Page 1 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide...
Page 2 Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall Applied Biosystems be liable for incidental, special, multiple, or consequential damages in connection with or arising from the use of this document.
Page 3: Table Of Contents
7900HT Instrument ........
Page 4 Powering On the 7900HT Instrument ........
Page 5 End-Point Runs on the 7900HT Instrument ....... . . 5-2...
Page 6 Real-Time Runs on the 7900HT Instrument ....... . . 6-4...
Page 7 Recommended Maintenance Schedule ........7-4 Section 7.1 Maintaining the 7900HT Instrument ......7-5 Replacing the Sample Block .
Page 8 Custom Oligonucleotide Synthesis ........C-6 DRAFT viii Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:40 am, 4351684ATOC.fm...
Page 9 Method of Relative Quantification ......D-8 Appendix E Limited Warranty Statement Bibliography Glossary Index DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:40 am, 4351684ATOC.fm...
Page 10 DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:40 am, 4351684ATOC.fm...
Page 11: Preface
Safety Alert Safety alert words also appear in user documentation. For more information, “Safety Alert Words” page xiv. Words DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, Preface.fm...
Page 12: How To Obtain More Information
In addition, the Services and Support page provides access to worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, Preface.fm...
Page 13 Safety and Electromagnetic Compatibility (EMC) Standards ....xxiii DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide xiii September 1, 2004 11:38 am, Safety.fm...
Page 14: Safety And Emc Compliance Information
ELECTRICAL HAZARD. Failure to ground the instrument properly can lead to an electrical shock. Ground the instrument according to the provided instructions. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, Safety.fm...
Page 15: Symbols On Instruments
Indicates the presence of a hot surface or other high-temperature hazard and to proceed with appropriate caution. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, Safety.fm...
Page 16: Safety Labels On Instruments
DANGER de Class 3B LED en cas d'ouverture. Eviter toute into the beam. exposition directe avec le faisceau. CAUTION Moving parts. ATTENTION Parties mobiles. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, Safety.fm...
Page 17: General Instrument Safety
Applied Biosystems. Using this instrument in a manner not specified by Applied Biosystems may result in personal injury or damage to the instrument. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide xvii September 1, 2004 11:38 am, Safety.fm...
Page 18: Chemical Safety
DOCUMENTS at the end of the document list. 5. After entering the required information, click View/Deliver Selected Documents Now. DRAFT xviii Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, Safety.fm...
Page 19: Chemical Waste Safety
• Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local, state/provincial, or national environmental and health regulations. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, Safety.fm...
Page 20: Electrical Safety
Safety and EMC Compliance Information Waste Profiles A waste profile for the Applied Biosystems 7900HT Fast Real-Time PCR System is provided in the Applied Biosystems 7900HT Fast Real-Time PCR System Site Preparation Guide. Waste profiles show the percentage compositions of the reagents in the waste stream generated during installation and during a typical user application, even though the typical application may not be used in your laboratory.
Page 21: Physical Hazard Safety
Laser Safety Laser The Applied Biosystems 7900HT Fast Real-Time PCR System uses a Class 3B laser. Under normal operating conditions, the instrument laser is categorized as a Class 3B Classification laser. When safety interlocks are disabled during certain servicing procedures, the laser can cause permanent eye damage, and, therefore, is classified under those conditions as a Class 3B laser.
Page 22: Bar Code Scanner Laser Safety
Safety and EMC Compliance Information The Applied Biosystems 7900HT Fast Real-Time PCR System has been tested to and complies with standard EN60825-1, “Radiation Safety of Laser Products, Equipment Classification, Requirements, and User’s Guide.” Laser Safety To ensure safe laser operation: Requirements •...
Page 23: Workstation Safety
Directive 89/336/EEC). This instrument has been tested to and complies with standard EN 61326 (Group 1, Class B), “Electrical Equipment for Measurement, Control and Laboratory Use – EMC Requirements.” DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide xxiii September 1, 2004 11:38 am, Safety.fm...
Page 24 This instrument has been tested to and complies with standard AS/NZS 2064, “Limits and Methods Measurement of Electromagnetic Disturbance Characteristics Standards of Industrial, Scientific, and Medical (ISM) Radio-frequency Equipment.” DRAFT xxiv Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, Safety.fm...
Page 25 7900HT Instrument ........
Page 26: Chapter 1 Product Overview
In an end-point run, the thermal cycling of prepared plates containing reagents and template is performed on a dedicated thermal cycler. Following the PCR, the 7900HT instrument is then used to collect a plate-read or a single reading of the fluorescence resulting from the completed reactions.
Page 27: Section 1.1 Getting To Know The Hardware
7900HT Instrument ........
Page 28: 7900Ht Instrument
Do not remove the cover to the Applied Biosystems 7900HT Fast Real-Time PCR System. Only a qualified Applied Biosystems service engineer may repair or adjust the internal components of the 7900HT instrument. Failure to comply can result in serious injury and/or damage to the instrument.
Page 29 Do not remove the cover to the 7900HT instrument. Only a qualified IMPORTANT! Applied Biosystems service engineer may repair or adjust the internal components. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, CH_Overview.fm...
Page 30: Computer
• (Optional) SDS Enterprise Database Client Software *Applied Biosystems recommends that you do not install programs to the C drive. The computer will boot faster if the C drive contains only the operating system. †Software required for the operation of the bar code reader.
Page 31: Bar Code Readers
Aligning the Fixed-Position Bar Code Reader ......7-49 DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, CH_Overview.fm...
Page 32: Zymark Twister Microplate Handler
Twister Microplate Handler coordinates plate handling for the 7900HT instrument, permitting 24-hour unattended operation. The arm provides a 310-degree rotational swing that permits access to the 7900HT instrument drawer, up to five plate stacking areas, and the fixed-position bar code reader.
Page 33: Compatible Consumables
Biosystems recommends that you load and unload the plates manually. Consumable Appendix C, “Kits, Reagents, and Consumables,” for a list of available consumables, requirements, and purchasing instructions. Requirements DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, CH_Overview.fm...
Page 34: Instrument Connections
Computer (ISA Card 1) Fixed-Position Bar Code Reader Comm/Power Bar Code Reader Cable Keyboard (not shown) *See Figure 1-9 on page 1-11. DRAFT 1-10 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, CH_Overview.fm...
Page 35 1. Use only the cord sets supplied in the bag marked “JAPAN ONLY.” 2. Discard all other unused cord sets. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 1-11 September 1, 2004 11:38 am, CH_Overview.fm...
Page 36 Chapter 1 Product Overview DRAFT 1-12 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, CH_Overview.fm...
Page 37: Section 1.2 Getting To Know The Software
Managing Sequence Detection System Data......1-17 DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 1-13...
Page 38: Software Components
Client Software on the network. Note: page 1-21 for more information on the software components of the SDS Enterprise Database. DRAFT 1-14 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, CH_Overview.fm...
Page 39: Sds Software Related Files And Formats
SDS Software The SDS software uses many different files and directories (Table 1-5). Some of these files store run data and calibration data: others are required to run the 7900HT instrument. Table 1-5 Common SDS Files File Type Ext.
Page 40 Because of memory constraints, the SDS Enterprise Database cannot IMPORTANT! save real-time plate documents greater than 40 MB. DRAFT 1-16 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, CH_Overview.fm...
Page 41: Managing Sequence Detection System Data
(see page 1-18) Stand-Alone Operation This mode uses the most basic configuration of the Applied Biosystems 7900HT Fast Real-Time PCR System (Figure 1-11) where the 7900HT instrument is used without any additional components. In this configuration, a technician runs plates individually using the SDS software which stores all data on the computer hard drives.
Page 42 Chapter 1 Product Overview Automated Operation In this mode, the Applied Biosystems 7900HT Fast Real-Time PCR System uses an Automation Accessory (Zymark Twister Microplate Handler and fixed-position bar code reader), to provide high-throughput operation suitable for small- to medium-scale studies. With this configuration...
Page 43: Section 1.3 Sds Enterprise Database
Analysis Sessions Analyzed as Studies Figure 1-13 Automated Operation of the of the 7900HT Instrument with SDS Enterprise Database DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 1-19 September 1, 2004 11:38 am, CH_Overview.fm...
Page 44 Chapter 1 Product Overview DRAFT 1-20 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, CH_Overview.fm...
Page 45 Supporting API Documentation ........1-28 DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 1-21...
Page 46: About The Sds Enterprise Database Feature
System. In addition to serving as a common repository for SDS data, the database provides user authentication, robust and scalable data management, and flexible archive capabilities. The database also provides the infrastructure and tools necessary to integrate a 7900HT instrument into a laboratory workflow made up of existing data management solutions. Access Control...
