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Chapter 6Analyzing Real-Time DataNotes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-2Real-Time Runs on the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-4Section 6.1 Absolute Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-5Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-6Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-8Analysis Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-9Opening the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-10Analyzing the Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-11Viewing Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-13After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-14Section 6.2 Relative Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-15Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-16Algorithmic Manipulation of Raw Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-17Automatic Outlier Removal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-19Essential Experimental Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-20Getting Started . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-21Analysis Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-22Options for Analyzing Relative Quantification Data . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-23Creating the Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-23Analyzing the Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-26About the Analysis Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-26Specifying the Analysis Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-29Analyzing the Study Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-30Viewing Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-31After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-35Section 6.3 Dissociation Curve Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-37Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-38Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-39Analysis Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-40Opening the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-41Analyzing the Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-41Determining T m Values for the Analyzed Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-42After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-44Section 6.4 Procedure Reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-45Setting the Baseline and Threshold Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-46Eliminating Outlying Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-49After the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-52viApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:40 am, 4351684ATOC.fm
Chapter 7Maintaining the InstrumentNotes for Database Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-2Recommended Maintenance Schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-4Section 7.1 Maintaining the 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-5Replacing the Sample Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-6Changing the Plate Adapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-12Decontaminating the Sample Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-14Performing a Background Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-16Preparing the Background Plate or TaqMan Low Density Array . . . . . . . . . . . . . 7-17Creating a Plate Document for the Background Run . . . . . . . . . . . . . . . . . . . . . . 7-17Running the Prepared Background Plate or TaqMan Low Density Array . . . . . . 7-18Analyzing the Background Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-19Performing a Pure Dye Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-20Preparing the Pure Dye Plates or Cards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-22Preparing a Plate Document for a Pure Dye Plate or Card . . . . . . . . . . . . . . . . . 7-22Running the Prepared Pure Dye Plate or Card . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-24Analyzing the Pure Dye Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-25Adding Custom Dyes to the Pure Dye Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-27Verifying Instrument Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-30Section 7.2 Maintaining the Plate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-35Automation Accessory Components and Stack Positions . . . . . . . . . . . . . . . . . . . . . 