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Affymetrix® Cytogenetics Copy Number Assay User Guide

1. OOOOOOOO OOOOOOOO OOOOOOOO OOOOOOOO OOOOOOQOO 12 p 13 i gt 14 d gt Do not pool negative control Nes PS Im Gel check for Add magnetic beads to PCR product each tube and incubate in tube rack Figure A 2 16 reaction workflow PCR to purification 110 Affymetrix Cytogenetics Copy Number Assay User Guide Purification continued to Fragmentation and Labeling After incubation centrifuge and place tubes on magnetic rack Transfer eluted sample to the appropriate well of a fresh 96 well plate h Spec Plate for Quantitation Fragment Label Plate OOC FOG OOC R OC A 0C 600C G i 600C Fragmentation gel 000000 OO00000Q OOOOOOOO OOOOOOOOQO OOOOOOOOQ OOOOOOOO OOOOOOOQ OOOOOOOQ OOOOOOOQ O0 G 9 G9 9 OOOOOOOQO OOOOOOOO Quantitate label and hyb samples onto arrays Figure A 3 16 reaction workflow Purification to Labeling t i Bi WERBEN i Nmp Hu JAM rag Ea m i i Appendix B 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 To avoid transfer mistakes keep all wells cappe
2. 30 Affymetrix Cytogenetics Copy Number Assay User Guide Prepare the Reagents Equipment and Consumables Thaw Reagents and Genomic DNA 1 Allow the following reagents to thaw on ice NE Buffer 2 NE Buffer 3 BSA 2 Ifthe plate of genomic DNA and controls is frozen allow it to thaw in a cooling chamber on ice Lu IMPORTANT Leave the Nspl and Styl enzymes at 20 C until ready to use Setup the Work Area Keep Sty master mix reagents on ice off to the side NE Buffer 3 Sty digest master mix tube j 4 Water 3ie alc i BSA SIC NE Buffer 2 OG 1 HOS S 16 Nsp digest master mix tube Na Figure 4 6 Setup for Nsp and Sty Digest Nspl and Styl enzymes not pictured still at 20 C To setup the work area Figure 4 6 1 Place a double cooling chamber and the water on ice 2 Labelthe 1 5 mL Eppendorf tubes as follows Using a blue marker label one tube NSP and place in the cooling chamber Using a red marker label one tube STY and set aside 3 Prepare the genomic DNA and controls as follows A Vortex at high speed for 3 sec B Spin down at 2000 rpm for 30 sec C Place in the cooling chamber 4 Prepare the NE Buffer 2 and BSA as follows A Vortex 3 times 1 sec each time B Pulse spin for 3 sec C Place in the cooling chamber on ice chapter 4 Affymetrix Cytogenetics Copy Number Assay 31 Preheat the Thermal Cycler Lid Power on the thermal cyc
3. Wash Buffer A non stringent wash buffer Wash Buffer B stringent wash buffer Hi IMPORTANT These wash and stain buffers differ from the GeneChip expression buffers Washing and Staining Arrays To wash and stain the arrays 1 Select your experiment GCOS or sample AGCC name The Probe Array Type appears automatically 2 Select the protocol GenomeWideSNP6_450 3 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 4 Insert an array into the designated module of the fluidics station while the cartridge lever is in the Down or Eject position 5 When finished verify that the cartridge lever is returned to the Up or Engaged position 6 Remove any vials remaining in the positions of the fluidics station module s being used 7 When prompted to Load Vials 1 2 3 place the three vials into positions 1 2 and 3 on the fluidics station A Place one vial containing 600 uL Streptavidin Phycoerythrin SAPE stain solution mix in position 1 B Place one vial containing 600 uL anti streptavidin biotinylated antibody stain solution in position 2 C Place one vial containing 1 mL Array Holding Buffer in position 3 90 Affymetrix Cytogenetics Copy Number Assay U
4. Affymetrix Cytogenetics Copy Number Assay User Guide P N 702607 Rev 2 For research use only Not for use in diagnostic procedures Trademarks s Affymetrixe AX GeneChips HuSNPs GenFlexe Flying Objective CustomExpresse CustomSeqe NetAffx Tools to Take You As Far As Your Visione The Way Ahead Powered by Affymetrix GeneChip compatible and Command Console are 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 that no right or license to any patent or other intellectual property owned or licensable by Affymetrix is conveyed or implied by this Affymetrix product In particular no right or license is conveyed or implied to use this Affymetrix product in combination with a product not provided licensed or specifically recommended by Affymetrix for such use Patents Arrays Products may be covered by one or more of the following patents U S Patent Nos 5 445 934 5 744 305 5 945 334 6 140 044 6 261 776 6 291 183 6 346 413 6 399 365 6 420 169 6 55
5. Reagent 1 Sample 4 Samples 8 Samples 12 Samples 24 Samples 15 extra 15 extra 15 extra 15 extra MES 12X 1 25 M 12 0 uL 55 2 uL 110 4 uL 165 6 uL 331 2 uL Denhardt s Solution 50X 13 0 uL 59 8 uL 119 6 uL 179 4 uL 358 8 uL EDTA 0 5 M 3 0 uL 13 8 uL 27 6 uL 41 4 uL 82 8 uL HSDNA 10 mg mL 3 0 uL 13 8 uL 27 6 uL 41 4 uL 82 8 uL OCR 0100 2 0 uL 9 2 uL 18 4 uL 27 6 uL 55 2 uL Human Cot 1 DNA 1 mg 3 0 uL 13 8 uL 27 6 uL 41 4 uL 82 8 uL mL Tween 20 396 1 0 uL 4 6 uL 9 2 uL 13 8 uL 27 6 uL DMSO 100 13 0 uL 59 8 uL 119 6 uL 179 4 uL 358 8 uL TMACL 5 M 140 0 uL 644 0 uL 1288 0 uL 1932 0 uL 3864 0 uL Total 190 uL 874 uL 1748 pL 2622 uL 5244 uL Using Premixed Hybridization Master Mix Hybridization Master Mix can be made ahead of time aliquoted and stored for 1 week at 20 C To prepare stored Hybridization Master Mix 1 Place the stored Hybridization Master Mix on the bench top and allow to warm to room temperature 2 Vortex at high speed until the mixture is homogeneous and without precipitates may take up to 5 min 3 Pulse spin for 3 sec 80 Affymetrix Cytogenetics Copy Number Assay User Guide Add Hybridization Master Mix and Denature To add Hybridization Master Mix and denature the samples 1 2 Optional pour the Hybridization Master Mix into a solution basin Using a P200 pipet add 190 uL of Hybridization Master Mix to each sample Total volume in each well is 262 5 uL Tig
6. OOOOOOO OOOOOOO OOOOOOO OOOOOOOO OOOOOOOOQO OOOOOOOO OOOOOOOOQO OOOOOOOOQO OOOOOOOQO OOO000000 OOOOOOOOQO Figure 4 23 96 well plate layout for NanoDrop 4 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 OD X 10 62 Affymetrix Cytogenetics Copy Number Assay User Guide Assess the OD Readings Acceptable OD Range and DNA Yield e An acceptable OD should fall within this range 0 9 to 1 4 DNA yield equivalent 4 5 to 7 0 ug uL This OD range is based on the use of a conventional UV spectrophotometer plate reader and assumes a path length of 1 cm The OD260 O0D280 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 gt 0 2 A Centrifuge the sample for 5 min B Place on the MagnaRack and pipet off the eluate C Requantitate the sample D If the OD320 reading is now lt 0 1 proceed If your OD readings are not within the acceptable range refer to OD Troubleshooting Guidelines on page 99 What To Do Next Do one of the following Proceed immediately to Stage 6 Fragmentation on page 63 If not proceeding immediately to the next step seal the plate of purifi
7. 1 Optional Tube 50 mL conical One per sample Tubes 2 0 mL Microcentrifuge Safe Lock minus neg control Must be round bottom Do NOT use conical tubes 1 Tube holder 1 Vortexer with foam tube adaptor attached 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 4 Reagents Required The following reagents are required for this stage Table 4 21 Reagents Required for Stage 4 PCR Product Purification Elution Buffer Buffer EB 75 EtOH ACS grade ethanol diluted to 75 using AccuGENE water Agencourt AMPure magnetic beads chapter 4 Affymetrix Cytogenetics Copy Number Assay 53 Important Information About This Stage To help ensure the best results carefully read the information below before you begin this stage of the protocol IMPORTANT e The storage temperature for Agencourt AMPure is 4 C refrigerator The pH should be 5 5 If not pH 5 5 discard and use fresh reagent To avoid cross contamination pipet very carefully when pooling the PCR reactions Prepare the 75 EtOH Dilute ACS grade ethanol to 75 using water AccuGENE Pool the PCR Products P 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
8. DNA must not be highly degraded For any particular SNP the genomic DNA fragment containing the SNP must have Nsp I or Sty 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 196 or 2 agarose gel using an appropriate size standard control Ref 103 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 Pre amplification methods or pre digestion with restriction enzymes other than Nsp I or Sty I have not been tested by Affymetrix If other methods are desired we recommend conducting experiments to evaluate their performance with this assay 16 Affymetrix Cytogenetics Copy Number Assay User Guide 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 on page 15 blood cell line Success with other types of samples such as saliva will depend on quality degree of degradation degree of inhibitors present etc quantity of genomic DNA extracted and purity of these types of samples as described under General Requirements on page 15 Genomic DNA Extraction Purification Methods Genomic DNA extraction and purification methods that meet the general requiremen
9. Pierce Biotechnology part of Thermo Fisher Scientific piercenet com Promega www promega com QIAGEN www1 qiagen com Rainin www rainin com Scientific Industries www scientificindustries com Sigma Aldrich www sigma aldrich com Stratagene stratagene com Teknova teknova com VWR vwr com Vector Labs vectorlabs com 122 Affymetrix Cytogenetics Copy Number Assay User Guide Li BER EB B EA EM ARI E BA et i un Ba Appendix THERMAL CYCLER PROGRAMS This appendix includes the thermal cycler programs required for the Affymetrix Cytogenetics Copy Number Assay Before you begin processing samples enter and save these programs into the appropriate thermal cyclers Cyto Digest Cyto Digest Program Temperature Time 37 C 120 min 65 C 20 min 4 C Hold Cyto Ligate Cyto Ligate Program Temperature Time 16 C 180 min 70 C 20 min 4 C Hold 124 Affymetrix Cytogenetics Copy Number Assay User Guide Cyto 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 use GeneAmp PCR System 9700 thermal cyclers with aluminum blocks Ramp speed Max Volume 100 uL Cyto PCR Program for GeneAmp PCR System 9700 Temperature Time Cycles 94 C 3 min 1X 94 C 30 sec 60 C 45 sec 30X 68 C 15 sec 68 C 7 min 1X 4 C HOLD Can be held
10. Daniel A Use of Buccal Cells Collected in Mouthwash as a Source of DNA for Clinical Testing Arch Pathol Lab Med 125 127 133 2001 King I B Satia Abouta J Thornquist M D Bigler J Patterson R E Kristal A R Shattuck A L Potter J D White E Abouta J S Buccal cell DNA yield quality and collection costs comparison of methods for large scale studies Cancer Epidemiol Biomarkers Prev 11 10 Pt 1 1130 3 2002 Lench N Stanier P Williamson R Simple non invasive method to obtain DNA for gene analysis Lancet Jun 18 1 8599 1356 1358 1988 Paez J G Lin M Beroukhim R Lee J C Zhao X Richter D J Gabriel S Herman P Sasaki H Altshuler D Li C Meyerson M Sellers W R Genome coverage and sequence fidelity of phi29 polymerase based multiple strand displacement whole genome amplification Nucleic Acids Research 32 9 2004 chapter 3 Genomic DNA General Requirements 17 Tzvetkov M V Becker C Kulle B Nurnberg P Brockmoller J Wojnowski L Genome wide single nucleotide polymorphism arrays demonstrate high fidelity of multiple displacement based whole genome amplification Electrophoresis Feb 26 3 710 5 2005 Wong K K Tsang Y T M Shen J Cheng R S Chang Y Man T Lau C C Allelic imbalance analysis by high density single nucleotide polymorphic allele SNP array with whole genome amplified DNA Nucleic Acids Res May 17 32 9 e69 2004 1
11. 1 sec each time Pulse spin for 3 sec 2 3 4 5 6 7 Table 4 33 Labeling Master Mix Immediately proceed to the next set of steps Add the Labeling Master Mix to the Samples Reagent 1 Sample 4 Samples 8 Samples 12 Samples 24 Samples 15 extra 15 extra 15 extra 15 extra TdT Buffer 5X 14 0 uL 64 4 uL 128 8 uL 193 2 uL 386 4 uL DNA Labeling Reagent 2 0 uL 9 2 uL 18 4 uL 27 6 uL 55 2 uL 30 mM TdT enzyme 30 U uL 3 5 uL 16 1 uL 32 2 uL 48 3 uL 96 6 uL Total 19 5 pL 89 5 uL 179 4 uL 269 1 pL 538 2 uL Add the Labeling Master Mix to the Samples To add the Labeling Master Mix to the samples Keep samples in the cooling chamber and all tubes on ice when making additions 1 Optional If processing 16 or more samples aliquot the Labeling Master Mix equally into strip tubes 2 Using a P20 single or 8 channel pipet A Aliquot 20 uL of Labeling Master Mix to each sample B Pipet up and down one time to ensure that all of the mix is added to the samples Fragmented DNA less 2 0 uL for gel analysis 53 0 uL Labeling Mix 19 5 uL Total 72 5 pL 74 Affymetrix Cytogenetics Copy Number Assay User Guide 3 Tightly seal the plate Vortex at high speed for 3 sec then spin down for 30 sec 5 Place on the pre heated thermal cycler block and run the Cyto Label program IMPORTANT Ensure that the seal is not pulled off any wells when you close the thermal cycler
12. For the controls aliquot 5 uL of A Ref 103 to wells G1 and G12 B Water to wells H1 and H12 5 Tightly seal the digest ligate plate Transfer two 5 uL aliquots of each diluted gDNA to the digest ligate plate one for Nsp reactions one for Sty reactions positive control 5 uL Ref 103 negative control 5 uL water Diluted gDNA samples Q 2 3 MU o Water AccuGENE 96 well plate labeled for Nsp and Sty digestion and ligation Ref 103 positive control Figure 4 5 Setup for aliquoting diluted gDNA and controls to a 96 well plate labeled for Nsp and Sty digest ligation What To Do Next Do one of the following Proceed to Stage 1 Nsp and Sty Restriction Enzyme Digest on page 28 Store the prepared digest ligate plate at 20 C 28 Affymetrix Cytogenetics Copy Number Assay User Guide Stage 1 Nsp and Sty Restriction Enzyme Digest About this Stage During this stage one aliquot of each sample is digested by the Nspl restriction enzyme the other aliquot by the Styl restriction enzyme You will 1 Prepare a Nsp Digest Master Mix and add it to the samples in column 1 2 Prepare a Sty Digest Master Mix and add it to the samples in column 12 3 Place the samples onto a thermal cycler and run the Cyto Digest program Location and Duration Pre PCR Clean Area Hands on time 30 min Cyto Digest thermal cycler program time 2 5 hr Input Required From
13. 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 7 4 Figure 7 4 Press down on the needle levers to start the bleach protocol 9 The fluidics station will begin the protocol emptying the lines and performing the cleaning cycles using bleach solution 10 After approximately 30 min the LCD will prompt you when the bleach cycle is over and the rinse cycle is about to begin chapter 7 Fluidics Station Care and Maintenance 105 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 traces 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 7 5 At this step there is no need to be concerned about the bleach remaining in the lines Figure 7 5 Immerse the three wash and water lines in the DI water bottle 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 d
14. This control meets the requirements outlined below The size of the starting genomic DNA can be compared with Ref103 DNA to assess the quality The control DNA should also be used as a routine experimental positive control and 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 DNA must be double stranded not single stranded 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 1 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 4 Affvmetrix Cytogenetics Copy Number Assay DNA must not be contaminated with other human genomic DNA sources or with genomic DNA from other organisms 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
15. it is then fragmented and end labeled using terminal deoxynucleotidyl transferase The stages involving enzymatic reactions are the most critical of the assay Thus it 1s important to carefully monitor and control any variables such as pH salt concentrations time and temperature all of which can adversely modulate enzyme activity Equipment and Calibration Keep dedicated equipment in each of the areas used for this protocol including pipettors ice buckets coolers etc To avoid contamination do not move equipment from one area to another Along with the enzymatic stages lab instrumentation plays an important role in the successful completion of this assay To aid in maintaining consistency across samples and operators all equipment should be well maintained and calibrated including All thermal cyclers GeneChip Hybridization Oven GeneChip Fluidics Station GeneChip Scanner 3000 7G Plate spectrophotometer or NanoDrop All multi channel pipets Pipetting chapter 1 Before You Start 3 Since the Cytogenetics Copy Number Assay involves a series of ordered stages the output of one stage directly impacts the performance of the subsequent stage For example the quantity and purity of the DNA after purification can affect the kinetics of the Fragmentation Reagent enzyme during the subsequent fragmentation stage To efficiently process samples Always use pipets that have been calibrated to 5 It is
16. 15 extra 15 extra 15 extra 15 extra H O 800 04 uL 3680 uL 7360 uL 11040 uL 22 08 mL SSPE 20X 360 uL 1656 uL 3312 uL 4968 uL 9 94 mL Tween 20 396 3 96 uL 18 2 uL 36 4 uL 54 6 uL 109 3 uL Denhardt s 50X 24 uL 110 4 uL 220 8 uL 331 2 uL 662 4 uL Total 1188 uL 5465 uL 10929 uL 16394 uL 32 79 mL 88 Affymetrix Cytogenetics Copy Number Assay User Guide SAPE Stain Solution Streptavidin Phycoerythrin SAPE should be stored in the dark at 4 C either foil wrapped or in an amber tube Remove SAPE from refrigerator and tap the tube to mix well before preparing stain solution Always prepare the SAPE stain solution immediately before use Mix well Do not freeze either concentrated SAPE or diluted SAPE stain solution A vial containing SAPE Stain Solution must be placed in position 1 for each module used Table 5 4 SAPE Stain Solution Reagent 4 Arrays 8 Arrays 12 Arrays 24 Arrays 10 extra 10 extra 10 extra 10 extra Stain Buffer 594 uL 2614 uL 5227 uL 7841 uL 15 68 mL 1 mg mL 6 uL 26 uL 53 uL 79 uL 158 4 uL Streptavidin Phycoerythrin SAPE Total 600 uL 2640 uL 5280 uL 7920 uL 15 84 mL Antibody Stain Solution Mix well A vial containing Antibody Stain Solution must be placed in position 2 for each module used Table 5 5 Antibody Solution Reagent 1 Array 4 Arrays 8 Arrays 12 Arrays 24 Arrays 10 extra 10 extra 10 extra 10 extra Stain Buffer 594 uL 2614 uL 5227 u