Page 47 RQ Manager Software, Controller Software or SNP Manager Software Router Figure 1-14 Basic LAN Architecture for the SDS Enterprise Database DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 1-23 September 1, 2004 11:38 am, CH_Overview.fm...
Page 48 RQ Manager Software, Controller Software or SNP Manager Software Router Laboratory Office Figure 1-15 WAN Architecture for the SDS Enterprise Database DRAFT 1-24 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, CH_Overview.fm...
Page 49: About The Sds Enterprise Database Software Suite
153,600 multiplexed reactions run on an Applied Biosystems 7900HT Fast Real-Time PCR System. See the RQ Manager Software User Guide (PN 4351670) for more information. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 1-25 September 1, 2004 11:38 am, CH_Overview.fm...
Page 50: Database Management Utilities
See the SDS Enterprise Database for the Applied Biosystems 7900HT Fast Real-Time PCR System Administrators Guide (PN 4351669) for more information. DRAFT 1-26 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, CH_Overview.fm...
Page 51: Database Design And Information Management
Figure 1-18 SDS Enterprise Database Data Flow Diagram DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 1-27 September 1, 2004 11:38 am, CH_Overview.fm...
Page 52: Supporting Api Documentation
Plate documents have a 1:1 correspondence with the optical plate or Low Density Array they represent. Each persisted plate documents consists of a collection of plate setup information for running a plate on the 7900HT instrument including: • Sample/well assignments, •...
Page 53 Powering On the 7900HT Instrument ........
Page 54: Chapter 2 Getting Started
Chapter 2 Getting Started Getting Started Before You Begin If this is your first time using the Applied Biosystems 7900HT Fast Real-Time PCR System, consider completing the “Basic Software Skills Tutorial” on page 2-10 before continuing. The tutorial will provide you with the fundamental knowledge required to operate the SDS software and will teach you timesaving techniques to allow you to use it quickly and efficiently.
Page 55 • Microsoft Windows Operating System Online Help • SDS Enterprise Database for the Applied Biosystems 7900HT Fast Real-Time PCR System Administrators Guide (PN 4351669) DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, CH_GetStart.fm...
Page 56: Powering On The 7900Ht Instrument
If operating normally, the Plate Handler moves the arm to the home position (over the output stack). 3. Power on the 7900HT instrument by pressing the power button located below the status lights on the front of the instrument (see Figure 2-1).
Page 57 Doing so prevents the instrument from uploading the firmware from the computer and causes the software to display an error. Reading the The 7900HT instrument contains three lights located on the lower-left side of the front panel to indicate the status of the instrument (see Figure 2-2).
Page 58 30 sec, and then restart. Note: If the problem persists, contact Applied Biosystems Technical Support. (Red) DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, CH_GetStart.fm...
Page 59: Using The Sds Software
7900HT the SDS software (see page 2-9) instrument (see page 2-9) Figure 2-3 Elements of the SDS Software User Interface DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, CH_GetStart.fm...
Page 60 2-12) – Zooms the plate grid (see page 2-12) – Opens the Display Settings dialog box (see page 2-12) DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, CH_GetStart.fm...
Page 61 The metered bar to the right of the message displays the progress of the action. About the The Instrument Status Icon indicates the status of the connection to the 7900HT instrument. Instrument Status Icon – The computer has successfully connected to the instrument and is awaiting a command –...
Page 62: Basic Software Skills Tutorial
3. In the SDS Online Help, click Basic Skills Tutorial from the list of options. 4. Follow the directions displayed on your screen. DRAFT 2-10 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, CH_GetStart.fm...
Page 63: Notes For Database Users
Enter your user name Enter your password Figure 2-5 Electronic Signature Verification Dialog Box Options DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 2-11 September 1, 2004 11:38 am, CH_GetStart.fm...
Page 64: Lesson 1: Using Plate Documents
Chapter 6, “Analyzing Real-Time Data.” The exercises in this lesson will familiarize you with the use of plate documents. DRAFT 2-12 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, CH_GetStart.fm...
Page 65 Basic Software Skills Tutorial Exercise 1: You will need to create a plate document for every plate you run on the 7900HT instrument. Creating a Plate Document The SDS software can handle multiple documents simultaneously, IMPORTANT! however the processing speed of your computer will decrease with each open document.
Page 66 The software opens the plate document template. 6. Export the plate setup information from the plate document template as explained on page 2-15. DRAFT 2-14 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, CH_GetStart.fm...
Page 67 Plate Document” on page 2-13. 9. Import the exported plate setup information into the plate document as explained on page 2-16. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 2-15 September 1, 2004 11:38 am, CH_GetStart.fm...
Page 68 For more information on importing and exporting setup table data using the SDS software, see Appendix A, “Software Reference.” 6. Save the plate document as explained on page 2-17. DRAFT 2-16 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, CH_GetStart.fm...
Page 69 The software saves the plate document to a file entitled Practice.sds. 6. Close the saved plate document file as explained on page 2-18. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 2-17 September 1, 2004 11:38 am, CH_GetStart.fm...
Page 70 The SDS software closes the file without saving it. 9. Open a plate document file as explained on page 2-19. DRAFT 2-18 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, CH_GetStart.fm...
Page 71 3. In the Look In field, select the Practice.sds file. Select 4. Click The software opens the Practice.sds file. 5. Select File > Close. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 2-19 September 1, 2004 11:38 am, CH_GetStart.fm...
Page 72 Click to open the document 5. Click 6. Go on to “Lesson 2: Viewing and Resizing Panes” on page 2-21. DRAFT 2-20 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, CH_GetStart.fm...
Page 73: Lesson 2: Viewing And Resizing Panes
3. Practice hiding and showing the other plate document elements by clicking other buttons in the Display toolbar until you are comfortable using the feature. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 2-21 September 1, 2004 11:38 am, CH_GetStart.fm...
Page 74 3. Using the sizing buttons, maximize/minimize other elements of the plate document until you are comfortable using the feature. DRAFT 2-22 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, CH_GetStart.fm...
Page 75: Lesson 3: Using The Plate Grid
The software displays the information for the well in a yellow pop-up window. Pop-up displays the information of the well to which the mouse cursor points DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 2-23 September 1, 2004 11:38 am, CH_GetStart.fm...
Page 76 The software outlines the selected wells with a black border. b. While holding down the Ctrl key, click the wells to de-select them. DRAFT 2-24 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, CH_GetStart.fm...
Page 77 5. Using the techniques illustrated in steps 1 to 4, practice selecting portions of the plate grid until you are comfortable using the feature. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 2-25 September 1, 2004 11:38 am, CH_GetStart.fm...
Page 78 The software resizes all wells in the plate grid to match the new width. Mouse cursor 3. Practice resizing the wells of the plate grid until you are comfortable using the feature. DRAFT 2-26 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, CH_GetStart.fm...
Page 79: Lesson 4: Using The Hand-Held Bar Code Reader
4. Click to close the Document Information dialog box. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 2-27 September 1, 2004 11:38 am, CH_GetStart.fm...
Page 80: Lesson 5: Using Contextual Menus
SDS software. To view the list, open the online help as explained in “Using the SDS Software Online Help” on page 2-3. DRAFT 2-28 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, CH_GetStart.fm...
Page 81: Using Sds Plate Documents
(see Appendix A, “Software Reference,” for more information). DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 2-29 September 1, 2004 11:38 am, CH_GetStart.fm...
Page 82 The tab is visible only in plate documents containing analyzed data from a real-time run with a programmed ramp. DRAFT 2-30 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, CH_GetStart.fm...
Page 83 • Passive Reference drop-down list – Displays the fluorescent dye used as a passive reference DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 2-31 September 1, 2004 11:38 am, CH_GetStart.fm...
Page 84 Chapter 2 Getting Started DRAFT 2-32 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, CH_GetStart.fm...
Page 85 Step 8 – Applying Sample and Plate Information ......3-28 Step 9 – Running the Plate on the 7900HT Instrument..... 3-29...
Page 86: Chapter 3 Preparing A Run
2. After the software validates your user name and password, it displays a message stating the success or failure of the signature. Click to continue. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Run.fm...
Page 87: Notes For Database Users
Description of the action that requires a signature Enter your user name Enter your password Figure 3-2 Electronic Signature Verification Dialog Box Options DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Run.fm...
Page 88: Before You Begin
Before You Begin Background Chapter 5, “Analyzing End-Point Data,” Chapter 6, “Analyzing Real-Time Data,” include brief discussions of the experiments that you can perform using the 7900HT Information instrument. Before beginning, you may want to review the appropriate chapter for your experiment: Allelic Discrimination .
Page 89: Workflow Overview
See “Importing Plate Document Setup Table Files” on page A-2 for more information. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Run.fm...
Page 90 3-5 and configure the Workflow method with a temperature ramp as explained on page 3-23. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Run.fm...
Page 91 See “Importing Plate Document Setup Table Files” on page A-2 for more information. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Run.fm...