7-36Adjusting the Sensitivity of the Plate Sensor Switch . . . . . . . . . . . . . . . . . . . . . . . . . 7-37Aligning the Plate Handler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-41Aligning the Fixed-Position Bar Code Reader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-49Cleaning and Replacing Gripper Finger Pads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-52Section 7.3 Maintaining the Computer and Software . . . . . . . . . . . . . . . . . . . . . . . 7-53General Computer Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-54Maintaining the SDS software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-55Chapter 8TroubleshootingTroubleshooting Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-2Low Precision or Irreproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-5Background Runs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-9Pure Dye Runs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-11Real-Time Runs (Quantitative PCR and Dissociation Curves) . . . . . . . . . . . . . . . . . . 8-12End-Point Runs (Allelic Discrimination) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-13Software and 7900HT Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-14Zymark Twister Microplate Handler and Fixed-Position Bar Code Reader . . . . . . . . 8-17TaqMan Low Density Array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-18SDS Enterprise Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-20Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:40 am, 4351684ATOC.fmvii
Page 1 and 2: Applied Biosystems7900HT Fast Real-
Page 3 and 4: ContentsPrefaceHow to Use This Guid
Page 5: Chapter 4Operating the InstrumentNo
Page 9 and 10: Appendix D Theory of OperationFluor
Page 11 and 12: PrefaceHow to Use This GuidePurpose
Page 13 and 14: Safety and EMC Compliance Informati
Page 15 and 16: Symbols on InstrumentsSymbols on In
Page 17 and 18: General Instrument SafetyGeneral In
Page 19 and 20: Chemical Waste SafetyChemical Safet
Page 21 and 22: Physical Hazard SafetyOvervoltageRa
Page 23 and 24: Workstation SafetyWorkstation Safet
Page 25 and 26: Product Overview 11In This Chapter
Page 27 and 28: 7900HT FAST Real-Time PCR SystemSec
Page 29 and 30: 7900HT InstrumentInterchangeableThe
Page 31 and 32: 7900HT FAST Real-Time PCR SystemBar
Page 33 and 34: Compatible ConsumablesCompatible Co
Page 35 and 36: Instrument ConnectionsFixed-Positio
Page 37 and 38: Section 1.2 Getting to Know the Sof
Page 39 and 40: SDS Software Related Files and Form
Page 41 and 42: 7900HT FAST Real-Time PCR SystemMan
Page 43 and 44: Esc F1 F2 F3 F4 F5 F6 F7 F8 F9 F10
Page 45 and 46: Section 1.3 SDS Enterprise Database
Page 47 and 48: * + Enter* +Enter3Down UpNumLockSys
Page 49 and 50: About the SDS Enterprise Database S
Page 51 and 52: Esc F1 F2 F3 F4 F5 F6 F7 F8 F9 F10
Page 53 and 54: Getting Started 22In This Chapter G
Page 55 and 56: Getting StartedUsing the SDSSoftwar
Page 57 and 58:
GR23967900HTPowering On the 7900HT
Page 59 and 60:
Using the SDS SoftwareUsing the SDS
Page 61 and 62:
Using the SDS SoftwareAbout theMess
Page 63 and 64:
Basic Software Skills TutorialNotes
Page 65 and 66:
Basic Software Skills TutorialExerc
Page 67 and 68:
Basic Software Skills TutorialExpor
Page 69 and 70:
Page 71 and 72:
Page 73 and 74:
Basic Software Skills TutorialLesso
Page 75 and 76:
Page 77 and 78:
Basic Software Skills Tutorial3. Se
Page 79 and 80:
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 97 and 98:
Page 99 and 100:
Page 101 and 102:
Step 3 - Configuring the Plate Docu
Page 103 and 104:
Step 5 - Programming the Plate Docu
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Page 107 and 108:
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:
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 127 and 128:
Page 129 and 130:
Page 131 and 132:
Page 133 and 134:
Page 135 and 136:
Page 137 and 138:
Section 4.2 Running an Individual P
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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 151 and 152:
Page 153 and 154:
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
Page 173 and 174:
Page 175 and 176:
Page 177 and 178:
After the AnalysisSaving theResults
Page 179 and 180:
Analyzing Real-Time Data 66In This
Page 181 and 182:
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
Page 209 and 210:
Viewing ResultsViewing ResultsDispl
Page 211 and 212:
Viewing ResultsAbout DownArrowsDown
Page 213 and 214:
After the AnalysisAfter the Analysi
Page 215 and 216:
Section 6.3 Dissociation Curve Anal
