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

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Chapter 4Operating the InstrumentPreparing Optical Plates for UsePreparing Platesfor Use on the7900HTInstrument1. Prepare the reactions in an optical plate by aliquoting reagents, enzyme, andsamples to the appropriate wells of an optical plate.IMPORTANT! The arrangement of the reactions (samples and assays) on theplate must match the configuration of information (sample names anddetectors/markers) on the corresponding plate document.2. Seal the optical plate with a seal that is compatible with the 7900HT instrument(Figures 4-3 and 4-4 on page 4-9).Note: See “Compatible Consumables” on page 4-6 for a list of seals for usewith the Applied Biosystems 7900HT Fast Real-Time PCR System.3. Briefly centrifuge the plate to collect the reactions at the bottom of the wells andto eliminate any air bubbles that may be present.4. Visually verify that each reaction is positioned at the bottom of its well.Correct PositionIncorrect PositionThe sample is positionedcorrectly in the bottom ofthe well.The sample lies on theside wall because theplate was not centrifuged.An air bubble lies at thebottom of the wellbecause the plate wasnot:• Centrifuged with enough force, or• Centrifuged for enough time5. If running an ABI PRISM 96-Well Optical Reaction Plate, apply the appropriatecompression pad to the sealed optical plate.If you are going to run the plate:• Individually using the SDS software, then apply a standard compressionpad (Figure 4-3)• As part of a batch using the Automation Controller Software, then apply anABI PRISM Snap-On Compression Pad (Figure 4-3)IMPORTANT! The ABI PRISM Snap-On Compression Pad/plate assemblymay still be hot after the Zymark ® Twister Microplate Handler loads it intothe Plate Handler’s output stack. Please wait at least 30 seconds beforemanually handling the Snap-On Compression Pad/plate assembly.6. Choose from the following. If performing:– Absolute or Relative Quantification, or Dissociation Curve AnalysisRun the plate as explained in “Running the Plate” on page 4-25.– Allelic Discrimination4-8 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm
Preparing Optical Plates for Usea. Load the plate onto a designated thermal cycler and perform the PCR.b. Briefly centrifuge the plate to draw the reactions to the bottom of the wells andto eliminate any air bubbles that may have formed during thermal cycling.c. Run the plate as explained in “Running the Plate” on page 4-25.‘Snap-On’Compression Pad(for automated runs)Standard compressionpad (for single-plateruns)Optical adhesivecoverOROptical Caps(flat caps only)Figure 4-396-Well Optical Reaction Plate AssemblyOptical adhesivecover384-wellplateFigure 4-4384-Well Optical Reaction Plate AssemblyApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-9DRAFTSeptember 1, 2004 11:39 am, CH_Basic.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 TutorialExerc
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Page 73 and 74: Basic Software Skills TutorialLesso
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Page 81 and 82: Using SDS Plate DocumentsUsing SDS
Page 83 and 84: Using SDS Plate DocumentsWell Inspe
Page 85 and 86: Preparing a Run 33In This Chapter N
Page 87 and 88: Notes for Database UsersDescription
Page 89 and 90: Workflow OverviewWorkflow OverviewE
Page 91 and 92: Workflow OverviewAllelicDiscriminat
Page 93 and 94: Step 1 - Creating a Plate DocumentS
Page 95 and 96: Step 2 - Applying Detectors and Mar
Page 101 and 102: Step 3 - Configuring the Plate Docu
Page 103 and 104: Step 5 - Programming the Plate Docu
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: Compatible ConsumablesTable 4-1Comp
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 and 158: Running Plates Using the Automation
Page 159 and 160: Analyzing End-Point Data 55In This
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
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Calling and Scrutinizing Allelic Di
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Calling and Scrutinizing Allelic Di
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After the AnalysisSaving theResults
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Analyzing Real-Time Data 66In This
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Notes for Database UsersDescription
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Section 6.1 Absolute Quantification
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OverviewAlgorithmicManipulation ofR
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Analysis ChecklistAnalysis Checklis
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Analyzing the DataAnalyzing the Dat
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Viewing ResultsViewing ResultsViewi
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Section 6.2 Relative Quantification
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OverviewAlgorithmic Manipulation of
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OverviewAutomatic Outlier RemovalAb
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Getting StartedGetting StartedUsing
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Options for Analyzing Relative Quan
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Creating the Studyd. In the Plate Q
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Analyzing the Study• Manual Ct -
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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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