Page 92: Quick Review: Powering On The 7900Ht Instrument
5. If using the SDS Enterprise Database, enter your user name and password in the appropriate fields of the Login dialog box, then click The SDS software displays the program window. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Run.fm...
Page 93: Step 1 - Creating A Plate Document
Administrator User Group to create plate documents. Requirement About ABI P Every plate run on the 7900HT instrument requires the creation of a plate document RISM within the SDS software. Each plate document is a virtual representation of a Plate Documents specific consumable (that is, a reaction plate or Low Density Array) containing samples and reagents for use on the 7900HT instrument.
Page 94 5. Create and copy detectors (and markers) to the new plate document as described “Step 2 – Applying Detectors and Markers” on page 3-11. DRAFT 3-10 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Run.fm...
Page 95: Step 2 - Applying Detectors And Markers
(up to 200 characters). Name field Group field Description field Reporter/Quencher drop-down lists Color box Notes field Creation and Modification date stamps DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 3-11 September 1, 2004 11:39 am, CH_Run.fm...
Page 96 • dissociation curve analysis allelic discrimination create and apply markers to the plate document as explained on page 3-14. DRAFT 3-12 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Run.fm...
Page 97 6. Configure the plate document with detector tasks as explained in “Step 3 – Configuring the Plate Document with Tasks” on page 3-16. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 3-13 September 1, 2004 11:39 am, CH_Run.fm...
Page 98 7. Apply the marker(s) to the allelic discrimination plate document as explained in “Copying and Applying Markers” on page 3-15. DRAFT 3-14 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Run.fm...
Page 99 6. Configure the plate document with detector tasks as explained in “Step 3 – Configuring the Plate Document with Tasks” on page 3-16. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 3-15 September 1, 2004 11:39 am, CH_Run.fm...
Page 100: Step 3 - Configuring The Plate Document With Tasks
The SDS software labels all selected wells with the task. Task drop-down list Selected wells (task applied to selection) DRAFT 3-16 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Run.fm...
Page 101 • Otherwise, program the method for your run as explained in “Step 5 – Programming the Plate Document Method” on page 3-19. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 3-17 September 1, 2004 11:39 am, CH_Run.fm...
Page 102: Step 4 - Setting The Passive Reference And Omitting Wells
(removed from use) Omit Well(s) check box (selected) 3. Program the method for the run as explained on page 3-19. DRAFT 3-18 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Run.fm...
Page 103: Step 5 - Programming The Plate Document Method
Longer runs and runs configured to collect data at multiple stages of the method can be considerably larger than the 15–25 MB average. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 3-19 September 1, 2004 11:39 am, CH_Run.fm...
Page 104 Performing Thermal Cycling of Allelic Discrimination Plates on the 7900HT Instrument To perform the thermal cycling and the plate read using the 7900HT instrument, run the plate first as a real-time plate document and then again as an allelic discrimination plate document as explained below.
Page 105 2. If necessary, select or de-select the 9600 Emulation check box. When the 9600 Emulation check box is checked, the SDS software Note: reduces the ramp rate of the 7900HT instrument to match that of the ABI P ® 7700 Sequence Detection System instrument.
Page 106 3-23. DRAFT 3-22 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Run.fm...
Page 107 Step 5 – Programming the Plate Document Method Programming a To generate the data required to perform a dissociation curve analysis, the 7900HT instrument must be programmed to run a ‘temperature ramp’ in which it slowly Temperature elevates the temperature of the samples while collecting fluorescence measurements...
Page 108: Step 6 - Saving The Plate Document As A Template
The SDS software applies the new display settings to the plate document. 4. Go to “Saving the Plate Document as a Template (Optional)” on page 3-25. DRAFT 3-24 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Run.fm...
Page 109 The SDS software closes the plate document template. 6. Create a plate document from the plate document template as explained on page 3-26. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 3-25 September 1, 2004 11:39 am, CH_Run.fm...
Page 110: Step 7 - Creating A Plate Document From The Template
– Scan the plate bar code using the hand-held bar code scanner. 3. Click to create a new plate document from the plate document template. DRAFT 3-26 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Run.fm...
Page 111 Step 7 – Creating a Plate Document from the Template 4. Configure the new plate document with sample names and plate information as explained on page 3-28. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 3-27 September 1, 2004 11:39 am, CH_Run.fm...
Page 112: Step 8 - Applying Sample And Plate Information
• Do not modify the bar code of a plate document that contains data from a run. 3. When finished, click 4. Run the plate document and associated plate as explained on page 3-29. DRAFT 3-28 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Run.fm...
Page 113: Step 9 - Running The Plate On The 7900Ht Instrument
A plate document must be saved to the database before a user IMPORTANT! belonging to the Operator User Group can run it. Options The 7900HT instrument can run prepared plates individually or in groups using the Zymark Twister Microplate Handler. for Running SDS Plates...
Page 114 Chapter 3 Preparing a Run DRAFT 3-30 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Run.fm...
Page 115 After the Run ........... . . 4-44 DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Basic.fm...
Page 116: Chapter 4 Operating The Instrument
2. After the software validates your user name and password, it displays a message stating the success or failure of the signature. Click to continue. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Basic.fm...
Page 117 Description of the action that requires a signature Enter your user name Enter your password Figure 4-2 Electronic Signature Verification Dialog Box Options DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Basic.fm...
Page 118: Before You Begin
Chapter 4 Operating the Instrument Before You Begin Pre-Run The following tasks must be complete to run plates on the 7900HT instrument. See the associated page number for details on each procedure. Checklist Done Check See Page A background run has been performed in the last month...
Page 119: Section 4.1 Consumable Preparation
Preparing TaqMan Low Density Arrays for Use......4-10 DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Basic.fm...
Page 120: Preventing Contamination
™ Optical Adhesive Covers 384-Well Septa RISM RISM 384-Well Optical (quantity 100) Reaction Plates • Optical Adhesive Covers (quantity 25) DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Basic.fm...
Page 121 (quantity 100) Cycling Plates • Optical Adhesive Covers (quantity 25) TaqMan ® None (self sealing) None (self sealing) Density Arrays DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Basic.fm...
Page 122: Preparing Optical Plates For Use
IMPORTANT! Instrument plate must match the configuration of information (sample names and detectors/markers) on the corresponding plate document. 2. Seal the optical plate with a seal that is compatible with the 7900HT instrument (Figures 4-3 page 4-9). “Compatible Consumables” on page 4-6...
Page 123 Figure 4-3 96-Well Optical Reaction Plate Assembly Optical adhesive cover 384-well plate Figure 4-4 384-Well Optical Reaction Plate Assembly DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Basic.fm...
Page 124: Preparing Taqman Low Density Arrays For Use
Barcode Provides coded access to GR2156a Low Density Array configuration databases Figure 4-5 The TaqMan Low Density Array DRAFT 4-10 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Basic.fm...
Page 125 Functional verification of the preloaded probes and primers inside the Low Density Array is performed as part of the Applied Biosystems manufacturing quality control process. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-11 September 1, 2004 11:39 am, CH_Basic.fm...
Page 126: About The Centrifuge System
Array’s fill reservoirs during centrifugation Bucket Contains the Low Density Arrays and card holder during centrifugation Figure 4-8 Centrifuge System Components DRAFT 4-12 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Basic.fm...
Page 127 Buttons 1–4 are programming buttons. Up ramp Down ramp Time rate control rate control controls controls 4. Power off the centrifuge. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-13 September 1, 2004 11:39 am, CH_Basic.fm...
Page 128: About The Sealer
Low Density Array’s fill reservoirs to its wells are open and undamaged. DRAFT 4-14 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Basic.fm...
Page 129 Low Density Array must be centrifuged. Continue with “Centrifuging the TaqMan Low Density Arrays” on page 4-16. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-15 September 1, 2004 11:39 am, CH_Basic.fm...
Page 130: Centrifuging The Taqman Low Density Arrays
4. Place a filled card holder in the bucket so that the “This Side Out” label faces the front of the bucket, which has the Sorvall emblem on it. Sorvall emblem here DRAFT 4-16 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Basic.fm...
Page 131 1-min spins to ensure complete distribution of the sample-specific PCR reaction mix. 3. Open the centrifuge cover by pressing the OPEN button. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-17 September 1, 2004 11:39 am, CH_Basic.fm...
Page 132 Do not exceed 1200 rpm or accumulated centrifugation times of IMPORTANT! more than 3 min. Excessive centrifugation speeds and times may deform the Low Density Array. DRAFT 4-18 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Basic.fm...
Page 133: Sealing The Taqman Low Density Arrays
If you move the carriage across the Low Density Array to return it to its starting position, serious damage to the Low Density Array will occur. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-19 September 1, 2004 11:39 am, CH_Basic.fm...
