ABI Prism® 7900HT Sequence Detection System ... - OpenWetWare

More documents

Recommendations

Info

Section: Dissociation Curve Analysis In This Section This section contains the following information: Topic See Page Overview 6-18 Before You Begin 6-19 Analysis Checklist 6-20 Analyzing the Run Data 6-21 Determining Tm Values for the Analyzed Run 6-22 After the Analysis 6-24 Real-Time Analysis 6-17
Overview About Dissociation Curve Analysis Employing the SYBR Green 1 Dye Mathematical Transformations 6-18 Real-Time Analysis The ABI PRISM 7900HT Sequence Detection System supports dissociation curve analysis of nucleic acids using SYBR® Green 1 double-stranded DNA binding dye chemistry. The objective of dissociation curve analysis is to accurately determine the melting temperature (T m ) of a single target nucleic acid sequence within an unknown PCR sample. Typical uses of dissociation curves include detection of non-specific products and primer concentration optimization. Dissociation curve analysis on the 7900HT instrument is made possible through the use of the fluorogenic SYBR Green 1 double-stranded DNA binding dye chemistry (see page A-3). Dissociation curves are commonly performed following the PCR stage of a SYBR Green experiment to screen for non-specific products. To generate the data needed to create a curve, the 7900HT instrument performs a programmed temperature ‘ramp’ in which it slowly elevates the temperature of the plate over several minutes. The specific binding characteristic of the SYBR Green 1 Dye permits the 7900HT instrument to monitor the hybridization activity of the nucleic acids present in the sample. During the run, the instrument records the decrease in SYBR Green fluorescence resulting from the dissociation of dsDNA. After the run, the SDS software processes the raw fluorescence data from the SYBR Green 1 Dye to generate a more meaningful representation of the relationship between spectral change and temperature for the dissociation curve run. Multicomponenting and Normalization The first mathematical transformation involves the conversion of the raw data, expressed in terms of Fluorescent Signal vs. Wavelength, using the extracted pure dye standards, to pure dye components. After the dye components have been identified, the software determines the contribution of each dye in the normalized data using the multicomponent algorithm (see “Multicomponenting” on page A-5 for a complete description of the process). Finally, the software normalizes the data using the component of the passive reference dye as shown below. R n R( SYBR) --------------------------------------------- R( PassiveReference) Derivation of Dissociation Curve Data The SDS software then computes the first derivative of the normalized data (Rn )for each reading taken by the 7900HT instrument during the temperature ramp. The resulting derivative data (Rn ´) is the rate of change in fluorescence as a function of temperature (see below). R n ′ = = dRn --------dT The software plots the negative of the resulting derivative data on graph of -R n ´ versus temperature (T) that visualizes the change in fluorescence at each temperature interval. The T m for the target nucleic acid can be determined from the graph by identifying the maximum for the rate of change (displayed as a peak) for the appropriate amplification curve.
Page 1 and 2:
ABI PRISM ® 7900HT Sequence Detect
Page 3 and 4:
© Copyright 2001, Applied Biosyste
Page 5 and 6:
iv Section:PlateDocumentSetup......
Page 7 and 8:
vi A Theory of Operation Fluorescen
Page 9 and 10:
Attention Words and Warning Labels
Page 11 and 12:
About Waste Profiles A waste profil
Page 13 and 14:
Safe Instrument Use Before Operatin
Page 16 and 17:
Product Overview 2 In This Chapter
Page 18 and 19:
Section: Getting to Know the Hardwa
Page 20 and 21:
Interchangeable Thermal Cycler Bloc
Page 22 and 23:
Computer Description The computer c
Page 24 and 25:
Zymark Twister Microplate Handler D
Page 26 and 27:
Instrument Connections Electrical C
Page 28 and 29:
Section: Getting to Know the Softwa
Page 30 and 31:
Managing Sequence Detection System
Page 32 and 33:
Getting Started 3 In This Chapter T
Page 34 and 35:
Using the SDS Software Online Help
Page 36 and 37:
Turning on the ABI PRISM 7900HT Seq
Page 38 and 39:
Using the SDS Software Workspace La
Page 40 and 41:
Using the General Toolbar Using the
Page 42 and 43:
Basic Software Skills Tutorial Abou
Page 44 and 45:
Exercise 3: Opening a Plate Documen
Page 46 and 47:
Lesson 2: Viewing and Resizing Pane
Page 48 and 49:
Exercise 2: Selecting Multiple Well
Page 50 and 51:
Lesson 4: Using the Hand-Held Bar C
Page 52 and 53:
Using SDS Plate Documents Using Mul
Page 54 and 55:
Run Setup and Basic Operation 4 In
Page 56 and 57:
Setup Checklists Experiments/Runs P
Page 58 and 59:
Section: Plate Document Setup In Th