17. 2 3 5 Oo Genomic DNA samples Reference Genomic DNA 103 do NOT dilute Figure 4 3 Diluting genomic DNA samples to 50 ng uL x NOTE The illustrations in this user guide 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 Appendix A Guidelines for Processing 16 Samples or Appendix B Guidelines for Processing 24 Samples for more information Dilute the Genomic DNA To dilute the genomic DNA 1 Thaw the genomic DNA gDNA and Ref 103 as follows A Place on the bench top at room temperature until thawed B Once thawed place in the cooling chamber on ice 2 Vortex the gDNA samples at high speed for 3 sec 3 Spin down for 30 sec then place back in the cooling chamber If sample concentration is unknown take an OD measurement of each sample now 26 Affymetrix Cytogenetics Copy Number Assay User Guide IMPORTANT To avoid contaminating samples with PCR product take an aliquot of each sample to the plate spectrophotometer or NanoDrop Apply the convention that 1 absorbance unit at 260 nm equals 50 ug mL for double stranded DNA This convention assumes a path length of 1 cm Consult your spectrophotometer handbook for more information If using a method other than UV absorbance correlate the reading to th
18. Care 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 prevent 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 hr and forty minutes to complete Affymetrix recommends running this protocol weekly regardless of
19. Cot 1 DNA 1 mg mL 500 uL per tube Invitrogen 15279 011 3 1096 Tween 20 Pierce 28320 a Herring Sperm DNA HSDNA 10 mg mL 100 mg Promega D1815 3 Absolute Ethanol for dilution to 7596 Sigma Aldrich 459844 qo Denhardt s Solution Sigma Aldrich D2532 3 DMSO Sigma Aldrich D5879 3 MES Hydrate SigmaUltra 50 g or 250 g Sigma Aldrich M5287 a MES Sodium Salt 10 g 25 g or 100 g Sigma Aldrich M5057 3 Tetramethyl Ammonium Chloride TMAC Sigma Aldrich T3411 qo 5 M NaCl RNase free DNase free Ambion 9760G o EDTA Ambion 9260G a R Phycoerythrin Streptavidin Molecular Probes S 866 3 Antistreptavidin antibody Vector Labs BA 0500 3 Bleach 5 2596 Sodium Hypochlorite VWR 21899 504 or equivalent 3 Distilled water Invitrogen 15230147 120 Affymetrix Cytogenetics Copy Number Assay User Guide Consumables Required from Other Suppliers Table C 9 Consumables Required from Other Suppliers Y item Vendor Part Number Adhesive film for 96 well plates use on of these i MicroAmp Clear Adhesive Film Applied Biosystems 4306311 Microseal B Film Bio Rad MSB1001 a DNA Marker All Purpose Hi Lo Bionexus BN2050 a Gel Loading Solution Sigma G2526 a Pipet tips 20 uL filter tips Rainin GP L10F 3 Pipet tips 200 pL filter tips Rainin GP L200F a Pipet tips 1000 pL filter tips Rainin GP L1000F m Plates 96 well unskirted PCR Bio Rad MLP 9601 3 Plate OD for UV spec 96 well E amp K Scientific EK 25
20. Repeat this process until all samples are loaded onto arrays and are placed in a hybridization oven All samples should be loaded within 30 min 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 ovens for 16 to 18 hr at 50 C and 60 rpm This temperature is optimized for this product and should be stringently followed 82 Affymetrix Cytogenetics Copy Number Assay User Guide EE E B L G5 Wi 5g fi i H i E Chapter WASHING STAINING AND SCANNING ARRAYS This chapter describes how to wash stain and scan the Affymetrix Genome Wide Human SNP Array 6 0 The instruments that you will use include the Fluidics Station 450 to wash and stain arrays GeneChip Scanner 3000 7G to scan 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 5 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 One of the following instrument control applications Affymetrix Affymetrix GeneChip Operating Software Affymetrix GeneChip Command Console Sterile RNase free microcentrifuge vials 1 5 mL USA Scientific 1415 2600 or e
21. Required The following equipment and consumables are required for this stage Table 4 31 Equipment and Consumables Required for Stage 7 Labeling Quantity Item As required Adhesive seals for 96 well plates 1 Centrifuge plate 1 Cooler chilled to 20 C 1 Cooling chamber single chilled to 4 C on ice do not freeze 1 Ice bucket filled with ice 1 Marker fine point permanent 1 Mini centrifuge microfuge 1 Pipet single channel P200 1 Pipet single channel P1000 1 Pipet 8 channel P20 accurate to within 5 As needed Pipet tips for pipets listed above 1 Thermal cycler 1 Tube centrifuge 1 5 mL 1 Vortexer 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 4 72 Affymetrix Cytogenetics Copy Number Assay User Guide Reagents Required The following reagents are required for this stage Table 4 32 Reagents Required for Stage 7 Labeling DNA Labeling Reagent 30 mM Terminal Deoxynucleotidyl Transferase TdT 30 U uL Terminal Deoxynucleotidyl Transferase Buffer TdT Buffer 5X Prepare the Reagents Consumables and Other Components Thaw Reagents Thaw the following reagents on ice 5X TdT Buffer DNA Labeling Reagent E IMPORTANT Leave the TdT enzyme at 20 C until ready to use Prepare Your Work Area To prepare the work
22. eb ebat RIAL de 0 Taba Ji J r na ede 53 Purify the Pooled PCR Products s keya deyl kn 2 Pdk Ke k era a eres 54 What TODS NE ut ceed DMMI M MIIIIEIEELMMIMIIIMJIBBDDMZMAM IMIMIMDBIMiMDMDM A D DNJJM J o IMTMADZEZEMZWMD 57 Stage b Quantitation l Al ss ete eto Me eic e M o W ata tate Y pla 58 About this Stage lt 25340 44 des eee y dd kbk ede beo eel LA e bbs D QA ees 58 Ocation and Duration zat irem EE Db D E RE gri Rare e STET re 58 Input Required from Previous Stage 0 les 58 Equipment and Consumables Required lle ee 58 Reagents RegulFed x RA de ice c eso ea AM WAA d US de e oce 58 Important Information About This Stage 0 0 eese 59 Prepare the Reagents Equipment and Consumables llle 59 Procedure if Using a Microplate Spectrophotometer KIR S 59 Procedure if Using a NanoDrop WW kk kk retra o Pee ae ne gated odes 61 Assess the OD Readings 0 0 ee KK KK ees 62 What TO DANE an rtt Det EP Aetas SIV laa EEG 62 Stage 6 Fragmentation 5 x 4 d Des de Be ond U wipe orl aed dish ERRARE VOR Ay An e 63 ADOUTITHIS Stage e 36 4 paar arene rl Bn ers rs e NE opi ed aU P o Fea a ETE A2 63 Location and Datio n 5 ues A at e dachte en ed o OM EE e lone a kd cea 63 Input Required from Previous Stage WWW kk kk kK KK KK eee 63 Equipment and Consumables Required Ak AV kk KK KK eee 64 Reagents Required c pessan es cati ae kun Re b d dizl ACE Ad did uk xu skin s i 64 G
23. ee wets UTERE ec 101 ARE BIGACH CY CIS Fs 6 uim BG N trt M MN Oy EEA eiei e wee ER ee 102 The Fulise Cycle Leto als Race eee HH cutee ad MS BR i testa et A 105 Guidelines for Processing 16 Samples nananana aeaea KK RR RR RSS 107 Digestion Ligation and POR 1 24248 fe See bee WG ees CREDI adem bes 108 Guidelines for Processing 24 Samples KK RR KK RR RR eee 111 Digestion Ligation and POR gt csc ah An ee day de toda E rotted ed Gs regole amp 112 Reagents Equipment and Consumables 00000000eeee 115 About thiS ADPONGIK a gt usta rr AMIL obo tate a oar ack NI MA BA DAKA dila Mb eya Ys 115 rot AJ VETO ULDG S i raya Sa S KA Gian alay ene cosi cte oes EYE di v ted cote Dan c linear as 115 Equipment Required from Other Suppliers 0 0 0 0 ee 117 Pre PCR Clean Area Equipment Required 00 0000 eee 117 Post PCR Area Equipment Required 0 0 0c ee 118 Reagents Required from Other Suppliers 2 0 kk eee 119 Consumables Required from Other Suppliers 00 0 0 eee ee 120 supplier GontactEISt 2c Rake eva diced KEY DY Eus aus 121 THermal amp yoeler Programs voce em dera th tee kalak ae EORR RR 123 Cyto DIGEST cesi Vi dete ett e p mm S RS 123 Cyto Ligate scm UG ae oe A ef PUE Eu pad Or eee ba 123 CVtO POR Lent sa Pele Tatts Ath dh DU HHHH HHHH 124 CROT E S9 n Dank ale ne N ARA DN De on mack Or a ETE 125 Gi ATO E n N E EE a E LECCE r r r GR E He r O tame asks DESEE 125 la
24. 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 The combination of Affymetrix SNP 6 0 arrays the Cytogenetics Copy Number Assay and Genotyping Console 2 1 software allows you to perform high resolution 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 Cytogenetics Solution a great tool for next generation cytogenetics studies Tips for Ensuring Successful Performance of the Protocol Successful performance of the Cytogenetics Copy Number Assay requires accuracy and attention to detail Many stages involve specific yet distinct enzymatic reactions For example in stage 1 genomic DNA is digested with the restriction enzymes Nspl and Styl In stage 2 it is ligated to a common adaptor with T4 DNA ligase Following ligation the template undergoes PCR using TITANIUM Taq DNA polymerase Once the product has been purified
25. gel to confirm restriction enzyme activity Use the correct concentration of BSA Failed adaptor ligation reaction Confirm enzyme activity Ligase buffer contains ATP and should be defrosted held at 4 C Vortex ligase buffer thoroughly before use to ensure precipitate is re suspended Avoid multiple freeze thaw cycles Try a fresh tube of buffer Reduced adaptor ligation efficiency due to adaptor self ligation DNA re ligation To prevent self ligation of adaptor work rapidly and add DNA ligase last Failed PCR reaction Check PCR reagents Take care with preparation of master mixes and ensure accurate pipetting and thorough mixing Reduced PCR reaction yield non optimal PCR conditions Use a calibrated thermal cycler and check PCR programs Use the recommended 96 well PCR plates Thoroughly mix PCR reaction Ligation mix not diluted prior to PCR reaction Ligation mixture diluted 1 4 with molecular biology grade water to remove potential inhibitors and maintain optimal pH and salt concentration Incorrect concentration of nucleotides Check dNTP stock concentration and vendor Used Nsp adaptor for Sty digest or vice versa Repeat ligation step with correct adaptors Samples affected but positive controls OK Non optimal reaction conditions Prepare master mixes as described and include a positive control to eliminate reagents and assay problems as detailed ab
26. lid 6 When the Cyto Label program is finished remove the plate from the thermal cycler and spin down for 30 sec Table 4 34 Cyto Thermal Cycler Program Cyto Label Program Temperature 37 C 4hr 95 C 15 min 4 C Hold OK to hold overnight What To Do Next Do one of the following Proceed to the next stage If not proceeding directly to the next stage you can Hold at 4 C on the thermal cycler overnight Freeze the samples at 20 C chapter 4 Affymetrix Cytogenetics Copy Number Assay 75 Stage 8 Target Hybridization About this Stage During this stage each sample will be hybridized onto a Genome Wide Human SNP Array 6 0 by Preparing a Hybridization Master Mix and adding it to each sample Denaturing the samples on a thermal cycler Loading each sample onto a Genome Wide Human SNP Array 6 0 Placing the arrays into a hybridization oven at 50 C for 16 to 18 hr Location and Duration Post PCR Area Hands on time 45 min Hybridization time 16 to 18 hr Input Required from Previous Stage The input required from Stage 7 Labeling is Item Labeled samples Equipment and Consumables Required The following equipment and consumables are required for this stage IMPORTANT Increased variability in Cytogenetics Copy Number Assay performance has been observed in GeneChip Hybridization Oven 640 models P N 800138 or 800189 manufactured prior to 2001 Chec
27. once you have successfully processed these samples 46 Affymetrix Cytogenetics Copy Number Assay User Guide Prepare the PCR Master Mix The same PCR master mix is used for both Nsp and Sty ligated samples IMPORTANT The PCR reaction is sensitive to the concentration of primer used It is critical that the correct amount of primer be added to the PCR Master Mix to achieve the correct distribution of fragments 200 to 1100 bp in the products Check the PCR reactions on a gel to ensure that the distribution is correct To prepare the PCR Master Mix 1 Keeping the 50 mL centrifuge tube in the cooling chamber add the reagents in the order shown in Table 4 17 on page 46 except for the Tag DNA polymerase Remove the TITANIUM Tag DNA Polymerase from the freezer and immediately place in a cooler Pulse spin the Tag DNA polymerase for 3 sec Immediately add the Tag DNA polymerase to the master mix then return the tube to the cooler Vortex the master mix at high speed 3 times 1 sec each time oa BB WRN Pour the master mix into the solution basin keeping the basin on ice Table 4 17 PCR Master Mix Volumes sufficient for processing both Nsp and Sty ligated samples Reagent 1 Sample 4 Samples 8 Samples 12 Samples 24 Samples 15 extra 15 extra 15 extra 15 extra AccuGENE water 39 5 uL 1272 uL 2544 uL 3816 uL 7632 uL TITANIUM 7aq PCR Buffer 10 uL 322 uL 644 uL 966 uL 1932 uL 10X GC Melt 5M 20
28. rooms greatly reduces the risk of sample contamination due to previously amplified PCR products These rooms are referred to as the Pre PCR Clean Room Post PCR Room The high level steps performed in each room is presented in Table 2 1 Table 2 1 Assay workflow when two separate rooms are used Room Template PCR Product Genomic DNA Pre PCR Clean Room Assay steps Genomic DNA preparation Digestion Ligation PCR setup only Post PCR Room Assay steps e PCR thermal cycling Fragmentation Labeling Hybridization e Washing and staining e Scanning 8 Affymetrix Cytogenetics Copy Number Assay User Guide 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 2 1 Activities that take place in this room include Preparation of non amplified genomic DNA Digestion and ligation reactions Preparation of PCR reactions 6 Equipment Shown Vortexer 2 Microfuge 3 Pipets on stand 4 Ice bucket 7 5 Thermal cycler 6 Plate centrifuge 7 Freezer Figure 2 1 Pre PCR Clean Room To help prevent sample contamination All of the 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 All of the equipment required for
29. top to room temperature 2 Vortex the plate at high speed for 3 sec then spin down at 2000 rpm for 30 sec 3 Mark each 2 0 mL microcentrifuge tube with a sample number such as 2 3 4 etc 4 Using a P200 single channel pipet transfer all 7 aliquots of each sample to the appropriately marked 2 0 mL tube Figure 4 16 on page 54 Do not pool the negative control Discard IMPORTANT Use round bottom tubes only Do NOT use conical tubes Change pipet tips after pooling each sample Sty PCR wells 3 100 uL from each well 300 uL Nsp PCR wells 4 100 uL from each well 400 uL Total Volume in Each 2 0 mL Microcentrifuge Tube 700 yuL tube 5 When finished examine the PCR plate and ensure that the total volume in each well has been transferred and pooled 54 Affymetrix Cytogenetics Copy Number Assay User Guide Pool PCR products for each sample in a 2 mL round bottom microcentrifuge tube Do not pool the negative control Discard Figure 4 16 Pooling PCR products Purify the Pooled PCR Products Add Agencourt AMPure Magnetic Beads and Incubate To add magnetic beads and incubate 1 Thoroughly mix the magnetic bead stock by vigorously shaking the bottle Examine the bottom of the bottle and ensure that the solution appears homogenous 2 Optional Pour magnetic beads into a 50 mL conical tube 3 Aliquot 1 mL of magnetic beads to each pooled sample IMPORTANT
30. uL 644 uL 1288 uL 1932 uL 3864 uL dNTP 2 5 mM each 14 uL 451 uL 902 uL 1352 uL 2704 uL PCR Primer 002 100 uM 4 5 uL 145 uL 290 uL 435 uL 870 uL TITANIUM 7aq DNA 2 uL 64 4 uL 129 uL 193 uL 386 uL Polymerase 50X do not add until ready to aliquot master mix to ligated samples Total 90 uL 2898 uL 5796 uL 8694 uL 17 4 mL Add PCR Master Mix to Each Sample To add the PCR Master Mix to samples 1 Aliquot 90 uL PCR Master Mix to each sample and control on the PCR plate To avoid contamination change pipet tips after each dispense For four samples you may have to tilt the solution basin for the last pickup dispense to ensure 90 uL picked up in each pipet tip Total volume in each well is 100 pL 2 Tightly seal the plate 3 Vortex at high speed for 3 sec then spin down at 2000 rpm for 30 sec 4 Keep in the cooling chamber on ice until ready to load onto a thermal cycler chapter 4 Affymetrix Cytogenetics Copy Number Assay 47 Load PCR Plate onto a Thermal Cycler Location Post PCR Area Procedure To load the plate and run the Cyto PCR program 1 Transfer the plate to the Post PCR Area 2 Ensure that the thermal cycler lid is preheated The block should be at room temperature 3 Load the plate onto the thermal cycler IMPORTANT Ensure that the seal is not pulled off any wells when you close the thermal cycler lid 4 Run the Cyto PCR program IMPORTANT PCR protocols for the MJ Te
31. 0X Fragmentation Buffer 16 00 uL 18 00 uL 20 00 uL 22 00 uL 24 00 uL Fragmentation Reagent enzyme 8 00 uL 8 00 uL 8 00 uL 8 00 uL 8 00 uL Total 160 uL 180 uL 200 pL 220 uL 240 uL 5 Remove the Fragmentation Reagent enzyme from the freezer and A Immediately pulse spin for 3 sec Spinning is required because the reagent tends to cling to the top of the tube making it warm quicker B Immediately place in a cooler Add the appropriate volume of Fragmentation Reagent Vortex the master mix at high speed 3 times 1 sec each time Pulse spin for 3 sec and immediately place on ice e 02 Proceed immediately to the next set of steps Add Fragmentation Master Mix to the Samples Add Fragmentation Master Mix to the Samples 5 uL to each sample Aliquot Frag m NIC YOY OCYCYCYCYCOCYCYCYCY LY Master Mix O MOO OOO O0O0OO0O 8Q CC C GO TNO OY OY caual ia strip DT 2 Q AZ NAM U AZ NAN N J O VS a f CSS N f 90OOOOOOOOOOO a f f C f T eDOOOOOOOOOO M 9 O O O O OO O O O O O BIY O JY ZIZ MZ MZ KUZ U U Ye OV ND amp iDOOOOOOOOOO o d s QOOOC QOOOOO O CYCYCOOOYOYC O 7 L AZ Na St VY VY VY Figure 4 25 Adding Fragmentation Master Mix to samples chapter 4 Affymetrix Cytogenetics Copy Number Assay 69 To add Fragmentation Master Mi