Page 134 Using force will not increase the sealing quality but only cause wear to the sealer. End position GR2176 Move in this direction: DRAFT 4-20 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Basic.fm...
Page 135 Use scissors rather than razor blades or other unprotected cutting devices. The Low Density Array is now ready to be run on the 7900HT instrument. 2. Choose from the following. If performing: – Absolute or Relative Quantification, or Dissociation Curve Analysis Run the plate as explained in “Running the Plate”...
Page 136 Chapter 4 Operating the Instrument DRAFT 4-22 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Basic.fm...
Page 137: Section 4.2 Running An Individual Plate
After the Run ........... . . 4-29 Pre-Run The following tasks must be complete to run plates on the 7900HT instrument. See the associated page number for details on each procedure.
Page 138: Saving The Plate Document
3. In the Saved Document dialog box, click 4. Run the plate document and associated plate as explained on page 4-25. DRAFT 4-24 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Basic.fm...
Page 139: Running A Single Plate (Using The Sds Software)
Well A1 Notched corner Bar code The A1 position is located in the top-left side of the instrument. IMPORTANT! DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-25 September 1, 2004 11:39 am, CH_Basic.fm...
Page 140 Open the instrument tray (after the run) 4-29 Analyze the run data after the run is complete 4-29 DRAFT 4-26 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Basic.fm...
Page 141 For more information on the elements of the Real-Time and Plate-Read tabs, Note: click and see the Sequence Detection Systems Software Online Help. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-27 September 1, 2004 11:39 am, CH_Basic.fm...
Page 142 4. If necessary, eject the plate and add the missing reaction component. 5. If desired, re-run the plate by re-creating the plate document. DRAFT 4-28 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Basic.fm...
Page 143: After The Run
Section 6.3 Dissociation Curve Analysis ....... 6-37 DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-29...
Page 144 SDS software and the Automation Controller Software. Because both programs the Instrument control the Applied Biosystems 7900HT Fast Real-Time PCR System, one program must relinquish control of the 7900HT instrument before the other can be used to operate it. To…...
Page 145: Section 4.3 Automated Operation
After the Run ........... . . 4-44 Pre-Run The following tasks must be complete to run plates on the 7900HT instrument. See the associated page number for details on each procedure.
Page 146: Operating The Software With An Sds Enterprise Database
5. Prepare and load the plates onto the Plate Handler as explained in “Loading Plates onto the Plate Handler” on page 4-41. DRAFT 4-32 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Basic.fm...
Page 147 To access the Save Analysis Results option by selecting Enterprise > Database Options. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-33 September 1, 2004 11:39 am, CH_Basic.fm...
Page 148: Operating The Software Without An Sds Enterprise Database
– Absolute Quantification Analysis (see page 6-5) – Relative Quantification Analysis (see page 6-15) – Dissociation Curve Analysis (see page 6-37) DRAFT 4-34 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Basic.fm...
Page 149: Adding A Plate Document To The Plate Queue (Using The Sds Software)
8. When you finish creating plate documents, run the queued plates as explained in “Starting and Configuring the Automation Controller Software for Operation” on page 4-39. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-35 September 1, 2004 11:39 am, CH_Basic.fm...
Page 150: Creating Plate Documents Using The Template Batch Utility
Select the Queue tab. 3. Click 4. Configure the Template Batch dialog box with Plate IDs: a. Click DRAFT 4-36 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Basic.fm...
Page 151 8. Click to close the Template Batch dialog box. 9. Select File > Close to close the plate document template. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-37 September 1, 2004 11:39 am, CH_Basic.fm...
Page 152 11. When finished creating and adding plate documents to the queue, run the queue as explained in “Starting and Configuring the Automation Controller Software for Operation” on page 4-39. DRAFT 4-38 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Basic.fm...
Page 153: Starting And Configuring The Automation Controller Software For Operation
The Applied Biosystems 7900HT Fast Real-Time PCR System employs the Automation Controller Software for automated operation of the 7900HT instrument. Configuring the The software coordinates the action of the 7900HT instrument, the bar code reader, Software and the Plate Handler while acquiring and saving raw data during each run.
Page 154 Clear All Plates. 2. Click The software removes all plate documents from the plate queue. DRAFT 4-40 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Basic.fm...
Page 155: Running Plates Using The Automation Controller Software
Zymark Twister Microplate Handler (top view) Bar code Well A1 Figure 4-11 Plate Positions of the Zymark Twister Microplate Handler DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-41 September 1, 2004 11:39 am, CH_Basic.fm...
Page 156 Use the Restack when finished function only when necessary. 4. Begin the plate queue as explained on page 4-43. DRAFT 4-42 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Basic.fm...
Page 157: Running The Instrument
If found, the Automation Controller Software loads the plate document information from the file system for the run. 4. The 7900HT instrument loads the instrument tray and runs the plate according to the parameters defined by the plate document file. Note: Before starting a real-time run, the instrument may pause (up to 15 minutes) to heat the heated cover to the appropriate temperature.
Page 158: After The Run
Section 6.3 Dissociation Curve Analysis....... .6-37 DRAFT 4-44 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Basic.fm...
Page 159 In This Chapter End-Point Runs on the 7900HT Instrument ......5-2 Notes for Database Users .
Page 160: Chapter 5 Analyzing End-Point Data
Currently, the SDS software supports one type of end-point analysis: Allelic Discrimination. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_End-Point.fm...
Page 161: Notes For Database Users
2. After the software validates your user name and password, it displays a message stating the success or failure of the signature. Click to continue. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_End-Point.fm...
Page 162 Description of the action that requires a signature Enter your user name Enter your password Figure 5-2 Electronic Signature Verification Dialog Box Options DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_End-Point.fm...
Page 163: Section 5.1 Allelic Discrimination
After the Analysis ..........5-18 DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_End-Point.fm...
Page 164: Overview
Allelic discrimination chemistry can be used for single-nucleotide polymorphism Instrument (SNP) detection. Employing the Allelic discrimination on the 7900HT instrument is made possible through the use of the fluorogenic 5´ nuclease assay (see page D-2). During the PCR, the fluorogenic 5´...
Page 165 The Maximum Likelihood Algorithm is designed to use No Template Controls, and autocalling is most effective when NTCs are tasked. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_End-Point.fm...
Page 166 • Contain rare sequence variations • Contain sequence duplications • Not contain a crucial reagent for amplification (the result of a pipetting error) DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_End-Point.fm...
Page 167: Before You Begin
“Assay Development Guidelines” on page B-2. The SDS software does not require that absolute quantification plates IMPORTANT! contain positive controls. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_End-Point.fm...
Page 168: Analysis Checklist
– Adjust the display settings for the plate document. – Print elements of the plate document. – Export the plate document results table or plots. DRAFT 5-10 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_End-Point.fm...
Page 169: Opening The Run Data
4. In the Search Results list, scroll to and select the session that you want to analyze. 5. Click 6. Click when finished. 7. Configure the Analysis Settings for each marker as explained below. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 5-11 September 1, 2004 11:39 am, CH_End-Point.fm...
Page 170: Analyzing An Allelic Discrimination Run
(or select Analysis > Analyze). The SDS software analyzes the run data and displays the results in the Results tab. DRAFT 5-12 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_End-Point.fm...
Page 171: Calling And Scrutinizing Allelic Discrimination Data
Zooms out on the plot by clicking the mouse button within the plot. • Scatterplot – A scatterplot of data points from the run. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 5-13 September 1, 2004 11:39 am, CH_End-Point.fm...
Page 172 Black border (surrounding selected wells) 3. Select the sample cluster exhibiting amplification of the first probe. Allele X homozygotes DRAFT 5-14 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_End-Point.fm...
Page 173 Repeat steps a and b until the alleles for each marker have been called. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 5-15 September 1, 2004 11:39 am, CH_End-Point.fm...
Page 174 • Contain sequence duplications • Contain contaminants • Be the result of errors in pipetting or sample preparation Undetermined samples DRAFT 5-16 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_End-Point.fm...
Page 175 Repeat steps a and b until the calls for each marker have been verified. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 5-17 September 1, 2004 11:39 am, CH_End-Point.fm...
Page 176: After The Analysis
To export a plot as a graphic file, see “Exporting Graphics” on page A-16 or click within the plot of interest for further instructions. DRAFT 5-18 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_End-Point.fm...
Page 177 In the Description field, enter a brief description of the study (up to 255 characters). c. Click d. Click DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 5-19 September 1, 2004 11:39 am, CH_End-Point.fm...
Page 178 The SDS software does not require that the file name match the bar code Note: of the corresponding plate. 4. Click The software saves the plate document to the specified directory. DRAFT 5-20 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_End-Point.fm...
Page 179 Notes for Database Users ..........6-2 Real-Time Runs on the 7900HT Instrument ......6-4 Section 6.1 Absolute Quantification .
