Applied Biosystems 7900HT Fast Real-Time PCR System and SDS ...

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Chapter 4Operating the InstrumentMonitoringInstrumentProgressThe Automation Controller Software displays the progress of the current run in theThermal Status tab. See page 4-27 for an explanation of the Thermal Status tab.Stopping theInstrumentTo stop the plate queue, clickbox at any time.in the Automation Controller Software dialogIf you clicked ... Then the instrument...while the Plate Handler ishandling a plateafter a plate has been loadedinto the instrument, butbefore the run has started.after the 7900HT instrumenthas started a run.aborts the run and moves the Plate Handler to the homeposition.aborts the current run, ejects the plate, and moves thePlate Handler to the home position.aborts the current run, ejects the plate, and moves thePlate Handler to the home position.IMPORTANT! Stopping a run during thermal cycling canaffect the chemistry of the reactions inside the plate.Before stopping a run, carefully read the guidelines onpage 4-28 to determine the best course of action.Ejecting a PlateTo eject a plate following a halted run (or to open/close the instrument tray),click from the Automation Controller Software window.After the RunAnalyzing theRun DataYou can analyze the run data immediately following the completion of the run.See the appropriate page for your application:Section 5.1 Allelic Discrimination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-5Section 6.1 Absolute Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-5Section 6.2 Relative Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-15Section 6.3 Dissociation Curve Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-374-44 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm
Analyzing End-Point Data 55In This Chapter End-Point Runs on the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-2Notes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-3Section 5.1 Allelic Discrimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-5Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-6Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-9Analysis Checklist. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-10Opening the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-11Analyzing an Allelic Discrimination Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-12Calling and Scrutinizing Allelic Discrimination Data . . . . . . . . . . . . . . . . . . . . . 5-13After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-18Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 5-1DRAFTSeptember 1, 2004 11:39 am, CH_End-Point.fm
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Applied Biosystems7900HT Fast Real-
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ContentsPrefaceHow to Use This Guid
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Chapter 4Operating the InstrumentNo
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Chapter 7Maintaining the Instrument
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Appendix D Theory of OperationFluor
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PrefaceHow to Use This GuidePurpose
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Safety and EMC Compliance Informati
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Symbols on InstrumentsSymbols on In
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General Instrument SafetyGeneral In
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Chemical Waste SafetyChemical Safet
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Physical Hazard SafetyOvervoltageRa
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Workstation SafetyWorkstation Safet
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Product Overview 11In This Chapter
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7900HT FAST Real-Time PCR SystemSec
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7900HT InstrumentInterchangeableThe
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7900HT FAST Real-Time PCR SystemBar
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Compatible ConsumablesCompatible Co
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Instrument ConnectionsFixed-Positio
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Section 1.2 Getting to Know the Sof
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SDS Software Related Files and Form
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7900HT FAST Real-Time PCR SystemMan
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Esc F1 F2 F3 F4 F5 F6 F7 F8 F9 F10
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Section 1.3 SDS Enterprise Database
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* + Enter* +Enter3Down UpNumLockSys
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About the SDS Enterprise Database S
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Esc F1 F2 F3 F4 F5 F6 F7 F8 F9 F10
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Getting Started 22In This Chapter G
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Getting StartedUsing the SDSSoftwar
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GR23967900HTPowering On the 7900HT
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Using the SDS SoftwareUsing the SDS
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Using the SDS SoftwareAbout theMess
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Basic Software Skills TutorialNotes
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Basic Software Skills TutorialExerc
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Basic Software Skills TutorialExpor
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Basic Software Skills TutorialLesso
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Basic Software Skills Tutorial3. Se
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Using SDS Plate DocumentsUsing SDS
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Using SDS Plate DocumentsWell Inspe
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Preparing a Run 33In This Chapter N
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Notes for Database UsersDescription
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Workflow OverviewWorkflow OverviewE
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Workflow OverviewAllelicDiscriminat
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Step 1 - Creating a Plate DocumentS
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Step 2 - Applying Detectors and Mar
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Step 3 - Configuring the Plate Docu
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Step 5 - Programming the Plate Docu
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Step 5 - Programming the Plate Docu
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Page 109 and 110: Step 6 - Saving the Plate Document
Page 111 and 112: Step 7 - Creating a Plate Document
Page 113 and 114: Step 9 - Running the Plate on the 7
Page 115 and 116: Operating the Instrument 44In This
Page 117 and 118: Notes for Database UsersDescription
Page 119 and 120: Section 4.1 Consumable PreparationS
Page 121 and 122: Compatible ConsumablesTable 4-1Comp
Page 123 and 124: Preparing Optical Plates for Usea.