32. 1 817 6 610 482 6 733 977 6 955 915 and D430 024 and other U S or foreign patents Products are manufactured and sold under license from OGT under 5 700 637 and 6 054 270 Reagents Products may be covered by one or more of the following patents U S Patent Nos 6 965 020 6 864 059 Copyright 2008 Affymetrix Inc All rights reserved Chapter 1 Chapter 2 Chapter 3 Chapter 4 ke i CONTENTS Berore YOU SIS uir es ed abandona a RC Re be rad a abd d fe red bal 1 About the Affymetrix Cytogenetics Solution kk KK KK KK KK sees 2 Tips for Ensuring Successful Performance of the Protocol liliis 2 Equipment and Calibration liiis 2 Fipettirig ce y HHHH HHH HHHH i putes ear eee d HHH 3 Reagent Handling and Storage kk kk kk kk kk KK KK KK RIK KK KK KK KK KI KIRI RIK KI KIRI KK eee 3 Master Mix Preparation Jk kk kk kk kk kk KK KK KK KK KK KK KI KI KI KK KK KK KK KIRI KI KI KIR KK 3 Laboratory WorktlOW l ama kk 6 n Kan k ka PER ER Y Wa Pes Phat PRP Te rd 4 Preparing the Work Area for Each Stage WWW kk kk kk KK KK KK eee 4 Thermal Cyclers 96 well Plate and Adhesive Seals 0 0 RR RR RR RR RIK 4 Program Your Thermal Cyclers vk kk kk kk kk kK KK KK KK KK KK KK KI KK KK KK K KK KK KK 5 Laboratory Setup and Recommendations ZAYE EE KK 7 Configuration 1 Two Separate Rooms kK KK KK KK KK KK KK eee 7 Pre POR Clearn ROOF s d irpo WA deere ws d d l Re E a ca RR a ee
33. 30 min hands on 16 18 hours 2 hours hands on 2 5 hours 1 hour hands on 15 min hands on 32 min per array to scan Figure 4 1 Workflow recommended for processing one to 24 samples 22 Affymetrix Cytogenetics Copy Number Assay User Guide Optional 3 Day Workflow Figure 4 2 illustrates the optional 3 day workflow The difference between the 3 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 lt 8 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 3 hours 30 min hands on 4 hours 30 min hands on 1 hour hands on Pre PCR Clean Room Post PCR Room Baan 1 5 hours QC Gel 1 Stage 4 1 5 hours hands on Stage 5 30 min hands on Day 2 1 5 hours 30 min hands on QC Gel 2 Stage 6 Stage 7 4 hours 30 min hands on 16 18 hours 2 hours hands on y lt gt E D RA RA t5 to D Q a uw D D NM zx L Stage 8 25 hours 1 hour hands on Day 3 Stage 9 r 15 min hands on 32 min per array to scan Figure 4 2 Optional 3 day workflow chapter 4 Affymetrix Cytogenetics Copy Number Assay 23 Genomic DNA
34. 4 Affymetrix Cytogenetics Copy Number Assay 51 Stage 4 PCR Product Purification About this Stage During this stage you will purify the PCR products by Pooling the Nsp and Sty PCR reactions Adding magnetic beads Agencourt AMPure to each pooled reaction and incubating the mix Adding 75 EtOH to wash DNA Adding Buffer EB to resuspend the beads and elute the DNA Location and Duration Post PCR Area Hands on time 1 hr DNA binding to magnetic bead 15 to 20 min EtOH wash approximately 10 to 20 min Elution 15 to 30 min Total time for this stage approximately 1 5 hr Input Required from Previous Stage The input required from Stage 3 Nsp and Sty PCR is Plate of Nsp and Sty PCR products 52 Affymetrix Cytogenetics Copy Number Assay User Guide Equipment and Consumables Required The following equipment and materials are required to perform this stage Table 4 20 Equipment and Consumables Required for Stage 4 PCR Product Purification Quantity Item 1 Adhesive seals for 96 well plates 1 Microcentrifuge Eppendorf 5415D with rotor for 24 tubes 2 0 mL 1 MagnaRack magnetic stand 1 Marker fine point permanent 1 Microtube Foam Insert for vortexing 2 0 mL tubes 1 Pipet single channel P20 1 Pipet single channel P200 1 Pipet single channel P1000 As needed Pipet tips for pipets listed above 1 Plate Bio Rad 96 well One per 96 well plate Plate holder
35. 8 Affymetrix Cytogenetics Copy Number Assay User Guide no Ses a r ra Chapter 4 AFFYMETRIX CYTOGENETICS COPY NUMBER ASSAY About the Protocol The Affymetrix Cytogenetics Copy Number Assay is designed for processing as few as four samples including controls The protocol is presented in the following stages Genomic DNA Preparation on page 23 Stage 1 Nsp and Sty Restriction Enzyme Digest on page 28 Stage 2 Nsp and Sty Ligation on page 34 Stage 3 Nsp and Sty PCR on page 41 Stage 4 PCR Product Purification on page 51 Stage 5 Quantitation on page 58 Stage 6 Fragmentation on page 63 Stage 7 Labeling on page 71 Stage 8 Target Hybridization on page 75 IMPORTANT The Cytogenetics Copy Number assay protocol is optimized for processing from 4 to 24 samples at a time to obtain copy number results This protocol is not intended for genome wide association studies An assay protocol for processing 48 samples is described in the Affymetrix Genome Wide Human SNP Nsp Sty 6 0 User Guide P N 702504 20 Affymetrix Cytogenetics Copy Number Assay User Guide About the Illustrations in this Chapter This protocol has been optimized for processing 4 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 9 to 24 samples refe
36. 801 required only if using microplate spectrophotometer Oo Solution Basin 55 mL sterile Labcor 730 004 3 TBE Gel 4 BMA Reliant precast Lonza Group LTD 54929 3 TBE Gel 2 BMA Reliant precast Lonza Group LTD 54939 a TBE for electrophoresis Any vendor or house made 3 Tough Spots 1 2 Diversified Biotech T SPOTS 50 a Tube Eppendorf Safe Lock Microcentrifuge 1 5 mL VWR 21008 959 3 Tube Eppendorf Safe Lock Microcentrifuge 2 0 mL VWR 20901 540 must be round bottom tubes m Tube centrifuge 50 mL VWR 21008 178 3 Tube strips 8 well 0 2 mL VWR 20170 004 appendix C Reagents Equipment and Consumables 121 Supplier Contact List Table C 10 Supplier Contact List Supplier Web Site Address Affymetrix www affymetrix com Agencourt Bioscience Corp agencourt com Ambion ambion com Applied Biosystems www appliedbiosystems com Bionexus Inc www bionexus net Bio Rad bio rad com Bio Smith biosmith com Clontech www clontech com Diversified Biotech divbio com E amp K Scientific eandkscientific com Eppendorf eppendorf com ESCO www escoglobal com Fisher Scientific www fishersci com Invitrogen Life Technologies invitrogen com Labcor labcorproducts com Lonza www lonza com Molecular Devices moleculardevices com Molecular Probes molecularprobes com NanoDrop nanodrop com New England Biolabs www neb com
37. Check that all of the beads have been pulled to the side in each tube If all of the beads have not been pulled to the side of the tubes leave the tubes on the stand an additional 3 min x NOTE The eluate will appear yellowish If you open the cap and look directly into the tube you will see that the eluate is clear 8 Transfer 47 uL of eluted sample to the appropriate well on a fresh 96 well plate Figure 4 21 on page 57 Brown residue on pipet tips is OK 9 Tightly seal the plate OOOOOOO OOOOOOO OOOOOOO OOOOOOOO OOOOOOOOQO O O O O O E O s O O Figure 4 21 Transferring each purified sample to a fresh 96 well plate What To Do Next Proceed to Stage 5 Quantitation on page 58 Here you will remove 2 uL from each sample for an OD measurement 58 Affymetrix Cytogenetics Copy Number Assay User Guide Stage 5 Quantitation About this Stage During this stage you will quantitate each sample Location and Duration Post PCR Room Hands on time 20 min Input Required from Previous Stage Input required from Stage 4 PCR Product Purification is Pooled purified PCR products 47 uL each sample Equipment and Consumables Required The following equipment and consumables are required for this stage Table 4 22 Equipment and Consumables Required for Stage 5 Quantitation Quantity Item As required Adhesive seals for 96 wel
38. Copy Number Assay 77 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 It is critical that the samples remain on the thermal cycler at 49 C after denaturation and while being loaded onto arrays About DMSO When adding to the Hybridization Master Mix pipet DMSO into the middle of the tube Do not touch the sides of the tube as the DMSO can leach particles out of the plastic which in turn may cause high background DMSO is light sensitive and must be stored in a dark glass bottle Do not store in a plastic container Be sure to equilibrate the arrays to room temperature otherwise the rubber septa may crack and the array may leak An accurate hybridization temperature is critical for this assay Therefore we recommend that your hybridization ovens be serviced at least once per year to ensure that they are operating within specifications Gloves safety glasses and lab coats must be worn when preparing the Hybridization Master Mix Consult the appropriate MSDS for reagent storage and handling requirements Prepare the Reagents Consumables and Other Components Prepare a 12X MES Stock Solution The 12X MES stock solution can be prepared in bulk and kept for at least one month if properly stored Proper storage Protect from light using aluminum foil Keepat 4 C IMPORTANT Do not au
39. KK KK K K K KI KIRI KI KII K 86 Prime the Fl rdics Station ac rs ES K et ee AA a sl ehe SS e eS 86 Wash and Stali Arrays suec Zik lar ae REGUM DR NUN Re REA die 87 Prepare Arrays for Washing and Staining kk kk kk kK kK KK KI KIR KIRI K KI RIK KIR KIRI KI 87 Prepare Buffers and Solutions psu mm Reds hte dendi UU d 87 Washing and Staining Arrays kk kk kk kK KK KK KK KK KI KIR KIRI KI KIR KI KR KK RI KK eens 89 Scanning AAV Sinko xx 4f sete e ben Rs A dol n de e e Sle n cde W qa dudo tetas 91 Prepare the Scanner kee at it HE E INS ee E et hot ede seio 91 Prepare Arrays for Scanning 2 ee ee eee 91 Seanrnigahie AEFay os Sorte a ahs Gays tates te acini e tas Bites a C SUPER ae sete deo 92 Shutting Down the Fluidics Station oe ceee cea 92 DR WAII DN N veto cc re nis SR ee AER 93 Chapter 6 Troubleshooting oc xway ayy E kara a QALA Au s que ERE eR ak AN d qat 95 General Assay Performance Recommendations KEK KK KK KII IK 95 Troubleshooting the Cytogenetics Copy Number Assay KK KIR cece eee 97 OD Troubleshooting Guidelines eee 99 Affymetrix Instruments kk kk kk kk kk KK KK KK KK KK ehh ss 100 contents v Fluidics Station Care and Maintenance llle 101 Chapter 7 Appendix A Appendix B Appendix C Appendix D General Fluidics Station Care kk KK KK KK KK KI KK KI KI KIR KI es 101 Fluidics Station Bleach PrOtO0GO eer ete edd a
40. L 7841 uL 15 68 mL 0 5 mg mL 6 uL 26 uL 53 uL 79 uL 158 4 uL biotinylated antibody Total 600 uL 2640 uL 5280 uL 7920 uL 15 84 mL Array Holding Buffer Mix well A vial containing Array Holding Buffer must be placed in position 3 for each module used Table 5 6 Array Holding Buffer Components Volume MES Stock Buffer 12X 8 3 mL 5 M NaCl 18 5 mL Tween 20 10 0 1 mL Water 73 1 mL Total 100 mL chapter 5 Washing Staining and Scanning Arrays 89 Washing and Staining Arrays Wash and Stain Protocol The GenomeWideSNP6_450 protocol is an antibody amplification protocol for mapping targets described in Table 5 7 Use it to wash and stain the Genome Wide Human SNP Array 6 0 Table 5 7 GenomeWideSNP6_450 protocol for the Fluidics Station 450 GenomeWideSNP6_450 Protocol for 49 Format Standard Arrays Post Hyb Wash 1 6 cycles of 5 mixes cycle with Wash Buffer A at 25 C Post Hyb Wash 2 24 cycles of 5 mixes cycle with Wash Buffer B at 45 C Stain Stain the array for 10 min in SAPE solution at 25 C Post Stain Wash 6 cycles of 5 mixes cycle with Wash Buffer A at 25 C 2nd Stain Stain the array for 10 min in Antibody Stain Solution at 25 C 3rd Stain Stain the array for 10 min in SAPE solution at 25 C Final Wash 10 cycles of 6 mixes cycle with Wash Buffer A at 30 C The final holding temperature is 25 C Filling Array Fill the array with Array Holding Buffer
41. Preparation About this Stage The human genomic DNA you will process using the Cytogenetics Copy Number Assay should meet the general requirements listed in Chapter 3 Genomic DNA General Requirements During this stage you will prepare the genomic DNA by 1 Determining the concentration of each sample if required 2 Diluting each sample to 50 ng uL using reduced EDTA TE buffer Location and Duration Pre PCR Clean Area Hands on time dependent upon number of samples to be processed Input Required The illustrations in this user guide depict the processing of eight samples six genomic DNA samples plus one positive and one negative control Table 4 1 Input Required for Genomic DNA Preparation Quantity Item 4 to 24 Genomic DNA samples that meet the requirements listed in Chapter 3 Genomic DNA General Requirements About Using Controls We recommend including one positive and one negative control with every set of samples processed For the positive control use the Ref 103 included in the Genome Wide Human SNP Nsp Sty Assay Kit 5 0 6 0 For the negative control use water AccuGENE 24 Affymetrix Cytogenetics Copy Number Assay User Guide Equipment and Consumables Required The equipment and consumables listed in Table 4 2 are required for this stage Table 4 2 Equipment and Consumables Required for Genomic DNA Preparation Quantity Item As required Adhesive seals f
42. Previous Stage The input required is shown below Plate containing two equal aliquots of each genomic DNA and each control prepared as instructed under Genomic DNA Preparation on page 23 5 uL at 50 ng uL in each well chapter 4 Affymetrix Cytogenetics Copy Number Assay 29 Equipment and Consumables Required The following equipment and consumables are required for this stage Table 4 4 Equipment and Consumables Required for Stage 1 Nsp and Sty Restriction Enzyme Digest Quantity Item As required Adhesive seals for 96 well plates 1 Centrifuge plate 1 Cooler chilled to 20 C 1 Cooling chamber double chilled to 4 C on ice do not freeze 1 Ice bucket filled with ice 1 Markers blue and red fine point permanent 1 Mini centrifuge microfuge 1 Pipet single channel P10 1 Pipet single channel P100 or P200 As required Pipet tips for pipets listed above 1 Thermal cycler 2 Tubes Eppendorf 1 5 mL 1 Vortexer 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 4 Reagents Required The following reagents are required for this stage Table 4 5 Reagents Required for Stage 1 Nsp and Sty Restriction Enzyme Digest BSA 100X 10 mg mL NE Buffer 2 10X NE Buffer 3 10X Nspl 10 U uL NEB Styl 10 U uL NEB AccuGENE Water molecular biology grade
43. Reactions To add Sty Ligation Master Mix to samples 1 Using a P20 pipet aliquot 5 25 uL of Sty Ligation Master Mix to each Sty digested sample and control Figure 4 11 The total volume in each well is now 25 uL 2 Tightly seal the plate SOON Add 5 25 uL Sty Ligation amp Master Mix to each sample and OloOOO OOOOO00lO control in column 1 OJO O OO OO O O O OlO OJOOOO0O60O00 600 0O OO ClO O O O O O O O O OlO JOlOOQO0600 0 0O O OO jJOJOOO amp CO000060 600 OO Figure 4 11 Adding Sty Ligation Master Mix to Sty digested samples and controls 40 Affymetrix Cytogenetics Copy Number Assay User Guide Load the Nsp and Sty Samples Onto the Thermal Cycler 1 2 3 Vortex the plate at high speed for 3 sec then spin down at 2000 rpm for 30 sec Ensure that the thermal cycler lid is preheated Load the plate onto the thermal cycler and run the Cyto Ligate program IMPORTANT Ensure that the seal is not pulled off the wells when you close the thermal cycler lid Return remaining reagents to the freezer and discard remaining master mix Table 4 13 Cyto Ligate Thermal Cycler Program Cyto Ligate Program Temperature Time 16 C 180 min 70 C 20 min 4 C Hold Dilute the Ligated Samples Li IMPORTANT It is crucial to dilute the ligated DNA with AccuGENE water prior to PCR To dilute the samples 1 2 3 4 Place th
44. Reagent tube label and record the concentration 2 Basedon the number of samples you are processing determine which Fragmentation Master Mix table to use 4to 7 samples Table 4 27 on page 67 8to 16 samples Table 4 28 on page 68 17 to 24 samples Table 4 29 on page 68 3 Add the appropriate volume of water and Fragmentation Buffer to the Frag tube on ice 4 Allow to cool on ice for 5 min Table 4 27 Fragmentation Master Mix for 4 to 7 Samples Reagent Fragmentation Reagent Concentration 2 25 U pL 2 5 U uL 2 75 U uL AccuGENE water 34 00 uL 38 50 uL 43 00 uL 48 50 uL 52 00 uL 10X Fragmentation Buffer 4 00 uL 4 50 uL 5 00 uL 4 50 uL 6 00 uL Fragmentation Reagent enzyme 2 00 uL 2 00 uL 2 00 uL 2 00 uL 2 00 uL Total 40 uL 45 uL 50 uL 55 uL 60 pL 68 Affymetrix Cytogenetics Copy Number Assay User Guide Table 4 28 Fragmentation Master Mix for 8 to 16 Samples Reagent Fragmentation Reagent Concentration 2 25 U pL 2 5 U uL 2 75 U pL AccuGENE water 85 00 uL 96 25 uL 107 50 uL 118 75 uL 130 00 uL 10X Fragmentation Buffer 10 00 uL 11 25 uL 12 50 uL 13 75 uL 15 00 uL Fragmentation Reagent enzyme 5 00 uL 5 00 uL 5 00 uL 5 00 uL 5 00 uL Total 100 uL 112 50 pL 125 uL 137 50 pL 150 uL Table 4 29 Fragmentation Master Mix for 17 to 24 Samples Reagent Fragmentation Reagent Concentration 2 25 U pL 2 5 U uL 2 75 U uL AccuGENE water 136 00 uL 154 00 uL 172 00 uL 190 00 uL 208 00 uL 1
45. SO Rhett te best Sn te ae das hec t Dub t ads a Re ROS naa le afe s 125 vi Affymetrix Cytogenetics Copy Number Assay User Guide Chapter l Topics in this chapter include About the Affymetrix Cytogenetics Solution on page 2 Tips for Ensuring Successful Performance of the Protocol on page 2 IMPORTANT The Cytogenetics Copy Number assay protocol is optimized for processing from 4 to 24 samples at a time to obtain copy number results This protocol is not intended for genome wide association studies An assay protocol for processing 48 samples is described in the Affymetrix Genome Wide Human SNP Nsp Sty 6 0 User Guide P N 702504 2 Affymetrix Cytogenetics Copy Number Assay User Guide About the Affymetrix Cytogenetics Solution 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
46. Station Care and Maintenance 103 3 As shown in Figure 7 2 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 water B Insert the waste line into the waste bottle C Immerse all three wash and water lines into the bleach solution Hi IMPORTANT Do NOT immerse the waste line into the bleach 4 Open the instrument control software GCOS or AGCC 5 Choose the current bleach protocol as of the writing of this manual it is BLEACHv2_450 for each module Figure 7 2 The bleach cycle Immerse the tubes into the 0 525 sodium hypochlorite solution The waste line remains in the waste bottle 104 Affymetrix Cytogenetics Copy Number Assay User Guide Fluidics Station 3 SBE Modulel Modue2 Module3 Module 4 Experiment Probe Array Type E View Protocol lidics Protocol BLEACHv2 450 Version Thoroughly clean F5450 by running then DI Water thru all lines and all ET 1hr 35min Current Stage Time Cycle Temp Time Remaining Close Figure 7 3 The Fluidics Station protocol window select all modules 6 In GCOS or AGCC run the protocol for all modules NOTE The fluidics station will not start until the needle lever is pressed down Figure 7 4 on page 104 The temperature will ramp up to 50 C 7 Follow the prompts on each LCD