Page 180: Chapter 6 Analyzing Real-Time Data
2. After the software validates your user name and password, it displays a message stating the success or failure of the signature. Click to continue. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 181 Description of the action that requires a signature Enter your user name Enter your password Figure 6-2 Electronic Signature Verification Dialog Box Options DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 182: Real-Time Runs On The 7900Ht Instrument
Instead of a standard curve, the Comparative Method calculates relative gene expression using the equation: –∆∆C Relative Quantity = 2 DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 183: Section 6.1 Absolute Quantification
After the Analysis ..........6-14 DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 184: Overview
TaqMan reagents targeting a specific nucleic acid sequence. The plate is then loaded into a 7900HT instrument which has been configured to perform a real-time run. During the thermal cycling, the instrument records the emission resulting from the ®...
Page 185 Results tab. The graph consists of a scatterplot of standard and unknown samples graphed on a linear-scale plot of Threshold Cycle (C ) versus quantity value. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 186: Before You Begin
20,000 copies) and two replicate populations (5000 and 10,000 copies). Population 1 Population 2 5000 10000 Figure 6-4 Sample Configuration of the Example Plate DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 187: Analysis Checklist
– Print elements of the plate document. – Export the plate document results table or plots. – Save the analysis. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 188: Opening The Run Data
10. When the software has loaded the plate document, click 11. Configure the Analysis Settings for each marker as explained in “Configuring the Analysis Settings” on page 6-11. DRAFT 6-10 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 189: Analyzing The Data
In the Threshold field, enter a threshold value to apply to the selected detector(s) or leave the field empty and set the threshold value manually as explained on page 6-46. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-11 September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 190 3. If you choose to remove outliers manually, visualize and eliminate outliers from the analyzed run data as explained on page 6-46. DRAFT 6-12 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 191: Viewing Results
• Standard curve plot – A scatterplot of datapoints from the absolute quantification run. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-13 September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 192: After The Analysis
..........6-53 DRAFT 6-14 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 193: Section 6.2 Relative Quantification
After the Analysis ..........6-35 DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-15...
Page 194: Overview
After the assays are verified, unknown samples are loaded onto an optical plate containing master mix and 5′-nuclease assays targeting a specific nucleic acid sequence. The reaction plate is then run on a 7900HT instrument that is configured for real-time analysis.
Page 195: Algorithmic Manipulation Of Raw Data
Equation, see: – ABI P 7700 Sequence Detection System User Bulletin #2: Relative RISM Quantification Of Gene Expression (PN 4303859) DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-17 September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 196 For this reason, Applied Biosystems strongly recommends reviewing all baseline and threshold parameters determined by the AutoC algorithm following analysis of the study data. DRAFT 6-18 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 197: Automatic Outlier Removal
Limits must be determined experimentally for each relative quantification assay (all targets and the endogenous control). DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-19 September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 198: Essential Experimental Components
(such as contamination, pipetting errors, and so on). DRAFT 6-20 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 199: Getting Started
For more information on the TaqMan Cytokine Gene Expression Plate I, see Note: the TaqMan Cytokine Gene Expression Plate I Protocol (PN 4306744). DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-21 September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 200: Analysis Checklist
– Adjust the display settings for the results table, plate grid, and plate document plots. – Print elements of the plate document. – Export the plate document results table or plots. DRAFT 6-22 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 201: Options For Analyzing Relative Quantification Data
4. Add the relative quantification plate documents to the study as explained in “Adding Plate Documents to the Study” on page 6-24. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-23 September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 202 (directory or hard drive) to search. c. If you want the SDS software to search all subdirectories in the chosen location, select Include Subdirectories. DRAFT 6-24 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 203 4. When you finish adding files to the study, click 5. Configure the analysis settings for the study as explained in “Configuring the Analysis Settings” on page 6-26. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-25 September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 204: Analyzing The Study
AutoCalling (see page 6-46). DRAFT 6-26 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 205 You can also identify and eliminate outliers manually, as explained in Note: “Eliminating Outlying Amplification” on page 6-49. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-27 September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 206 – Instructs the software to export the ∆C values of individual samples in the analysis instead of the average ∆C values for the replicate group. DRAFT 6-28 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 207: Specifying The Analysis Settings
7. In the Calibrator Sample drop-down list, select the sample that you want to use as the calibrator for your study. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-29 September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 208: Analyzing The Study Data
3. If you choose to remove outliers manually, visualize and eliminate outliers from the analyzed run data as explained on page 6-46. DRAFT 6-30 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 209: Viewing Results
The expression minimum ( ) and maximum ( ) symbols are described in Note: detail on the page 6-33. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-31 September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 210 (or —): Indicates that the amplification curves for the associated sample replicate group never crossed the threshold during the duration of the run (‘undetermined’ values). DRAFT 6-32 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 211 (if applicable), and averages the resulting data. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-33 September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 212 • Rejected – When autocalling is enabled, indicates if the sample data was rejected. DRAFT 6-34 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 213: After The Analysis
......... . (see below) Saving the After an analysis, the SDS software allows you to save the results as an SDS 7900HT Multiple Plate Document (*.sdm).
Page 214 Chapter 6 Analyzing Real-Time Data DRAFT 6-36 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 215: Section 6.3 Dissociation Curve Analysis
After the Analysis ..........6-44 DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-37...
Page 216: Overview
D-3). Dissociation curves are commonly performed following the PCR stage of a SYBR Green dye 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.
Page 217: Before You Begin
Each well of the plate contains SYBR Green 1 dye, forward and reverse primers, and genomic DNA known to contain complimentary binding sites. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-39 September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 218: Analysis Checklist
– Adjust the display settings for the results table, plate grid, and plate document plots. – Print elements of the plate document. – Export the plate document results table or plots. DRAFT 6-40 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 219: Opening The Run Data
3. Determine the T for the dissociation curves displayed within the Dissociation Curve tab as explained on page 6-44. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-41 September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 220: Determining T Values For The Analyzed Run
Step drop-down list allows you to displays the data from each by selecting the position of the ramp in the thermal profile. DRAFT 6-42 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 221 Figure 6-16 Plots of the Dissociation Curve Tab • Detector drop-down list – Chooses the data displayed within the plot based on detector name. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-43 September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 222: After The Analysis
..........6-53 DRAFT 6-44 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 223: Section 6.4 Procedure Reference
After the Analysis ..........6-52 DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-45...
Page 224: Setting The Baseline And Threshold Values
The software displays the data for the selected wells within the Amplification plot. 4. In the Plot drop-down list, select ∆R vs. Cycle. 5. Double-click the Amplification plot, or click DRAFT 6-46 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 225 Increase the Stop baseline value by dragging the right range marker ( ) to a later cycle. 8. Double-click the Amplification plot or click DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-47 September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 226 • Manually – Visualize and eliminate outliers from the analyzed run data as explained in “Eliminating Outlying Amplification” on page 6-49. DRAFT 6-48 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 227: Eliminating Outlying Amplification
• If analyzing an Absolute Quantification Plate Document, view the results of the run displayed within the Standard Curve plot and Results table. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-49 September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 228 Omit Well(s) check box (selected) To reinstate an omitted well, select the well, then click the Omit Well(s) Note: check box. DRAFT 6-50 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 229 7. Repeat the procedures in “Visualizing Outliers” on page 6-49 to verify that all outliers have been removed from the analysis. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-51 September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 230: After The Analysis
To export a plot as a graphic file, see “Exporting Graphics” on page A-16 or click in the plot of interest for further instructions. DRAFT 6-52 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 231 In the Description field, enter a brief description of the study (up to 255 characters). c. Click d. Click DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 6-53 September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 232 The SDS software does not require that the file name match the bar code Note: of the corresponding plate. 4. Click . The software saves the plate document to the specified directory. DRAFT 6-54 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Real-Time.fm...
Page 233 Recommended Maintenance Schedule ........7-4 Section 7.1 Maintaining the 7900HT Instrument ......7-5 Replacing the Sample Block .