Page 217 and 218:
Before You BeginExample ResultsFigu
Page 219 and 220:
Opening the Run DataOpening the Run
Page 221 and 222:
Determining T m Values for the Anal
Page 223 and 224:
Section 6.4 Procedure ReferenceSect
Page 225 and 226:
Setting the Baseline and Threshold
Page 227 and 228:
Eliminating Outlying AmplificationE
Page 229 and 230:
Eliminating Outlying Amplification6
Page 231 and 232:
After the AnalysisSaving theResults
Page 233 and 234:
Maintaining the Instrument 77In Thi
Page 235 and 236:
Page 237 and 238:
Section 7.1 Maintaining the 7900HT
Page 239 and 240:
Replacing the Sample BlockMaterials
Page 241 and 242:
Replacing the Sample Block9. Loosen
Page 243 and 244:
7900HT FAST Real-Time PCR SystemRep
Page 245 and 246:
Changing the Plate Adapter4. Place
Page 247 and 248:
Decontaminating the Sample BlockCle
Page 249 and 250:
Performing a Background RunPreparin
Page 251 and 252:
Performing a Background RunAnalyzin
Page 253 and 254:
Performing a Pure Dye RunAfter a ru
Page 255 and 256:
Performing a Pure Dye Run• Fast 9
Page 257 and 258:
Performing a Pure Dye RunAnalyzing
Page 259 and 260:
Adding Custom Dyes to the Pure Dye
Page 261 and 262:
Adding Custom Dyes to the Pure Dye
Page 263 and 264:
Verifying Instrument PerformanceTab
Page 265 and 266:
Verifying Instrument PerformancePre
Page 267 and 268:
Section 7.2 Maintaining the Plate H
Page 269 and 270:
Adjusting the Sensitivity of the Pl
Page 271 and 272:
Adjusting the Sensitivity of the Pl
Page 273 and 274:
7900HT FAST Real-Time PCR SystemAli
Page 275 and 276:
Aligning the Plate Handler5. Using
Page 277 and 278:
Aligning the Plate Handler7. Using
Page 279 and 280:
Aligning the Plate Handler13. Click
Page 281 and 282:
7900HT FAST Real-Time PCR SystemAli
Page 283 and 284:
Aligning the Fixed-Position Bar Cod
Page 285 and 286:
Section 7.3 Maintaining the Compute
Page 287 and 288:
Maintaining the SDS softwareSeveral
Page 289 and 290:
Troubleshooting 88In This Chapter T
Page 291 and 292:
Troubleshooting TableTable 8-1Troub
Page 293 and 294:
Low Precision or IrreproducibilityL
Page 295 and 296:
Low Precision or IrreproducibilityD
Page 297 and 298:
Background RunsBackground RunsBackg
Page 299 and 300:
Pure Dye Runs6. Repeat step 4 until
Page 301 and 302:
End-Point Runs (Allelic Discriminat
Page 303 and 304:
Software and 7900HT InstrumentTable
Page 305 and 306:
Zymark Twister Microplate Handler a
Page 307 and 308:
TaqMan Low Density ArrayTable 8-6Tr
Page 309 and 310:
Software ReferenceAAIn This Appendi
Page 311 and 312:
Importing Plate Document Setup Tabl
Page 313 and 314:
Setup Table Files1. File VersionDes
Page 315 and 316:
Setup Table Files6. Detectors ListD
Page 317 and 318:
Setup Table Files9. Markers List (A
Page 319 and 320:
Setup Table FilesFormat(for a singl
Page 321 and 322:
Setup Table FilesFormat(for a singl
Page 323 and 324:
Setup Table FilesExample Code A-16A
Page 325 and 326:
Exporting Plate Document Data4. In
Page 327 and 328:
Operating the SDS Software from a C
Page 329 and 330:
Using the Search ToolLogical Operat
Page 331 and 332:
Connecting SDS Software to the Data
Page 333 and 334:
Designing TaqMan Reagent-BasedAssay
Page 335 and 336:
Assay Development Guidelines• For
Page 337 and 338:
Design Tips for Allelic Discriminat
Page 339 and 340:
Kits, Reagents, and ConsumablesCCIn
Page 341 and 342:
Consumables and DisposablesConsumab
Page 343 and 344:
Instrument Maintenance and Verifica
Page 345 and 346:
Custom Oligonucleotide SynthesisTab
Page 347 and 348:
Theory of OperationDDIn This Append
Page 349 and 350:
Fluorescent-Based ChemistriesBasics
Page 351 and 352:
Real-Time Data AnalysisPhase 2: Lin
Page 353 and 354:
Real-Time Data AnalysisSignificance
Page 355 and 356:
A similar equation for the endogeno
Page 357 and 358:
Limited Warranty StatementEEWarrant
Page 359 and 360:
BibliographyGeneral Paperson TaqMan
Page 361 and 362:
Klein, D., Janda, P., Steinborn, R.
Page 363 and 364:
Dolken, L., F. , Schuler, and G. ,
Page 365 and 366:
AllelicDiscriminationVirtanen M., H
Page 367 and 368:
Glossary.sdd.sdm.sds.sdt∆C T∆
Page 369 and 370:
ackgroundbaselinecalibratorcDNA(com
Page 371 and 372:
minor groovebinder (MGB)multicompon
Page 373 and 374:
R nrunsessionsingle nucleotidepolym
Page 375 and 376:
IndexSymbols- 6-31↑ 6-33Numerics7
Page 377 and 378:
fluorescent, common sources 8-7isol
Page 379 and 380:
adjusting step parameters 3-21confi
Page 381 and 382:
sample block locking bar 7-8sample
Page 384:
Worldwide Sales and SupportApplied
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