47. The solution is viscous and sticky Pipet carefully to ensure that you aspirate and dispense 1 mL Thorough mixing is critical to ensure that the PCR products bind to the beads Securely cap each tube and mix well by inverting 10X 5 Incubate at room temperature for 10 min During incubation the DNA binds to the magnetic beads 6 Load the tubes cap hinge facing out onto the microcentrifuge and spin for 3 min at maximum speed 16 100 rcf Figure 4 17 on page 55 7 Place the tubes on the magnetic stand Figure 4 18 on page 55 Leaving the tubes in the rack pipet off the supernatant without disturbing the bead pellet and discard 55 chapter 4 Affymetrix Cytogenetics Copy Number Assay ba I Position tubes with the cap hinges facing out Bead pellet will be spun to the bottom and back of the tube Y P 7 22 F max 2449 ey jo e 2 o je pY Avoid contact with the bead pellet when pipetting off the supernatant Figure 4 19 Bead pulled to back and side of tube in magnetic stand 56 Affymetrix Cytogenetics Copy Number Assay User Guide Add Ethanol To add ethanol 1 Using a P1000 pipet add 1 5 mL of 75 EtOH to each tube 2 Cap the tubes and load them into the foam tube adaptor Figure 4 20 Fully insert tubes into the foam to ensure they are secure Space tubes adequately to balance 3 Vortex at 75 power for 2 min Centrifug
48. absorbance unit at 260 nm equals 50 pg 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 Measure the OD of each sample at 260 280 and 320 nm OD280 and OD320 are used as controls 2 Determine the OD260 measurement for the water blank and average 3 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 ug L Undiluted sample concentration OD X 0 05 ug uL X 100 chapter 4 Affymetrix Cytogenetics Copy Number Assay 61 Procedure if Using a NanoDrop Lu IMPORTANT The P20 pipet must be accurate to within 5 To prepare diluted aliquots of the purified samples 1 Using a P20 pipet aliquot 18 uL of water to the corresponding wells of a 96 well plate 2 Using a P20 pipet A Transfer 2 uL of each purified sample to the corresponding well of the 96 well plate B Pipette up and down 2 times to ensure that all of the sample is dispensed The result 1s a 10 fold dilution 3 Do one of the following to mix the samples Seta P20 pipet to 17 uL and pipet up and down 5 times Seal the plate vortex and spin down at 2000 rpm for 30 sec 18 uL water AccuGENE 2 uL purified sample in each well
49. are the Fragmentation Master Mix on page 67 Ensure fragmentation reagent is kept at 20 C Do not reuse diluted working stock CEL file can not be generated GCOS or AGCC is unable to Unable to place a grid on the dat file due to Hybridization controls including oligo B2 must be align grid the absence of B2 signal added to hybridization cocktail for grid alignment dat image is dim Insufficient signal intensity or staining Make fresh stain buffers failure Incorrect wash buffers used on fluidics station Prime the fluidics station with the correct buffers prior to running the assay Incorrect wash buffers will disrupt hybridization of the labeled fragmented DNA High MAPD or Low Contrast OC Values Gel images and Over fragmentation of DNA sample due to spectrophotometric incorrect dilution of Fragmentation Reagent quantitation indicate successful stock PCR reaction Check that you have selected the correct activity of DNase to add to fragmentation reaction See Prepare the Fragmentation Master Mix on page 67 Work quickly and on ice mix thoroughly Transfer reactions to pre heated thermal cycler 37 C Extremely high MAPD or low Labeling reaction suboptimal Contrast OC values Sample hybridization is absent on cel and dat images but B2 grid is bright Use a new vial of Terminal Dideoxynucleotidyl Transferase Verify the labeling reagents and repeat labeling Positive con
50. area 1 Place a cooling chamber on ice Figure 4 27 on page 72 2 Prepare the reagents as follows A Vortex each reagent at high speed 3 times 1 sec each time B Pulse spin for 3 sec then place in the cooling chamber 3 Label the 1 5 mL centrifuge tube LBL and place in the cooling chamber TdT Buffer e DNA Labeling Reagent A y P L1 o ey Ce yo hS MONOD Erre yen a OIIO IT j Label master mix tube mR p E a m S n ms HE Wi 2 YA amp V2 EM N sx Figure 4 27 Setup for labeling chapter 4 Affymetrix Cytogenetics Copy Number Assay 73 Preheat the Thermal Cycler Block The block must be heated to 37 C before samples are loaded To preheat the thermal cycler block 1 Turn on the thermal cycler and preheat the block to 37 C 2 Allow it to heat for 10 min before loading samples Prepare the Labeling Master Mix Preparation Keep all reagents and tubes on ice while preparing the Labeling Master Mix To prepare the Labeling Master Mix 1 Add the following to the 1 5 mL centrifuge tube on ice using the volumes shown in Table 4 33 on page 73 5X TdT Buffer DNA Labeling Reagent Remove the TdT enzyme from the freezer and immediately place in the cooler Pulse spin the enzyme for 3 sec then immediately place back in the cooler Add the TdT enzyme to the master mix Vortex the master mix at high speed 3 times
51. block at room temperature Prepare the Arrays To prepare the arrays 1 Unwrap the arrays and place on the bench top septa side up 2 Mark the front or back of each array with a designation that will identify which sample is loaded onto each array Figure 4 28 3 Allow the arrays to warm to room temperature on the bench top 10 to 15 min 4 Insert a 200 pL pipet tip into the upper right septum of each array 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 Figure 4 28 Arrays prepared for sample loading chapter 4 Affymetrix Cytogenetics Copy Number Assay 79 Prepare the Hybridization Master Mix As an option you can prepare a larger volume of Hybridization Master Mix than required The extra mix can be aliquoted and stored at 20 C for up to one week Preparing Fresh Hybridization Master Mix To prepare the Hybridization Master Mix 1 To the 50 mL centrifuge tube add the appropriate volume of each reagent in the order shown in Table 4 37 DMSO addition pipet directly into the solution of other reagents Avoid pipetting along the side of the tube 2 Mix well 3 If making a larger volume aliquot out the volume required and store the remainder at 20 C for up to one week Table 4 37 Hybridization Master Mix
52. ce in a cooling chamber on ice or keep at 4 C 4 Labela fresh 96 well plate as shown in Figure 4 14 on page 49 This plate is referred to as the gel plate Aliquot 3 uL of 2X Gel Loading Dye to each well to be used Load 10 uL BioNexus Hi Lo Ladder to the first and last lanes of the gel 7 Transfer 3 uL of Nsp PCR product from each well in one column only to the corresponding wells of the gel plate 9 g 8 Transfer 3 uL of Sty PCR product from each well in one column only to the corresponding wells of the gel plate chapter 4 Affymetrix Cytogenetics Copy Number Assay 49 PCR Plate 9 Seal both plates 10 Vortex the gel plate then spin them down at 2000 rpm for 30 sec 11 Load the total volume from each well of the gel plate onto a 2 TBE gel 12 Run the gel at 120V for 40 min to 1 hr 13 Verify that the PCR product distribution is between 250 bp to 1100 bp Figure 4 15 on page 50 50 Affymetrix Cytogenetics Copy Number Assay User Guide 2000 1000 750 500 300 d om an 100 Figure 4 15 Example of PCR products run on 2 TBE agarose gel at 120V for 1 hr Average product distribution is between 200to 1100 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 51 If not proceeding directly to the next stage seal the plate with PCR product and store at 20 C chapter
53. d aed 8 PPOSEPCR BOOFnD sida sl nln se Geen e ete E uan dons ce PB re UU RR de ERN RR eget ace 9 Configuration 2 One Room kk kk kk kK KK KK KK KK KK KI KK KK KK KK KK KK KK KK KK KK 10 Pre PCR Clean Area 4 kk kk kk kK KK KK KK KI KK KI KIR KI KI KI K KI KI KI KI K KK KI KIR K KI KIRI KK KK KK Kl 11 POStEPCR ALG 2d sa4 4 usce Pad dd wd a a n wana r dd dta i ge or a di di oA poe ha 11 Single Direction Workflow kk kk kk kk kk KK KK KK KK KK KK KK KK KK KK KK KK KK 13 Contamination Prevention hha sa bib dad bb kb hh 14 Safety Precautions c lt WA E20 trepat 2 22d sa a Benes L j W Ee 4 din 14 Genomic DNA General Requirements l l KK RR RR eee 15 General Requirements kk kk kk kk kk kk kk kK kK kK KK a 15 Sources of Human Genomic DNA Jk kk kk kk kk kk KK KK KK eee 16 Genomic DNA Extraction Purification Methods kk kk kk KK KK KK KK RR KK KK IK 16 DNA Cleanup ee e _e_ _ e e e e e e r _ _e ree _rhr r _ _ Keph_ ha r eee Des bao oe ees ete 16 PAST OV CTI COS ia se Bees a eee sted pide ge det ETT 16 Affymetrix Cytogenetics Copy Number Assay 0000000e eee 19 About the Protocol 1 uu kusa cue allkl ka a ie WW Rd ae i CR OU ed o we ee 19 About the Illustrations in this Chapter kk kk kk kk kK KK KK KK es 20 About the Reagents Equipment and Consumables Specified in this Chapter 20 Mein ello ao ce Ate actin an a tthe as acs tits
54. d except for 112 Affymetrix Cytogenetics Copy Number Assay User Guide Digestion Ligation and PCR N Es 960909000 a zc A NSW U Second Transfer Digest Ligate Plate Example When transferring samples from Digest Ligate Cap all wells in columns 5 through 12 on PCR Plate 1 plate column 1 to PCR plate 1 Cap all wells in columns 2 through 12 on the Digest The columns to which you are transferring to on the Ligate plate One column on the Digest Ligate plate PCR plate po II II NS 9999999 axx e e ee 7 gt WC j j 3 ED IDI J _ KW NN NV NU PCR Plate 1 666 amp amp O0 amp 6 amp 9 amp C C Third Transfer Digest Ligate Plate First Transfer Digest Ligate Plate Figure B 1 24 reaction workflow Digest Ligate plate to PCR plates appendix B Guidelines for Processing 24 Samples 113 PCR to Purification PCR Plate 1 Gel check for PCR product PCR Plate 2 Ne lO0600o0j GF 16 do w 6 W O OOOO PCR Plate 3 Do not pool negative control Im 4 eRe 1 SN T v lt r f geo oO 22 oD 9 QS oo C Oc So Do ES m EE to Fi
55. d out in the Post 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 Cytogenetics Copy Number 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 AccuGENE water from Cambrex and ligase and restriction enzymes from New England Biolabs Additional recommendations are as follows 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 DNA Ligase and the Fragmentation Reagent DNase I enzyme Both of these enzymes are sensitive to temperatures exceeding 20 C To prevent loss of enzyme activity Immediately place enzymes in a cooler chilled to 20 C when removed from the freezer Immediately return the enzyme to 20 C after use Take care when pipetting enzymes stored in glycerol which is viscous Do not store at 80 C Because Fragmentation Reagent act
56. dd Nsp Digest Master Mix to samples 1 Aliquot 14 75 uL of Nsp Digest Master Mix to each sample and controls in column 1 2 Return remaining NE Buffer 2 and Nspl enzyme to the freezer 3 Discard remaining Nsp Digest Master Mix 32 Affymetrix Cytogenetics Copy Number Assay User Guide Helclclelctetetetetetels Master Mixto each sare TH ODOOOOOOOOOQOO and controls in column 1 OlO O O O O O OO O OlO OJOOOOOOOOOQOO OIOOOOOOOQOO OlO sOOOOooooooQO0OQlo JOJdOOoOoOooooooOJ o Figure 4 7 Adding Nsp Digest Master Mix to gDNA samples and controls Prepare the Sty Digest Master Mix Keeping all reagents and tubes on ice prepare the Sty Digest Master Mix as follows 1 Tothe 1 5 mL Eppendorf tube labeled STY add the appropriate volumes of the following reagents as shown in Table 4 7 Water AccuGENE NE Buffer 3 BSA Place the master mix in the cooling chamber Remove the StyI enzyme from the freezer and immediately place in a cooler Pulse spin the enzyme for 3 sec Immediately add the enzyme to the master mix Return remaining enzyme to the cooler Vortex the master mix at high speed 3 times 1 sec each time Pulse spin for 3 sec e Anon hh N Place in the cooling chamber 10 Proceed immediately to Add Sty Digest Master Mix to Samples on page 33 Table 4 7 Styl Digest Master Mix Reagent 1 Sample 4 Samples 8 Samples 12 Samples 24 Samples 25 extra 15 extra 15
57. e Scanning the Array NOTE Customers using the Autoloader should refer to the Autoloader User s Guide To scan arrays 1 Select the experiment name GCOS or sample name AGCC that corresponds to the array being scanned Following the GCOS or AGCC instructions as appropriate load the array into the scanner and begin the scan Only one scan per array is required Pixel resolution and wavelength are preset and cannot be changed WARNING The scanner door will open and close automatically Do not attempt to manually open or close the scanner door as this may damage the instrument Do not force the array into the holder 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 GCOS or 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 Guid
58. e Table 4 9 Equipment and Consumables Required for Stage 2 Nsp and Sty Ligation Quantity Item 1 Adhesive seals for 96 well plates 1 Centrifuge plate 1 Cooler chilled to 20 C 1 Cooling chamber double chilled to 4 C on ice do not freeze 1 Ice bucket filled with ice 1 Marker blue and red fine point permanent 1 Mini centrifuge microfuge 1 Pipet single channel P10 1 Pipet single channel P20 1 Pipet single channel P100 or P200 As needed Pipet tips for pipets listed above 1 Thermal cycler 3 Tubes Eppendorf 1 5 mL 1 Vortexer 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 4 Reagents Required The following reagents are required for this stage Table 4 10 Reagents Required for Stage 2 Nsp and Sty Ligation T4 DNA Ligase 400 U uL NEB T4 DNA Ligase Buffer 10X Adaptor Nsp 50 uM Adaptor Sty 50 uM Water AccuGENE molecular biology grade 36 Affymetrix Cytogenetics Copy Number Assay User Guide Prepare the Reagents Consumables and Other Components Thaw the Reagents and Digested Samples To thaw the reagents and digested samples 1 Allow the following reagents to thaw on ice Adaptor Nsp Adaptor Sty TA DNA Ligase Buffer 10X requires approximately 20 min to thaw 2 If the digested samples were frozen allow them to
59. e Be sure to allow all reagents to reach equilibrium before adding new fluid About the Fragmentation Reagent Enzyme This enzyme is extremely temperature sensitive and rapidly loses activity at higher temperatures To avoid loss of activity 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 Dilute immediately prior to use Keep at 20 C until ready to use Transport and hold in a 20 C cooler Return to the cooler immediately after use Spin down so that the contents of the tube are uniform Perform all steps rapidly and without interruption This enzyme is sticky and may adhere to the walls of some microfuge tubes and 96 well plates This enzyme is viscous and requires extra care when pipetting Follow these guidelines Pipet slowly to allow enough time for the correct volume of solution to enter the pipet tip Avoid excess solution on the outside of the pipet tip 66 Affymetrix Cytogenetics Copy Number Assay User Guide Prepare the Reagents Consumables and Other Components Thaw Reagents Thaw the Fragmentation Buffer 10X on ice Hi IMPORTANT Leave the Fragmentation Reagent at 20 C until ready to use Setup Your Work Area To setup your work area Figure 4 24 1 Place a cooling chamber and the water on ice 2 Place the plate of purified quantitated samples in the cooling chamber 3 Prepare the Fragme
60. e causes include Magnetic beads may have been carried over into purified sample Action Spin down the sample for 5 min Place on the MagnaRack and pipet out the eluate Precipitate may be present in the eluted samples There may be defects in the OD plate e Air bubbles in the OD plate or in solutions Affymetrix Instruments Under any of the following conditions unplug the instrument from the power source and contact Affymetrix Technical Support When the power cord is damaged or frayed If any liquid has penetrated the instrument If after service or calibration the instrument does not perform to specifications x 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 E mail supporteurope affymetrix com Voyager Mercury Park Wycombe Lane Wooburn Green UK and Others Tel 44 0 1628 552550 France Tel 0800919505 High Wycombe pene Germany Tel 01803001334 United Kingdom Fax 44 0 1628 552585 Affymetrix Japan K K NENA Tel 03 5730 8200 16 Floor 4 1 23 Shiba Fax 03 5730 8201 Minato ku Tokyo 108 0014 ax Japan m u p E HH u SEKIR j Chapter FLUIDICS STATION CARE AND MAINTENANCE General Fluidics Station
61. e equivalent UV absorbance reading Based on OD measurements dilute each sample in a separate well of the 96 well plate to 50 ng uL using reduced EDTA TE buffer IMPORTANT Do NOT dilute Ref 103 it is already at a working concentration An elevated EDTA level may interfere with subsequent reactions 6 Seal the plate vortex at high speed for 3 sec then spin down for 30 sec 7 Place back on the cooling chamber Aliquoting the Prepared Genomic DNA and Controls Setup the Work Area To setup the work area 1 Mark a 96 well plate as shown in Figure 4 4 use a blue marker for Nsp N and a red marker for Sty 5 The Nsp and Sty digestion and ligation reactions will be performed in this plate Place the plate on the bottom half of the cooling chamber Figure 4 5 on page 27 Place at least 5 uL of water on ice negative control Figure 4 4 Marking a 96 well plate for Nsp and Sty digestion and ligation chapter 4 Affymetrix Cytogenetics Copy Number Assay 27 Aliquot the gDNA and Controls NOTE 5 uL of the 50 ng uL working stock is equivalent to 250 ng genomic DNA per well To aliquot the prepared genomic DNA and controls 1 Vortex the Ref 103 for 3 sec then spin down for 30 sec 2 Transfer two 5 uL aliquots of the first sample to wells Al and A12 of the digest ligate plate Figure 4 5 on page 27 3 Transfer two 5 uL aliquots of each remaining gDNA sample in the same manner
