Purified Sample Lu IMPORTANT The P20 pipette must be calibrated as per the manufacturer s specifications To prepare diluted aliquots of the purified samples 1 Using a multi channel P200 pipette aliquot 198 uL of water to the corresponding wells of a UV plate 2 Pipet 200 uL of water into each well of an empty row to be used as a BLANK Figure 6 20 on page 76 3 Using a multi channel P20 pipette A Transfer 2 uL of each purified sample to the corresponding well of the UV plate B Pipet up and down 2 times to ensure that all of the sample is dispensed The result is a 100 fold dilution Seal the plate with purified samples tightly with a new seal and store at 20 C 5 Seal the UV plate and using a Kimwipe on the adaptor surface vortex and spin down at 2000 rpm for 1 minute Figure 6 20 UV Plate Layout 198 uL Attymetrix Nuclease Free water 2 uL purified sample in each well P p 09069909900O0OCO 200 HL water for blank 000O00O0OoOOoOoQQ eCHDOODOOOOOOCQOCQOLU OOOOO0OO0O0O0O0O00 EOOOOOOOOOZGO QQU QCQOOOODUOOCQOLU OOOOOOOODOOOQO K OODOOOOOOOOQ Quantitate the Diluted PCR Product Apply the convention that 1 absorbance unit at 260 nm equals 50 ug mL equivalent to 0 05 ug uL for double stranded PCR products This convention assumes a path length of 1 cm Consult your spectrophotometer handbook for further information To quantitate the diluted purified PCR product 1 Mea
45. Scan Assay has an inherent risk of contamination with PCR product from previous reactions In Chapter 3 Laboratory Setup and Recommendations on page 11 we strongly recommend two separate work areas be used to minimize the risk of cross contamination during the assay procedure It is essential to adhere to workflow recommendations PCR reactions should be set up in the Pre PCR Area only Personnel should not re enter the Pre PCR Clean Area once exposed to PCR products without first showering and changing into clean clothes Carefully reading and following the protocol as written is essential The CytoScan Assay has been validated using the reagents and suppliers listed Substitution of reagents and taking shortcuts are not recommended as your results could be suboptimal For example always use Affymetrix Nuclease Free water and PCR reagents from Clontech Additional recommendations are as follows a Think ahead to ensure that the reagents and equipment you require including pipettes are in the correct work area Ensuring the proper equipment is available in the proper laboratory areas will make the workflow easier and will help reduce the risk of sample contamination Pay particular attention to the storage and handling of reagents Proper storage and handling is particularly important for enzymes such as T4 DNA Ligase and the Fragmentation Reagent Both of these enzymes are sensitive to temperatures exceeding 20 C To prevent
46. Using a multi channel P20 pipette transfer 10 uL of each ligated and diluted sample to the corresponding four wells of the PCR plate Seal the plate tightly with a new seal and spin down at 2000 rpm for 1 minute If not proceeding immediately to PCR stage store the plate with the remaining samples at 20 C 1 2 3 4 5 6 00000000 K2 KHa Ks KeK 00000000 Chapter 6 CytoScan Assay Protocol 61 Thaw the Reagents and Samples IMPORTANT Leave the 50X TITANIUM Tag DNA Polymerase at 20 C until ready to use Allow the following reagents to thaw at room temperature Immediately place on the cooling block on ice when reagents are thawed a 10X TITANIUM Taq PCR Buffer a dNTP Mixture 2 5 mM each PCR Primer 002 Prepare the Samples and Reagents To prepare the ligated samples and reagents 1 Labelthe 15 mL centrifuge tube PCR For more than 8 samples use a 50 mL tube 2 Place on ice a Chilled Affymetrix Nuclease Free water GC Melt Reagent Reagent reservoir should be placed on the upper half of the cooling block on ice 3 Ifthe diluted ligated samples aliquoted into the PCR plate are frozen thaw them at room temperature Make sure the plate is sealed tightly and vortex at high speed for 1 second in all corners and in the center according to the guidelines in Seal Vortex and Spin on page 5 then spin down at 2000 rpm for 1 minute 4 Immediately p
47. a 30 mM DNA Labeling Reagent Table 6 9 Labeling Master Mix Reagent 1 Sample 8 Samples 16 Samples 24 Samples 20 overage 20 overage 20 overage 5X TdT Buffer 14 0 uL 134 4 uL 268 8 HL 403 2 uL 30 mM DNA Labeling Reagent 2 0 HL 19 2 uL 38 4 uL 57 6 uL TdT 3 5 uL 33 6 uL 67 2 uL 100 8 uL Total 19 5 uL 187 2 pL 374 4 uL 561 6 pL 2 Remove the TdT enzyme from the freezer and immediately place in the cooler chilled to 20 C 3 Vortex the enzyme at high speed one time for 1 second Pulse spin the enzyme for 3 seconds then immediately place back in the 20 C cooler Add the TdT enzyme to the master mix Place the enzyme back in the 20 C cooler Vortex the master mix at high speed 3 times 1 second each time Pulse spin for 3 seconds Add the Labeling Master Mix to the Samples To add the Labeling Master Mix to the samples Keep samples in the cooling block and all tubes on ice when making additions 1 Aliquot the Labeling Master Mix equally into strip tubes that are pre chilled on the cooling block on ice Seal the strip with an adhesive seal strip or strip caps and pulse spin Place back in the cooling block remove the seal and discard 90 Affymetrix CytoScan Assay User Manual s NOTE When working with more than 8 samples we strongly recommend dividing the master mix into strip tubes and dispensing the master mix from the strip tubes into the samples using a multi channel
48. ace the tubes on the magnetic rack multiple racks may be needed depending on model chosen and work method UV Spec Plate for Quantitation OO000900900000 G 6 amp Gaiueoooooo OOOOOOOOOOOO OOOOOOOOOOOO OOOOOOOOOOOO OOOOOOOOOOOO OOOOOOOOOOOO OOOOOOOOOOOO Fragmentation gel 500 bp 130 Affymetrix CytoScan Assay User Manual Guidelines for Processing 24 Samples This appendix illustrates the plate layouts recommended for processing 24 reactions 22 samples plus one positive and one negative control It also provides a high level overview of the workflow Digestion Ligation and PCR Figure B 1 24 Reaction Workflow Digest Ligate Plate to PCR Plates First Transfer Digest Ligate Plate 60 02 G 0 s W A amp 2 C G G 0 9 C s 9 WO 3 amp 65 6 7 amp amp 9 9 65 62 CO CO C 2 8 4 9 9 9 2 0000000060000 PG OO 4 8 9 7 W GD 2 c s 0 5 6 D 9 9 amp 9 amp amp 2 5 OF s 4 45 66 17 amp amp 49 9 6 amp 2 C C 63 4 is de 62 ie d5 9 amp 62 C 3 4 65 e 17 9 9 6 amp 62 C O To avoid transfer mistakes keep all wells capped except for a One row on the Digest Ligate plate a The rows to which you are transferring to on the PCR plate Example When transferring samples from Digest Ligate plate row A to PCR plate a Cap all w
49. ach Cycle ot epe hr De eR o og CPP RR E Ed s 120 The Rinse Cycle 2 oar oO Ce ERR Qr Oed Sedit WE 124 Appendix A Appendix B Appendix C Appendix D Appendix E Contents v Guidelines for Processing 16 Samples 127 Digestion Ligation and PCR 0 00 00 cece es 127 PCR tO PUNMCATION Lisa optas esp X Ebo tees etse edu ss 128 Purification Continued to Fragmentation and Labeling 129 Guidelines for Processing 24 Samples Ls 131 Digestion Ligation and PER i ccccece c ERR we Eee PESE 131 PCR tO PUNINCAHON 2244 cs nani deeded Pee REPAS aided dumis 132 Purification Continued to Fragmentation and Labeling 133 Analyzing Sample Fragmentation Using the Agilent 2100 BIOS DU Zen o4 Vida a Rr quedar Se do doa A shane pes 135 Burning E G lS sace n hic aci 09 OP RR e ea aE ene EREE 137 Equipment E Gels and Reagents Required 4 137 Genomic DNA on 196 E Gel 0 0 22 es 137 Diluting Genomic DNA Samples 00 000000 eee eee 137 Running the E Gel 0 2 naana aaaea 138 PCR Product on 2 E Gel s 0 eee 138 Diluting the Tracklt Cyan Orange Loading Buffer 138 Diluting PCR Product see ARRIERE RRRREES Y I PPS 138 R nning the Esel amp ai de dO RE dor eda tee v ETE 139 Fragmented Product on 496 E Gel 4 140 Diluting the Tracklt Cyan Orange Loading Buffer 140 Diluting Fragmented Product lle 140 Running the E Gel
50. al 1 Plate centrifuge 1 Plate spectrophotometer or NanoDrop required only if no OD measurements available for samples 1 Vortexer 2 GeneMate 96 Well PCR Tube Storage Rack IMPORTANT Use only the thermal cyclers 96 well plate and adhesive films and listed under Thermal Cyclers 96 Well Plate and Adhesive Seals on page 8 Reagents Required The following reagents are required for this stage Table 5 2 Reagents Required for Preparing the Genomic DNA Reagent Low EDTA TE Buffer Genomic DNA positive control About Stage 1 Restriction Enzyme Digestion During this stage sample is digested by the Nsp I restriction enzyme You will 1 Prepare a Digestion Master Mix and add it to the samples 2 Place the samples onto a thermal cycler and run the CytoScan Digest program For the detailed protocol see Stage 1 Restriction Enzyme Digestion on page 48 Location and Duration Pre PCR Clean Area Hands on time 30 minutes a CytoScan Digest thermal cycler program time 2 5 hr 28 Affymetrix CytoScan Assay User Manual Input Required From Previous Stage This stage requires a plate containing aliquots of each genomic DNA and each control prepared as instructed under Preparing the Genomic DNA on page 43 5 uL at 50 ng uL in each well Equipment and Consumables Required The following equipment and consumables are required for this stage Table 5 3 Equipment and Consumabl
51. and discard the seal Chilled Affymetrix Nuclease Free water Ligated samples Chapter 6 CytoScan Assay Protocol 59 00000000 8 Using a P200 pipette add 75 uL of water to each reaction Ligated DNA 25 HL Chilled Affymetrix 75 HL Nuclease Free water Total 100 pL 9 Tightly seal the plate with a new seal 10 Vortex at high speed for 1 second in all corners and in the center according to the guidelines in Seal Vortex and Spin on page 5 then spin down at 2000 rpm for minute 11 If not proceeding with PCR set up store the plate at 20 C 60 Affymetrix CytoScan Assay User Manual Transfer Diluted Ligated Samples to the PCR Plate To transfer the diluted ligated samples to the PCR plate 1 2 Diluted A2X3X4X 5X6 Y ligated samples Place a double cooling block on ice Keep the diluted ligated sample plate on the upper half of the cooling block If the diluted ligated samples are frozen thaw them at room temperature Make sure the plate is sealed tightly and vortex at high speed for 1 second in all corners and in the center according to the guidelines in Seal Vortex and Spin on page 5 then spin down at 2000 rpm for 1 minute Immediately place the plate on the upper half of the cooling block Place a new PCR plate in the lower half as shown in Figure 6 9 and label as PCR Unseal the ligated and diluted sample plate and discard the seal
52. anisms PCR amplification of the ligated genomic DNA is not human specific so sufficient quantities of non human DNA may also be amplified and could potentially result in compromised genotype calls Contaminated or mixed DNA may manifest as high detection rates and low call rates DNA must not be degraded The genomic DNA fragment must have Nsp I restriction sites intact so that ligation can occur on both ends of the fragment and PCR can be successful The approximate average size of genomic DNA may be assessed on a 0 8 or 1 agarose gel using an appropriate size standard control Control Genomic DNA can be run on the same gel for side by side comparison High quality genomic DNA will run as a major band at approximately 10 20 kb on the gel 22 Affymetrix CytoScan Assay User Manual Pre amplification methods or pre digestion with restriction enzymes other than Nsp I have not been tested by Affymetrix If other methods are desired we recommend conducting experiments to evaluate their performance with this assay Sources of Human Genomic DNA The following sources of human genomic DNA have been successfully tested in the laboratories at Affymetrix for DNA that meets the requirements described in the section General Requirements and Recommendations a Blood Cell line Blood Collection Methods The two blood collection methods that have been shown to be compatible with the assay are EDTA and Heparin Genomic DNA Extraction Purif
53. apter 5 Assay Overview 31 Reagents Required The following reagents are required for this stage Table 5 6 Reagents Required for Stage 2 Ligation Reagent T4 DNA Ligase 10X T4 DNA Ligase Buffer 50 uM Adaptor Nsp About Stage 3 PCR During this stage you will Dilute the ligated DNA by adding 75uL of chilled nuclease free water Transfer 10 ul of each diluted ligated sample into four wells of a 96 well plate Prepare a PCR Master Mix and add it to each ligated sample Place the samples onto a thermal cycler and run the CytoScan PCR program wv Pw NM Confirm each PCR reaction by running 3 uL of each PCR product on a gel For the detailed protocol see Stage 3 PCR on page 58 Location and Duration Pre PCR Clean Area u PCR Master Mix preparation n PCR set up Post PCR Area samples placed on thermal cycler Hands on time 1 hr CytoScan PCR thermal cycler program time 1 5 hr Samples can be held overnight at 4 C in the thermal cycler 32 Affymetrix CytoScan Assay User Manual Equipment and Materials Required The following equipment and materials are required to perform this stage Table 5 7 Equipment and Consumables Required for Stage 3 PCR Quantity Item As required Adhesive seals for 96 well plates 1 Plate centrifuge 1 Cooler chilled to 20 C Cooling chamber double block chilled to 4 C placed on ice do not freeze Ice bu
54. aring the hybridization setup leave the samples on the cooling block on ice The following table lists the equipment and consumables required Table 5 19 Equipment and Consumables Required for Stage 8 Target Hybridization Quantity Item 1 Adhesive seals for 96 well plates 1 Cooling chamber double block chilled to 4 C placed on ice do not freeze One array per sample CytoScan Array 1 GeneChip Hybridization Oven 645 1 Ice bucket filled with ice 1 Pipette single channel P200 1 Pipette single channel P1000 1 Pipette 12 channel 20 200 HL As needed Pipette tips for pipettes listed above 1 Reagent reservoir 55 mL 1 Thermal cycler 2 per array Tough Spots 1 2 diameter PN Spot 2200 Diversified Biotech 1 Tube centrifuge 15 mL 1 Vortexer 4 GeneMate 96 Well PCR Tube Storage Rack IMPORTANT Use only the thermal cyclers tubes 96 well plate and adhesive film and listed under Thermal Cyclers 96 Well Plate and Adhesive Seals on page 8 42 Affymetrix CytoScan Assay User Manual Reagents Required The following reagents are required for this stage Table 5 20 Reagents Required for Stage 8 Target Hybridization Reagent Hyb Buffer Part 1 Hyb Buffer Part 2 Hyb Buffer Part 3 Hyb Buffer Part 4 Oligo Control Reagent 0100 CytoScan Assay Protocol