Page 125 and 126: Preparing TaqMan Low Density Arrays
Page 137 and 138: Section 4.2 Running an Individual P
Page 139 and 140: Running a Single Plate (Using the S
Page 141 and 142: Running a Single Plate (Using the S
Page 143 and 144: After the RunAfter the RunUser Acce
Page 145 and 146: Section 4.3 Automated OperationSect
Page 147 and 148: Operating the Software with an SDS
Page 149 and 150: Operating the Software without an S
Page 155 and 156: Running Plates Using the Automation
Page 157: Running Plates Using the Automation
Page 161 and 162: Notes for Database UsersNotes for D
Page 163 and 164: Section 5.1 Allelic DiscriminationS
Page 165 and 166: OverviewTable 5-1Signal and Sequenc
Page 167 and 168: Before You BeginBefore You BeginUsi
Page 169 and 170: Opening the Run DataOpening the Run
Page 171 and 172: Calling and Scrutinizing Allelic Di
Page 177 and 178: After the AnalysisSaving theResults
Page 179 and 180: Analyzing Real-Time Data 66In This
Page 181 and 182: Notes for Database UsersDescription
Page 183 and 184: Section 6.1 Absolute Quantification
Page 185 and 186: OverviewAlgorithmicManipulation ofR
Page 187 and 188: Analysis ChecklistAnalysis Checklis
Page 189 and 190: Analyzing the DataAnalyzing the Dat
Page 191 and 192: Viewing ResultsViewing ResultsViewi
Page 193 and 194: Section 6.2 Relative Quantification
Page 195 and 196: OverviewAlgorithmic Manipulation of
Page 197 and 198: OverviewAutomatic Outlier RemovalAb
Page 199 and 200: Getting StartedGetting StartedUsing
Page 201 and 202: Options for Analyzing Relative Quan
Page 203 and 204: Creating the Studyd. In the Plate Q
Page 205 and 206: Analyzing the Study• Manual Ct -
Page 207 and 208: Analyzing the StudySpecifying the A
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Viewing ResultsViewing ResultsDispl
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Viewing ResultsAbout DownArrowsDown
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After the AnalysisAfter the Analysi
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Section 6.3 Dissociation Curve Anal
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Before You BeginExample ResultsFigu
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Opening the Run DataOpening the Run
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Determining T m Values for the Anal
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Section 6.4 Procedure ReferenceSect
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Setting the Baseline and Threshold
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Eliminating Outlying AmplificationE
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Eliminating Outlying Amplification6
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After the AnalysisSaving theResults
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Maintaining the Instrument 77In Thi
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Notes for Database UsersDescription
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Section 7.1 Maintaining the 7900HT
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Replacing the Sample BlockMaterials
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Replacing the Sample Block9. Loosen
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7900HT FAST Real-Time PCR SystemRep
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Changing the Plate Adapter4. Place
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Decontaminating the Sample BlockCle
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Performing a Background RunPreparin
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Performing a Background RunAnalyzin
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Performing a Pure Dye RunAfter a ru
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Performing a Pure Dye Run• Fast 9
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Performing a Pure Dye RunAnalyzing
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Adding Custom Dyes to the Pure Dye
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Adding Custom Dyes to the Pure Dye
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Verifying Instrument PerformanceTab
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Verifying Instrument PerformancePre
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Section 7.2 Maintaining the Plate H
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Adjusting the Sensitivity of the Pl
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Adjusting the Sensitivity of the Pl
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7900HT FAST Real-Time PCR SystemAli
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Aligning the Plate Handler5. Using
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Aligning the Plate Handler7. Using
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Aligning the Plate Handler13. Click
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7900HT FAST Real-Time PCR SystemAli
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Aligning the Fixed-Position Bar Cod
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Section 7.3 Maintaining the Compute
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Maintaining the SDS softwareSeveral
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Troubleshooting 88In This Chapter T
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Troubleshooting TableTable 8-1Troub
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Low Precision or IrreproducibilityL
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Low Precision or IrreproducibilityD
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Background RunsBackground RunsBackg
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Pure Dye Runs6. Repeat step 4 until
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End-Point Runs (Allelic Discriminat
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Software and 7900HT InstrumentTable
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Zymark Twister Microplate Handler a
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TaqMan Low Density ArrayTable 8-6Tr
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Software ReferenceAAIn This Appendi
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Importing Plate Document Setup Tabl
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Setup Table Files1. File VersionDes
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Setup Table Files6. Detectors ListD
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Setup Table Files9. Markers List (A
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Setup Table FilesFormat(for a singl
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Setup Table FilesFormat(for a singl
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Setup Table FilesExample Code A-16A
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Exporting Plate Document Data4. In
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Operating the SDS Software from a C
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Using the Search ToolLogical Operat
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Connecting SDS Software to the Data
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Designing TaqMan Reagent-BasedAssay
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Assay Development Guidelines• For
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Design Tips for Allelic Discriminat
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Kits, Reagents, and ConsumablesCCIn
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Consumables and DisposablesConsumab
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Instrument Maintenance and Verifica
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Custom Oligonucleotide SynthesisTab
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Theory of OperationDDIn This Append
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Fluorescent-Based ChemistriesBasics
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Real-Time Data AnalysisPhase 2: Lin
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Real-Time Data AnalysisSignificance
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A similar equation for the endogeno
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Limited Warranty StatementEEWarrant
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BibliographyGeneral Paperson TaqMan
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Klein, D., Janda, P., Steinborn, R.
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Dolken, L., F. , Schuler, and G. ,
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AllelicDiscriminationVirtanen M., H
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Glossary.sdd.sdm.sds.sdt∆C T∆
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ackgroundbaselinecalibratorcDNA(com
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minor groovebinder (MGB)multicompon
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R nrunsessionsingle nucleotidepolym
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IndexSymbols- 6-31↑ 6-33Numerics7
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fluorescent, common sources 8-7isol
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adjusting step parameters 3-21confi
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sample block locking bar 7-8sample
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Worldwide Sales and SupportApplied
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