62. e for more information When the protocol is complete turn the instrument off Place the wash lines in a different bottle of deionized water than the one used for the shutdown protocol 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 chapter 5 Washing Staining and Scanning Arrays 93 Data Analysis To analyze the data collected by the scanner use Affymetrix Genotyping Console version 2 1 or later Genotyping Console includes copy number and LOH algorithms for the SNP Array 6 0 It also includes the Genotyping Console Browser and Segment Reporting Tool Affymetrix Genotyping Console Version 2 1 AFFYMETRIX a 2008 Affymetrix Inc All Rights Reserved LJ Figure 5 2 Use Genotyping Console version 2 1 or later to analyze your data 94 Affymetrix Cytogenetics Copy Number Assay User Guide Chapter TROUBLESHOOTING General Assay Performance Recommendations As with any assay using PCR the Cytogenetics Copy Number Assay has an inherent risk of contamination with PCR product from previous reactions In Chapter 2 Laboratory Setup and Recommendations 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 only be carrie
63. e plate and spin down at 2000 rpm for 30 sec Table 4 8 Cyto Digest Program Cyto Digest Program Temperature Time 37 C 120 min 65 C 20 min 4 C Hold What To Do Next Do one of the following If following the recommended workflow Figure 4 1 on page 21 place the plate in a cooling chamber on ice and proceed immediately to Stage 2 Nsp and Sty Ligation on page 34 If not proceeding directly to the next step store the plate at 20 C 34 Affymetrix Cytogenetics Copy Number Assay User Guide Stage 2 Nsp and Sty Ligation About this Stage During this stage the Nsp digested samples are ligated using the Nsp Adaptor the Sty digested samples are ligated using the Sty Adaptor You will 1 2 3 4 Prepare a Nsp Ligation Master Mix and add it to the Nsp digested samples Prepare a Sty Ligation Master Mix and add it to the Sty digested samples Place samples onto a thermal cycler and run the Cyto Ligate program Dilute the ligated samples with water Location and Duration Pre PCR Clean Area Hands on time 30 min Cyto Ligate thermal cycler program time 3 3 hr Input Required From Previous Stage The input required from Stage 1 Nsp and Sty Restriction Enzyme Digest is Plate of Nsp and Sty digested samples chapter 4 Affymetrix Cytogenetics Copy Number Assay 35 Equipment and Consumables Required The following equipment and consumables are required for this stag
64. e the tubes for 3 min at maximum speed hinges facing out 16 100 rcf 5 Place the tubes on the magnetic stand Figure 4 20 Resuspended bead clump and vortexer with foam tube adaptor 6 Leaving the tubes in the rack pipet off the supernatant without disturbing the bead pellet and discard Spin the tubes for 30 sec at maximum speed hinges facing out 16 100 rcf Place the tubes back on the magnetic stand o 0 M Using a P20 pipet remove remaining drops of EtOH from the bottom of each tube Lu IMPORTANT Be careful not to disturb or break off any of the bead pellet 10 Allow the remaining EtOH to evaporate by leaving the tubes uncapped at room temperature for 15 min Add Buffer EB To add Buffer EB to each sample 1 Using a P200 pipet add 55 uL of Buffer EB to each tube 2 Cap the tubes and load them into the foam tube adaptor 3 Vortex at 75 power for 10 min Vortexing will resuspend the magnetic beads 4 Examine each tube to ensure that the beads are resuspended in a homogeneous slurry IMPORTANT If the beads are not fully resuspended flick the tube to dislodge the pellet and vortex an additional 2 min Re examine 5 Centrifuge the tubes for 5 min at maximum speed hinges facing out 16 100 rcf 6 Place the tubes on the magnetic stand for 5 min The magnetic beads are pulled to the side of the tube Figure 4 19 chapter 4 Affymetrix Cytogenetics Copy Number Assay 57 7
65. e water on ice 20 min prior to use When the Cyto Ligate program is finished remove the plate and spin down at 2000 rpm for 30 sec Place in a cooling chamber on ice Using a P200 pipet add 75 uL of water to each reaction Nsp and Sty Ligated DNA 25 uL Water AccuGENE 75 uL Total 100 uL Tightly seal the plate Vortex at high speed for 3 sec then spin at 2000 rpm for 30 sec What To Do Next Do one of the following If following the recommended workflow Figure 4 1 on page 21 proceed immediately to Stage 3 Nsp and Sty PCR on page 41 Samples can be stored in a cooling chamber on ice for up to 60 min If not proceeding directly to the next step store the plate at 20 C chapter 4 Affymetrix Cytogenetics Copy Number Assay 41 Stage 3 Nsp and Sty PCR About this Stage During this stage you will 1 Transfer equal aliquots of each Nsp diluted ligated sample into four wells of a 96 well plate Sty diluted ligated sample into three wells of the same 96 well plate 2 Prepare a PCR Master Mix and add it to each ligated sample 3 Place the samples onto a thermal cycler and run the Cyto PCR program 4 Confirm each PCR reaction by running 3 uL of each PCR product on a gel Location and Duration Pre PCR Clean Area PCR Master Mix preparation PCR set up Post PCR Area samples placed on thermal cycler Hands on time 1 hr Cyto PCR thermal cycler program time 1 5 hr Sa
66. ed samples and store at 20 C chapter 4 Affymetrix Cytogenetics Copy Number Assay 63 Stage 6 Fragmentation About this Stage During this stage the purified samples are fragmented using Fragmentation Reagent enzyme by Preparing a Fragmentation Master Mix Quickly adding the mix to each sample Placing the samples onto a thermal cycler and running the Cyto Fragment program Checking each reaction on a gel Location and Duration Post PCR Area Hands on time 30 min Cyto Fragment thermal cycler program time 1 hr Input Required from Previous Stage The input required from Stage 5 Quantitation is Item Plate of purified samples that have been quantitated 64 Affymetrix Cytogenetics Copy Number Assay User Guide Equipment and Consumables Required The following equipment and consumables are required for this stage Table 4 24 Equipment and Consumables Required for Stage 6 Fragmentation Quantity Item As required Adhesive seals for 96 well plates 1 Centrifuge plate 1 Cooler chilled to 20 C 1 Cooling chamber single chilled to 4 C on ice do not freeze 1 Ice bucket filled with ice 1 Marker fine point permanent 1 Mini centrifuge microfuge 1 Pipet single channel P20 1 Pipet single channel P100 1 Pipet single channel P1000 1 Pipet 8 channel P20 accurate to within 596 As needed Pipet tips for pipets listed above 1 Plate B
67. el 20 200 uL Rainin L8 200 3 Pipet 8 channel 100 1000 uL Rainin L8 1000 a Plate centrifuge multipurpose Eppendorf 5804 or 5810 Oo Refrigerator 4 C 6 cu ft Any vendor _ Spectrophotometer select one of the following SpectraMax spectrophotometer Molecular Devices Spectramax Plus384 NanoDrop NanoDrop ND 1000 Select one of these thermal cyclers if routinely processing gt 8 samples you may to use additional thermal cyclers for PCR a GeneAmp PCR System 9700 gold silver block Applied Biosystems 4314878 MJ Tetrad PTC 255 Bio Rad _ DNA Engine Tetrad 2 Bio Rad PTC 0240G n Vortexer 2 required VWR 58816 1212 One vortexer must have a plate pad The Microtube Foam Insert listed above will be attached to the other vortexer appendix C Reagents Equipment and Consumables 119 Reagents Required from Other Suppliers Table C 8 Reagents Required from Other Suppliers Y item Vendor Part Number Oo Nsp Enzyme New England Biolabs RO602L NE Buffer 2 10X BSA 100X 3 Sty Enzyme New England Biolabs R0500S NE Buffer 3 10X BSA 100X a T4 DNA Ligase New England Biolabs MO202L 3 Magnetic Beads Agencourt 000130 60 mL 000132 450 mL 3 TITANIUM DNA Amplification Kit 300 rxn Clontech 639240 3 Reduced EDTA TE Buffer pH 8 0 TekNova T0223 qo AccuGENE Water Lonza Group LTD 51200 3 SSPE Lonza Group LTD 51214 3 EB Buffer Qiagen 19086 a Human
68. els and Related Materials Required AWA kk kk kk KK KK eee een ee 65 Important Information About This Stage 0 KK RR KK RR RR RR RR RIK KI 65 Prepare the Reagents Consumables and Other Components 66 Prepare the Samples for Fragmentation kk kk kK KK KII K K K KIRI ee 67 What ko DO N E X A ny at area eh GaN neri aM e pU IER e N as a tie Anos 69 Check the Fragmentation Reaction by Running a Gel Wu RR RR eee eee 70 otad SWAIN Gira wen A na Ka KA thee eee Ra e Drain awan 71 A ere B BIS Stages ptm d o YR dea eden eate een dd HHHH HHH 71 location and DUFatiOn x paiete ceste ERI e d dh kal al E de defe eR e oi RG ded 71 Input Required from Previous Stage kk kk kK kK KK KK KK eee 71 Equipment and Consumables Required Ak A kk kK KK KK KIRI eee 71 Reagents REGUIFEM i mied ect wA ler dedo detis estes ha ava YEE RA te anak dod don 72 Prepare the Reagents Consumables and Other Components 72 Prepare the Labeling Master MIX mae KI KII K K K K KIRI KI KIRI UI ated Mea 4 73 iv Affymetrix Cytogenetics Copy Number Assay User Guide Add the Labeling Master Mix to the Samples 0 000000 RR RR RR eee 73 What TODO NEI xs rubor x esee A Dr r r ate en ta Ae A Ane e Ted ETT Nee S 74 Stage 8 Target HyVbridizatioD 22 ou s k x oes t senectt eS eee e ES 75 AbOUtthiS Stage a aa a o s catu oov pb enc Ed aer he b b Ib d VXAP dA eR ae Ed ode 75 lF ca
69. enetics Copy Number Assay User Guide Check the Fragmentation Reaction by Running a Gel The instructions below are for running 4 TBE gels To ensure that fragmentation was successful 1 NO PWN When the Cyto Fragment program is finished A Remove the samples from the thermal cycler B Spin down for 30 sec and place in a cooling chamber on ice Remove 2 0 uL of each sample and place in a 96 well plate Add 4 uL gel loading dye to each sample Load the samples onto the gel Load 10 uL BioNexus All Purpose Hi Lo Ladder to the first and last lanes Run the samples on a 4 TBE gel at 120V for 30 min to 1 hr Inspect the gel and compare it against the example shown in Figure 4 26 below Figure 4 26 Typical example of fragmented PCR products run on 4 TBE agarose gel at 120V for 30 min to 1 hr Average fragment size is 180 bp chapter 4 Affymetrix Cytogenetics Copy Number Assay 71 Stage 7 Labeling About this Stage During this stage you will label the fragmented samples using the DNA Labeling Reagent by Preparing a Labeling Master Mix Adding the mix to each sample Placing the samples onto a thermal cycler and running the Cyto Label program Location and Duration Post PCR Area Hands on time 30 min Cyto Label thermal cycler program time 4 25 hr Input Required from Previous Stage The input required from Stage 6 Fragmentation is Fragmented samples Equipment and Consumables
70. enetics Copy Number 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 NOTE The illustrations in this user guide depict the recommended setup for 8 samples 6 genomic DNA samples plus 1 positive and 1 negative control Thermal Cyclers 96 well Plate and Adhesive Seals The Cytogenetics Copy Number Assay has been optimized using the following thermal cyclers 96 well plate and adhesive films IMPORTANT Use only the 96 well plate and adhesive seals listed in Table 1 1 and only the thermal cyclers listed in Table 1 2 Using other plates and seals that are incompatible with these thermal cyclers can result in loss of sample or poor results Table 1 1 96 well plate and adhesive seals optimized for use with this protocol Item Vendor Part Number Multiplate 96 well unskirted PCR plate Bio Rad MLP 9601 Adhesive seals Microseal B Adhesive Seal Bio Rad MSB1001 MicroAmp Clear Adhesive Film Applied Biosystems 4306311 chapter 1 Before You Start 5 Table 1 2 Thermal cyclers optimized for use with this protocol Laboratory Thermal Cyclers Validated for Use Applied Biosystems units Pre PCR Clean Area 2720 Thermal Cycler GeneAmp PCR System 9700 Use one of these units Bio Rad units MJ Tet
71. er Wash B Stringent Wash Buffer Anti streptavidin Antibody 0 5 mg mL MES Stock Buffer Array Holding Buffer Wash A Non Stringent Wash Buffer 6X SSPE 0 0196 Tween 20 For 1000 mL 300 mL of 20X SSPE 1 0 mL of 10 Tween 20 699 mL of water Filter through a 0 2 um filter Store at room temperature chapter 5 Washing Staining and Scanning Arrays 85 Wash B Stringent Wash Buffer 0 6X SSPE 0 01 Tween 20 For 1000 mL 30 mL of 20X SSPE 1 0 mL of 10 Tween 20 969 mL of water Filter through a 0 2 um filter Store at room temperature The pH should be 8 IMPORTANT Prepare Wash B in smaller quantities to avoid long term storage Tightly seal the container to avoid changes in salt concentration due to evaporation 0 5 mg mL Anti Streptavidin Antibody Resuspend 0 5 mg in 1 mL of water Store at 4 C 12X MES Stock Buffer 1 25 M MES 0 89 M Na For 1000 mL 70 4g of MES hydrate 193 3g of MES Sodium Salt 800 mL of Molecular Biology Grade Water Mix and adjust volume to 1000 mL The pH should be between 6 5 and 6 7 Filter through a 0 2 um filter IMPORTANT Do not autoclave Store at 2 C to 8 C and shield from light Discard solution if yellow 1X Array Holding Buffer Final 1X concentration is 100 mM MES 1M Nar 0 0196 Tween 20 For 100 mL 8 3 mL of 12X MES Stock Buffer 18 5 mL of 5 M NaCI 0 1 mL of 10 Tween 20 73 1 mL of water Store a
72. essential that you be proficient with the use of single and multi channel pipets To familiarize yourself with the use of multi channel pipets 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 Reagent Handling and Storage IMPORTANT Always use the 30 reaction Genome Wide Human SNP Nsp Sty Assay Kit 5 0 6 0 P N 901013 for this protocol This kit has been tested for multiple freeze thaw cycles You can freeze thaw the reagents in the 30 reaction kit lt 8 times Successful sample processing can be achieved by incorporating the following principles Use only fresh reagents from the recommended vendors to help eliminate changes in pH or the salt concentration of buffers Properly store all enzyme reagents Storage methods can profoundly impact activity Store the reagents used for the digestion ligation and PCR in the Pre PCR Clean Area Consult the appropriate MSDS for reagent storage and handling requirements When Using Reagents at the Lab Bench Properly chill essential equipment such as cooling chambers and reagent coolers before use Unless otherwise indicated keep all reagents except enzymes on ice or in a cooling chamber block that has been chilled to 4 C on ice or in a refrigerator Ensure that enzymes are kept at 20 C until needed When removed from the freezer im
73. extra 15 extra AccuGENE Water 11 55 uL 57 8 uL 106 3 uL 159 4 uL 318 8 uL NE Buffer 3 10X 2 uL 10 uL 18 4 uL 27 6 uL 55 2 uL BSA 100X 10 mg mL 0 2 uL 1 pL 1 8 uL 2 8 uL 5 5 uL Styl 10 U uL 1 uL 5 uL 9 2 uL 13 8 uL 27 6 uL Total 14 75 uL 73 8 uL 135 7 uL 203 6 uL 407 1 uL 2596 extra is required for 4 samples only If processing 8 samples 15 extra is sufficient chapter 4 Affymetrix Cytogenetics Copy Number Assay 33 Add Sty Digest Master Mix to Samples To add the Sty Digest Master Mix to samples 1 Aliquot 14 75 uL of Sty Digest Master Mix to each sample and control in column 12 The total volume in each well is now 19 75 uL 2 Tightly seal the plate lOOOOOCOOOOOOlIO OIOOOOOOOOO OlO Add 14 75 uL Sty Digest Master OlO O OO O O O O O OlO sample and control in OJIOOOOOOOOO COO OJOOOO0O0600O00O 0O lO OIOOOOOOOOOO O OIOOOOOOOOOO O sIOooooooooQOIO Figure 4 8 Adding Sty Digest Master Mix Load Nsp and Sty Samples onto the Thermal Cycler 1 Vortex the plate at high speed for 3 sec then spin down at 2000 rpm for 30 sec 2 Ensure that the lid of thermal cycler is preheated 3 Load the plate onto the thermal cycler and run the Cyto Digest program Table 4 8 IMPORTANT Ensure that the seal is not pulled off the wells when you close the thermal cycler lid 4 Return any remaining reagents to the freezer 5 When the program is finished remove th
74. gure B 2 24 reaction workflow PCR to purification 114 Affymetrix Cytogenetics Copy Number Assay User Guide Purification continued to Fragmentation and Labeling 3329 59 After incubation centrifuge and place tubes on magnetic rack Transfer eluted sample to the appropriate well of a fresh 96 well plate Fragment Label Plate rGG amp jaIOOOOOOOO UV Spec Plate for Quantitation Fragmentation gel R ho0O00000000 6G9OOOOOOOOC e jQ09999999 99999999c e j000000000 gt 888990000906 8960 OOOOOOOOO eGeeOOoO0000006 amp eaeeooooooooo 9999999996 Qeeooooooooo amp eoloooooooOo Quantitate label and hyb samples onto arrays Figure B 3 24 reaction workflow PCR to purification Tu EROS Hu LE T EBI Appendix 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 Cytogenetics Copy Number Assay m 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 From Affymetrix Affymetrix Equipment Required Table C 1 Affymetrix Equipment Required Y tem Pa
75. his 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 108 Affymetrix Cytogenetics Copy Number Assay User Guide Digestion Ligation and PCR First Transfer Digest Ligate Plate s To avoid transfer mistakes keep all wells capped except for One column on the Digest Ligate plate The columns to which you are transferring to on the PCR plate Example When transferring samples from Digest Ligate plate column 1 to PCR plate 1 Cap all wells in columns 2 through 12 on the Digest Ligate plate Cap all wells in columns 5 through 12 on PCR Plate 1 co N s 5 N co N 91 N a PCR Plate 1 Second Transfer A NO OOO C CD QOIS Digest Ligate Plate DODO T e OOP 9 RO 0 JR 10000 0001 2000 2 E S L NV iO NS i D m NJOVU I S b m M c S m i Yy E j poc NV SS NS NS 0 6 9 G S 9 eax o Figure A 1 16 reaction workflow Digest Ligate plate to 96 well plates appendix A Guidelines for Processing 16 Samples 109 PCR to Purification 2 mL Tubes 1 per Reaction PCR Plate 1 il
76. htly seal the plate Vortex the plate for 30 sec then spin down for 30 sec Place the plate onto the thermal cycler and run the Cyto Hyb program IMPORTANT Ensure that the seal is not pulled off any wells when you close the thermal cycler lid Table 4 38 Cyto Hyb Thermal Cycler Program Cyto Hyb Program Temperature 95 C 10 min 49 C Hold Load the Samples onto Arrays To load the samples onto arrays 1 2 When the thermal cycler reaches 49 C open the lid If gt 7 samples cut and remove the film from one column of samples Leave the remaining wells covered Keeping these wells covered will help to prevent cross contamination and evaporation Using a P200 pipet remove 200 uL of the first sample and immediately inject it into an array Cover the septa on the array with a Tough Spot Figure 4 29 Press firmly to ensure a tight seal to prevent evaporation and leakage Septa covered with Tough Spots Figure 4 29 Loading samples onto arrays chapter 4 Affymetrix Cytogenetics Copy Number Assay 81 5 When 4 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 min Ensure that the oven is balanced as the trays are loaded and ensure that the trays are rotating at 60 rpm at all times 6