Page 234: Chapter 7 Maintaining The Instrument
2. After the software validates your user name and password, it displays a message stating the success or failure of the signature. Click to continue. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 235 Description of the action that requires a signature Enter your user name Enter your password Figure 7-2 Electronic Signature Verification Dialog Box Options DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 236: Recommended Maintenance Schedule
Check Applied Biosystems Web Site for Software 7-55 Updates Semi-Annually (6 Months) *Perform a background run before each Pure Dye Run. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 237: Section 7.1 Maintaining The 7900Ht Instrument
Verifying Instrument Performance ........7-30 DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 238: Replacing The Sample Block
(Zymark position 2) as explained on pages 7-41 to 7-44. 10. Align the fixed-position bar code reader for the new plate format (see page 7-49). DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 239 100 °C. Before performing this procedure, wait until the sample block reaches room temperature. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 240 Some instruments may require the use of an adjustable crescent IMPORTANT! wrench to loosen the sample block locking bolt. Sample block locking bar Sample block locking bolt DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 241 Being careful not to damage the heat sinks on the bottom of the sample block, slide the sample block out of the instrument and place it on a clean, level surface. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 242 Failure to do so prevents the instrument from uploading the firmware from the computer and causes the SDS software to display an error. DRAFT 7-10 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 243 Perform pure dye and background runs after replacing the sample IMPORTANT! block, even if you are replacing a sample block module with another block of the same format. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-11 September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 244: Changing The Plate Adapter
– Fast 96-Well Plate Adapter – TaqMan ® Low Density Array Adapter Changing the 1. If the instrument tray is inside the 7900HT instrument, move the instrument tray to the OUT position: Plate Adapter a. Start the SDS software. b. Click (or select File >...
Page 245 5. Replace and tighten the four screws in the order shown below: The order in which the screws are tightened is important to IMPORTANT! ensure proper alignment of the plate to the sample block inside the 7900HT instrument. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-13 September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 246: Decontaminating The Sample Block
If you plan to use a cleaning or decontamination method other than IMPORTANT! the one in this manual, check with Applied Biosystems first to ensure that the method will not damage the sample block module or the 7900HT instrument. Materials •...
Page 247 “Replacing the Sample Block” on page 7-10. 9. Run a background plate to confirm that the contamination has been removed. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-15 September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 248: Performing A Background Run
• Components for making a TaqMan Low Density Array background plate: – Microfluidic card spectral calibration reagents – Centrifuge – Microfluidic card sealer DRAFT 7-16 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 249: Preparing The Background Plate Or Taqman Low Density Array
4. If the background plate or Low Density Array is labeled with a bar code, click the Barcode field, then scan the bar code number using the hand-held bar code reader. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-17 September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 250: Running The Prepared Background Plate Or Taqman Low Density Array
The A1 position is in the top-left side of the instrument tray. Note: DRAFT 7-18 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 251: Analyzing The Background Data
(or select File > Save). The software saves the plate document. 3. Select File > Close. The software closes the plate document. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-19 September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 252: Performing A Pure Dye Run
The 7900HT instrument supports the detection of custom pure dyes (dyes Note: other than those provided by Applied Biosystems). To add custom pure dyes to the Pure Dye set for your instrument, see “Adding Custom Dyes to the Pure Dye Set” on page 7-27.
Page 253 • Centrifuge, with plate adapter Microfluidic Pure Dye Cards • Microfluidic card spectral calibration reagents • Centrifuge • Microfluidic card sealer DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-21 September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 254: Preparing The Pure Dye Plates Or Cards
96 Well Pure Dyes Plate 1.sdt. To run Plate 2 (containing SYBR, TAMRA, TET, and VIC dyes), select 96 Well Pure Dyes Plate 2.sdt. DRAFT 7-22 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 255 The software saves the plate document. 7. Prepare and run the pure dye plate or card as explained below. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-23 September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 256: Running The Prepared Pure Dye Plate Or Card
Note: 2. Click The 7900HT instrument begins the pure dye run. The method for a pure dye run is coded into the software and consists of a single 2-min hold at 60 °C. Before starting the run, the instrument may pause (up to 15 min) to heat Note: the heated cover to the appropriate temperature.
Page 257: Analyzing The Pure Dye Run
Click here to remove it Example spectra from a Microfluidic Pure Dye Card: Wavelength shift Click here to remove it DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-25 September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 258 “Running the Prepared Pure Dye Plate or Card” on page 7-24 – “Analyzing the Pure Dye Run” on page 7-25 DRAFT 7-26 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 259: Adding Custom Dyes To The Pure Dye Set
Adding Custom Dyes to the Pure Dye Set When to Perform The Applied Biosystems 7900HT Fast Real-Time PCR System can be used to run assays designed with custom dyes (dyes not manufactured by Applied Biosystems). However, before using custom dyes with the 7900HT instrument, you must create and run a custom pure dye plate.
Page 260 Container – Select the appropriate plate format. Template – Select Blank Template. c. Click . The software creates a new plate document. DRAFT 7-28 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 261 6. Run the custom Pure Dye plate as explained in “Preparing the Pure Dye Plates” on page 7-22. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-29 September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 262: Verifying Instrument Performance
Purpose of The TaqMan RNase P Instrument Verification Plates are experiments run to verify the performance of the 7900HT instrument for use with optical plates of the same RNase P Runs format (384-Well, 96-Well, or 96-Well Fast). The TaqMan RNase P Instrument...
Page 263 The TaqMan ® Low Density 7900HT Installation Array (TGF-β card) is an experiment run to verify the performance of the 7900HT instrument for use with TGF-β Runs TaqMan Low Density Arrays. The TGF-β card is preloaded with the reagents necessary for the detection and quantification of genomic copies of the TGF-β gene.
Page 264 7. Pipette 100 µL of the reaction mix into each of the eight fill reservoirs. 8. Continue with “Preparing the Plate Document” on page 7-33. DRAFT 7-32 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 265 Select Files of type > SDS 7900HT Document (*.sds). d. Click The software saves the plate document. The software is now configured for the RNase P run. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-33 September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 266 Up to 10 wells from each replicate group in a 384-well TaqMan RNase P Note: Instrument Verification Plate can be ignored to meet specifications. DRAFT 7-34 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 267: Section 7.2 Maintaining The Plate Handler
Cleaning and Replacing Gripper Finger Pads ......7-52 DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-35...
Page 268: Automation Accessory Components And Stack Positions
(front of instrument) Well A1 Position 4 Stack 1 Figure 7-6 Positions of the Automation Controller and Zymark Twister Software DRAFT 7-36 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 269: Adjusting The Sensitivity Of The Plate Sensor Switch
To make the adjustment easier, place your ear close to the sensor switch while making the adjustment and listen for the switch to engage. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-37 September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 270 If you lose count, begin again from step 4 and identify the zero point for the switch. 6. Test the adjustment as explained on page 7-39. DRAFT 7-38 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 271 9. Grasp the plate with one hand and, from the Zymark Twister Software, click to release the plate. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-39 September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 272 Close to exit the remaining software. d. Select File > Exit. DRAFT 7-40 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 273: Aligning The Plate Handler
The software quits the Automation Controller Software. 4. Select > Programs > Zymark Twister Plate Handler > Twister. The Zymark Twister Software starts. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-41 September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 274 4. Check the rotary position of the Plate Handler arm to confirm that the gripper: • is centered over the stack • will not contact the sides of the stack when lowered DRAFT 7-42 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 275 The software records the rotary position for the Zymark position 4 (input stack 1). 12. Click The gripper releases the plate. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-43 September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 276 6. Again, using the software to move the Plate Handler arm, center the gripper and plate along the Y-axis of the instrument tray as explained in step 4. DRAFT 7-44 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 277 Contact between the plate and the stack or all stacks may be unavoidable. Note: However, try to minimize the contact as much as possible. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-45 September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 278 12. Lower the Plate Handler arm and click , and click The software records the rotary position for position 0 (the output stack). DRAFT 7-46 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 279 8. Using the Vertical Positioning commands, raise and lower Plate Handler arm several times to check the alignment. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-47 September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 280 7900HT Front view with Robot 7900HT FAST Real-Time PCR System GR2009 Fixed-position bar code reader and underlying platform covers DRAFT 7-48 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 281: Aligning The Fixed-Position Bar Code Reader
2. Select > Programs > PSC Laser Data > LDHOST. The LDHOST software starts and displays the LDHOST window. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-49 September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 282 The software updates the Terminal dialog box every 0.5 sec indicating the percentage of accurate reads completed during the 0.5 sec interval. DRAFT 7-50 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 283 The LDHOST window closes. 10. Replace the cover for the fixed-position bar code reader (from step 1 on page 7-49). DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-51 September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 284: Cleaning And Replacing Gripper Finger Pads
The screws do not automatically align the grippers. Make sure that the Note: finger pads are making good contact with the plate when the arm grips a plate. DRAFT 7-52 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 285: Section 7.3 Maintaining The Computer And Software
System. Although an Applied Biosystems service engineer will complete most of the maintenance, this section discusses important issues that you should understand. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 7-53 September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 286: General Computer Maintenance
Applied Biosystems 7900HT Fast Real-Time PCR System can generate over 200 MB of data. Without a strategy for distributing and archiving SDS-related files, the 7900HT instrument can easily fill the hard drive of the computer within just a few weeks of operation. See “Managing Sequence Detection System Data”...
Page 287: Maintaining The Sds Software
Upgrading the Do not upgrade the operating system of the computer connected to the 7900HT instrument unless instructed to do otherwise by an Applied Biosystems Operating representative.