Page 60 and 61:
Step 2 - Applying Detectors and Mar
Page 62 and 63:
Creating an Allelic Discrimination
Page 64 and 65:
Step 3 - Configuring the Plate Docu
Page 66 and 67:
Step 4 - Programming the Plate Docu
Page 68 and 69:
To create a method for the absolute
Page 70 and 71:
Step 5 - Saving the Plate Document
Page 72 and 73:
Step 7 - Applying Sample and Plate
Page 74 and 75: Section: Running an Individual Plat
Page 76 and 77: Preparing and Running a Single Plat
Page 78 and 79: Operating the 7900HT Instrument Usi
Page 80 and 81: After the Run Analyzing the Run Dat
Page 82 and 83: Section: Running Multiple Plates Us
Page 84 and 85: Adding a Plate Document to the Plat
Page 86 and 87: To create plate documents from a te
Page 88 and 89: Adding Plates to the Plate Queue Re
Page 90 and 91: Loading Plates To load plates onto
Page 92 and 93: End-Point Analysis 5 In This Chapte
Page 94 and 95: Section: Allelic Discrimination In
Page 96 and 97: Algorithmic Manipulation of Raw All
Page 98 and 99: Before You Begin Using SDS Online H
Page 100 and 101: Analyzing a Completed Allelic Discr
Page 102 and 103: Calling Allele Types To call allele
Page 104 and 105: Scrutinizing the Allele Calls To an
Page 106: After the Analysis Changing the Pla
Page 109 and 110: Real-Time Runs on the 7900HT Instru
Page 111 and 112: Overview About Absolute Quantificat
Page 113 and 114: Before You Begin Using SDS Online H
Page 115 and 116: Analyzing the Run Data Configuring
Page 117 and 118: Setting the Baseline and Threshold
Page 119 and 120: Eliminating Outliers For any PCR, e
Page 121 and 122: Viewing the Standard Curve 6-14 Rea
Page 123: 6-16 Real-Time Analysis
Page 127 and 128: Analysis Checklist WhereYouArein th
Page 129 and 130: Determining T m Values for the Anal
Page 131 and 132: After the Analysis Changing the Pla
Page 133 and 134: Recommended Maintenance Schedule Ma
Page 135 and 136: Replacing the Sample Block When to
Page 137 and 138: 7-6 System Maintenance To remove th
Page 139 and 140: 7-8 System Maintenance To replace t
Page 141 and 142: 7-10 System Maintenance To replace
Page 143 and 144: Cleaning the Sample Block Wells 7-1
Page 145 and 146: Prepare a Background Plate Document
Page 147 and 148: Extracting the Background 7-16 Syst
Page 149 and 150: Materials Required The following ma
Page 151 and 152: Extracting Pure Dye Information fro
Page 153 and 154: Constructing a Custom Pure Dye Plat
Page 155 and 156: Verifying Instrument Performance Us
Page 157 and 158: Preparing and Running an RNase P Pl
Page 159 and 160: Adjusting the Sensitivity of the Pl
Page 161 and 162: Testing the Adjustment 7-30 System
Page 163 and 164: Aligning the Plate Handler When to
Page 165 and 166: 7-34 System Maintenance To align th
Page 167 and 168: Re-checking the Input Stack 1 7-36
Page 169 and 170: Defining the Positions of the Remai
Page 171 and 172: Aligning the Fixed-Position Bar Cod
Page 173 and 174: 7-42 System Maintenance To position
Page 175 and 176:
7-44 System Maintenance
Page 177 and 178:
General Computer Maintenance Mainte
Page 179 and 180:
Maintaining the SDS software Admini
Page 181 and 182:
Troubleshooting Table 8-2 Troublesh
Page 183 and 184:
Low Precision or Irreproducibility
Page 185 and 186:
Writing on the Reaction Plates Fluo
Page 187 and 188:
Background Runs Background Troubles
Page 189 and 190:
Pure Dye Runs Pure Dye Troubleshoot
Page 191 and 192:
8-12 Troubleshooting Troubleshootin
Page 193 and 194:
Software and 7900HT Instrument Trou
Page 195 and 196:
8-16 Troubleshooting Troubleshootin
Page 198:
User Bulletins 9 9 About This Chapt
Page 201 and 202:
Fluorescent-Based Chemistries Funda
Page 203 and 204:
Fluorescence Detection and Data Col
Page 205 and 206:
Normalization of Reporter Signals A
Page 207 and 208:
Determining Initial Template Concen
Page 209 and 210:
Calculating Threshold Cycles A-10 T
Page 212 and 213:
Importing and Exporting Plate Docum
Page 214 and 215:
Configuring the Setup Table File wi
Page 216 and 217:
About the Setup Table File Format S
Page 218 and 219:
Setup Table Elements (continued) Nu
Page 220 and 221:
Exporting Plate Document Data Expor
Page 222 and 223:
Designing TaqMan Assays C In This A
Page 224 and 225:
Design Probes and Primers The follo
Page 226 and 227:
Design Tips for Allelic Discriminat
Page 228 and 229:
Kits, Reagents and Consumables D In
Page 230 and 231:
Consumables and Disposables The ABI
Page 232:
TaqMan Pre-Developed Assays and Rea
Page 236:
Contacting Technical Support F Serv
Page 239 and 240:
G-2 Limited Warranty Statement No a
Page 241 and 242:
A(continued) Automation Controller
Page 243 and 244:
I(continued) installing plate adapt
Page 245 and 246:
Q quantifying probes and primers C-
Page 248 and 249:
Headquarters 850 Lincoln Centre Dri
show all

ABI Prism® 7900HT Sequence Detection System ... - OpenWetWare

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?