77. igation Master Mix Keeping all reagents and tubes on ice prepare the Nsp Ligation Master Mix as follows 1 To the 1 5 mL Eppendorf tube labeled NSP add the following reagents based on the volumes shown in Table 4 11 T4 DNA Ligase Buffer 10X Adaptor Nsp Remove the T4 DNA Ligase from the freezer and immediately place in the cooler Pulse spin the T4 DNA Ligase for 3 sec Immediately add the T4 DNA Ligase to the master mix then place back in the cooler Vortex the master mix at high speed 3 times 1 sec each time Pulse spin for 3 sec Place the master mix on ice e Mog amp N Proceed immediately to 4d4 Nsp Ligation Master Mix to Reactions Table 4 11 Nspl Ligation Master Mix Reagent 1 Sample 4 Samples 8 Samples 12 Samples 24 Samples 15 extra 15 extra 15 extra 15 extra T4 DNA Ligase Buffer 2 5 uL 11 5 uL 23 0 uL 34 5 uL 69 uL 10X Adaptor Nsp 0 75 uL 3 45 uL 6 90 uL 10 35 uL 20 7 uL 50 uM T4 DNA Ligase 2 uL 9 2 uL 18 4 uL 27 6 uL 55 2 uL 400 U uL Total 5 25 uL 24 15 uL 48 30 uL 72 45 pL 144 90 uL 38 Affymetrix Cytogenetics Copy Number Assay User Guide Add Nsp Ligation Master Mix to Reactions To add Nsp Ligation Master Mix to samples 1 Using a P20 pipet aliquot 5 25 uL of Nsp Ligation Master Mix to each Nsp digested sample and control Figure 4 10 Discard any remaining Nsp Ligation Master Mix Nsp Digested DNA 19 75 uL Nsp Ligation Master Mi
78. inar 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 Store master stocks of PCR primer and adaptor 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 pipets and tips the thermal cycler and vortexer Post PCR Area 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 2 2 on page 10 consists of Computer monitor and keyboard Fluidics station Scanner Ice bucket Magnetic stand 9 g amp N Vortexer with plate stand 12 Affymetrix Cytogenetics Copy Number Assay User Guide 10 11 12 13 14 15 16 17 18 Microfuge Pipets on stand Vortexer with tube mixer attachment Thermal cycler one to three Hybridi
79. io Rad 96 well 1 Thermal cycler 1 Tube Eppendorf 1 5 mL 1 Tubes strip of 8 with caps 0 2 mL 1 Vortexer 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 4 Reagents Required The following reagents are required for this stage Table 4 25 Reagents Required for Stage 6 Fragmentation Fragmentation Buffer 10X Fragmentation Reagent enzyme DNase AccuGENE water molecular biology grade chapter 4 Affymetrix Cytogenetics Copy Number Assay 65 Gels and Related Materials Required Verifying the fragmentation reaction is required for this stage You can use the following gels and related materials Table 4 26 Gels and Related Materials Required Item Reagent 4 TBE Gel precast or house made DNA Markers 5 pL per well Gel loading solution Important Information About This Stage To help ensure the best results carefully read the information below before you begin this stage of the protocol IMPORTANT The degree of fragmentation is critical Perform this stage carefully to ensure uniform reproducible fragmentation Use only the AccuGENE water Using in house ddH O or other water can negatively affect your results The fragmentation reaction is particularly sensitive to pH and metal ion contamination All additions dilutions and mixing must be performed on ic
80. ivity can decline over time after dilution on ice add it to the samples as quickly as possible Preparing master mixes with a 15 excess 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 The PCR reaction for this assay has been validated using the specified thermal cyclers These thermal cyclers were chosen because of their ramping times 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 200to 1100 bp size range See Check the PCR Reaction by Running a Gel on page 48 for more information 96 Affymetrix Cytogenetics Copy Number Assay User Guide Following fragmentation run your samples on a gel Successful fragmentation is confirmed by the presence of a smear of lt 180 bp in size See Check the Fragmentation Reaction by Running a Gel on page 70 for more information Run controls in parallel with each group of samples Substitute water for DNA as a negative control The absence of bands on your PCR gel for this control confirms no previou
81. k the serial number of your hybridization oven s If the serial numbers are 11214 or lower contact Affymetrix for an upgrade 76 Affymetrix Cytogenetics Copy Number Assay User Guide The following table lists the equipment and consumables required Table 4 35 Equipment and Consumables Required for Stage 8 Target Hybridization Quantity Item 1 Adhesive seals for 96 well plates 1 Cooling chamber chilled to 4 C on ice do not freeze One array per sample Genome Wide Human SNP Array 6 0 1 GeneChip Hybridization Oven 640 or 645 1 Ice bucket filled with ice 1 Pipet single channel P200 1 Pipet single channel P1000 As needed Pipet tips for pipets listed above 1 Solution basin 55 mL 1 Thermal cycler 2 per array Tough Spots 1 Tube centrifuge 50 mL 1 Vortexer 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 4 Reagents Required The following reagents are required for this stage Table 4 36 Reagents Required for Stage 8 Target Hybridization Denhardt s Solution 50X DMSO 100 EDTA 0 5 M Herring Sperm DNA HSDNA 10 mg mL Human Cot 1 DNA 1 mg mL MES Hydrate SigmaUltra MES Sodium Salt Tetramethyl Ammonium Chloride TMACL 5M Tween 20 1096 Oligo Control Reagent OCR 0100 chapter 4 Affymetrix Cytogenetics
82. l plates 1 Marker fine point permanent 1 Mini centrifuge microfuge 1 Pipette single channel P20 1 Pipette single channel P200 As needed Pipette tips for pipets 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 Optional Tube 50 mL conical or solution basin 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 4 Reagents Required The following reagents are required for this stage Table 4 23 Reagents Required for Stage 5 Quantitation Reagent Water AccuGENE molecular biology grade chapter 4 Affymetrix Cytogenetics Copy Number Assay 59 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 quantitative linear range of the instrument The spectrophotometer should be calibrated regularly to ensure correct readings 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 10 min before
83. ler to preheat the lid Leave the block at room temperature Prepare the Nsp Digest Master Mix Keeping all reagents and tubes on ice prepare the Nsp Digest Master Mix as follows 1 To the 1 5 mL Eppendorf tube labeled NSP add the appropriate volumes of the following reagents see Table 4 6 Water AccuGENE NE Buffer 2 BSA Place the master mix in the cooling chamber Remove the NspI enzyme from the freezer and immediately place in a cooler Pulse spin the enzyme for 3 sec Immediately add the enzyme to the master mix Return the enzyme to the cooler Vortex the master mix at high speed 3 times 1 sec each time g9 N OAR N Pulse spin for 3 sec Place in the cooling chamber 10 Proceed immediately to 44d Nsp Digest Master Mix to Samples on page 31 Table 4 6 Nspl Digest Master Mix Reagent 1 Sample 4 Samples 8 Samples 12 Samples 24 Samples 25 extra 15 extra 15 extra 15 extra AccuGENE Water 11 55 uL 57 8 uL 106 3 uL 159 4 uL 318 8 uL NE Buffer 2 10X 2 uL 10 uL 18 4 uL 27 6 uL 55 2 uL BSA 100X 10 mg mL 0 2 uL 1 uL 1 8 uL 2 8 uL 5 5 uL Nspl 10 U uL 1 uL 5 uL 9 2 uL 13 8 uL 27 6 uL Total 14 75 uL 73 8 uL 135 7 uL 203 6 pL 407 1 pL To avoid pipetting lt 1 pL of BSA prepare 25 extra when processing lt 4 samples Add Nsp Digest Master Mix to Samples Genomic DNA 50 ng uL 5 00 uL Nsp Digest Master Mix 14 75 uL Total Volume 19 75 uL To a
84. mediately place in a cooler that has been chilled to 20 C Keep all tubes master mixes and working solutions in chilled cooling chambers on ice Since enzyme activity is a function of temperature ensure that all temperature transitions are rapid and or well controlled to help maintain consistency across samples Master Mix Preparation Carefully follow each master mix recipe Use pipets that have been calibrated to 5 When molecular biology grade water is specified be sure to use the AccuGENE water listed in Appendix C Using in house ddH5O or other water can negatively affect your results The enzymatic reaction in Stage 6 Fragmentation is particularly sensitive to pH and metal ion contamination If you run out of master mix during any of these procedures a volume error has been made or the pipets are not accurate We recommend that you stop and repeat the experiment 4 Affymetrix Cytogenetics Copy Number Assay User Guide 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 Preparing the Work Area for Each Stage Many of the stages in the Cytog
85. mples can be held overnight at 4 C Input Required from Previous Stage The input required from Stage 2 Nsp and Sty Ligation is Diluted ligated Nsp and Sty samples 42 Affymetrix Cytogenetics Copy Number Assay User Guide Equipment and Materials Required The following equipment and materials are required to perform this stage Table 4 14 Equipment and Consumables Required for Stage 3 Nsp and Sty PCR Quantity Item As required Adhesive seals for 96 well plates 1 Centrifuge plate 1 Cooler chilled to 20 C 1 Cooling chamber double chilled to 4 C on ice do not freeze 1 Ice bucket filled with ice 2 Markers blue and red fine point permanent 1 Mini centrifuge microfuge 1 Pipet single channel P20 1 Pipet single channel P100 1 Pipet single channel P200 1 Pipet single channel P1000 1 Optional Pipet 8 channel P20 1 Optional Pipet 8 channel P200 As required Pipet tips for pipets listed above 1to3 Plate Bio Rad 96 well PCR 1 to 8 samples 1 plate e 9 to 16 samples 2 plates 17 to 24 samples 3 plates As required Plate holder 96 well PCR 1 Solution basin 5b mL 1 Thermal cycler If routinely processing 8 samples you may want to use more than one thermal cycler 1 Tube centrifuge 50 mL 1 Vortexer IMPORTANT Use only the thermal cyclers 96 well plate and adhesive films and listed under Thermal Cyclers 96 well Plate and Adhesive Seals
86. n 645 00 0331 appendix C Reagents Equipment and Consumables 117 Equipment Required from Other Suppliers Pre PCR Clean Area Equipment Required When performing the pre PCR stages of the Cytogenetics Copy Number Assay great care should be taken to avoid sample contamination with PCR products If the assay is to be run in a single room we strongly recommend that the pre PCR stages be performed in a laminar flow or PCR cabinet Table C 6 Pre PCR Clean Area Equipment Required v Item Vendor Part Number o Recommended if protocol is to be performed in one room only Laminar Cabinet ESCO SVE 6A or Laminar Flow Cabinet 6 ft ESCO SVE 6A equivalent PCR Cabinet PCR Cabinet C B S Scientific P 048 02 or equivalent 1 Benchtop Cooler 20 C Stratagene 400012 m Biocooler aluminum block 96 well Bio Smith 81001 Required if processing 8 samples 1 for 9 to 16 samples 2 for 17 to 24 samples H Cooling Chamber double block Diversified Biotech CHAM 1020 3 Freezer 20 C deep freeze manual defrost 17 cu ft Any vendor 3 Ice Bucket 4 to 9 liters Magic Touch Icewares Fisher Scientific 3 Microfuge for tubes and strip tubes Any vendor a Pipet single channel 2 20 uL Rainin L 20 3 Pipet single channel 20 200 uL Rainin L 200 3 Pipet single channel 100 1000 uL Rainin L 1000 3 Pipet 8 channel 2 20 uL Rainin L8 20 a Pipet 8 channel 20 200 uL Rainin L8 200 a Plate centrif
87. n on a gel About Controls To assess the presence of contamination always include one PCR negative control with every set of samples run Use water as the negative control 44 Affymetrix Cytogenetics Copy Number Assay User Guide Prepare the Reagents Consumables and Other Components Thaw Reagents and Samples To thaw the reagents and samples 1 Allow the following reagents to thaw on ice TITANIUM Tag PCR Buffer dNTPs PCR Primer 002 Lt IMPORTANT Leave the TITANIUM Taq DNA Polymerase at 20 C until ready to use 2 Ifthe diluted ligated samples are frozen thaw them in a cooling chamber on ice Setup Your Work Area Pre PCR Clean Area To setup your work area Figure 4 13 on page 45 1 Place a double cooling chamber on ice 2 Place the plate of diluted ligated samples in the top half of the cooling chamber 3 Label a fresh 96 well plate as shown in Figure 4 12 blue for Nsp red for Sty 4 Place the plate in the lower half of the cooling chamber OOOOOOO OOOOOOO OOOOOOO OOOOOOQO0 Figure 4 12 Labeling the 96 well plate for PCR Prepare the Ligated Samples and Reagents To prepare the ligated samples and reagents 1 Labelthe 50 mL centrifuge tubes PCR 2 Place on ice Water AccuGENE GC Melt Solution basin 3 Prepare the diluted ligated samples as follows A Vortex at high speed for 3 sec then spin down at 2000 rpm for 30 sec B Place in the top half of
88. ntation Buffer as follows A Vortex 3 times 1 sec each time B Pulse spin for 3 sec C Place in the cooling chamber 4 Label the 1 5 mL Eppendorf tube Frag and place in the cooling chamber Purified samples Fragmentation 1 Buffer 2 3 Water 4 AccuGENE 5 O J AK Fragmentation master mix tube Figure 4 24 Setup for fragmentation Preheat the Thermal Cycler Block The block must be heated to 37 C before samples are loaded To preheat the thermal cycler 1 Power on the thermal cycler and preheat the block to 37 C 2 Allow it to heat for 10 min before loading samples chapter 4 Affymetrix Cytogenetics Copy Number Assay 67 Prepare the Samples for Fragmentation Add Fragmentation Buffer to Samples Lu IMPORTANT All additions in this procedure must be performed on ice To prepare the samples for Fragmentation 1 Add 5 uL of Fragmentation Buffer to each sample 2 Check your pipet tips each time to ensure that all of the buffer has been dispensed The total volume in each well is now 50 uL Prepare the Fragmentation Master Mix IMPORTANT The concentration of stock Fragmentation Reagent U L may vary from lot to lot Therefore read the label on the tube and record the stock concentration before diluting this reagent To prepare the Fragmentation Master Mix The Fragmentation Reagent must be diluted to 0 1 U uL in the master mix 1 Read the Fragmentation
89. o Samples kk KK ee eee 33 Load Nsp and Sty Samples onto the Thermal Cycler XW RR RR 00 000 33 What TODO NexXt x usage utu a lade rae eg ED MDMMMRMH N NN ERNnNnHnHnNH HJRIDJM 33 Stage 22 Nsp arid Sty Ligation e ce DR ob ge wore a Se e de Id go 34 AboutthiS Stage 4 ceste dan d dr antena EQ du C ca add hs dar det d stent EO sa 34 Location ma Duran aie ona vue ERIT eme W MEN e eee oh iTi 34 Input Required From Previous Stage kk kk KK KK KK KIRI KI KII KI KI KII 34 Equipment and Consumables Required Ak kk kk kK KII KK ee 35 Reagents Required 4 5 2 ed dut fhe daw eh E IRE Tp ea VIDEA s 35 Prepare the Reagents Consumables and Other Components 36 Prepare the Nsp Ligation Master MiX 4k kk kk KK KK KI KI KK K KIRI eee 37 Add Nsp Ligation Master Mix to Reactions 0 000 000 cc KIRI KII KI 38 Prepare the Sty Ligation Master Mix kk kk kk kK ee 38 Add Sty Ligation Master Mix to Reactions 0 0 0 0 KEK eee 39 Load the Nsp and Sty Samples Onto the Thermal Cycler 00 40 Dilute the Ligated Samples 0 0 0 0 0 eee 40 What TODON sec le etie t E ee eie Sed ase reete SE eda 40 Stage 933Nsp and Sty POR AW W y fes SEA A W n Lae eed dpud eben I 41 ADOULXHIS Stage oodd anter taa rtt od PU PB D e psa a ea de KR A 41 I ocationzaridiburatiom gt i ee 28 SA et tet tere Sit 0 A AA ln ets 41 Input Required from Previous Stage kk kk kK KK RR eee 41 Equipmen
90. on page 4 chapter 4 Affymetrix Cytogenetics Copy Number Assay 43 Reagents Required The following reagents are required for this stage Table 4 15 Reagents Required for Stage 3 Nsp and Sty PCR AccuGENE water molecular biology grade PCR Primer 002 100 uM From the Clontech TITANIUM DNA Amplification Kit e dNTPs 2 5 mM each GC Melt 5M e TITANIUM Taq DNA Polymerase 50X e TITANIUM Taq PCR Buffer 10X Gels and Related Materials Required Verifying the PCR reaction is required for this stage Table 4 16 Gels and Related Materials Required for Stage 3 Nsp and Sty PCR DNA Marker BioNexus All Purpose Hi Lo Ladder Gels 296 TBE precast or house made Gel loading solution Plates 96 well reaction Important Information About This Stage To help ensure the best results carefully read the information below before you begin this stage of the protocol IMPORTANT e Ensure that the Nsp and Sty ligated samples were diluted to 100 uL with AccuGENE water Prepare the PCR Master Mix immediately prior to use in Pre PCR Clean Area ideally in a laminar flow or PCR cabinet To help ensure the correct distribution of fragments be sure to add the correct amount of primer Mix well to ensure the even distribution of primers Ensure that the PCR product distribution is between 250 bp to 1100 bp by running 3 pL aliquots of each PCR reactio
91. ontamination All of the 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 of the equipment required for the steps performed in this area should be dedicated Do not move any equipment including ice buckets and pipets between the Pre and Post PCR Rooms Always wear a fresh gown and gloves to minimize sample contamination Configuration 2 One Room One room with two distinctly separ Post PCR Area chapter 2 Laboratory Setup and Recommendations 11 Pre PCR Clean Area For the best results adhere to the following guidelines Keep the Pre PCR Clean Area Free of DNA template and PCR amplicons Protected from contaminants by performing all steps inside a laminar flow cabinet or a PCR cabinet If using a laminar flow cabinet 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 2 2 on page 10 is listed below Laminar flow cabinet or PCR cabinet Vortexer Microfuge Pipets on stand Ice bucket Thermal cycler Plate centrifuge g N 9 g amp N Freezer About Lam