55. as been tested for multiple freeze thaw cycles You can freeze thaw the reagents in the 24 reaction kit 5 times 24 Affymetrix CytoScan Assay User Manual Equipment Consumables and Other Reagents This protocol has been optimized using the equipment consumables and reagents listed herein For best results we strongly recommend that you adhere to the described protocol without any deviation do not substitute reagents Workflows Recommended 4 Day Workflow Figure 5 1 shows the recommended 4 day workflow for one operator processing 8 to 24 samples including controls Figure 5 1 Recommended 4 Day Workflow HE DNA mm 1 3hr mm 30 min hands on Stage 2 4hr Ligation 30 min hands on 1 hr hands on Day 1 Ends at Pre PCR Room Main Lab stage 3B oy lt ES gt Starts with PCR and ends with Quantitation QC Gel 1 2 hr hands on 30 min hands on Stage 6 1 5 hr Fragmentation 30 min hands on E QC Gel 2 Stage 7 5hr Day s lt Labeling 30 min hands on Starts with Fragmentation and ends with Hybridization H ETE e En ies NEN yybridization min hands on Stage 9A 3hr Wash and Stain 30 min hands on Day 4 Y Wash stain Stage 9B 15 min hands on eon Scanning 32 min per array to scan Chapter 5 Assay Overview 25 Optional 3 Day Workflow Figure 5 2 illustrates the optional 3 day workflow The difference between the 3
56. ck the Fragmentation Reaction by Running a Gel If not proceeding directly to the next stage store the samples at 20 C Chapter 6 CytoScan Assay Protocol 85 Check the Fragmentation Reaction by Running a Gel The instructions below are for running 4 TBE gels To ensure that fragmentation was successful 1 10 11 12 13 When the CytoScan Fragment program is finished A Remove the samples from the thermal cycler B Make sure the plate is sealed tightly then spin down at 2000 rpm for 1 minute Place on the lower half of the cooling block on ice Label two 8 strip tubes one as Fragmentation QC Samples and the other as Gel Analysis Remove and discard the plate seal Remove 4 ul of fragmented samples into strip tubes labeled as Fragmentation QC Samples Seal the fragmented DNA plate with a new seal and keep it on the lower half of the cooling block on ice If not proceeding immediately to Labeling step store the plate at 20 C Add 28 uL water to the strip tubes labeled as Fragmentation QC Samples Seal the strip vortex and spin down Remove 8 uL of the diluted Fragmented QC samples and dispense into respective wells of the strip tubes labeled as Gel Analysis NOTE Do not forget to add ethidium bromide to the gel running buffer in the gel box Add 2 drops of the ethidium bromide per 1L of 1X TBE Seal and store the remaining Fragmentation QC Sample strip tubes at 20 C for further analys
57. cket filled with ice Marker fine point permanent Mini centrifuge microfuge Pipette single channel P20 Pipette single channel P100 Pipette single channel P200 Pipette single channel P1000 1 Pipette 12 channel 2 20 uL 1 Pipette 12 channel 20 200 HL As required Pipette tips for pipettes listed above 1 Plate Bio Rad 96 well PCR 2 GeneMate 96 Well PCR Rube Storage Rack Reagent reservoir 25 mL Thermal cycler Tube centrifuge 15 or 50 mL Vortexer 1 Electrophoresis gel box 1 Electrophoresis power supply IIMPORTANT Use only the thermal cyclers 96 well plate and adhesive films listed under Thermal Cyclers 96 Well Plate and Adhesive Seals on page 8 Chapter 5 Assay Overview 33 Reagents Required The following reagents are required for this stage Table 5 8 Reagents Required for Stage 3 PCR Reagent Chilled Affymetrix Nuclease Free Water PCR Primer 002 From the Clontech TITANIUM DNA Amplification Kit 300 or 400 rxn a dNTP Mixture 2 5 mM each a GC Melt Reagent a 50X TITANIUM Taq DNA Polymerase a 10X TITANIUM Taq PCR Buffer Gels and Related Materials Required Verifying the PCR reaction is required for this stage Table 5 9 Gels and Related Materials Required for Stage 3 PCR Reagent DNA Marker USB PCR Markers 50 2000bp Gels
58. commendations 11 Configuration 1 Two Separate Rooms 00000 20s 11 Pre PCR Clean Room 2 eel 12 PoOSEPCR ROOM uta ack Bb equ lt vae ete te KO Rol ee Beek ees 13 Configuration 2 One Room aasa anaana 15 Pre PCR Clean AAS 16 Post PCR Area llis 17 Affymetrix CytoScan Assay User Manual Chapter 4 Chapter 5 Chapter 6 Single Direction Workflow lisse 18 Contamination Prevention 0 200 00 anaana es 19 Satety Precautioris ves site ws zeit aea a be kode Rake EE DIE 19 Genomic DNA General Requirements 21 General Requirements and Recommendations 0 21 Sources of Human Genomic DNA lsseeee ee 22 Blood Collection Methods 2 22 Genomic DNA Extraction Purification Methods 00 22 RNase Treatment lllisieeeee ees 22 Assay Overview s l eacus REG Rura dee Ra RE E 23 Assay and Reagent Configuration 0 0 0 0 asana caaan raau 23 WOTKIlOWS e fase se teeta ak ee on heh REESE Es ase EHE ops 24 Recommended 4 Day Workflow lisse lens 24 Overview and List of Required Reagents Equipment and Consumables 26 About Genomic DNA Preparation 0 26 About Stage 1 Restriction Enzyme Digestion 9 27 About Stage 2 Ligation 1 0 0 0 ees 29 About Stages RCR 31 About Stage 4 PCR Product Purification 00000 34 About Stage 5 Quantitation 0 nee 35 About Stage 6
59. ctivity on the label of the Fragmentation Reagent tube and formulate the master mix appropriately Thermal cycler was not programmed correctly or is out of calibration a Confirm that the fragmentation reaction temperature and incubation time are correctly programmed on the thermal cycler Confirm that the fragmentation mix is made correctly as per the guidelines a Verify that the thermal cycler is within calibration Chapter 8 Troubleshooting 115 CEL File Generation Likely Cause Solution CEL file is not generated Signal from the corner checkerboards is absent Verify that the Oligo Control Reagent was added to the Hybridization Master Mix during assembly The Oligo Control Reagent must be present during hybridization to ensure proper grid alignment Signal from corner checkerboards is dim a Verify that the correct amount of the Oligo Control Reagent was added to the Hybridization Master Mix during assembly a Ensure that GeneChip Hybridization Oven 645 is calibrated and set to the correct temperature a Ensure that Hybridization Master Mix was correctly assembled and added at the correct volume to the fragmented samples a Confirm that Stain Buffer 1 and Stain Buffer 2 are placed in the correct order on the fluidics station Stain Buffer 1 is light sensitive Be sure to store Stain Buffer 1 in the dark when not in use a Use only those staining reagents provided by Affymetrix
60. d sample three at a time Organize your tips to enable multi channel pipetting into the tubes to match the tube and pipette 4 Securely cap each tube and mix well by inverting 10 times Thorough mixing is critical to ensure that the PCR products bind to the beads 5 Incubate at room temperature for 10 minutes During incubation the DNA binds to the Purification Beads 6 Load the tubes cap hinge facing out onto the microcentrifuge and spin for 3 minutes at maximum speed 16 100 rcf Figure 6 14 Figure 6 14 Position tubes with cap hinges out Position tubes with the cap hinges facing out Bead pellet will be spun to the bottom and back of the tube 7 Place the tubes on the magnetic stand until all of the pellets move to the magnet Figure 6 15 Chapter 6 CytoScan Assay Protocol 71 Figure 6 15 Samples with Purification Beads MagnaRack Shown as an Example 8 Usea P1000 pipette to pipet off the supernatant without disturbing the bead pellet Discard the supernatant For gt 8 samples use a multi channel P1000 pipette to pipet off the supernatant without disturbing the bead pellet Discard the supernatant Ensure that the pipette tips are arranged to enable this It is recommended that you remove supernatant from at the most 3 samples at a time Figure 6 16 Bead Pulled to Back and Side of Tube in Magnetic Stand Note The bead pellet will form and be pulled aside in
61. d silver block Applied N8050200 If routinely processing gt 8 samples you may to use Biosystems additional thermal cyclers for PCR Vortex Genie 2 USA Scientific 7404 5600 Richter Anti vibration Pad ISC BioExpress S 7350 25 The microtube foam insert listed above will be attached to one of the vortexers The vortex used with the foam insert may migrate across the bench top during operation We recommend the use of a pad such as the one listed here to prevent movement 152 Affymetrix CytoScan Assay User Manual Consumables Required from Other Suppliers Table F 11 Consumables Required From Other Suppliers v Item Vendor Part Number 1 MicroAmp Clear Adhesive Film for 96 well plates Applied 4306311 Biosystems 1 Pipette tips 20 pL filter tips Rainin GP L10F 1 Pipette tips 200 HL filter tips Rainin GP L200F C1 Pipette tips 1000 pL filter tips Rainin GP L1000F 1 Plates 96 well unskirted PCR Bio Rad MLP 9601 1 Plate OD for UV spec 96 well E amp K Scientific EK 25801 required only if using microplate spectrophotometer 1 Reagent Reservoir 25 mL Diversified RESE 3000 Biotech 1 TBE Gel 4 BMA Reliant precast Lonza Group LTD 54929 1 TBE Gel 2 BMA Reliant precast Lonza Group LTD 54939 1 TBE for electrophoresis Any vendor or house made 1 Tracklt 25 bp DNA Ladder Life Technologies 10488 022 1 Tough Spots 1 2 Diversified Spot 2200
62. day workflow and the 4 day workflow is that you will hybridize your samples onto arrays at the end of day 2 This workflow may be an option if you are processing a small number of samples If processing gt 8 samples the length of time required to complete all Day 2 activities will likely require more than an 8 hr work day Figure 5 2 Optional 3 Day Workflow eem 3 hr 30 min hands on 4 hr 30 min hands on mi Gel 1 1 hr hands on Pre PCR Room Main Lab Stage 3B R PCI Essa R L Gea Stage 4 PCR Purification 2 hr hands on dudum 30 min hands on Stage 6 Feed 2 P lt TO E 1 5 hr 30 min hands on tart ith PCR iea QC Gel 2 Purification and ends with Hybridization SEDE Labeling Stage 8 IW Hybridization Stage 9A 3hr Wash and Stain 30 min hands on Day 3 Wash stain scan Stage 9B 15 min hands on Scanning 32 min per array to scan 5 hr 30 min hands on 16 18 hr 45 min hands on 26 Affymetrix CytoScan Assay User Manual Overview and List of Required Reagents Equipment and Consumables About Genomic DNA Preparation The human genomic DNA you will process using the CytoScan Assay should meet the general requirements listed in Genomic DNA General Requirements on page 21 During this stage you will 1 Determine the concentration of each genomic DNA sample if required 2 Dilute each genomic DNA sample to 50 ng uL using Low EDTA TE buffer
63. dered stages the output of one stage directly impacts the performance of the subsequent stage To efficiently process samples Always use pipettes that have been calibrated as per the manufacturer s specifications a It is essential that operators be proficient with the use of single and multi channel pipettes Always use filter tips for pipetting This is essential to reduce sample contamination To familiarize yourself with the use of multi channel pipettes we strongly recommend practicing several times before processing actual samples You can use water to get a feel for aspirating and dispensing solutions to multiple wells simultaneously Take special care to observe complete evacuation of liquid from all pipette tips when using a multi channel pipette Reagent Handling and Storage IMPORTANT Always use the 24 reaction CytoScan Assay Kit P N 901808 for this protocol You can freeze thaw the reagents in the 24 reaction kit lt 5 times Proper storage and handling of reagents is essential for robust performance Follow these guidelines to ensure best results Use reagents from the recommended vendors only Store all reagents at the recommended temperatures and conditions Do not use reagents that have been improperly stored Storage methods can profoundly impact activity Upon receipt of the reagent kit store the Affymetrix Nuclease Free water at 4 C and the Low EDTA TE Buffer at room temperature for
64. ding dye is recommended in order to improve visualization 1 Dilute the RediLoad dye to 0 1X concentration and use 3 uL of the diluted dye 0 1X concentration for each sample 138 Affymetrix CytoScan Assay User Manual 2 Bring each sample to a total volume of 20 uL using Nuclease Free water For example if the volume of genomic DNA required for 25 ng is 5 uL add 3 uL of 0 1X RediLoad and 12 uL of Nuclease Free water for a total volume of 20 uL Strip tubes or 96 well PCR plates can be used for diluting genomic DNA samples 3 Briefly vortex and spin down the diluted DNA samples before loading onto the E Gel Running the E Gel 1 Turn on the power for the E Base red light 2 Push the Power Prg button to make sure the program is set to EG mode not EP 3 Remove the comb s from a 48 well 1 Agarose E Gel and wipe away any buffer that comes out of the gel or is on the surface 4 Insert the E Gel into the slot 12 well E Gels can also be used if running a smaller number of genomic DNA samples 5 Load 20 uL of genomic DNA sample onto the 48 well 1 agarose E Gel 6 Dilute the High Range DNA Marker 1 3 dilution 5 uL of Marker in 10 uL of Nuclease Free water and load all 15 uL into each of the marker wells as needed 7 Fill all empty wells with 20 uL water 8 Set the run time to 27 minutes 9 Push the Power Prg button again it will change from red to green When the run time is reached the system will au
65. e labeled for digestion and ligation Genomic DNA from CytoScan Reagent Kit positive control What To Do Next Do one of the following a Proceed to Stage 1 Restriction Enzyme Digestion o Store the prepared sample plate at 20 C 48 Affymetrix CytoScan Assay User Manual Stage 1 Restriction Enzyme Digestion Prepare the Reagents Equipment and Consumables Turn On the Thermal Cycler Power on the thermal cycler to preheat the lid Leave the block at room temperature Set Up the Work Area To set up the work area J 1 Place a double cooling block and the water on ice 2 3 4 Place an 8 tube strip as shown in on the upper half of the cooling block Label a 1 5 mL Eppendorf tube as and place in the cooling block Cut adhesive seal into strips wide enough to seal 8 or 12 strip tubes Figure 6 4 Setup for Digestion Nsp enzyme not pictured still at 20 C Strip tubes to aliquot Digestion Master Mix 10X Nsp Buffer 100X BSA Digestion Master Mix tube Chapter 6 CytoScan Assay Protocol 49 Thaw the Reagents and Genomic DNA 1 Prepare the genomic DNA and controls as follows A Vortex at high speed 3 times 1 second each time B Spin down at 2000 rpm for 1 minute If the plate of genomic DNA and controls is frozen allow it to thaw at room temperature Immediately spin down the plate in the centrifuge at 2000 rpm for 1 minute and
66. e Fragmentation Reagent as minimally as possible holding the vial at the cap rather than the center Return the Fragmentation Reagent to the cooler as soon asthe reagent has been dispensed We recommend storing the Fragmentation Reagent at 20 C inside a cooler to preserve its activity Do not over vortex the Fragmentation Reagent 114 Affymetrix CytoScan Assay User Manual Fragmentation QC Step Gel or Bioanalyzer Likely Cause a Insufficient Fragmentation Reagent or 10X Fragmentation Buffer was added during assembly of the Fragmentation Master Mix Improper mixing of the Fragmentation Master Mix Solution Verify pipette calibration and function Take care when preparing the master mix to ensure accurate pipetting and thorough mixing The Fragmentation Master Mix was not made fresh or was allowed to warm to room temperature before use Samples were frozen during fragmentation reaction assembly or centrifugation Incorrect Fragmentation Reagent units were used to prepare the master mix Keep the Fragmentation Master Mix on ice at all times to preserve activity Work quickly during reaction assembly Do not save or reuse a previously assembled Fragmentation Master Mix Make sure that cold blocks are not chilled to 20 C as sample freezing can occur Before centrifugation ensure that the interior of the chilled plate centrifuge is not lower than 2 C Verify the unit a