Page 288 Chapter 7 Maintaining the Instrument DRAFT 7-56 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Maintenance.fm...
Page 289 Software and 7900HT Instrument ........
Page 290: Chapter 8 Troubleshooting
More than two outliers per dye in a single row Real-Time Runs (Quantitative PCR and Dissociation Curves) 8-12 End-Point Runs (Allelic Discrimination) 8-13 DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Trouble.fm...
Page 291 Handler arm Plate Handler does not restack plates in original locations Fixed-position bar code reader not reading plate bar codes DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Trouble.fm...
Page 292 Archive or restore operation was terminated 8-20 SDS Enterprise Database prematurely Cannot Connect to the Database DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Trouble.fm...
Page 293: Low Precision Or Irreproducibility
Low Precision or Irreproducibility Low Precision or Irreproducibility Overview There are many reasons why an assay run with the Applied Biosystems 7900HT Fast Real-Time PCR System can have less than optimal precision. Factors that can affect precision are described in detail below.
Page 294 If splashing Reagents occurs, briefly centrifuge the reaction plate to remove all traces of liquid from the caps. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Trouble.fm...
Page 295: Low Precision Or Irreproducibility
Therefore, carefully monitor the reaction plate as it is being transferred into the thermal cycler or 7900HT instrument. If you observe any drops, take steps to remove them, such as centrifugation.
Page 296 If running non-Applied Biosystems chemistry, be sure to set the passive reference for your experiment as explained on “Setting the Passive Reference” on page 3-18. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Trouble.fm...
Page 297: Background Runs
The following figure illustrates the raw data produced by a run on a sample block module containing a contaminated well. Possible contamination DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Trouble.fm...
Page 298 10-12, the location of the contaminated well (G10) is determined. DRAFT 8-10 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Trouble.fm...
Page 299: Pure Dye Runs
If the problem • Contamination persists, discard the pure dye plate or card and run a new one. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 8-11 September 1, 2004 11:39 am, CH_Trouble.fm...
Page 300: Real-Time Runs (Quantitative Pcr And Dissociation Curves)
The higher the MSE value, the greater the deviation between the multicomponented data and the raw data. DRAFT 8-12 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Trouble.fm...
Page 301: End-Point Runs (Allelic Discrimination)
• Signal Plateaus – Do any of the signals plateau? Signal plateaus or saturation can be an indication that a well contains too much template or fluorescent signal. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 8-13 September 1, 2004 11:39 am, CH_Trouble.fm...
Page 302: Software And 7900Ht Instrument
You may have to reload the OS from the CDs. Contact Dell for troubleshooting the computer hardware. DRAFT 8-14 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Trouble.fm...
Page 303 Remove all plates from the a plate or plates output stack of the Plate Handler before starting the queue. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 8-15 September 1, 2004 11:39 am, CH_Trouble.fm...
Page 304 After the above Contact Dell for troubleshooting solution has been the computer hardware. tried, the problem is still not fixed DRAFT 8-16 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Trouble.fm...
Page 305: Zymark Twister Microplate Handler And Fixed-Position Bar Code Reader
“Aligning the Fixed-Position Bar Code Bar code reader is broken Reader” on page 7-49. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 8-17 September 1, 2004 11:39 am, CH_Trouble.fm...
Page 306: Taqman Low Density Array
The exterior surface of the reaction wells (optical side of the Low Density Array) must be free of water condensation. DRAFT 8-18 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Trouble.fm...
Page 307 Delete the outlier calculated, a specific and reanalyze. replicate appears to be an outlier. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 8-19 September 1, 2004 11:39 am, CH_Trouble.fm...
Page 308: Sds Enterprise Database
Reattempt the archive or archiving or restoring a restoration task. java.sql.SQLException: large data set Io exception: End of TNS data channel DRAFT 8-20 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, CH_Trouble.fm...
Page 309 Connecting SDS Software to the Database ......A-22 DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_SWReference.fm...
Page 310: Importing Plate Document Setup Table Files
11. Configure the setup table file with plate document information (detector, task, marker, and sample data) as explained on page A-3. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_SWReference.fm...
Page 311 The software imports the setup table information from the text file and automatically configures the plate document plate grid and setup table with detector, detector task, marker, and sample data. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_SWReference.fm...
Page 312: Setup Table Files
DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_SWReference.fm...
Page 313: File Version
Format [ *** Output Plate ID <tab> plate id <cr> ] Example *** Output Plate ID 384N75822034 DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_SWReference.fm...
Page 314: Detector Definitions
[ Detector <tab> Reporter <tab> Quencher <tab> Description <tab> Comments <cr> ] Example Example Code A-1 Detectors List Header Detector Reporter Quencher Description Comments DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_SWReference.fm...
Page 315: Detectors List
GAPDH Probe Example Probe SYBR Green SYBR SYBR Green I Example Probe RNase P TAMRA RNase P Probe Example Probe DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_SWReference.fm...
Page 316: Marker Definitions
[ Marker <tab> AlleleX <tab> AlleleY <tab> Description <tab> Comments <cr> ] Example Example Code A-4 Marker AlleleX AlleleY Description Comments DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_SWReference.fm...
Page 317: Markers List (Allelic Discrimination Only
GAPDH Probe Example Probe SYBR Green SYBR SYBR Green I Example Probe RNase P TAMRA RNase P Probe Example Probe DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_SWReference.fm...
Page 318: Well-Detector Information
All detectors that appear in this section must have been previously IMPORTANT! defined in the Detector Definitions section (elements 4–6). DRAFT A-10 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_SWReference.fm...
Page 319 Task Quantity Calibrator GAPDH ENDO TNF-α UNKN Sample 1 GAPDH ENDO TNF-α UNKN Sample 12 GAPDH ENDO TNF-α UNKN DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide A-11 September 1, 2004 11:39 am, App_SWReference.fm...
Page 320: Well-Marker Information
All markers that appear in this section must have been previously IMPORTANT! defined in the Marker Definitions section (elements 7–9). DRAFT A-12 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_SWReference.fm...
Page 321 Task Quantity Calibrator GAPDH ENDO TNF-α UNKN Sample 1 GAPDH ENDO TNF-α UNKN Sample 12 GAPDH ENDO TNF-α UNKN DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide A-13 September 1, 2004 11:39 am, App_SWReference.fm...
Page 322: Example Setup Table Files
Liver 18S-VIC-mgb ENDO ISG56 TARG Information for wells 17 through 380 has been removed from this example for brevity. DRAFT A-14 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_SWReference.fm...
Page 323 UNKN SNP4 UNKN SNP4 UNKN Information for wells 11 through 381 has been removed from this example for brevity. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide A-15 September 1, 2004 11:39 am, App_SWReference.fm...
Page 324: Exporting Graphics
2. Click (or select File > Export). 3. Navigate to the directory you would like to receive the exported file(s). DRAFT A-16 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_SWReference.fm...
Page 325 7. Click the File name text box, and enter a name for the exported file. 8. Click The software saves the exported data to the designated location. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide A-17 September 1, 2004 11:39 am, App_SWReference.fm...
Page 326: Operating The Sds Software From A Command Line
– Is the SDS plate document file that you have targeted for sdsfile.sds analysis. • – Is the name of the exported results file. sdsresultsfile.txt DRAFT A-18 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_SWReference.fm...
Page 327 Applied Biosystems recommends the following settings: • End-point runs (allelic discrimination): -Xms20m -Xmx60m • Real-time runs (absolute or relative quantifiation): -Xms80m -Xmx90m DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide A-19 September 1, 2004 11:39 am, App_SWReference.fm...
Page 328: Using The Search Tool
Contains the condition (IF, AND, or OR) for the evaluation the evaluation for the evaluation Figure A-1 Add Files dialog box DRAFT A-20 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_SWReference.fm...
Page 329 • contains : Contains the alphanumeric string found in Value 1 Value 1 and 2 The Value 1 and Value 2 criteria define the condition(s) for the evaluation. Criteria DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide A-21 September 1, 2004 11:39 am, App_SWReference.fm...
Page 330: Connecting Sds Software To The Database
Note: connection is unavailable. By disabling the database connection, the SDS software does not require users to log in before using the 7900HT instrument. After the database becomes available again, the data collected in the interim can be uploaded to the SDS Enterprise Database using the SDS Document Loader. For more information, see the SDS Enterprise Database for the Applied Biosystems 7900HT Fast Real-Time PCR System Administrators Guide (P/N 4351669).
Page 331 • If the SDS software is unable to successfully connect to the database, the software displays the error message shown below. 7. In the Options dialog box, click DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide A-23 September 1, 2004 11:39 am, App_SWReference.fm...
Page 332 Appendix A Software Reference 8. Log into the database by entering your user name and password, then click DRAFT A-24 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_SWReference.fm...