92. or 96 well plates 1 Cooling chamber double chilled to 4 C on ice do not freeze 1 Ice bucket filled with ice 2 Markers red and blue fine point permanent 1 Mini microcentrifuge microfuge 1 Pipet single channel P20 1 Pipet single channel P100 or P200 As needed Pipet tips 2 Plate Bio Rad 96 well unskirted 1 Plate centrifuge 1 Plate spectrophotometer or NanoDrop required only if no OD measurements available for samples 1 Vortexer 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 4 Reagents Required The following reagents are required for this stage Table 4 3 Reagents Required for Genomic DNA Preparation Reduced EDTA TE Buffer 0 1 mM EDTA 10 mM Tris HCL pH 8 0 Reference Genomic DNA 103 positive control AccuGENE water negative control chapter 4 Affymetrix Cytogenetics Copy Number Assay 25 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 Therefore you should correlate readings from other methods to the equivalent UV absorbance reading Setup the Work Area To setup the work area 1 Place a double cooling chamber on ice Figure 4 3 2 Place a 96 well plate in the top half of the cooling chamber Reduced EDTA TE Buffer o
93. ormed If the LCD window instructs the user to prime chapter 5 Washing Staining and Scanning Arrays 87 Wash and Stain Arrays The staining protocol for mapping arrays is a three stage process 1 A Streptavidin Phycoerythin SAPE stain 2 Anantibody amplification step 3 A final stain with SAPE Once stained each array is filled with Array Holding Buffer prior to scanning Prepare Arrays for Washing and Staining To prepare the arrays for washing and staining 1 After 16 to 18 hr of hybridization remove the arrays from the oven 2 Extract the hybridization cocktail from each array and transfer it to the corresponding well of a 96 well plate Store on ice during the procedure or at 80 C for long term storage 3 Fill each array completely with 270 uL of Array Holding Buffer See Array Holding Buffer on page 88 for buffer recipe 4 Allow the arrays to equilibrate to room temperature before washing and staining x NOTE Arrays can be stored in the Array Holding Buffer at 4 C for up to 3 hr before proceeding with washing and staining Equilibrate arrays to room temperature before washing and staining Prepare Buffers and Solutions Prepare the following buffers and solutions recipes follow Mix well Stain Buffer SAPE Stain Solution Antibody Stain Solution Array Holding Buffer Stain Buffer Mix well Table 5 3 Stain Buffer Reagent 1 Array 4 Arrays 8 Arrays 12 Arrays 24 Arrays
94. ove Insufficient starting material 250 ng genomic DNA should be used Confirm concentration using calibrated spectrophotometer Sample DNA contains enzymatic or chemical inhibitors Ensure genomic DNA is purified and diluted in low EDTA 0 1mM TE buffer Use the DNA cleanup procedure on page 16 to remove inhibitors Degraded sample DNA Confirm quality of genomic DNA sample Low PCR yield DNA lost during purification Gel images show PCR product but low OD Beads not fully resuspended during DNA elution step Ensure that beads are fully resuspended and mixed during the elution step before pelleting and transfer of the eluate 98 Affymetrix Cytogenetics Copy Number Assay User Guide Problem Likely Cause Insufficient purified PCR product for fragmentation Solution Volume of eluate for particular samples is lt 47 uL Do the following in this order 1 Measure the actual volume using a pipettor 2 Add Buffer EB to a final volume of 47 uL 3 Mix by pipetting up and down 4 Transfer 2 uL to the corresponding well s in the OD plate 5 Proceed to fragmentation with 45 uL in each well Fragmented PCR product is not the correct size PCR product is still visible in Failed or incomplete fragmentation due to 200 1100 bp size region reduced DNase activity Check that you have selected the correct activity of DNase to add to fragmentation reaction See Prep
95. overnight For the MJ Tetrad PTC 225 and Tetrad 2 Use Heated Lid and Calculated Temperature Volume 100 uL Cyto PCR Program for MJ Tetrad PTC 225 and Tetrad 2 Temperature Time Cycles 94 C 3 min 1X 94 C 30 sec 60 C 30 sec 30X 68 C 15 sec 68 C 7 min 1X 4 C HOLD Can be held overnight Cyto Fragment Cyto Fragment Program Temperature 37 C 35 min 95 C 15 min 4 C Hold appendix D Thermal Cycler Programs 125 Cyto Label Cyto Label Program Temperature 37 C 4hr 95 C 15 min 4 C Hold Samples can remain at 4 C overnight Cyto Hyb Cyto Hyb Program Temperature 95 C 49 C Hold 126 Affymetrix Cytogenetics Copy Number Assay User Guide
96. quivalent Pipets P 2 P 20 P 200 P 1000 Rainin Pipetman 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 USA Scientific 9185 0000 84 Affymetrix Cytogenetics Copy Number Assay User Guide 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 Information and part numbers listed are based on U S catalog information Table 5 2 Reagents Required for Washing and Staining Arrays Reagent Vendor Part Number AccuGENE Molecular Biology Grade Water 1 L Lonza 51200 Distilled water Invitrogen 15230147 20X SSPE 3 M NaCl 0 2 M NaH2POA 0 02 M EDTA Lonza 51214 Anti streptavidin antibody goat biotinylated reconstitute Vector Laboratories BA 0500 according to product instructions R Phycoerythrin Streptavidin Molecular Probes S 866 10 Surfact Amps 20 Tween 20 Pierce Chemical 28320 Bleach 5 25 Sodium Hypochlorite VWR Scientific 21899 504 or equivalent Denhardt s Solution 50X concentrate Sigma Aldrich D2532 MES hydrate Sigma Aldrich M5287 MES Sodium Salt Sigma Aldrich M5057 5 M NaCl RNase free DNase free Ambion 9760G Reagent Preparation Prepare the following buffers and antibody Wash A Non Stringent Wash Buff
97. r 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 About the Reagents Equipment and Consumables Specified in this Chapter Genome Wide Human SNP Nsp Sty Assay Kit 5 0 6 0 30 Reactions IMPORTANT Always use the 30 reaction Genome Wide Human SNP Nsp Sty Assay Kit 5 0 6 0 for this protocol This kit has been tested for multiple freeze thaw cycles You can freeze thaw the reagents in the 30 reaction kit lt 8 times Equipment Consumables and Other Reagents IMPORTANT This protocol has been optimized using the equipment consumables and reagents listed herein For the best results we strongly recommend that you adhere to the protocol as described no deviations do not substitute reagents chapter 4 Affymetrix Cytogenetics Copy Number Assay 21 Workflows Recommended 4 Day Workflow Figure 4 1 shows the recommended 4 day workflow for one operator processing four to 24 samples including controls Stage 1 8 hours 30 min hands on Stage 2 4 hours 30 min hands on Day 1 fhour hands on Pre PCR Clean Room Sagas Er QEDERE E LES Qui DL dC ECC ODE REC Post PCR Room eser 1 5 hours QC Gel 1 Stage 4 1 5 hours hands on Stage 5 30 min hands on Day 2 Stage 6 15 hours 30 min hands on QC Gel 2 Stage 7 4 hours
98. rad PTC 225 DNA Engine Tetrad 2 Applied Biosystems Post PCR Area GeneAmp PCR System 9700 silver block or gold plated silver block Use one of these units Bio Rad units if processing 8 samples you may want to use MJ Tetrad PTC 225 additional thermal cyclers for PCR DNA Engine Tetrad 2 Program Your Thermal Cyclers The thermal cycler programs listed in Table 1 3 and Table 1 4 are used during 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 D Thermal Cycler Programs Table 1 3 Pre PCR Clean Area of Thermal Cyclers Required Program Name Cyto Digest Cyto Ligate Table 1 4 Post PCR Area of Thermal Cyclers Required Program Name Cyto PCR 1 Cyto Fragment if routinely processing gt 8 samples you may want to use additional thermal cyclers for PCR eye Label Cyto Hyb 6 Affymetrix Cytogenetics Copy Number Assay User Guide Chapter u ERO HUE RR LABORATORY SETUP AND RECOMMENDATIONS This chapter provides an overview of two laboratory setups that can used when performing the Affymetrix Cytogenetics Copy Number Assay IMPORTANT If possible we strongly recommend using two separate rooms when performing this protocol Configuration 1 Two Separate Rooms The use of two separate
99. rain the lines with air The LCD display will read CLEANING DONE 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 7 2 below 106 Affymetrix Cytogenetics Copy Number Assay User Guide Table 7 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 Perform 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 Remove the DI water and perform a dry protocol period of time longer than one week 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 t i Bi WERBEN i Emp Hu JAM rag Ea m i i Appendix GUIDELINES FOR PROCESSING 16 SAMPLES T
100. rate copies of the protocol for each room Safety Precautions The Affymetrix Genome Wide Human SNP Nsp Sty Assay Kit 5 0 6 0 as well as the Affymetrix Genome Wide Human SNP Array 6 0 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 x NOTE 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 Chapter 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 200 and 1100 bp in size throughout the genome To achieve this the genomic DNA must be of high quality and must be free of contaminants that would affect the enzymatic reactions carried out For this protocol you will use the Affymetrix Genome Wide Human SNP Nsp Sty Assay Kit 5 0 6 0 30 reaction P N 901013 This kit contains the genomic DNA control Reference Genomic DNA 103 Ref 103
101. rt Number m GeneChip Fluidics Station 450 00 0079 Oo GeneChip Hybridization Oven 640 or 645 800139 640 00 0331 645 3 GeneChip 3000 Scanner with 7G upgrade 901153 Affymetrix Software Required Table C 2 Affymetrix Software Required Y Item Part Number a One of the following instrument control applications Latest version GeneChip Operating Software GeneChip Command Console a Genotyping Console Latest version 116 Affymetrix Cytogenetics Copy Number Assay User Guide Affymetrix Arrays Required Table C 3 Affymetrix Genome Wide Human SNP Array 6 0 Part Number 100 arrays 901150 4 50 arrays 901153 5 arrays 901182 Affymetrix Reagents Required Table C 4 Affymetrix Genome Wide Human SNP Nsp Sty Assay Kit 5 0 6 0 30 reaction kit Y item Part Number o 30 reactions 901013 Components contained in this kit e Adaptor Nsp 50 uM e Adaptor Sty 50 uM PCR Primer 002 100 uM GeneChip Fragmentation Reagent 10X Fragmentation Buffer GeneChip DNA Labeling Reagent 30 mM e Terminal Deoxynucleotidyl Transferase 30 U uL 5X Terminal Deoxynucleotidyl Transferase Buffer Oligonucleotide Control Reagent 0100 e Reference Genomic DNA 103 50 ng uL Optional Affymetrix Equipment Table C 5 Optional Affymetrix Equipment Part Number n GeneChip System 3000Dx v 2 with Data Transfer Server 00 0349 3 GeneChip Hybridization Ove
102. ser Guide 10 11 12 D Press down on the needle lever to snap needles into position and to start the run Once these steps are complete the fluidics protocol begins The Fluidics Station dialog box at the workstation terminal and the LCD window displays the status of the washing and staining steps When staining is finished remove the microcentrifuge vials containing stain and replace with three empty vials as prompted Remove the arrays from the fluidics station by first pressing down the cartridge lever to the eject position Check the array window for large bubbles or air pockets If bubbles are present 1 use a pipette to manually fill the array with Array Holding Buffer 2 remove one half of the solution then 3 manually fill the array with Array Holding Buffer IMPORTANT If a bubble is present do not return the array to the array holder The array must be filled manually with Array Holding Buffer If the array has no large bubbles it is ready for scanning Pull up on the cartridge lever to engage wash block and proceed to Scanning Arrays on page 91 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 When finished washing and staining shut down the fluidics station following the procedure listed under Shutting Down the Fluidics Station on page 92 chapter 5 Washing Staining and Scanning Arrays 91 Scanning Arra
103. sly amplified PCR product has contaminated your samples Use Reference Genomic DNA 103 as a positive control included in the reagent kit 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 sensitivity of the array The oligonucleotide control reagents contain oligo B2 which is used for grid alignment Regularly calibrate all multichannel pipettes Check that your spectrophotometer is accurately calibrated and be sure the OD measurement is within the quantitative linear range of the instrument 0 2 to 2 0 OD Hybridization ovens should be serviced at least once per year to ensure that they are operating within the manufacturer s specifications chapter 6 Troubleshooting 97 Troubleshooting the Cytogenetics Copy Number Assay Problem Likely Cause Faint absent bands on PCR gel Solution Both samples amp positive control affected Problem with master mixes or individual reagents Ensure all reagents added to master mixes and enzymes are stored at 20 C Work quickly with enzymes and return to 20 C directly after use to prevent loss of activity Failed restriction digest Use restriction enzyme to digest a known good DNA sample Run
104. t 2 C to 8 C and shield from light 86 Affymetrix Cytogenetics Copy Number Assay User Guide Fluidics Station and Scanner Control Software You will use one of the instrument control applications listed below to operate the fluidics station and the scanner For more information on these applications refer to the appropriate user s guide Affymetrix GeneChip Operating Software GCOS Affymetrix GeneChip Operating Software User s Guide Affymetrix GeneChip Command Console AGCC Affymetrix GeneChip Command Console User s Guide Register a New Experiment or Sample To register a new experiment or sample If using GCOS register a new Experiment If using AGCC register a new Sample Prime the Fluidics Station The Fluidics Station 450 is used to wash and stain the arrays it is operated using either GCOS or AGCC software To prime the Fluidics Station 1 Turn on the Fluidics Station 2 Prime the Fluidics Station Select protocol Prime 450 for each module ntake buffer reservoir use Non Stringent Wash Buffer ntake buffer reservoir B use Stringent Wash Buffer About Priming the Fluidics Station Priming ensures the lines of the 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 perf
105. t and Materials Required KK KK ee 42 Reagents Required si samimiy apru mitna E ee Gand eh abc DI p ath ede oko tp we ale ed 43 Gels and Related Materials Required llle 43 Important Information About This Stage Wak kK KK eee 43 Prepare the Reagents Consumables and Other Components 44 Transfer Diluted Ligated Samples to the PCR Plate AA KK KIRI III K K KII 45 Prepare the PCR Master MIX or oU XEIRid4edeh OD DEEX ote ees Malad 46 contents iii Add PCR Master Mix to Each Sample kk kk kk KK KK KK KIRR KIR KK RR KR KK KK KK 46 Load PCR Plate onto a Thermal Cycler 2 20 kk kk kk KK KK KK KI KIRI KIRI KI KIR KIR KIIR 47 Check the PCR Reaction by Running a Gel kk kk kk KK KK K R 48 What Tol DO NEXE nirera a KAMA AN Hila Sept eat QNI Db Y ou Me AE DA tt nih at k r 50 Stage 4 PCR Product PUrlflGatiODi ue lx yl kk Wi a Doe WERT RR Ae ee Fo 51 About this Stage 2 1 3 ts c bulan y e kb se ebrei px een eee edt e dede 51 LOCATION and D rati n J 33 2y t are QA Qa D N geek e D tres Rede 51 Input Required from Previous Stage eee 51 Equipment and Consumables Required llle 52 Reagents Reg irsd S sx a sal a hh kal dk lea lk al ai wal aleke rhe 52 Important Information About This Stage W kk kk KK KK eee 53 Prepare the 75 EtOH c a4 EE ele Mi tuc ec OR ka e A d RE lek be chad gd 53 Poolthe PCR PrOQUGtS x the ba eed tts Eh eo
106. thaw in a cooling chamber on ice Lu IMPORTANT Leave the T4 DNA Ligase at 20 C until ready to use Setup the Work Area Keep Sty master mix reagents on ice off to the side Sty Adaptor Sty ligation master mix tube T4 DNA Ligase Buffer Nsp Adaptor o Nsp ligation master mix tube Tor 216 Ole aJe le SIE J lt Figure 4 9 Setup for Nsp and Sty Ligation T4 DNA Ligase enzyme not pictured still at 20 C To setup the work area Figure 4 9 1 Place a double cooling chamber on ice 2 Label the 1 5 mL Eppendorf tubes as follows Label one tube NSP and place in the cooling chamber Label one tube STY and set aside 3 Prepare the digested samples as follows A Vortex at high speed for 3 sec then spin down at 2000 rpm for 30 sec B Place in the cooling chamber on ice 4 To prepare the reagents A Vortex at high speed 3 times sec each time except for the enzyme B Pulse spin for 3 sec C Place in the cooling chamber chapter 4 Affymetrix Cytogenetics Copy Number Assay 37 IMPORTANT T4 DNA Ligase Buffer 10X contains ATP and should be thawed on ice Vortex the buffer as long as necessary before use to ensure precipitate is re suspended and that the buffer is clear Preheat the Thermal Cycler Lid Power on the thermal cycler to preheat the lid Leave the block at room temperature The lid must be preheated before samples are loaded Prepare the Nsp L
107. the chamber as shown in Figure 4 13 on page 45 chapter 4 Affymetrix Cytogenetics Copy Number Assay 45 Solution basin Nsp diluted ligated Sty diluted ligated samples samples PCR master mix tube Water AccuGENE 1 1 2 5 5 2 Titanium Taq PCR Buffer GC Melt T oa 8 WN 4 IDOOOGOOO OOOOGOOO Dee oases 00000000 dNTPs 00000000 00000000 PCR Primer 002 Figure 4 13 Setup for PCR Titanium Taq DNA Polymerase not shown still at 20 C 4 To prepare the reagents A Vortex at high speed 3 times 1 sec each time except for the enzyme B Pulse spin for 3 sec C Place on ice or in the cooling chamber Preheat the Thermal Cycler Lids Post PCR Area Have someone in the Post PCR Area power on the thermal cycler s to preheat the lid Leave the blocks at room temperature To avoid contamination do not go from the Pre PCR Clean Area to the Post PCR Area and back again Transfer Diluted Ligated Samples to the PCR Plate To transfer the diluted ligated samples to the PCR plate 1 Using a P20 pipet single or multichannel A Transfer 10 uL of each Nsp ligated sample to the corresponding four wells of the PCR plate Figure 4 13 on page 45 B Transfer 10 uL of each Sty ligated sample to the corresponding three wells of the PCR plate 2 Seal label and store the plate with the remaining ligated Nsp and Sty samples at 20 C Discard this plate