67. e sure the Hybridization Master Mix is adequately vortexed Add Hybridization Master Mix and Denature f NOTE When working with more than 8 samples we strongly recommend transferring the master mix to a reservoir and dispensing the master mix from the reservoir into the samples using a multi channel pipette To add Hybridization Master Mix and denature the samples 1 Remove and discard the plate seal 2 Pour the Hybridization Master Mix into a reagent reservoir placed on the upper half of the cooling chamber on ice Use a multi channel pipette to add 190 uL of Hybridization mix to the samples Chapter 6 CytoScan Assay Protocol 97 p important The Hybridization Master Mix is viscous pipette carefully when dispensing to samples 3 Tightly seal the plate with a new seal and carefully check to confirm that the plate is well sealed Lu IMPORTANT The volume in the hybridizaton plate is full Ensure that the plate is vortexed to mix sample and hybridization buffer well Vortex the plate at high speed for 1 second each in all corners and in the center REPEAT vortexing to ensure that the plate is well mixed then spin down for 1 minute Place the plate onto the pre heated thermal cycler and run the CytoScan Hyb program Table 6 12 CytoScan Hyb Thermal Cycler Program CytoScan Hyb Program Temperature Time 95 C 10 minutes 49 C Hold Load the Samples onto Arrays To load the
68. eChip Fluidics Station 450 00 0079 1 Tubing Silicone peristaltic for GeneChip Fluidics Station 450 400110 1 GeneChip Hybridization Oven 645 00 0331 1 GeneChip 3000 Scanner with 7G upgrade Contact Affymetrix Affymetrix Software Required Table F 2 Affymetrix Software Required v item Part Number 1 GeneChip Command Console Version 3 2 2 or higher 146 Affymetrix CytoScan Assay User Manual Affymetrix Arrays Required Table F 3 Affymetrix CytoScan HD Reagents and Array Kits v Item Part Number 1 CytoScan Reagent Kit 24 rxns per kit 901808 1 CytoScan HD Array Kit 6 pack 901833 1 CytoScan HD Array and Reagent Kit Bundle 24 arrays rxns 901835 CytoScan HD Training Kit 901834 Table F 4 Affymetrix CytoScan 750K Reagents and Array Kits v Item Part Number 1 CytoScan Reagent Kit 24 rxns per kit 901808 C CytoScan 750K Array Kit 6 pack 901858 1 CytoScan 750K Array and Reagent Kit Bundle 24 arrays rxns 901859 1 CytoScan 750K Training Kit 901860 Affymetrix Reagents Required Appendix F Reagents Equipment and Consumables 147 Table F 5 Affymetrix CytoScan Assay Kit 24 Reaction Kit Components v Item Qty Part Number C1 Affymetrix GeneChip Restriction and Ligation Reagents 1 901803 o Nsp 1 901718 o 10X Nsp 1 Buffer 1 901719 o 100X BSA 1 901720 o Low EDTA TE Buff
69. ed into freshly laundered clothing Maintain an ambient laboratory environment throughout the procedure Store reagents under appropriate conditions according to the box label and reagent kit insert Use proper gowning procedures Print separate copies of the protocol for each room Use filter tips for all pipetting steps Safety Precautions The Affymetrix CytoScan Assay Kit as well as the Affymetrix CytoScan Arrays are for research use only All blood and other potentially infectious materials should be handled as if capable of transmitting infection and disposed of with proper precautions in accordance with federal state and local regulations Some components required for this assay may pose significant health risks Follow prudent laboratory practices when handling and disposing of carcinogens and toxins Refer to the manufacturer s Material Safety Data Sheet for additional information Wear appropriate personal protective equipment when performing this assay At a minimum safety glasses and chemical resistant gloves should be worn 20 Affymetrix CytoScan Assay User Manual Genomic DNA General Requirements The general requirements for genomic DNA sources and extraction methods are described in this chapter The success of this assay requires the amplification of PCR fragments between 150 to 2000 bp in size throughout the genome To achieve this the genomic DNA must be of high quality and must be free of con
70. ells in row B through row H on the Digest Ligate plate a Cap all wells in row E through row H on PCR Plate 132 Affymetrix CytoScan Assay User Manual PCR to Purification Figure B 2 24 Reaction Workflow PCR to Purification EEE amen AAAAAAAAA A AA CU 2 G7 G2 8 6 G2 Aa we o 2 8 43 G2 9 9 OM Gel check for KO G2 8 9 S KD 9 8 ie 9 O p ds eO G2 8 6 8 9 CD 8 9 MMA C fas 24 45 6 17 48 49 20 21 22 Ka 4 G5 86 67 48 49 89 23 23 CX 7 Ka 8 as Go 87 8 as a9 AD IO 2 L Ke 4 8s 6 67 88 is 20 23 22 J VVYVVYVVVYYVYY i d d q d f i i TT Ea Ea T Do not pool dgHEHHHH negative control Add purification beads to each tube and incubate in the tube rack 2000 bp 1500 bp Www 1000 bp 750 bp 9500 bp S 300 bp Ww 150 bp 50 bp Appendix B Guidelines for Processing 24 Samples 133 Purification Continued to Fragmentation and Labeling Figure B 3 24 Reaction Workflow Purification Continued to Fragmentation and Labeling After incubation centrifuge and place the tubes on the magnetic rack multiple racks ma
71. eparation of the Fragmentation Master Mix Carefully observe the pipette tip and the shaft during pipetting of the fragmentation reagent Touch the tip to the inside of the vial to help remove any droplets of enzyme clinging to the exterior of the tip Make sure pipettes are calibrated Purified samples or assembled reactions were allowed to warm to room temperature during reaction assembly or prior to incubation Ensure that the plate centrifuge is completely chilled to 4 C before spinning the assembled fragmentation plate Keep the master mix samples and reaction components on ice or in a cooling block at all times during master mix assembly and dispensing of the master mix to the samples Check that the fragmentation reaction temperature and incubation time are correctly programmed on the thermal cycler and that the fragmentation mix is made correctly as per the guidelines Ensure that the master mix tube and strip tubes are pre chilled before reaction setup Incorrect fragmentation reagent unit was used to prepare the master mix Verify the unit activity on the label of the Fragmentation Reagent tube and formulate the master mix appropriately Under fragmentation PCR product is still visible in 150 2000 bp size region on a 496 agarose gel Improper storage or handling of the Fragmentation Reagent The Fragmentation Reagent should be stored at 20 C at all times Handle th
72. er 1 901697 o 10X T4 DNA Ligase Buffer 1 901722 o T4 DNA Ligase 1 901723 o Water Nuclease Free 1 901781 C1 Affymetrix GeneChip Ligation Adaptors and 1 901749 Fragmentation Reagents Pouch 1 Store in the Pre PCR Room a Genomic DNA 1 900421 a 50 uM Adaptor Nsp 1 900697 o PCR Primer 002 1 901016 Pouch 2 Store in the Post PCR Room n GeneChip Fragmentation Reagent 1 901010 o 10X Fragmentation Buffer 1 900422 n TdT 1 901154 a 5X TdT Buffer 1 900696 o 30 mM DNA Labeling Reagent 1 900699 o Oligo Control Reagent 0100 1 900541 C1 Affymetrix GeneChip Hybridization Reagents 1 901804 o Hyb Buffer Part 1 1 901725 o Hyb Buffer Part 2 1 901726 o Hyb Buffer Part 3 1 901727 o Hyb Buffer Part 4 1 901728 C1 Affymetrix GeneChip Stain Reagents 1 901805 n Stain Buffer 1 1 901751 n Stain Buffer 2 1 901752 a Affymetrix GeneChip Array Holding Buffer 1 901733 n Purification Beads 1 901807 1 Affymetrix GeneChip Purification Reagents 1 901826 o Elution Buffer 1 901738 n Water Nuclease Free 1 901781 o Purification Wash Buffer 901372 1 Affymetrix GeneChip Wash A 2 901680 1 Affymetrix GeneChip Wash B 2 901681 148 Affymetrix CytoScan Assay User Manual Table F 6 Other Affymetrix Reagents Required Item Part Number 1 DNA Marker PCR Markers 50 2000 bp USB 76710 1 TBE Buffer 5X Solution USB 75891 1 1mL RapidRun Loading Dye USB 77524 1 ML 5 mL RapidRun Loading Dye USB 77524 5 ML 1 Ethidium Bromide
73. er Manual The Bleach Cycle To avoid carryover or cross contamination from the bleach protocol Affymetrix recommends the use of dedicated bottles for bleach and DI water Additional bottles can be obtained from Affymetrix Table 9 1 Affymetrix Recommended Bottles 1 Disengage the washblock for each module by pressing down on the cartridge lever Remove any probe array cartridge Figure 9 1 on page 121 2 Prepare 500 mL of 0 52596 sodium hypochlorite solution using deionized water You can follow these directions to make 500 mL of bleach Ina 1 liter plastic or glass graduated cylinder combine 43 75 mL of commercial bleach such as Clorox bleach which is 6 15 sodium hypochlorite with 456 25 mL of DI HO mix well Pour the solution into a 500 mL plastic bottle and place the Part Number Description 400118 Media Bottle SQ 500 mL 400119 Media Bottle SQ 1000 mL plastic bottle on fluidics station Lu IMPORTANT The shelf life of this solution is 24 hr After this period you must prepare fresh solution a Each fluidics station with 4 modules requires 500 mL of 0 525 sodium hypochlorite solution Chapter 9 Fluidics Station Care and Maintenance 121 3 As shown in Figure 9 2 on page 122 A Place on the fluidics station an empty one liter waste bottle a 500 mL bottle of bleach and a one liter bottle of DI water The Bleach protocol requires approximately one liter of DI wate
74. er stocks of PCR primer and adaptor only in the laminar flow cabinet IMPORTANT We strongly recommend that each pre PCR step be performed in a laminar flow or PCR cabinet including reagent and master mix preparation The use of this cabinet is essential for preventing sample contamination due to the introduction of PCR products from the Post PCR Area and DNA template All of the equipment required for the pre PCR steps should be dedicated for pre PCR and kept in the laminar flow or PCR cabinet This equipment includes pipettes and tips the thermal cycler and vortexer Post PCR Area Chapter 3 Laboratory Setup and Recommendations 17 The Post PCR Area has airborne contamination with PCR product and template After entering the Post PCR Area it is inadvisable to re enter the Pre PCR Clean Area without first showering and changing into freshly laundered clothes The equipment shown for the Post PCR Area in Figure 3 3 on page 15 consists of WRN 0 Um RES wW Noa A 2 o2 o2 ol 2 2 a O ON DU PWN O Computer monitor and keyboard Fluidics station Scanner Ice bucket Magnetic stand Vortexer Microfuge Pipettes on stand Vortexer with foam tube adaptor Thermal cycler one to three GeneChip Hybridization Oven 645 Refrigerated plate centrifuge Microcentrifuge Plate spectrophotometer Gel imager Electrophoresis gel box Electrophoresis power supply Refrigerator Freezer 18 Affymetr
75. es Required for Stage 1 Restriction Enzyme Digestion Quantity Item As required Adhesive seals for 96 well plates 1 Plate centrifuge 1 Cooler chilled to 20 C Cooling chamber double block chilled to 4 C placed on ice do not freeze Ice bucket filled with ice Marker fine point permanent Mini centrifuge microfuge Pipette single channel P10 Pipette single channel P100 or P200 1 Pipette 12 channel 2 20 uL 1 Pipette 12 channel 20 200 HL As required Pipette tips for pipettes listed above 1 Thermal cycler 1 8 12 well strip tubes 0 2 mL As required 8 12 tube strip caps 2 Tubes Eppendorf Safe Lock Tubes 1 5 mL Natural 1 Vortexer 2 GeneMate 96 Well PCR Tube Storage Rack IMPORTANT Use only the thermal cyclers 96 well plate and adhesive films and listed under Thermal Cyclers 96 Well Plate and Adhesive Seals on page 8 Chapter 5 Assay Overview 29 Reagents Required The following reagents are required for this stage Table 5 4 Reagents Required for Stage 1 Restriction Enzyme Digestion Reagent 100X BSA 10X Nsp Buffer Nsp Chilled Affymetrix Nuclease Free Water About Stage 2 Ligation During this stage the digested samples are ligated using the Nsp I Adaptor You will 1 Prepare a Ligation Master Mix and add it to the Nsp I digested sam
76. event salt crystals from forming within the fluidics system To ensure proper functioning of the instrument perform periodic maintenance When not using the instrument leave the sample needles in the lowered position Each needle should extend into an empty vial This will protect them from accidental damage Always use deionized water to prevent contamination of the lines Change buffers with freshly prepared buffer at each system startup The fluidics station should be positioned on a sturdy level bench away from extremes in temperature and away from moving air WARNING Before performing any maintenance turn off power to the fluidics station to avoid injury in case of a pump or electrical malfunction Fluidics Station Bleach Protocol Affymetrix recommends a weekly cleaning protocol for the fluidics station This protocol uses commonly purchased sodium hypochlorite bleach This protocol is designed to eliminate any residual SAPE antibody complex that may be present in the fluidics station tubing and needles The protocol runs a bleach solution through the system followed by a rinse cycle with deionized DI water This protocol takes approximately one hour and forty minutes to complete Affymetrix recommends running this protocol weekly regardless of the frequency of use The current version of the protocol can be found at www affymetrix com support technical fluidics scripts affx 120 Affymetrix CytoScan Assay Us
77. ever a plate is taken out of the freezer first thaw the plate ensure that the seal is tight centrifuge and only then remove the plate seal When reaction setup is completed always use a new seal to seal the plate When applying the seal to a plate press the seal tightly onto the plate using an adhesive film applicator Using a plastic lid or a plastic tube rack is a potential source of contamination Make sure that the seal is tight around all plate well edges 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 NEVER REUSE A SEAL ALWAYS USE A NEW SEAL Sealing Strip Tubes Vortex Cut adhesive seal into strips wide enough to seal 8 or 12 strip tubes Alternatively strip caps can also be used for sealing Seal the strip tubes containing master mix with the adhesive strips or strip caps before spinning in the bench top quick spin microfuge Master Mix tubes Vortex the master mix at high speed 3 times 1 second each time Vortex reagents 3 times 1 second each time Vortex enzyme Quick vortex 1 second Vortex plates High speed for 1 second in all corners and in the center Figure 2 1 Chapter 2 Best Practices 7 Figure 2 1 Vortex Plates at the Corners and Center OCOOODQOOCOCO Spin When instructed to spin down plates or reagent vials follow these guidelines
78. fication Wash Buffer 1 Add 45 mL of absolute ethanol to the Purification Wash Buffer bottle IMPORTANT Ensure that the correct amount of ethanol has been added to the Purification Wash Buffer bottle 2 Cap the bottle tightly and shake 3 Enter the preparation date on the bottle label and put a check mark in the check box Pool the PCR Products ZZ CAUTION Be very careful when pooling PCR products Avoid cross contaminating neighboring wells with small droplets To pool the PCR products 1 If frozen thaw the PCR products in a plate holder on the bench top to room temperature 2 Ensure the plate seal is tight Vortex the plate at high speed for 1 second in all corners andin the center according to the guidelines in Seal Vortex and Spin on page 5 then spin down at 2000 rpm for 1 minute 3 Mark each 1 5 mL Eppendorf tube with a sample number such as 2 3 4 etc Remove and discard the plate seal 5 Using a P200 single or multi channel pipette transfer all 4 aliquots of each sample to the appropriately marked 1 5 mL tube Figure 6 13 on page 69 Do not pool the negative control Discard Ls IMPORTANT Change pipette tips after each transfer PCR wells 4 100 uL from each well 400 HL Total Volume in Each 1 5 mL Eppendorf Tube 400 pL tube 3 ul aliquoted for PCR gel 6 When finished examine the PCR plate and ensure that the total volume in each well has been transferred and
79. follows A Vortex 3 times 1 second each time B Pulse spin for 3 seconds C Place in the cooling block Prepare the Fragmentation Master Mix Ls IMPORTANT All additions in this procedure must be performed on ice We strongly recommend preparing the Fragmentation Master Mix following Table 6 7 only The Fragmentation Master Mix is sufficient for 1 to 24 samples Lu IMPORTANT Check the Fragmentation Reagent concentration before making the Master Mix Do not make less than the recommended volume of Master Mix 1 Check the concentration of the Fragmentation Reagent stated on the tube label then add the required volume of the water and fragmentation buffer by following Table 6 7 2 Mix by vortexing for 1 second and pulse spin Place back in the cooling block on ice 82 Affymetrix CytoScan Assay User Manual Table 6 7 Fragmentation Master Mix Reagent Fragmentation Reagent Concentration 2 0 U uL 2 25 U HL 2 5 U uL 2 75 U HL 3 0 U uL Chilled Affymetrix Nuclease Free 122 4 uL 123 2 uL 123 8 uL 124 4 uL 124 8 uL Water 10X Fragmentation Buffer 158 4 uL 158 4 uL 158 4 uL 158 4 uL 158 4 uL Fragmentation Reagent 7 2 uL 6 4 uL 5 8 uL 5 2 uL 4 8 uL Total 288 0 pL 288 0 HL 288 0 pL 288 0 HL 288 0 HL 3 Remove the Fragmentation Reagent from the freezer and immediately place it in the cooler chilled to 20 C A Vortex the Fragmentation Reagent at high speed one time for 1 second B Immedia
80. ge 8 Washing Arrays on page 8 a Preparing the Work Area for Each Stage on page 8 a Thermal Cyclers 96 Well Plate and Adhesive Seals on page 8 a Hybridization Oven on page 10 Using positive and negative controls is recommended to assess the performance of each run We recommend using the Genomic DNA Control supplied in the CytoScan Reagent Kit as a positive control carried through the entire assay up to hybridization on the arrays We recommend using Low EDTA TE Buffer as a negative control through the PCR gel QC stage only Equipment and Calibration Keep dedicated equipment in each ofthe areas used for this protocol including pipettors ice buckets coolers etc It is critical to use equipment that conforms to the guidelines and specifications detailed in this manual To avoid contamination do not move equipment back and forth from the Post PCR Room to Pre PCR Clean Room Lab instrumentation plays an important role in the successful execution of this assay To help maintain consistency across samples and operators all equipment must be well maintained and routinely calibrated per manufacturer recommendations including All thermal cyclers a GeneChip Hybridization Oven 645 Pipetting Affymetrix CytoScan Assay User Manual a GeneChip Fluidics Station a GeneChip Scanner 3000 7G Plate spectrophotometer or NanoDrop a All single and multi channel pipettes Since the CytoScan Assay involves a series of or
81. here the majority ofthe area under the curve lies either above or below 15 bp respectively Figure C 1 Example of Correctly Fragmented DNA Sample FU S S e 2004 PUTA PEU p 1E T T s bp is 50 100 150 200 300 400 S00 700 1500 136 Affymetrix CytoScan Assay User Manual Running E Gels Equipment E Gels and Reagents Required Table D 1 Equipment E Gels and Reagents Required Item Supplier Part No Mother E Base Device Life Technologies EB M03 Daughter E Base Device optional for EB D03 running multiple gels simultaneously E Gel 48 1 Agarose Gels G8008 01 E Gel 48 2 Agarose Gels G8008 02 E Gel 48 4 Agarose Gels G8008 04 RediLoad Loading Buffer 750026 E Gel 96 High Range DNA Marker 12352 019 Tracklt 25 bp DNA Ladder 25 500 bp 10488 022 Tracklt Cyan Orange Loading Buffer 10482 028 PCR Marker 50 2000 bp Affymetrix 76710 f NOTE The E Gel contains ethidium bromide Review the manufacturer s Material Safety Data Sheet for proper handling and disposal Use good laboratory practices and always wear gloves when handling E Gels Dispose of the gel and gloves in accordance with national state and local regulations Genomic DNA on 1 E Gel Diluting Genomic DNA Samples Loading a DNA mass of approximately 25 ng per well is recommended If lower amounts are loaded omission of the loa
82. ication 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 Genomic DNA extracted using the following methods have been tested at Affymetrix QIAGEN Gentra Puregene Kit 5 PRIME PerfectPure DNA Blood Kit Lu IMPORTANT The CytoScan Assay requires genomic DNA concentration gt 50 ng uL Therefore the elution volumes for each of the kits will need to be adjusted accordingly to achieve the desired concentration RNase Treatment The presence of RNA and free nucleotides can interfere with some quantitation methods using specrophotometer or a NanoDrop instrument To eliminate RNA contamination perform RNase treatment during extraction as follows QIAGEN Gentra Puregene Kit Perform RNase treatment as recommended in the extraction kit manual prior to elution of genomic DNA a 5 PRIME PerfectPure DNA Blood Kit Use only RNase treated purification columns for extraction of genomic DNA The purified genomic DNA extracted using the two methods above should meet the DNA quality specifications per the manufacturer s kit extraction manual Assay Overview This chapter provides an overview of the Affymetrix CytoScan Assay including information about assay configuration and workflows It briefly explains each ste
83. icking the tab to create new sample ARR files A new window opens 6 Click Save to save the new sample files Prepare the Arrays 1 Place the arrays on a clean bench top area designated for hybridization 2 Insert a 200 uL pipette tip into the upper right septum of each array 3 Paste two 1 2 Tough Spots on the top edge of the array for later use Figure 6 27 IMPORTANT To ensure that the data collected during scanning is associated with the correct sample mark each array in a meaningful way It is critical that you know which sample is loaded onto each array 94 Affymetrix CytoScan Assay User Manual Figure 6 27 Arrays Prepared for Sample Loading Prepare the Reagents and Consumables Set Up the Work Area To set up the work area 1 Place a double cooling block on ice Figure 6 28 on page 95 2 Place a reagent reservoir on the upper half of the cooling block on ice 3 Labelthe 15 mL centrifuge tube as Hyb Master Mix and place on the ice Prepare the Samples 1 Ifthe labeled samples from the previous stage were frozen allow them to thaw on the bench top to room temperature and spin down at 2000 rpm for 1 minute 2 Immediately place the plate in the lower half of the cooling block on ice Thaw and Prepare the Reagents Thaw the following reagents at room temperature Immediately place on cooling block on ice when thawed Hyb Buffer Part 1 Hyb Buffer Part 2 Hyb
84. ightly seal the plate with a new seal according to the guidelines in Seal Vortex and Spin on page 5 Vortex at high speed for 1 second in all corners and in the center according to the guidelines in Seal Vortex and Spin on page 5 REPEAT vortexing one more time then spin down at 2000 rpm for 1 minute Keep the plate in the cooling block on ice until ready to load onto a thermal cycler Load PCR Plate onto a Thermal Cycler s IMPORTANT Ensure that the GeneAmp PCR System 9700 thermal cycler you are using is equipped with silver or gold plated silver blocks Do NOT use thermal cyclers with aluminum blocks It is difficult to visually distinguish silver and aluminum blocks Instead confirm the block type by checking the part number Location Post PCR Area Procedure To load the plate and run the CytoScan PCR program 1 2 Transfer the plate on ice to the Post PCR Area Ensure that the thermal cycler lid is preheated 3 Chapter 6 CytoScan Assay Protocol 65 The block should be at room temperature Load the plate onto the thermal cycler 4 Run the CytoScan PCR program Table 6 6 CytoScan PCR Thermal Cycler Program for the GeneAmp PCR System 9700 use only silver or gold plated silver blocks CytoScan PCR Program for GeneAmp PCR System 9700 Temperature Time Cycles 94 C 3 minutes 1X 94 C 30 seconds 60 C 45 seconds 30X 68 C 15 seconds 68 C 7 minutes 1X 4 C HOLD
85. iming and array washing a Ensure that the Fluidics Stations are maintained according to the guidelines in the Fluidics Station User s Guide 118 Affymetrix CytoScan Assay User Manual Affymetrix Instruments Under any ofthe following conditions unplug the instrument from the power source and contact Affymetrix Technical Support When the power cord is damaged or frayed a If any liquid has penetrated the instrument a If after service or calibration the instrument does not perform to specifications NOTE Make sure you have the model and serial number available when calling Affymetrix Technical Support Affymetrix Inc 3420 Central Expressway E mail support affymetrix com Santa Clara CA 95051 Tel 1 888 362 2447 1 888 DNA CHIP USA Fax 1 408 731 5441 Affymetrix UK Ltd Voyager Mercury Park E mail supporteurope affymetrix com Wycombe Lane Wooburn Green UK and Others Tel 44 0 1628 552550 High Wycombe HP10 OHH France Tel 0800919505 United Kingdom Germany Tel 01803001334 Fax 44 0 1628 552585 Affymetrix Japan K K Mita NN Bldg Tel 03 5730 8200 16 Floor 4 1 23 Shiba Fax 03 5730 8201 Minato ku Tokyo 108 0014 Japan Fluidics Station Care and Maintenance General Fluidics Station Care a Use a surge protector on the power line to the fluidics station Always run a Shutdown protocol when the instrument will be off or unused overnight or longer This will pr
86. is using the Agilent 2100 Bioanalyzer For more details see Appendix C on page 135 Add 2 uL of USB 5X RapidRun Loading Dye to each sample in the Gel Analysis QC strip Seal the strip tubes tightly with an adhesive seal strip vortex and spin down Load 8 uL of the samples onto the gel Load 2 uL TrackIt 25 bp DNA Ladder to the first and last lanes Run the samples on a 4 TBE gel at 5 V cm for 45 minutes or until the dye front reaches at least 75 of distance down the gel NOTE Run gels at 5V cm 5 volts x Distance in cm between electrodes For example run a 33 cm electrophoresis box at 165 V run a 16 cm electrophoresis box at 80 V 86 Affymetrix CytoScan Assay User Manual 14 Inspect the gel and compare it against the example shown in Figure 6 24 The majority of fragment distribution should be between 25 to 125 bp Figure 6 24 Example of Fragmented PCR Products Run on 496 TBE Agarose Gel at 5 V cm for 45 minutes Fragmentation is confirmed by majority of distribution between 25 to 125 bp 1 2 3 4 5 6 Wi 500 bp 500 bp 25 bp ies 125 bp 25 bp D n H T D T 25 bp Chapter 6 CytoScan Assay Protocol 87 Stage 7 Labeling Prepare the Reagents Equipment and Consumables Turn on the Thermal Cycler Power on the thermal cycler to preheat the lid Leave the block at room temperature Set Up the Work Area 1 2 3 4 Place a double cooling block on ice Figu
87. ix CytoScan Assay User Manual Single Direction Workflow To keep the Pre PCR Clean Area as free from PCR amplicons and other contaminants as possible always maintain a single direction workflow Figure 3 4 Single Direction Workflow 1 Laminar flow or PCR cabinet aa Q EN Enter laboratory Note Entering and exiting through the Pre PCR Clean Pre PCR Clean Area Area only is permissible as long as the Post PCR Area has not been entered 9 8 Post PCR Area Chapter 3 Laboratory Setup and Recommendations 19 Contamination Prevention Care should be taken to minimize possible sources of contamination that could interfere with copy number and genotyping analysis To reduce the possibility of cross contamination Affymetrix strongly recommends that you maintain a single direction workflow from the Pre PCR Clean Area to the Post PCR Area Do not re enter the Pre PCR Clean Area from the Post PCR Area The most likely potential source of contamination for the CytoScan Assay is previously amplified PCR product Precautions that you can take to minimize contaminating pre PCR steps with amplified PCR product include the following Each area should contain dedicated equipment such as thermal cyclers microfuges pipettes and tips ice buckets etc Once you enter the Post PCR Area do not return to the Pre PCR Clean Area until you have showered and chang
88. lace in the lower half of the chamber as shown in Figure 6 10 62 Affymetrix CytoScan Assay User Manual Reagent reservoir L2 3f a I 5To T I oo00o000000 089000000 00000000 PCR Master Mix tube PCR Primer 002 dNTP Mixture 2 5 mM each GC Melt Reagent TITANIUM Taq PCR Buffer 5 To prepare the reagents except enzyme A Vortex the reagents at high speed 3 times 1 second each time Pulse spin for 3 seconds B Place in the cooling block Turn On the Thermal Cycler Post PCR Area Have someone in the Post PCR Area power on the thermal cycler to preheat the lid Leave the block at room temperature To avoid contamination do not go from the Pre PCR Clean Area to the Post PCR Area and back again Chapter 6 CytoScan Assay Protocol 63 Prepare the PCR Master Mix Lu IMPORTANT Accurate pipetting of all components is critical for obtaining the correct size distribution of PCR products Keeping the 15 mL centrifuge tube on ice add the reagents in the order shown in Table 6 5 and Figure 6 10 on page 62 except for the 50X TITANIUM Taq DNA polymerase Remove the 50X TITANIUM Tag DNA Polymerase from the freezer and immediately place in a cooler chilled to 20 C Vortex at high speed for 1 second Pulse spin the 50X TITANIUM Tag DNA polymerase for 3 seconds Immediately add the 50X TITANIUM Taq DNA polymerase to the master mix then return the tube to
89. minar flow cabinet or a PCR cabinet when the entire assay is to be performed in one room Pre PCR Clean Area 9 8 Em mm EE o NEM S EE S Oe Em mm E E NERO 8 Ce m MARKING ON FLOOR TO DELINEATE PRE PCR CLEAN AREA FROM POST PCR AREA m umm Nm NEM NE NM Nm Post PCR Area GED 809 og 0507 16 Affymetrix CytoScan Assay User Manual Pre PCR Clean Area For the best results adhere to the following guidelines Keep the Pre PCR Clean Area free of PCR amplicons a If both pre and post PCR operations are performed in the same room and a laminar flow cabinet is used keep it turned on at all times Keep the UV light in the laminar flow or PCR cabinet turned on when not in use Always wear a gown booties and gloves to prevent PCR carryover and to minimize the risk of trace levels of contaminants being brought into this area Equipment in Pre PCR Clean Area The equipment shown for the Pre PCR Clean Area in Figure 3 3 on page 15 is listed below Laminar flow cabinet or PCR cabinet Vortexer Microfuge Pipettes on stand Ice bucket with ice Thermal cycler Plate centrifuge Freezer wp en auw PWN Refrigerator About Laminar Flow Cabinets The air curtain from the laminar flow cabinet prevents the introduction of contaminants from the surrounding air into work area particularly PCR products from the Post PCR Area Open mast