108. the frequency of use The current version of the protocol can be found at www affymetrix com support technical fluidics_scripts affx 102 Affymetrix Cytogenetics Copy Number Assay User Guide 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 7 1 Affymetrix Recommended Bottles Part Number Description 400118 Media Bottle SO 500 mL 400119 Media Bottle SO 1000 mL 1 Disengage the washblock for each module by pressing down on the cartridge lever Remove any probe array cartridge Figure 7 1 on page 102 2 Prepare 500 mL of 0 525 sodium hypochlorite solution using deionized water You can follow these directions to make 500 mL of bleach In a 1 liter plastic or glass graduated cylinder combine 43 75 mL of commercial bleach such as Clorox bleach which is 6 sodium hypochlorite with 456 25 mL of DI H O mix well Pour the solution into a 500 mL plastic bottle and place the plastic bottle on fluidics station IMPORTANT The shelf life of this solution is 24 hr After this period you must prepare fresh solution e Each fluidics station with 4 modules requires 500 mL of 0 525 sodium hypochlorite solution Figure 7 1 Disengaged washblocks showing cartridge levers in the down position Remove any cartridges chapter 7 Fluidics
109. the software may be incorrect OD calculations may be incorrect and should be checked Table 6 2 Sample OD lt 0 9 4 5 ug uL If the sample OD is less than 0 8 calculated concentration less than 4 ug pL a problem may exist with either the genomic DNA the PCR reaction the elution of purified PCR products or the OD readings Possible problems with input genomic DNA that would lead to reduced yield include The presence of inhibitors heme EDTA etc Severely degraded genomic DNA naccurate concentration of genomic DNA Check the OD reading for the PCR products derived from Reference DNA 103 as a control for these issues Troubleshooting possible problems with the elution or OD readings Possible causes include The purified PCR product was eluted in a volume greater than 55 uL The purified PCR product was not mixed adequately before making the 1 100 dilution The diluted PCR product was not mixed adequately before taking the OD reading Table 6 3 OD260 OD280 ratio is not between 1 8 and 2 0 Possible causes include The PCR product may be not be sufficiently purified Ensure the vortexer is working properly An error may have been made while taking the OD readings The PCR product may not have been adequately washed Check the 7596 EtOH wash solution 100 Affymetrix Cytogenetics Copy Number Assay User Guide Table 6 4 The OD320 measurement is gt 0 2 Possibl
110. the steps performed in this room should be dedicated Do not move any equipment including ice buckets and pipets 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 chapter 2 Laboratory Setup and Recommendations 9 Post PCR Room 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 PCR amplification PCR product purification and quantitation PCR product fragmentation and labeling Sample hybridization onto arrays Scanning of arrays 60 005 o 0409 o0 OP Oo o0 Equipment Shown Vortexer with plate pad Microfuge Pipets on stand Ice bucket Magnetic stand Hyb oven ooo N 9g e N Plate centrifuge e MicroCentrifuge Spectrophotometer N Gel Imager m N mh _ 0 Q Fluidics Station o Scanner _ N Refrigerator _ oo Freezer Vortexer with tube mixer attachment Thermal cycler one to three Gel box for electrophoresis Computer monitor keyboard To help prevent sample c
111. tion ANG Duration k ce rere eee e tet d Kela NE eee e UM p irs 75 Input Required from Previous Stage WW kk kk KK KK KK IIIIII KI KIRI KI KI KIRI KI KII 75 Equipment and Consumables Required Ak kk kK KK KK KK eee 75 REAGENTS REGULE s PA K a eet W e k k fed we m c n Beat sg Eve N A Te 76 Important Information About This Stage kk kK KK eae 77 Prepare the Reagents Consumables and Other Components 77 Prepare the Arrays kk kk kk kk kK kK KK KK KK KK KK KK KK KK KK KK KK KK KK KK KK KK KK 78 Prepare the Hybridization Master Mix kk kk kk KK KK K K K K nee 79 Chapter 5 Washing Staining and Scanning Arrays 00 kK kK KK KK KK KK 83 Equipment and Consumables Required A ky kk kK KK IK eee 83 Reagents Required i k kk kk kk kk kk kk kK KK KK KK KK KK KK KK KI KIR KK KK KK KK KK KK KK KK 84 Reagent Preparatlons ee Xi Wana e KRE aos auod ala KOSE WA tet Xw deum 84 Wash A Non Stringent Wash Buffer kk kk a kk kk kK KK KK KK KI KIRI K KIR KIR KI KI KI KK KK 84 Wash B Stringent Wash Buffer kk kk kk kk kk kK KK K KK KI KK KK KI KI KI KIRI K KI K KI KI KK KK YR 85 0 5 mg mL Anti Streptavidin Antibody 0 0 kk kk KK KK KK KK KK RR RR RY RY KK 85 T12 MIES AStOCKIBUTIGT si o yl cts w K Mate LA E eS edt ides 85 IX Array Holding BUTS a So Xwa lA A E i Caedm o aya CHA E quet 85 Fluidics Station and Scanner Control Software WW KK eae 86 Register a New Experiment or Sample 00 kK
112. toclave Store between 2 C and 8 C and shield from light using aluminum foil Discard solution if it turns yellow To prepare 500 mL of 12X MES Stock Solution 1 25 M MES 0 89 M Na 1 Combine 35 2 g MES hydrate 96 65 g MES sodium salt 400 mL AccuGENE water Mix and adjust volume to 475 mL Test the pH The pH should be between 6 5 and 6 7 Adjust the pH so it falls between 6 5 and 6 7 Adjust the volume to 500 mL Filter the solution through a 0 2 um filter ON Nome Protect from light using aluminum foil and store at 4 C 78 Affymetrix Cytogenetics Copy Number Assay User Guide Preheat the Hybridization Ovens To preheat the hybridization ovens 1 Turn on each oven and set the temperature to 50 C 2 Set the rpm to 60 3 Turn the rotation on and allow to preheat for 1 hr before loading arrays IMPORTANT An accurate hybridization temperature is critical for this assay Therefore we recommend that your hybridization ovens be serviced at least once per year to ensure that they are operating within the manufacturer s specifications Prepare the Samples To prepare the samples 1 Ifthe labeled samples from the previous stage were frozen allow them to thaw on the bench top to room temperature Vortex at high speed for 3 sec then spin down for 30 sec 3 Place in a cooling chamber on ice Preheat the Thermal Cycler Lid Power on the thermal cycler to preheat the lid Leave the
113. trad PTC 225 and Applied Biosystems thermal cyclers are different See Table 4 18 and Table 4 19 on page 48 If using GeneAmp PCR System 9700 thermal cyclers be sure the blocks are silver or gold plated silver Do NOT use thermal cyclers with aluminum blocks It is not easy to visually distinguish between silver and aluminum blocks Table 4 18 Cyto PCR Thermal Cycler Program for the GeneAmp PCR System 9700 silver or gold plated silver blocks Cyto PCR Program for GeneAmp PCR System 9700 Temperature Time Cycles 94 C 3 min 1X 94 C 30 sec 60 C 45 sec 30X 68 C 15 sec 68 C 7 min 1X 4 C HOLD Can be held overnight Volume 100 uL Specify Maximum mode 48 Affymetrix Cytogenetics Copy Number Assay User Guide Table 4 19 Cyto PCR Thermal Cycler Program for the MJ Tetrad PTC 225 Cyto PCR Program for MJ Tetrad PTC 225 Temperature Time Cycles 94 C 3 min 1X 94 C 30 sec 60 C 30 sec 30X 68 C 15 sec 68 C 7 min 1X 4 C HOLD Can be held overnight Volume 100 uL Use Heated Lid and Calculated Temperature 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 Wear the appropriate personal protective equipment when handling ethidium bromide Run the Gels When the Cyto PCR program is finished 1 Remove the plate from the thermal cycler 2 Spin down at 2000 rpm for 30 sec 3 Pla
114. trol has good MAPD Genomic DNA not optimal and Contrast OC values but samples do not Ensure DNA samples are of high quality i e run in a 1 to 296 gel and compare to Reference 103 DNA control Use positive control sample as a reference guide for assay procedures Prepare master mixes for samples and controls Very high MAPD or low Contrast Mixed up Nsp and Sty enzymes during the QC values digestion or ligation stages Repeat the experiment making sure the correct reagents are used for each digestion and ligation stage chapter 6 Troubleshooting 99 OD Troubleshooting Guidelines Table 6 1 Sample OD gt 1 4 gt 7 ug uL If the sample OD is greater than 1 4 calculated concentration greater than 7 ug uL a problem may exist with either PCR product elution or the OD reading The limit on PCR yield is approximately 7 ug uL as observed in practice and as predicted by the mass of dNTPs in the reaction Possible causes include Check the OD320 reading If it is 0 1 you may have bead slurry carryover OK to proceed The purified PCR product was eluted in a volume less than 55 uL The purified PCR product was not mixed adequately before making the 1 100 dilution The diluted PCR product was not mixed adequately before taking the OD reading The plate spectrophotometer or NanoDrop may require calibration Pipettes may require calibration The settings on the plate spectrophotometer NanoDrop or
115. ts 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 1 SDS ProK digestion phenol chloroform extraction Microcon or Centricon Millipore ultrapurification and concentration 2 QIAGEN QIAamp DNA Blood Maxi Kit DNA Cleanup If a genomic DNA preparation is suspected to contain inhibitors the following cleanup procedure can be used 1 Add 0 5 volumes of 7 5 M NH OAc 2 5 volumes of absolute ethanol stored at 20 C and 0 5 uL of glycogen 5 mg mL to 250 ng genomic DNA 2 Vortex and incubate at 20 C for 1 hr 3 Centrifuge at 12 000 X x g in a microcentrifuge at room temperature for 20 min 4 Remove supernatant and wash pellet with 0 5 mL of 80 ethanol 5 Centrifuge at 12 000 X g at room temperature for 5 min 6 Remove the 80 ethanol and repeat the 80 ethanol wash one more time 7 Resuspend the pellet in reduced EDTA TE buffer 10 mM Tris pH 8 0 0 1 mM EDTA pH 8 0 References Feigelson H S Rodriguez C Robertson A S Jacobs E J Calle E E Reid Y A Thun M J Determinants of DNA yield and quality from buccal cell samples collected with mouthwash Cancer Epidemiol Biomarkers Prev 10 9 1005 8 2001 Heath Ellen M Morken Nathaniel W Campbell Kristen A Tkach Dennis Boyd Erin A Strom
116. uge multipurpose Eppendorf 5804 or 5810 3 Vortexer with plate pad VWR 58816 1212 Select one of these thermal cyclers a GeneAmp PCR System 9700 gold silver block Applied Biosystems 4314878 e 2720 Thermal Cycler Applied Biosystems 4359659 e MJ Tetrad PTC 255 Bio Rad DNA Engine Tetrad 2 Bio Rad PTC 0240G 118 Affymetrix Cytogenetics Copy Number Assay User Guide Post PCR Area Equipment Required Table C 7 Post PCR Area Equipment Required v Item Vendor Part Number Oo Benchtop Cooler 20 C Stratagene 400012 3 Cooling Chamber single Diversified Biotech CHAM 1020 m Freezer 20 C deep freeze manual defrost 17 cu ft Any vendor 3 Gel box for electrophoresis Any vendor 3 Gel imager Any vendor H Ice Bucket 4 to 9 liters Magic Touch Icewares Fisher Scientific _ 3 MagnaRack magnetic stand Invitrogen CS15000 3 Microcentrifuge 5414D or R Eppendorf 022621408 3 Microcentrifuge Standard Rotor F 45 24 11 24 bores Eppendorf 022621502 3 Microfuge for tubes and strip tubes Any vendor qo Microtube Foam Insert with stem Scientific Industries 504 0233 00 Multiple Sample Starter Set Model H301 you will attach to one of the vortexers 3 Pipet single channel 2 20 uL Rainin L 20 3 Pipet single channel 20 200 uL Rainin L 200 a Pipet single channel 100 1000 uL Rainin L 1000 m Pipet 8 channel 2 20 uL Rainin L8 20 a Pipet 8 chann
117. use Prepare Your Work Area To prepare the work area 1 Place the following on the bench top Optional conical tube or solution basin Water UV or 96 well plate 2 Spin down the purified samples at 2000 rpm for 30 sec and put in a plate holder Procedure if Using a Microplate Spectrophotometer Prepare Diluted Aliquots of Purified Sample Lu IMPORTANT The P20 pipet must be accurate to within 596 To prepare diluted aliquots of the purified samples 1 2 3 Using a P200 pipet aliquot 198 uL of water to the corresponding wells of a UV plate Pipet 200 uL of water into each well of an empty column Figure 4 22 below Using a P20 pipet A Transfer 2 uL of each purified sample to the corresponding well of the UV plate B Pipette up and down 2 times to ensure that all of the sample is dispensed The result is a 100 fold dilution Do one of the following to mix the samples Set a P200 pipet to 170 uL and pipet up and down 5 times Seal the plate vortex and spin down at 2000 rpm for 30 sec 60 Affymetrix Cytogenetics Copy Number Assay User Guide 198 uL water AccuGENE 2 uL purified sample in each well 200 uL water for blank O amp O 9 21 00600000 OOOOOOOO OOOOOOOO OOOOOOOQ OOO00000Q 30 09 0000 OODOODCUCQCLD OOOOOOOO QOQOQQOQ OOOOOOOO OOOOOOOO Figure 4 22 UV plate layout Quantitate the Diluted PCR Product Apply the convention that 1
118. uted Guach en aul Qa a hic eit aren aoa E 21 ii Affymetrix Cytogenetics Copy Number Assay User Guide Genomic DNA Preparation kk kk kk kk kk kK kk KK KK KI KK KI KK KK KK KIR KI KIRI KI KK KK KK KK KK 23 ADOUTTAIS Stage 3x 4 Mo Ma UE eo ee ee attics Va E UE 23 LOCATON ANG Duration ttm oa ex exte o e A od ad etm cau Ex opt AT RT 23 IriputeRequlled s as ax ne ge toh t d ADAE Tot EAD Rated Ge Bah BR eae de XA BUR edo dl Ex Ces d 23 Equipment and Consumables Required llle 24 Reagents Required i Jk kk kk kk kk kk kk KK KK KK KK KK KK KK KK KK KK KK KI KK KK KK KK KK 24 Preparing the Genomic DNA i 4444 Qala l k a W oe aa aw B s n ws 25 Aliquoting the Prepared Genomic DNA and Controls llle 26 What ToDo Nax A c Ka e ane n REEEFT De ie epe W Y KAWANA d Dese end dala 27 Stage 1 Nsp and Sty Restriction Enzyme Digest AVIK KIR RIK ee 28 ADOUTTAIS Stage o SR ark ek Rana de RR ER api pot eec did RN A ce ae 28 Bel sat N Ms el ot fe ose Oe Sette e t SE Nm e as 28 Input Required From Previous Stage 0 eee 28 Equipment and Consumables Required kk kk kK KK KIIR eee 29 Reagents Required JLL kk kk kk kk kK kK nee eee 29 Prepare the Reagents Equipment and Consumables AR K KIL 30 Prepare the Nsp Digest Master MIX 4k kk kk KK KK KK K KI eee 31 Add Nsp Digest Master Mix to Samples 0 00000 anaana raran 31 Prepare the Sty Digest Master Mix kk kk kk kK KK KII K K K K ee 32 Add Sty Digest Master Mix t
119. x 5 25 uL Total 25 00 pL Contains ATP and DTT Keep on ice MOI O O O O O O O O OJO Add 5 25 uL Nsp Ligation OJODOOOOOOOOQO O Master Mixto each sample MOOOOOOQOOOQO OIlOOOOOOOOOO O OIlOOOOOOOOOO O OJOOOO60600000l0O sOoOoooooooOoO J 8sogoooooooooOQjJgp Figure 4 10 Adding Nsp ligate master mix to Nsp digested samples and controls Prepare the Sty Ligation Master Mix Keeping all reagents and tubes on ice prepare the Sty Ligation Master Mix as follows 1 oon RON To the 1 5 mL Eppendorf tube labeled STY add the following reagents based on the volumes shown in Table 4 12 on page 39 T4 DNA Ligase Buffer 10X Adaptor Sty Immediately add the T4 DNA Ligase to the master mix then place back in the cooler Vortex the master mix at high speed 3 times 1 sec each time Pulse spin for 3 sec Place the master mix on ice Proceed immediately to Add Sty Ligation Master Mix to Reactions chapter 4 Affymetrix Cytogenetics Copy Number Assay 39 Table 4 12 Sty Ligation Master Mix Reagent 1 Sample 4 Samples 8 Samples 12 Samples 24 Samples 15 extra 15 extra 15 extra 15 extra T4 DNA Ligase Buffer 2 5 uL 11 5 uL 23 0 uL 34 5 uL 69 uL 10X Adaptor Sty 0 75 uL 3 45 uL 6 90 uL 10 35 uL 20 7 uL 50 uM T4 DNA Ligase 2 uL 9 2 uL 18 4 uL 27 6 uL 55 2 uL 400 U uL Total 5 25 uL 24 15 uL 48 30 uL 72 45 pL 144 90 uL Add Sty Ligation Master Mix to
120. x to the samples 1 Quickly and on ice aliquot out the Fragmentation Master Mix equally to the strip tubes Figure 4 25 2 Spin down the strip tubes 3 Using an 8 channel P20 pipet transfer 5 uL of Fragmentation Master Mix to each sample do not pipet up and down Avoid introducing air bubbles at the bottom of the tubes to ensure the accurate transfer of 5 uL to each sample IMPORTANT To help ensure the same amount of fragmentation for each sample add the master mix to the samples as quickly as possible Sample with Fragmentation Buffer 50 uL Fragmentation Master Mix 5 uL Total 55 uL 4 Remove and discard any remaining Fragmentation Master Mix Never re use Fragmentation Master Mix 5 Tightly seal the plate 6 Vortex at high speed for 3 sec then spin down for 30 sec 7 Immediately load the samples onto the pre heated block of the thermal cycler 37 C and run the Cyto Fragment program Table 4 30 IMPORTANT Ensure that the seal is not pulled off any wells when you close the thermal cycler lid Table 4 30 Cyto Fragment Thermal Cycler Program Cyto Fragment Program Temperature Time 37 C 35 min 95 C 15 min 4 C Hold What To Do Next Proceed directly to the next stage While the labeling reaction is taking place check the fragmentation reaction by running gels as described under Check the Fragmentation Reaction by Running a Gel on page 70 70 Affymetrix Cytog
121. ys The GeneChip Scanner 3000 7G is controlled by GCOS or AGCC software Prepare the Scanner Turn on the scanner at least 10 min before use WARNING The scanner 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 2 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 On the back of the array cartridge clean excess fluid from around the septa Carefully cover both septa with Tough Spots See Figure 5 1 on page 91 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 Insert an array into the scanner and test the autofocus to ensure the spots do not interfere with the focus If a focus error message is observed remove the spot and apply a new spot Ensure that the spots lie flat Figure 5 1 Applying Tough Spots to Arrays 92 Affymetrix Cytogenetics Copy Number Assay User Guid
122. zation oven Plate centrifuge MicroCentrifuge Spectrophotometer Gel imager Gel box for electrophoresis Refrigerator Freezer Eu l Figure 2 3 Single direction workflow 14 Affymetrix Cytogenetics Copy Number Assay User Guide Contamination Prevention Care should be taken to minimize possible sources of contamination that would reduce genotyping accuracy call rate and consequently genetic power 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 IMPORTANT The most likely potential source of contamination for the Cytogenetics Copy Number Assay is previously amplified PCR product Each area should contain dedicated equipment such as thermal cyclers microfuges pipets 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 changed into freshly laundered clothing e Maintain an ambient laboratory environment throughout the procedure Precautions that you can take to minimize contaminating pre PCR steps with amplified PCR product include the following Store reagents in the proper area according to the box label and reagent kit insert Use proper gowning procedures Print sepa

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Affymetrix® Cytogenetics Copy Number Assay User Guide