90. mples 16 Samples 24 Samples 20 20 20 overage overage overage Chilled Affymetrix 11 55 uL 110 9 uL 221 8 uL 332 6 uL Nuclease Free Water 10X Nsp Buffer 2 00 uL 19 2 uL 38 4 uL 57 6 HL 100X BSA 0 20 uL 1 9 uL 3 8 uL 5 8 uL Nsp 1 00 uL 9 6 uL 19 2 uL 28 8 uL Total 14 75 HL 141 6 HL 283 2 HL 424 8 pL Chapter 6 CytoScan Assay Protocol 51 Add Digestion Master Mix to Samples To add Digestion Master Mix to samples s NOTE When working with more than 8 samples we strongly recommend dividing the master mix into strip tubes and dispensing the master mix from the strip tubes into the samples using a multi channel pipette 1 Divide the Digestion Master Mix equally into the 8 12 strip tubes on ice Seal the strip tube with an adhesive seal strip or strip caps Spin and place back on a cooling block on ice Remove the seal and discard Unseal the plate and discard the seal Using a multi channel P20 pipette aliquot 14 75 uL of Digestion Master Mix to each sample and controls in row A 4 Sealthe plate tightly with a new seal Genomic DNA 50 ng uL 5 00 uL Digestion Master Mix 14 75 uL Total Volume 19 75 uL Figure 6 5 Adding Digestion Master Mix to Samples and Controls NONE TET Add 14 75 HL Digestion Master Mix to each sample and control in row A JO O 5505800 0000000 00000000 0101010101010 OOOOOOOQQ OOOOOOOQQ 01010101010100 01010101010100 OO
91. n 645 is calibrated before starting the hybridization step Accurate hybridization temperature is critical for this assay We recommend servicing hybridization ovens at least once per year to ensure that they are operating within the manufacturer s specifications Laboratory Setup and Recommendations This chapter provides an overview of two laboratory setups that can be used when performing the Affymetrix CytoScan Assay Lu IMPORTANT If possible we strongly recommend using two separate rooms when performing this protocol Configuration 1 Two Separate Rooms The use of two separate rooms greatly reduces the risk of sample contamination due to previously amplified PCR products These rooms are referred to as the a Pre PCR Clean Room a Post PCR Room The high level steps performed in each room are presented in Table 3 1 Table 3 1 Assay Workflow When Two Separate Rooms are Used Room Template PCR Product Genomic DNA Pre PCR Clean Room o Ligation o PCR setup only Post PCR Room Assay steps n PCR thermal cycling o Fragmentation n Labeling a Hybridization a Washing and staining o Scanning Assay steps o Genomic DNA preparation o Digestion 12 Affymetrix CytoScan Assay User Manual Pre PCR Clean Room The Pre PCR Clean Room should be a low copy DNA template lab and should be free of PCR product amplicons The major pieces of equipment required for this room are shown in Figure
92. n contamination 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 Laboratory Workflow Maintain a single direction workflow Do not re enter the Pre PCR Clean Area after entering the Post PCR Area until you have showered and changed into freshly laundered clothing Never bring amplified products into the Pre PCR Clean Area Keep dedicated equipment in each room or area used for this protocol To avoid contamination do not move equipment between the Pre PCR Clean Area and the Post PCR Area Seal Vortex and Spin Unless otherwise noted follow the instructions below when the protocol instructs you to seal vortex and spin 6 Affymetrix CytoScan Assay User Manual Handling the Plate Seal NOTE We recommend using MicroAmp Clear Adhesive Films to seal your plates To minimize sample cross contamination and to ensure tight seals use each seal only once NEVER REUSE A SEAL Discard used seals immediately to avoid contaminating equipment or working surfaces with DNA The seal may become loose due to high temperature in the thermal cycler Always ensure tight sealing before vortexing a plate Whenever a plate is taken out of the thermal cycler before continuing on to the next step ensure that the seal is tight spin the plate in the centrifuge then remove the seal and discard When
93. nadequate bead washing prior to elution Repeat purification with attention towards complete removal of the binding eluate before the bead wash Excess Elution Buffer added to beads Verify pipette calibration and function Incorrect buffer was used for elution Verify that the Elution Buffer was used during the elution step and not the Purification Wash Buffer Purification Beads were over dried Do not dry Purification Beads longer than the recommended time The eluted DNA plate was inadequately vortexed before taking an aliquot for an OD reading Eluted DNA can be heterogeneous Repeat the dilution followed by an OD reading making sure to vortex the eluted DNA and the OD plate thoroughly at each step 112 Affymetrix CytoScan Assay User Manual Purification Yield QC Step Likely Cause Solution PCR reaction volume was inaccurate Repeat the assay and confirm that the PCR reaction is set up correctly High yields gt 4 5 ug ul Too little Elution Buffer added to the Purification Beads Eluted DNA plate inadequately vortexed before OD reading is taken Instruments or pipettes may be out of calibration or incorrectly set Yield calculation formula within the software template may be incorrect Verify pipette calibration and function Make sure 52 HL of Elution Buffer is added to the Purification Beads for elution Eluted DNA can be heterogeneous Repeat
94. nd workflows available in the Command Console software If you would like to learn more about Command Console please refer to the Affymetrix GeneChip Command Console 3 2 User Manual P N 702569 Prepare the Equipment Turn On the Thermal Cycler Power on the thermal cycler to preheat the lid Leave the block at room temperature Preheat the Hybridization Oven 645 ES note Confirm that the Hybridization Oven 645 is calibrated The hybridization oven should be serviced at least once per year to ensure operation within specification To preheat the hybridization ovens 1 Turn on the oven at least 1 hour before hybridization with the temperature set to 50 C 2 Set the rpm to 60 3 Turn the rotation on and allow to preheat for 1 hr before loading arrays Prepare the Arrays and Create a Batch Registration File To prepare the arrays 1 Unwrap the arrays and place on the bench top septa side up 2 Mark the front and back of each array with a designation that will identify which sample is loaded onto each array Figure 6 27 Allow the arrays to warm to room temperature on the bench top for 10 to 15 minutes During this time you can scan the barcode which will be used in batch registration 92 Affymetrix CytoScan Assay User Manual Create a Batch Registration File To register a new sample using AGCC 1 From the Command Console launch the AGCC Portal Lu IMPORTANT Confirm that you are running AGCC v
95. ndow display the status of the washing and staining steps When the wash and stain procedure is completed remove the arrays from the fluidics station by first pressing down the cartridge lever to the Eject position Check the array window for bubbles or air pockets If air bubbles are present return the array to the fluidics station Follow the instructions on the LCD panel of the fluidics station Pull the lever up and load to remove bubbles If air bubbles are still present after repeating the above process a few times use the manual process A Insert a 200 uL pipette tip into the upper right septum of the array B Using a pipette remove half of the solution C Manually fill the array with Array Holding Buffer If the array has no bubble it is ready for scanning Proceed to Scanning Arrays on page 102 If the arrays cannot be scanned promptly store them at 4 C in the dark until ready for scanning Scan must be performed within 24 hr Pull up on the cartridge lever to engage wash block Remove the microcentrifuge vials containing stain and replace with three empty vials as prompted When washing and staining are complete shut down the fluidics station following the procedure on page 105 Scanning Arrays The GeneChip Scanner 3000 7G is controlled by AGCC software Prepare the Scanner Turn on the scanner at least 10 minutes before use Chapter 7 Washing Staining and Scanning Arrays 103 WARNING The scanne
96. ng MAPD Assay drift due to variation in assay execution Over fragmentation Degraded starting material Reference is inappropriate for the sample a Recalibrate pipettes to ensure accurate delivery of reagent volumes Consider operator retraining or review by an Affymetrix Field Applications Scientist if the problem persists a Review Chapter 2 Best Practices on page 3 See above a Perform a QC gel of input DNAs to assess samples for degradation Ensure that the DNA samples are of high quality for example run in a 1 to 296 agarose gel and compare to the Genomic DNA Control provided in the CytoScan Reagent Kit Use only the recommended sample types peripheral blood and cell line DNA Chapter 8 Troubleshooting 117 Data QC Failures Likely Cause Solution High waviness SD Degraded genomic DNA Incompatible sample type Incompatible genomic DNA extraction method used Sample specific effect Confirm that the genomic DNA sample meets the quality and integrity guidelines in Chapter 4 on page 21 Use only cell line or blood derived genomic DNA Only use the recommended extraction methods See Chapter 4 on page 22 See the Chromosome Analysis Suite User Manual P N 702943 High MAPD with low SNPQC Error during washing the array Ensure that the Wash A and B lines of the Fluidics Station are placed in the correct wash buffers during pr
97. nning of arrays The major pieces of equipment required for this room are shown in Figure 3 2 14 Affymetrix CytoScan Assay User Manual Figure 3 2 Post PCR Room Equipment Shown 1 PWN Seat gr 11 17 10 Vortexer Microfuge Pipettes on stand Vortexer with foam tube adaptor Ice bucket Magnetic stand Thermal cycler GeneChip Hybridization Oven 645 Refrigerated plate centrifuge Microcentrifuge Plate spectrophotometer 12 13 14 15 16 Gel Imager Electrophoresis gel box Computer monitor keyboard Fluidics Station Scanner Refrigerator 18 Freezer To help prevent sample contamination All ofthe reagents and master stocks required for the steps performed in the Post PCR Room should be stored in this room under the appropriate conditions All ofthe equipment required for the steps performed in this area should be dedicated Do not move any equipment including ice buckets and pipettes between the Pre and Post PCR Rooms Chapter 3 Laboratory Setup and Recommendations 15 Always wear a fresh gown and gloves to minimize sample contamination Configuration 2 One Room One room with two distinctly separated areas Pre PCR Clean Area and Post PCR Area Figure 3 3 One Room Configuration 1 Laminar Flow or PCR Cabinet uu o We strongly recommend the use of a la
98. o position all tubes with the cap hinges facing out as shown in Figure 6 14 6 Place the tubes on the magnetic stand for 10 minutes The purification beads are pulled to the side of the tube Figure 6 18 Figure 6 18 Bead Pulled to Back and Side of Tube in Magnetic Stand Note The bead pellet will form and be pulled aside in all supported racks This is an example of pellet formation in the MagnaRack Avoid contact with the bead pellet when pipetting off the supernatant 7 Check that all of the beads have been pulled to the side in each tube If not vortex the tubes to resuspend the pellet Centrifuge the tubes for 3 minutes at maximum speed position the tubes with the cap hinges facing out 16 100 rcf Place the tubes on the magnetic stand for 10 minutes 74 Affymetrix CytoScan Assay User Manual NOTE The eluate may appear yellowish 8 Transfer 47 uL of eluted sample to the appropriate well on a fresh 96 well plate Figure 6 19 on page 74 Sometimes at this step a brown residue is observed at the end of the pipette tip It will usually remain behind on the tip when the sample is pipetted out Figure 6 19 Transfer Each Purified Sample to a Fresh 96 well Plate MagnaRack Shown as an Example OOOOOOOOQO OOOOOO6 00000006 OOOOOOOQ OOOOOOQ OOOOOOOQ 20000000 e OOOOOOO0 OQ 9 After transferring the eluted samples to the plate tightl
99. oScan Assay User Manual To set up the work area Figure 6 22 1 Turn down the plate centrifuge to 4 C at least 15 to 20 minutes prior to proceeding into the fragmentation step Remember to close the centrifuge lid to facilitate effective cooling Place a double cooling block and the Affymetrix Nuclease Free water on ice 3 Place an 8 tube strip in the cooling block as shown in Figure 6 22 and chill it for at least 10 minutes prior to use 4 Labela 1 5 mL Eppendorf tube as FRAG for the Fragmentation Master Mix and keep it chilled in the cooling block 5 Cutadhesive seal into strips wide enough to seal 8 or 12 strip tubes Ensure that the plate centrifuge is at 4 C Strip tubes to aliquot Fragmentation Master Mix Fragmentation Master Mix tube Purified 112 3 4 5 6 a ee ean samples Chapter 6 CytoScan Assay Protocol 81 Thaw and Prepare the Reagents Lu IMPORTANT Leave the Fragmentation Reagent at 20 C until ready to use 1 Remove the plate of purified quantitated samples from the 20 C freezer and thaw at room temperature Once thawed completely make sure the plate is sealed tightly then vortex and spin down the plate Place the plate on lower half of the cooling block on ice and chill for 10 minutes prior to use 2 Thaw the Fragmentation Buffer 10X at room temperature Immediately place on cooling block on ice when thawed 3 Prepare the Fragmentation Buffer as
100. oScanHD Array 450 a If using the CytoScan 750K Array select CytoScan750K Array 450 Start the protocol and follow the instructions in the LCD on the Fluidics Station If you are unfamiliar with inserting and removing arrays from the fluidics station modules refer to the appropriate Fluidics Station User s Guide or Quick Reference Card P N 08 0093 for the Fluidics Station 450 Eject the wash block to avoid sensor time out Remove any previously loaded empty vials When prompted to Load vials 1 2 3 A Place one vial containing 500 uL Stain Buffer 1 in position 1 B Place one vial containing 500 uL Stain Buffer 2 in position 2 C Place one vial containing 800 uL Array Holding Buffer in position 3 After 16 to 18 hrs of hybridization remove no more than 8 arrays at a time from the oven Remove the Tough Spots from the arrays 102 Affymetrix CytoScan Assay User Manual 8 10 1 12 13 14 15 Lu IMPORTANT Once the arrays are removed from the hybridization oven quickly load them onto the Fluidics Station Delays during this step will impact data quality Immediately insert the arrays into the designated modules of the fluidics station while the cartridge lever is in the Down or Eject position Press down on the needle lever to snap needles into position and to start the run The fluidics protocol begins The Fluidics Station dialog box at the workstation terminal and the LCD wi
101. ocol 55 Prepare the Ligation Master Mix Keeping all reagents and tubes on ice prepare the Ligation Master Mix as follows 1 To the 1 5 mL Eppendorf tube labeled Lig add the following reagents based on the volumes shown in Table 6 3 a 10X T4 DNA Ligase Buffer 50 uM Adaptor Nsp I Remove the T4 DNA Ligase from the freezer and immediately place in the cooler chilled to 20 C Vortex at high speed for 1 second Pulse spin the T4 DNA Ligase for 3 seconds and place it in the 20 C cooler Immediately add the T4 DNA Ligase to the master mix then place back in the 20 C cooler Table 6 3 Ligation Master Mix Reagent 1 Sample 8 Samples 16 Samples 24 Samples 25 overage 25 overage 25 overage 10X T4 DNA Ligase Buffer 2 50 uL 25 0 uL 50 0 uL 75 0 uL 50 uM Adaptor Nsp I 0 75 uL 7 5 uL 15 0 uL 22 5 uL T4 DNA Ligase 2 00 uL 20 0 uL 40 0 uL 60 0 uL Total 5 25 HL 52 5 uL 105 0 pL 157 5 pL pe Ono Vortex the master mix at high speed 3 times second each time Pulse spin for 3 seconds Place the master mix in the cooling block on ice Proceed immediately to Add Ligation Master Mix to Reactions 56 Affymetrix CytoScan Assay User Manual Add Ligation Master Mix to Reactions To add Ligation Master Mix to samples E NOTE When working with more than 8 samples we strongly recommend dividing the master mix into strip tubes and dispensing the master mix from the strip
102. on genome wide DNA copy number analysis The Affymetrix solution for cytogenetics also provides genotyping information enabling detection of loss of heterozygosity LOH which can be used to detect UPDs The combined high resolution DNA copy number data and the ability to detect gains losses and UPDs on a single array makes the Affymetrix CytoScan Solution a great tool for next generation cytogenetics studies 2 Affymetrix CytoScan Assay User Manual About This Manual This manual is a guide for technical personnel conducting the Affymetrix CytoScan Assay experiments in the laboratory It contains Best practices that Affymetrix recommends Laboratory setup Sample preparation Equipment and consumables required for each step Step by step protocols for the assay Protocols for washing staining and scanning arrays Troubleshooting information Fluidics Station care and maintenance Guidelines for processing 16 and 24 sample formats Protocol for fragmentation QC using the Agilent 2100 Bioanalyzer Protocols for E Gels Best Practices Controls This chapter provides tips for ensuring successful performance of the protocol Topics in this chapter include Controls a Equipment and Calibration a Pipetting on page 4 Reagent Handling and Storage on page 4 a Laboratory Workflow on page 5 Seal Vortex and Spin on page 5 a Fragmentation Step on page 7 a Running Gels on page 7 a Hybridization on pa
103. or Use With the CytoScan Assay Protocol Item Vendor Part Number Multiplate 96 well unskirted PCR plate Bio Rad MLP 9601 MicroAmp Clear Adhesive Film Applied Biosystems 4306311 Table 2 2 Thermal Cyclers Optimized For Use With the CytoScan Assay Protocol Laboratory Thermal Cyclers Validated for Use Pre PCR Clean Area Applied Biosystems Units Use one of these units 2720 Thermal Cycler GeneAmp PCR System 9700 Post PCR Area Applied Biosystems GeneAmp PCR System 9700 silver block or gold plated silver block Program Your Thermal Cyclers Use only calibrated thermal cyclers We recommend that thermal cyclers be serviced at least once per year to ensure that they are operating within the manufacturer s specifications The thermal cycler programs listed in Table 2 3 and Table 2 4 are used in this protocol Enter and store these programs on the appropriate thermal cycler in the Pre PCR Clean Area and the Post PCR Area Thermal cycler program details are listed in Appendix E Thermal Cycler Programs Table 2 3 Pre PCR Clean Area of Thermal Cyclers Required Program Name CytoScan Digest CytoScan Ligate 10 Affymetrix CytoScan Assay User Manual Table 2 4 Post PCR Area of Thermal Cyclers Required Program Name CytoScan PCR CytoScan Fragment CytoScan Label CytoScan Hyb Hybridization Oven Confirm that the GeneChip Hybridization Ove
104. p of the assay and lists the required equipment and consumables a Assay and Reagent Configuration a Workflows on page 24 Overview and List of Required Reagents Equipment and Consumables on page 26 o About Genomic DNA Preparation on page 26 u About Stage 1 Restriction Enzyme Digestion on page 27 u About Stage 2 Ligation on page 29 u About Stage 3 PCR on page 31 u About Stage 4 PCR Product Purification on page 34 u About Stage 5 Quantitation on page 35 u About Stage 6 Fragmentation on page 36 u About Stage 7 Labeling on page 39 u About Stage 8 Target Hybridization on page 40 The CytoScan Assay protocol is optimized for processing 8 to 24 samples at a time to obtain whole genome copy number results and SNP information This protocol 1s not intended for genome wide association studies Assay and Reagent Configuration This protocol has been optimized for processing 8 to 24 samples The illustrations in this chapter are based on running 8 samples 6 genomic DNA samples plus 1 positive and 1 negative control Use these illustrations as guidelines when processing 8 or fewer samples If processing more than 8 samples refer to Appendix A Guidelines for Processing 16 Samples or Appendix B Guidelines for Processing 24 Samples Important guidelines for plate layouts are included in these appendices CytoScan Assay Kit 24 Reactions Always use the 24 reaction CytoScan Reagent Kit P N 901808 for this protocol This kit h
105. p tubes tightly with an adhesive seal strip 10 Vortex the gel strip tubes then spin them down briefly in the strip tube microfuge NOTE Do not forget to add ethidium bromide to the gel running buffer Add two drops of ethidium bromide per liter of 1X TBE 11 Load 8 uL from each well of the gel strip tube onto a 2 TBE gel 12 Load 5 uL USB PCR marker 50 2000bp to the first and last wells of the gel 13 Run the gel at 5V cm for 45 minutes or until the dye front reaches at least 7596 of distance down the gel Chapter 6 CytoScan Assay Protocol 67 NOTE Run gels at 5V cm 5 volts x Distance in cm between electrodes For example run a 33 cm electrophoresis box at 165 V run a 16 cm electrophoresis box at 80 V 14 Verify that the PCR product distribution is between 150 bp to 2000 bp Figure 6 12 on page 67 Figure 6 12 Example of PCR products run on 296 TBE agarose gel at 5 v cm for 45 minutes Average product distribution is between 150 to 2000 bp 2000 bp 1500 bp 1000 bp 750 bp 500 bp 300 bp 150 bp Dye front 50 bp What To Do Next Do one of the following If the PCR has been confirmed proceed to Stage 4 PCR Product Purification on page 68 If not proceeding directly to the next stage seal the plate with PCR product and store at 20 C 68 Affymetrix CytoScan Assay User Manual Stage 4 PCR Product Purification Prepare Puri
106. perated using AGCC software To prime the Fluidics Station 1 Turn on the Fluidics Station 2 Prime the Fluidics Station From Affymetrix Command Console application start the Affymetrix Launcher From the Affymetrix Launcher open AGCC Fluidics Control application Chapter 7 Washing Staining and Scanning Arrays 101 a From the AGCC Fluidics Control panel select PRIME 450 script for the specific fluidics stations and the modules Lu IMPORTANT Use the Affymetrix GeneChip Wash A and Wash B buffers that are designated for the CytoScan Assay only These wash and stain buffers differ from the GeneChip expression buffers Intake buffer reservoir A use Wash A Intake buffer reservoir B use Wash B To initiate the fluidics script click the Run icon for each module or click the Run All icon for all the selected stations and modules Washing and Staining Arrays 1 2 Briefly vortex the stain bottles before aliquoting the reagents Aliquot the following reagents into 1 5 mL microfuge tubes for each array A Aliquot 500 uL Stain Buffer 1 into 1 5 mL microfuge tubes use amber color tubes as Stain Buffer 1 is light sensitive B Aliquot 500 uL Stain Buffer 2 into 1 5 mL microfuge tubes clear natural tubes C Aliquot 800 uL Array Holding Buffer into 1 5 mL microfuge tubes blue tubes Select a protocol from the AGCC Fluidics Control Panel a If using the CytoScan HD Array select Cyt
107. pipette Remove and discard the plate seal Using a P20 multi channel pipette aliquot 19 5 uL of Labeling Master Mix to each sample Fragmented DNA less 4 0 HL for gel analysis 51 0 uL Labeling Mix 19 5 uL Total 70 5 pL Seal the plate tightly with a new seal 5 Vortex at high speed for 1 second each in all corners and in the center according to the guidelines in Seal Vortex and Spin on page 5 then spin down for 1 minute at 2000 rpm 6 Place the labeling plate in the pre heated thermal cycler block and run the CytoScan Label program Table 6 10 CytoScan Thermal Cycler Program CytoScan Label Program Temperature Time 37 C 4 hr 95 C 15 minutes 4 C Hold OK to hold overnight 7 When the CytoScan Label program is finished remove the plate from the thermal cycler and spin down at 2000 rpm for 1 minute What To Do Next Do one of the following Proceed to the next stage If not proceeding directly to the next stage you can o Hold at 4 C on the thermal cycler overnight o Freeze the samples at 20 C Chapter 6 CytoScan Assay Protocol 91 Stage 8 Target Hybridization Important Information About This Stage To help ensure the best results carefully read the information below before you begin this stage of the protocol Since this manual is intended as an assay protocol manual there is no specific section on all of the various features a
108. ples 2 Place samples onto a thermal cycler and run the CytoScan Ligate program For the detailed protocol see Stage 2 Ligation on page 53 Location and Duration a Pre PCR Clean Area Hands on time 30 minutes CytoScan Ligate thermal cycler program time 3 3 hr 30 Affymetrix CytoScan Assay User Manual Equipment and Consumables Required The following equipment and consumables are required for this stage Table 5 5 Equipment and Consumables Required for Stage 2 Ligation Quantity Item 1 Adhesive seals for 96 well plates 1 Plate centrifuge Cooler chilled to 20 C Cooling chamber double block chilled to 4 C placed on ice do not freeze Ice bucket filled with ice Marker fine point permanent Mini centrifuge microfuge Pipette single channel P10 Pipette single channel P20 Pipette single channel P100 or P200 1 Pipette 12 channel 2 20 uL 1 Pipette 12 channel 20 200 HL As needed Pipette tips for pipettes listed above 1 Thermal cycler 1 8 12 well strip tubes 0 2 mL As required 8 12 tube strip caps 1 Tubes Eppendorf Safe Lock Tubes 1 5 mL Natural 1 Vortexer 2 GeneMate 96 Well PCR Tube Storage Rack IMPORTANT Use only the thermal cyclers 96 well plate and adhesive films and listed under Thermal Cyclers 96 Well Plate and Adhesive Seals on page 8 Ch
109. processing from 8 to 24 samples at a time to obtain whole genome copy number and SNP information from Affymetrix CytoScan Arrays This protocol is not intended for Genome Wide Association studies Cytogenetics studies are performed to identify structural changes in DNA such as copy number changes Individuals typically have two copies of the genome in each of their cells one inherited from the mother and one inherited from the father Chromosomal abnormalities are common in several disease states such as Deletions When one or both copies of a particular chromosome region are lost Gains When a chromosome or chromosomal region is duplicated or multiplied Uniparental Disomies UPDs When two copies of a chromosome or chromosomal region are present but both have been inherited from a single parent Traditional cytogenetics techniques such as karyotyping and fluorescent in situ hybridization FISH have been used to study chromosomal abnormalities for decades However karyotyping only detects abnormalities at low resolutions larger than 5 Mb and FISH is a more focused and targeted approach without the benefit of genome wide analysis Further these techniques are limited to only providing copy number information so that UPDs cannot be identified Together Affymetrix CytoScan Arrays and the CytoScan Assay along with the Command Console and Chromosome Analysis Suite software enable you to perform high resoluti
110. r B Insert the waste line into the waste bottle C Immerse all three wash and water lines into the bleach solution Ls IMPORTANT Do NOT immerse the waste line into the bleach 4 Open the instrument control software AGCC 5 Choose the current bleach protocol for each module 122 Affymetrix CytoScan Assay User Manual Immerse the tubes into the 0 5296 sodium hypochlorite solution The waste line remains in the waste bottle Fluidics Station 3 9 BLEACHy2 450 6 In AGCC run the protocol for all modules Chapter 9 Fluidics Station Care and Maintenance 123 NOTE The fluidics station will not start until the needle lever is pressed down Figure 9 4 on page 123 The temperature will ramp up to 50 C 7 Follow the prompts on each LCD Load empty 1 5 mL vials onto each module if not already done so 8 Press down on each of the needle levers to start the bleach protocol Figure 9 4 9 The fluidics station will begin the protocol emptying the lines and performing the cleaning cycles using bleach solution 10 After approximately 30 minutes the LCD will prompt you when the bleach cycle is over and the rinse cycle is about to begin 124 Affymetrix CytoScan Assay User Manual The Rinse Cycle Once the bleach cycle has finished the second part of the protocol is a rinse step This step is essential to remove all t
111. r 8 Chapter 9 Stage 8 Target Hybridization 4 91 Important Information About This Stage 4 91 Prepare the Equipment 00 0000 ce eee 91 Prepare the Arrays and Create a Batch Registration File 91 Prepare the Reagents and Consumables aaa aaa anana aaau 94 Prepare the Hybridization Master Mix llle 96 Add Hybridization Master Mix and Denature 0 96 Load the Samples onto Arrays llli ee 97 Washing Staining and Scanning ArraySs 99 Equipment and Consumables Required 000000 ce eee 99 Reagents Required 0 000000 eee 100 Fluidics Station and Scanner Control Software 0 100 Prime the Fluidics Stationr seek 420664 0204444 thang Shi i 100 Washing and Staining Arrays 0 0 0 0 0 eee 101 Scanning Arrays a ae iaaa op s 102 Prepare the Scanner 4 102 Prepare Arrays for Scanning 4 103 Scanning the Atay oss stipe s sae cee see EE fa deo aoe Raa Ld 104 Adding Arrays During an Autoloader Run aaaaaa ananuna auaa 104 Shutting Down the Fluidics Station 0 0 200 000 cee ee 105 Troubleshooting rrr Rh nns 107 General Assay Performance Recommendations 0 107 Troubleshooting the CytoScan Assay 9 109 Affymetrix Instruments lisse 118 Fluidics Station Care and Maintenance 119 General Fluidics Station Care 4 119 Fluidics Station Bleach Protocol 0 2 eee 119 The Ble
112. r uses a laser and is equipped with a safety interlock system Defeating the interlock system may result in exposure to hazardous laser light Read and be familiar with the operation of the scanner before attempting to scan an array Refer to the GeneChip Scanner 3000 Quick Reference Card P N 08 0075 Prepare Arrays for Scanning To prepare arrays for scanning 1 If the arrays were stored at 4 C allow them to warm to room temperature before scanning If necessary clean the glass surface of the array with a non abrasive towel or tissue before scanning Do not use alcohol to clean the glass surface On the back of the array cartridge clean excess fluid from around the septa Carefully cover both septa with Tough Spots Figure 7 1 Press to ensure the spots remain flat If the spots do not apply smoothly e g if you see bumps bubbles tears or curled edges do not attempt to smooth out the spot Remove the spot and apply a new spot Figure 7 1 Applyilng Tough Spots to Arrays 104 Affymetrix CytoScan Assay User Manual Scanning the Array NOTE Customers using the Autoloader should refer to the Autoloader User s Guide To scan arrays 1 Open the AGCC Scan Control application from the Affymetrix Launcher 2 Load the arrays onto the Autoloader of the scanner 3 Once all the arrays are loaded click the Start icon to initiate the scan 4 Select the check box arrays in
113. races of bleach from the system Failure to complete this step can result in damaged arrays 1 Follow the prompts on the LCD for each module Lift up on the needle levers and remove the bleach vials Load clean empty vials onto each module 2 Remove the three wash and water lines from the bleach bottle and transfer them to the DI water bottle Figure 9 5 At this step there is no need to be concerned about the bleach remaining in the lines Figure 9 5 Immerse the Three Wash and Water Lines in the DI Water Bottle 1 4 s oco mlt 3 Press down on the needle levers to begin the rinse cycle The fluidics station will empty the lines and rinse the needles 4 When the rinse is completed after approximately one hour the fluidics station will bring the temperature back to 25 C and drain the lines with air The LCD display will read CLEANING DONE Chapter 9 Fluidics Station Care and Maintenance 125 5 Discard the vials used for the bleach protocol 6 After completing the bleach protocol follow the suggestions for storage of the Fluidics Station 450 in Table 9 2 Table 9 2 Storage Suggestions for the Fluidics Station 450 If Then do this Planning to use the system immediately After running the bleach protocol remove the DI water supply used in the rinse phase and install the appropriate reagents for use in the next staining and washing protocol including fresh DI water Perfo
114. re 6 25 on page 88 Place an 8 tube strip in the upper half of the cooling block on ice Label the 1 5 mL eppendorf tube as LBL and place in the cooling block Cut adhesive seal into strips wide enough to seal 8 or 12 strip tubes Thaw and Prepare the Reagents 1 Thaw the following reagents at room temperature Immediately place on cooling block on ice when thawed n 5X TdT Buffer n 30 mM DNA Labeling Reagent Lu IMPORTANT Leave the TdT enzyme at 20 C until ready to use 2 Prepare the 5X TdT Buffer and 30 mM DNA Labeling Reagent as follows A Vortex each reagent at high speed 3 times 1 second each time B Pulse spin for 3 seconds then place in the cooling block If the fragmented samples were frozen allow them to thaw at room temperature Immediately spin down the plate at 2000 rpm for 1 minute and place on the lower half of the cooling block on ice 88 Affymetrix CytoScan Assay User Manual Strip tubes to aliquot Labeling Master Mix Label Master Mix tube samples Fragmented 1 2 3 4 516 5X TdT Buffer 30 mM DNA Labeling Reagent Chapter 6 CytoScan Assay Protocol 89 Prepare the Labeling Master Mix Preparation Keep all reagents and tubes in the cooling block on ice while preparing the Labeling Master Mix To prepare the Labeling Master Mix 1 Add the following to the 1 5 mL Eppendorf tube on ice using the volumes shown in Table 6 9 5X TdT Buffer
115. refully to ensure uniform reproducible fragmentation To help ensure the best results carefully read the information below before you begin this stage of the protocol EJ IMPORTANT All additions dilutions and mixing must be performed on ice Ensure all reagents reach equilibrium before use About the Fragmentation Reagent This enzyme is extremely temperature sensitive and rapidly loses activity at higher temperatures To avoid loss of activity o Handle the tube by the cap only Do not touch the sides of the tube as the heat from your fingers will raise the reagent temperature o Keep at 20 C until ready to use Transport and hold in a 20 C cooler Return to the cooler immediately after use n Spin down so that the contents of the tube are uniform u Perform all steps rapidly and without interruption Add enzyme to the fragmentation master mix last This enzyme is viscous and requires extra care when pipetting Follow these guidelines u Pipet slowly to allow enough time for the correct volume of solution to enter the pipette tip o Avoid excess solution on the outside of the pipette tip Prepare the Reagents Equipment and Consumables Turn on the Thermal Cycler Power on the thermal cycler to preheat the lid Leave the block at room temperature Set Up the Work Area Lu IMPORTANT Always spin down the fragmentation plate in a centrifuge that has been cooled down to 4 C 80 Affymetrix Cyt
116. rm a prime protocol without loading your probe arrays Failure to run a prime protocol will result in irreparable damage to the loaded hybridized probe arrays Not planning to use the system immediately Since the system is already well purged with water there is no need to run an additional shutdown protocol Remove the old DI water bottle and replace it with a fresh bottle Not planning to use the system for an extended period of time longer than one week Remove the DI water and perform a dry protocol shutdown This will remove most of the water from the system and prevent unwanted microbial growth in the supply lines Also remove the pump tubing from the peristaltic pump rollers 126 Affymetrix CytoScan Assay User Manual Guidelines for Processing 16 Samples This appendix illustrates the plate layouts recommended for processing 16 reactions 14 samples plus one positive and one negative control It also provides a high level overview of the workflow Digestion Ligation and PCR First Transfer Digest Ligate Plate PCR Plate 09000006 5G 690 6 6 6 6 5 69 6 62 OOOO 0 0 8 0 G 9 D s 8 0 9 9 D i Q5 9 9 9 0 9 E OM D 3 4 C F OM OOO e OM 3 4 C n amp 9 amp 2 62 6 9 CO O To avoid transfer mistakes keep all wells capped except for a One row on the Digest Ligate plate a The rows to which
117. s Table 5 16 Gels and Related Materials Required Item Reagent 496 TBE Gel precast or house made 1X TBE Buffer Ethidium Bromide Solution 5X RapidRun Loading Dye TrackIt 25 bp DNA Ladder Chapter 5 Assay Overview 39 About Stage 7 Labeling During this stage you will label the fragmented samples using the DNA Labeling Reagent as follows 1 Prepare a Labeling Master Mix 2 Add the mix to each sample 3 Place the samples onto a thermal cycler and run the CytoScan Label program For the detailed protocol see Stage 7 Labeling on page 87 Location and Duration Post PCR Area Hands on time 30 minutes CytoScan Label thermal cycler program time 4 25 hr Equipment and Consumables Required The following equipment and consumables are required for this stage Table 5 17 Equipment and Consumables Required for Stage 7 Labeling Quantity Item As required Adhesive seals for 96 well plates 1 Plate centrifuge 1 Cooler chilled to 20 C 1 Cooling chamber double block chilled to 4 C placed on ice do not freeze 1 Ice bucket filled with ice 1 Marker fine point permanent 1 Mini centrifuge microfuge 1 Pipette single channel P200 1 Pipette single channel P1000 1 Pipette 12 channel P20 accurate to within 5 As needed Pipette tips for pipettes listed above 1 Thermal cycler 1 Tube Eppendorf Safe Lock Tubes 1 5 mL Natural
118. samples onto arrays 1 When the thermal cycler reaches 49 C leave the samples at 49 C for at least one minute and then open the lid Lu IMPORTANT Load only 6 to 8 arrays at a time Remove the seal from the hybridization plate for only 6 to 8 samples at a time If you are hybridizing more than eight samples cut and remove the seal from 6 to 8 samples at a time only Leave the remaining wells covered Keeping these wells covered helps prevent cross contamination and evaporation 98 Affymetrix CytoScan Assay User Manual Figure 6 29 Loading Samples onto Arrays Septa covered with Tough Spots Lu IMPORTANT The hybridization mix is very viscous Pipette slowly to ensure that all of the volume is loaded into the chip 3 Using a P200 pipette remove 200 uL of the first sample and immediately inject it into an array 4 Cover the septa on the array with the 1 2 Tough Spots that were previously placed on the top edge of the array Figure 6 29 Press firmly to ensure a tight seal to prevent evaporation and leakage 5 When 6 to 8 arrays are loaded and the septa are covered A Load the arrays into an oven tray evenly spaced B Immediately place the tray into the hybridization oven Do not allow loaded arrays to sit at room temperature for more than approximately 1 minute Ensure that the oven is balanced as the trays are loaded and ensure that the trays are rotating at 60 rpm at all
119. se GeneAmp PCR System 9700 thermal cyclers with aluminum blocks Ramp speed Max 144 Affymetrix CytoScan Assay User Manual Volume 100 uL Table E 3 CytoScan PCR Program for GeneAmp PCR System 9700 CytoScan Fragment Temperature Time Cycles 94 C 3 minutes 1X 94 C 30 seconds 60 C 45 seconds 30X 68 C 15 seconds 68 C 7 minutes 1X 4 C HOLD Can be held overnight Table E 4 CytoScan Fragment Program Temperature Time 37 C 35 minutes 95 C 15 minutes 4 C Hold CytoScan Label Table E 5 CytoScan Label Program CytoScan Hyb Temperature Time 37 C 4hr 95 C 15 minutes 4 C Hold Samples can remain at 4 C overnight Table E 6 CytoScan Hyb Program Temperature Time 95 C 10 minutes 49 C Hold Reagents Equipment and Consumables About this Appendix This appendix includes the vendor and part number information for the reagents equipment and consumables that have been validated for use with the Affymetrix CytoScan Assay Lu IMPORTANT This protocol has been optimized using the equipment consumables and reagents listed in this user guide For the best results we strongly recommend that you adhere to the protocol as described Do not deviate from the protocol do not substitute reagents Affymetrix Equipment Required Table F 1 Affymetrix Equipment Required Item Part Number Gen
120. sure the OD of each sample at 260 280 and 320 nm Chapter 6 CytoScan Assay Protocol 77 OD280 and OD320 are used as controls Determine the OD260 measurement for the water blank and average Determine the concentration of each PCR product as follows A Calculate one OD reading for every sample OD sample OD average water blank OD B Calculate the undiluted concentration for each sample in pg L Undiluted sample concentration OD x 0 05 ug uL x 100 Procedure if Using a NanoDrop IMPORTANT The P20 pipette must be calibrated as per the manufacturer s specifications To prepare diluted aliquots of the purified samples 1 Using a P20 pipette aliquot 18 uL of water to the corresponding wells of a 96 well plate 2 Using a P20 pipette A Transfer 2 uL of each purified sample to the corresponding well of the 96 well plate B Pipet up and down 2 times to ensure that all of the sample is dispensed The result is a 10 fold dilution 3 Do one of the following to mix the samples Set a P20 pipette to 17 uL and pipet up and down 5 times Seal the plate tightly vortex and spin down at 2000 rpm for 1 minute Figure 6 21 96 well Plate Layout for NanoDrop 18 uL Affymetrix Nuclease a Free water 2 uL purified sample in each well 2 OOOOOOOQ DIULODOUL US OOOOOOOO OOOOOOO OCIO ECC OOOOOOO CS C16 6 RA OOOOOO Ole QUOOODCUD OOOOOOOQ 7
121. taminants that would affect the enzymatic reactions carried out For this protocol you will use the Affymetrix CytoScan Assay Kit 24 sample P N 901808 This kit contains the control Genomic DNA This control meets the requirements outlined below The size of the starting genomic DNA can be compared with the control Genomic DNA to assess the quality The control Genomic DNA should also be used as a routine experimental positive control for troubleshooting Assay performance may vary for genomic DNA samples that do not meet the general requirements described below However the reliability of any given result should be assessed in the context of overall experimental design and goals General Requirements and Recommendations DNA must be double stranded not single stranded This can be verified using PicoGreen quantitation This requirement relates to the restriction enzyme digestion step in the protocol DNA must be free of PCR inhibitors Examples of inhibitors include high concentrations of heme from blood and high concentrations of chelating agents i e EDTA The genomic DNA extraction purification method should render DNA that is generally salt free because high concentrations of certain salts can also inhibit PCR and other enzyme reactions DNA should be prepared as described in Chapter 6 CytoScan Assay Protocol DNA must not be contaminated with other human genomic DNA sources or with genomic DNA from other org
122. tely pulse spin for 3 seconds to bring down any reagent that may be clinging to the top of the tube C Immediately place in the 20 C cooler 4 Addthe appropriate volume of Fragmentation Reagent from Table 6 7 Immediately place it back in the 20 C cooler Vortex the master mix at high speed 3 times 1 second each time Pulse spin for 3 seconds and immediately place in the cooling block Proceed immediately to the next set of steps Add Fragmentation Master Mix to the Samples Add Fragmentation Master Mix to the Samples Figure 6 23 Adding Fragmentation Master Mix to Samples Aliquoted Fragmentation Master Mix TII 2 Add 10 uL of Fragmentation Master CYCYOOQOC OL O OO Mix to each sample DOOODODOOOOOO OQOOOOOOQOGQOOQOQO0 OOOOOOOOQOOOQO OOOOODOUQO QDOCQOQ OOOOOOOOOOOO LIODDOOOCOCUD VOLHOOOOQCIOOOCC Chapter 6 CytoScan Assay Protocol 83 To add Fragmentation Master Mix to the samples 1 Quickly aliquot out the Fragmentation Master Mix equally to the strip tubes placed in the cooling block on ice Figure 6 23 Seal the strip tubes with an adhesive seal strip or strip caps Spin down the strip tubes and place them back in the cooling block on ice Remove the seal and discard Remove and discard the plate seal Using a multi channel P20 pipette transfer 10 uL of Fragmentation Master Mix to each sample do not pipet up and down Avoid introducing air bubbles at the bottom of
123. th 81001 Required if processing 8 samples 1 for 9 to 16 samples 2 for 17 to 24 samples O Cooling Chamber double block Diversified CHAM 1020 Biotech Freezer 20 C deep freeze manual defrost 17 cu ft Any vendor 1 Rectangular Ice Tray Large 9L 16 x 13 in 41 x 33cm LabScientific RECB1202 1 Microfuge for tubes and strip tubes Any vendor 1 96 well Tube Storage Racks GeneMate R 7909 2 1 Pipette single channel 2 20 HL Rainin L 20 1 Pipette single channel 20 200 HL Rainin L 200 1 Pipette single channel 100 1000 HL Rainin L 1000 1 Pipette 12 channel 2 20 uL Rainin L12 20 Pipette 12 channel 20 200 HL Rainin L12 200 1 Plate centrifuge multipurpose Eppendorf 5804 or 5810 1 Vortexer Any vendor Select one of these thermal cyclers 3 n GeneAmp PCR System 9700 gold silver block Applied N8050200 Biosystems a 2720 Thermal Cycler Applied 4359659 Biosystems 150 Affymetrix CytoScan Assay User Manual Post PCR Area Equipment Required Table F 10 Post PCR Area Equipment Required v item Vendor Part Number Benchtop Cooler 20 C Agilent 401349 Technologies 1 Cooling Chamber double block Diversified CHAM 1020 Biotech 1 Freezer 20 C deep freeze manual defrost 17 cu ft Any vendor 1 Electrophoresis gel box Any vendor Electrophoresis power supply VWR VWR105 1 Gel imager Any vendor 1 Rectangular ice tray
124. the 20 C cooler Vortex the master mix at high speed 3 times 1 second each time 7 Pour the master mix into the reagent reservoir keeping the cooling block on ice Table 6 5 PCR Master Mix Reagent 1 Sample 8 Samples 16 Samples 24 Samples 15 overage 15 overage 15 overage Chilled Affymetrix Nuclease Free 39 5 uL 1453 6 uL 2907 2 HL 4360 8 HL Water 10X TITANIUM Tag PCR Buffer 10 0 uL 368 0 uL 736 0 uL 1104 0 uL GC Melt Reagent 20 0 uL 736 0 uL 1472 0 uL 2208 0 uL dNTP Mixture 2 5 mM each 14 0 uL 515 2 uL 1030 4 HL 1545 6 uL PCR Primer 002 4 5 uL 165 6 uL 331 2 uL 496 8 uL 50X TITANIUM Tag DNA Polymerase 2 0 uL 73 6 uL 147 2 uL 220 8 uL Do not add until ready to aliquot master mix to ligated samples Total 90 0 pL 3312 0 pL 6624 0 pL 9936 0 pL 64 Affymetrix CytoScan Assay User Manual Add PCR Master Mix to Each Sample To add the PCR Master Mix to samples 1 2 Unseal the PCR sample plate and discard the seal Using a multi channel P200 pipette aliquot 90 uL PCR Master Mix to each sample and control on the PCR plate To avoid contamination change pipette tips after each dispensing For eight samples you may have to tilt the reagent reservoir to ensure that each pipette tip picks up 90 uL After adding the master mix the total volume in each well is 100 uL Ligated and diluted DNA 10 uL PCR Master Mix 90 uL Total 100 pL T
125. the slot 12 well E gels can also be used if running a smaller number of samples Load all 20 uL of the diluted fragmented product from above onto the 48 well 4 agarose E Gel Dilute the TrackIt 25 bp DNA Marker 1 15 dilution 2 uL in 28 uL of Nuclease Free water and load 15 uL into each of the marker wells as needed Fill empty wells with 20 uL water Set the run time to 19 minutes Push the Power Prg button again it will change from red to green When the run time is reached the system will automatically shut off the dye should be near the end of the lane The gel is then ready for imaging Appendix D Running E Gels 141 Figure D 2 Gel Image of Fragmented Product from Ref103 Genomic DNA on 496 E Gel 142 Affymetrix CytoScan Assay User Manual Thermal Cycler Programs This appendix includes the thermal cycler programs required for the Affymetrix CytoScan Assay Before you begin processing samples enter and save these programs into the appropriate thermal cyclers CytoScan Digest Table E 1 CytoScan Digest Program Temperature Time 37 C 2 hours 65 C 20 minutes 4 C Hold CytoScan Ligate Table E 2 CytoScan Ligate Program Temperature Time 16 C 3 hours 70 C 20 minutes 4 C Hold CytoScan PCR For the GeneAmp PCR System 9700 You must use GeneAmp PCR System 9700 thermal cyclers with silver or gold plated silver blocks Do not u
126. ting within specification Troubleshooting the CytoScan Assay Chapter 8 Troubleshooting 109 PCR Gel QC Step Likely Cause Solution Faint or no PCR product visible Failed restriction digest or on gel Both samples and adapter ligation positive control affected Repeat the assay from the beginning with Genomic Control DNA after reviewing best practices ensuring that all equipment is correctly calibrated and reagents are handled and stored properly If available include ligated material from a previous successful experiment as a positive control for the PCR step If it fails again repeat with fresh reagents Ensure that the ligation buffer is thoroughly resuspended before use Ensure that the reaction plates are sealed tightly in all steps Non optimal PCR conditions Use only calibrated thermal cyclers Double check PCR programs to ensure that they have been entered correctly Check the PCR reagents Use only those reagents recommended by Affymetrix Verify pipette calibration and function Repeat PCR from the remaining digestion ligation material if available otherwise restart from the beginning Take care with preparation of master mixes Ensure accurate pipetting and thorough mixing a Use the recommended 96 well PCR plates and plate seals a Ensure that the plates are sealed tightly in all steps Ligation reaction not diluted or diluted ligation reaction not mi
127. toScan Assay User Manual Table F 12 Supplier Contact List Continued Supplier Web Site Address Scientific Industries www scientificindustries com Sigma Aldrich www sigma aldrich com USB www usb affymetrix com Teknova www teknova com VWR www vwr com
128. tomatically shut off the dye should be near the end of the lane The gel is now ready for imaging PCR Product on 2 E Gel Diluting the Tracklt Cyan Orange Loading Buffer The following instructions prepare a 1000 fold dilution of the TrackIt Cyan Orange Loading Buffer 1 Add 50 uL of TrackIt Cyan Orange Loading Buffer to 49 95 mL Nuclease Free water total volume is 50 mL 2 Mix well and store at room temperature Diluting PCR Product Dilutions can be prepared in strip tubes or 96 well plates 1 After the PCR step is complete aliquot 3 uL from the first row of the PCR product to 17 uL of the 1 1000 fold diluted Loading Buffer to give a total volume of 20 uL 2 Running the 1 2 3 Appendix D Running E Gels 139 Briefly vortex and spin down the diluted samples before loading onto the E Gel E Gel Turn on the power for the E Base red light Push the Power Prg button to make sure the program is set to EG mode not EP Remove the comb s from the E Gel and wipe away any buffer that comes out of the gel or is on the surface Insert the 48 well 2 Agarose E Gel into the slot 12 well E Gels can also be used if running a smaller number of samples Load all 20 uL of the diluted PCR product from above onto the 48 well 2 agarose E Gel Dilute the PCR marker 1 3 dilution 5 uL in 10 uL of Nuclease Free water and load all 15 uL into each of the marker wells as needed Fill empty wells with 20 uL water
129. trifuge 1 Cooler chilled to 20 C 1 Cooling chamber double block chilled to 4 C placed on ice do not freeze 1 Ice bucket filled with ice 1 Marker fine point permanent 1 Mini centrifuge microfuge 1 Pipette single channel P20 1 Pipette single channel P100 1 Pipette single channel P200 1 Pipette 12 channel P20 accurate to within 5 As needed Pipette tips for pipettes listed above 1 Plate Bio Rad 96 well 1 Thermal cycler 1 Tube Eppendorf Safe Lock Tubes 1 5 mL Natural 1 8 12 well strip tubes 0 2 mL As required 8 12 tube strip caps 38 Affymetrix CytoScan Assay User Manual Table 5 14 Equipment and Consumables Required for Stage 6 Fragmentation Continued Quantity Item 1 Vortexer 1 Electrophoresis gel box 1 Electrophoresis power supply 4 GeneMate 96 Well PCR Tube Storage Rack IMPORTANT Use only the thermal cyclers 96 well plate and adhesive films and listed under Thermal Cyclers 96 Well Plate and Adhesive Seals on page 8 Reagents Required The following reagents are required for this stage Table 5 15 Reagents Required for Stage 6 Fragmentation Reagent 10X Fragmentation Buffer Fragmentation Reagent Chilled Affymetrix Nuclease Free Water Gels and Related Materials Required Verifying the fragmentation reaction is required for this stage You can use the following gels and related material
130. tubes into the samples using a multi channel pipette 1 Divide the Ligation Master Mix equally into the 8 12 strip tubes on ice Seal the strip tube with an adhesive seal strip or strip caps and pulse spin Place back in the cooling block on ice remove the seal and discard Unseal the digested sample plate and discard the seal Using a multi channel P20 pipette aliquot 5 25 uL of Ligation Master Mix to each digested sample and control Figure 6 7 Digested DNA 19 75 uL Ligation Master Mix 5 25 uL Total 25 00 pL Figure 6 7 Adding Ligation Master Mix to Digested Samples and Controls Aliquoted Ligation Master Mix Add 5 25 uL Ligation Master Mix to each sample and control in row A HO OOGOOOOOOCIOOCOO 000060000000 SDODODOQOOQGOQOQOLU SOOCODOLDOOOQOOCtQO FSOODOODOOGOCUOQOQO FOODOOCOOOUCIOU SODOCODOLDO QCDO COCHQUIDOQOOUCCOC Chapter 6 CytoScan Assay Protocol 57 Load the Samples Onto the Thermal Cycler 1 Seal the plate tightly with a new seal 2 Vortex at high speed for 1 second in all corners and in the center according to the guidelines in Seal Vortex and Spin on page 5 then spin down at 2000 rpm for 1 minute Ensure that the thermal cycler lid is preheated Load the plate onto the thermal cycler and run the CytoScan Ligate program Return the remaining reagents to the freezer and discard the remaining master mix Table 6 4 CytoScan Ligate Thermal
131. w the heading row contains the information for one Sample ARR file Additional columns for new attributes can be added to the spreadsheet at any time W Step 3 Upload the batch registration file to create new sample ARR files Enter the path or click Browse to find the batch registration file XLS format or Tab delimited TXT DAllow Custom Barcodes Click Upload to upload the Sample information tecn fa Wi 2 3 Within Step 1 A Enter the number of samples for which a spreadsheet needs to be created under Create a Spreadsheet for B Select Default from the Project Set to drop down list Chapter 6 CytoScan Assay Protocol 93 C Select the appropriate array type from the Probe Array type set to drop down list a If using the CytoScan HD Array select CytoScanHD Array a If using the CytoScan 750K Array select CytoScan750K Array D Click Download An Excel spreadsheet will open 4 Within Step 2 A Name the experiment file using the following convention SampleName PlateCoordinate ExperimentDescriptionString ArrayType Ope ratorInitials_yyyymmdd B The sample file name and the Array name would be identical C Scan the corresponding barcodes for each Sample name D Save the Excel file in Excel 97 2003 workbook format 5 Within Step 3 A Browse to the location of the Batch registration file that was saved B Upload the Batch registration file by cl
132. xed properly prior to PCR Be sure to correctly dilute the ligation reaction with the water provided in the kit and mix properly before proceeding with PCR 110 Affymetrix CytoScan Assay User Manual PCR Gel QC Step Likely Cause Solution Faint or no PCR product visible on the gel Samples are affected but positive control is OK Insufficient or degraded genomic DNA Sample DNA contains enzymatic or chemical inhibitors Nsp can be inhibited by high concentrations of salts a Starting amount of 250 ng genomic DNA should be used a Confirm the concentration using a calibrated spectrophotometer a Confirm that the genomic DNA sample meets the quality and integrity guidelines See Chapter 4 on page 21 Ensure that genomic DNA is extracted using one of the recommended procedures See Chapter 4 on page 22 Wrong size distribution of PCR product Mispipetting of PCR primer volume in the master mix Mispipetting of Taq polymerase in the master mix Verify pipette calibration and function Repeat PCR from the remaining digestion ligation material if available otherwise restart from the beginning PCR product evident in the negative control Reagents or equipment contaminated with ligated product or amplified product a Always use filter tips Clean the pre PCR lab area and equipment thoroughly using 1096 bleach Decontaminate the pipettes following
133. y be needed depending on model chosen and work method Ec UV Spec Plate for Quantitation mae ai 000000000000 amp 3 6 5 69 2 amp 6 69 6 62 OO Fragment Label Plate QOOO00000000 vy eee a 0 0G 0600 6 amp 9 69 d 69606966069 69 69 60 CO O E UE Eo00000000000 000000000000 EDO OOO OOOO A OQOOOOOOOOOOO Fragmentation gel 00000O0O0O0O0O0O00 kOOOOODOOOOOQQO jj BOOOOOOOOOOQOO UM L gt Quantitate label and hyb samples onto arrays 1 1 25 bp m 25 bp u n D H T j Hi ded 134 Affymetrix CytoScan Assay User Manual Analyzing Sample Fragmentation Using the Agilent 2100 Bioanalyzer 1 Thaw the fragmentation aliquot prepared in Step 8 of Check the Fragmentation Reaction by Running a Gel on page 85 at room temperature Mix sample by vortexing Use luL per sample as input volume for the bioanalyzer 2 Use the Agilent DNA 1000 Assay Kit Cat 5067 1504 Refer to the user guide for instructions on sample preparation and sample analysis on the bioanalyzer 3 Evaluate the DNA fragmentation distribution using the sample elution profile shown in the electropherogram Figure C 1 4 To evaluate each profile the 15bp control peak should be identified and assigned correctly tallest peak elution time around 43 seconds Correctly fragmented DNA samples have profiles which stretch out below and above I5bp Under or over fragmentation is indicated by profiles w
134. y seal the plate and vortex at high speed for 1 second in all corners and in the center according to the guidelines in Seal Vortex and Spin on page 5 then spin down at 2000 rpm for 1 minute What To Do Next Proceed to Stage 5 Quantitation on page 75 Chapter 6 CytoScan Assay Protocol 75 Stage 5 Quantitation Important Information About This Stage To help ensure the best results carefully read the information below before you begin this stage of the protocol Lu IMPORTANT The accuracy of the OD measurement is critical Carefully follow this procedure and be sure the OD measurement is within the linear range of the instrument The spectrophotometer should be calibrated regularly to ensure correct readings a This protocol has been optimized using a UV spectrophotometer for quantitation Prepare the Reagents Equipment and Consumables Turn on the Spectrophotometer Turn the instrument ON and allow it to warm for at least 10 minutes before use Prepare the Work Area To prepare the work area 1 Place the following on the bench top a Optional conical tube or reagent reservoir a Affymetrix Nuclease Free Water a UV or 96 well plate 2 Ensure that the plate is sealed tightly Vortex and spin down the purified samples at 2000 rpm for 1 minute and put in a plate holder 76 Affymetrix CytoScan Assay User Manual Procedure if Using a Microplate Spectrophotometer Prepare Diluted Aliquots of
135. your convenience Do not use expired reagents or reagents that have undergone more than the recommended number of freeze thaw cycles Seal all vials and bottle caps well after use to prevent evaporation Do not store enzymes in a frost free freezer Chapter 2 Best Practices 5 Store the reagents used for digestion ligation and PCR only in the Pre PCR Clean Area When Using Reagents at the Lab Bench Properly chill essential equipment such as cooling blocks and reagent coolers before use a Unless otherwise indicated keep all reagents except enzymes on ice or in a cooling block that has been chilled to 4 C and placed on ice during use a Ensure that enzymes are kept at 20 C until needed When removed from the freezer immediately place in a bench top reagent cooler that has been chilled to 20 C Keep all tubes master mixes and working solutions in chilled cooling blocks on ice a Since enzyme activity is a function of temperature ensure that all temperature transitions to incubation temperatures are rapid and or well controlled to help maintain consistency across samples Master Mix Preparation Carefully follow each master mix recipe Use pipettes that have been calibrated as per the manufacturer s specifications Use only the Affymetrix Nuclease Free water that is supplied with the kit Do not use any other water The enzymatic reaction in Stage 6 Fragmentation is particularly sensitive to pH and metal io

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