Page 333 Design Tips for Quantitative PCR Assays....... .B-6 DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_P&PDesign.fm...
Page 334: Appendix B Designing Taqman Reagent-Based Assays
– Position the polymorphism site approximately in the center of each probe. • For absolute or relative quantification (see page B-6) DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_P&PDesign.fm...
Page 335 Applied Biosystems protocols are available on the Applied Biosystems Note: Company Web Site. See “How to Obtain Services and Support” page xii for more information. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_P&PDesign.fm...
Page 336 If conducting a quantitative PCR experiment, consider the use of replicate Note: assays to enhance the precision of you data. Assay DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_P&PDesign.fm...
Page 337: Design Tips For Allelic Discrimination Assays
N N N N N N N N N N N N N N N N N N N N If necessary, place the Polymorphism polymorphism here. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_P&PDesign.fm...
Page 338: Design Tips For Quantitative Pcr Assays
RNA must then be diluted 10 –10 -fold to be at a concentration similar to the target in biological samples. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_P&PDesign.fm...
Page 339 States. Contact your Regional Sales Office for local Part numbers and prices. See “How to Obtain Services and Support” page xii. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_Consumables.fm...
Page 340: Appendix C Kits, Reagents, And Consumables
Verification Plate (PN 4351979), and a TaqMan ® Fast Reagents Starter Kit (PN 4352407) 4329012 7900HT System TaqMan ® Low Density Array Upgrade 1 kit DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_Consumables.fm...
Page 341: Consumables And Disposables
Barcode (code 128), 10-Pack Includes 10 of PN 4309849, ABI P ™ 384-Well Clear RISM Optical Reaction Plates with Barcode DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_Consumables.fm...
Page 342 Optical 96-Well Fast Thermal Cycling Plate with Barcode 20 Plates (code 128) ® 4312063 MicroAmp Splash Free Support Base for 96-Well Reaction 10 Bases Plates DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_Consumables.fm...
Page 343: Instrument Maintenance And Verification
RNase P primers, and FAM ™ dye-labeled probe) and template to detect and quantitate genomic copies of the human RNase P gene. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_Consumables.fm...
Page 344: Taqman Pre-Developed Assays And Reagents
® TaqMan Probes (5’-Fluorescent label: 6-FAM, VIC or TET; 3’-label: TAMRA) 450025 5,000-6,000 pmols 450024 15,000-25,000 pmols 450023 50,000-100,000 pmols DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_Consumables.fm...
Page 345 Minimum 4,000 pmols purified for sequence detection 4304971 Minimum 40,000 pmols purified for sequence detection 4304972 Minimum 130,000 pmols purified for sequence detection DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_Consumables.fm...
Page 346 Appendix C Kits, Reagents, and Consumables DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_Consumables.fm...
Page 347 Method of Relative Quantification..... . . D-8 DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_Theory.fm...
Page 348: Fluorescent-Based Chemistries
The increase in fluorescence signal is detected only if the target sequence is complementary to the probe and is amplified during PCR. Because of these requirements, any nonspecific amplification is not detected. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_Theory.fm...
Page 349 Figure D-2 Binding Activity of the SYBR Green 1 Dye during the PCR DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_Theory.fm...
Page 350: Real-Time Data Analysis
Appendix D Theory of Operation Real-Time Data Analysis Kinetic Analysis/ The 7900HT instrument can be used to determine the absolute or relative quantity of a target nucleic acid sequence in a test sample by analyzing the cycle-to-cycle change Quantitative PCR in fluorescence signal as a result of amplification during a PCR.
Page 351 5000 2500 1250 Cycle Number Figure D-4 Amplification Plot from a Real-Time Run of an RNase P Instrument Verification Plate DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_Theory.fm...
Page 352 PCR appears to have three stages. Initially, R appears as a flat line because the fluorescent signal is below the detection limit of the 7900HT instrument. In the second stage, the signal can be detected as it continues to increase in direct proportion to the increase in the products of PCR.
Page 353 Likewise, a change in threshold cycle number of one must equate to a two-fold difference in initial template concentration. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_Theory.fm...
Page 354: Comparative C Method Of Relative Quantification
Thus, × where: = threshold number of target molecules = threshold cycle for target amplification = constant DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_Theory.fm...
Page 355 A similar equation for the endogenous reference reaction is: × where: = threshold number of reference molecules = initial number of reference molecules = efficiency of reference amplification = threshold cycle for reference amplification T, R = constant Dividing X by R gives the following expression: ×...
Page 356 (amplicon size <150 bp), the efficiency is close to 1. Therefore, the amount of target, normalized to an endogenous reference and relative to a calibrator, is given by: ∆ ∆ – DRAFT D-10 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, App_Theory.fm...
Page 357: Appendix E Limited Warranty Statement
(b) modified or repaired by a party other than Applied Biosystems, or (c) used in a manner not in accordance with the instructions contained in the Instrument User's Manual. This Warranty does not cover the customer-installable accessories or customer-installable consumable parts for the Instrument that are listed in the Instrument User's Manual.
Page 358 WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE AND IS IN LIEU OF ANY OTHER OBLIGATION ON THE PART OF APPLIED BIOSYSTEMS. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, App_Warranty.fm...
Page 359 PCR standardisation for molecular diagnostics and real-time quantitative PCR [letter]. Leukemia 12(8):1324-1325, 1998. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide Bibliography-1 September 1, 2004 11:38 am, Bibliography.fm...
Page 360 General Papers Arnold, B.A., Hepler, R.W., and Keller, P.M. One-Step Fluorescent Probe Product-Enhanced Reverse Transcriptase Assay. BioTechniques 25(1):98-106, 1998. on Absolute Quantitation Ambs S., S. Dennis, J. Fairman, M. Wright and J. Papkoff. 1999. Inhibition of tumor growth correlates with the expression level of a human angiostatin transgene in transfected B16F10 melanoma cells [In Process Citation].
Page 361 Experimental Transmission of Human Granulocytic Ehrlichia Agent by Intravenous Inoculation with Infected Leukocytes and by Infected Ticks. J.Clin.Microbiol. 37(12):4042-4044, 1999. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide Bibliography-3 September 1, 2004 11:38 am, Bibliography.fm...
Page 362 McCune and D. Ganem. 1999. Experimental Transmission of Kaposi's Sarcoma-associated Herpesvirus (KSHV/HHV-8) to SCID-hu Thy/Liv Mice. J. Exp. Med. 190:1857-1868. DRAFT Bibliography-4 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, Bibliography.fm...
Page 363 [In Process Citation]. Int.J.Cancer 81(5):700-704, 1999. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide Bibliography-5 September 1, 2004 11:38 am, Bibliography.fm...
Page 364 Lantz, O. Quantitative ELISA-polymerase chain reaction at saturation using homologous internal DNA standards and chemiluminescence revelation. Eur.Cytokine.Netw. 9(2):197-204, 1998. DRAFT Bibliography-6 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, Bibliography.fm...
Page 365 G., and Witte, J.S. Association between a CYP3A4 genetic variant and clinical presentation in African-American prostate cancer patients [In Process Citation]. Cancer Epidemiol.Biomarkers.Prev. 8(10):901-905, 1999. DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide Bibliography-7 September 1, 2004 11:38 am, Bibliography.fm...
Page 366 S-transferase T1 and M1 gene deletions using TaqMan probes. Res. Commun. Mol. Pathol. Pharmacol. 103:3-15, 1999. DRAFT Bibliography-8 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, Bibliography.fm...
Page 367 SDS software). SDS 7900HT Multiple plate documents are used for the analysis of relative quantification data. Multiple Plate Document (*.sdm) DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide Glossary-1 September 1, 2004 11:39 am, Glossary.fm...
Page 368 Method setting that increases or decreases the value for any PCR set-point parameter value (time or temperature) by a fixed amount every cycle. DRAFT Glossary-2 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, Glossary.fm...
Page 369 Often these command line parameters are filenames, directory paths, or flag settings. consumable (1) A vessel used to contain reactions run on the 7900HT instrument (a microplate or a TaqMan ® Low Density Array). (2) A reagent kit, chemical, or disposable labware used as part of an assay.
Page 370 7900HT instrument make use of the following information: thermal cycling times and temperatures, ramp rates, auto-increment values, and data collection settings. DRAFT Glossary-4 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:39 am, Glossary.fm...
Page 371 The analysis performed by the SDS software used to distinguish the contribution of individual dyes to the raw spectral data collected by the 7900HT instrument. The overlapping spectra from the individual dye components in a well generate the composite spectrum that represents a raw data fluorescent reading.
Page 372 ® Low Density Array. real-time Refers to real-time PCR processing of individual plates by the 7900HT instrument for run/analysis absolute and relative quantification. The 7900HT instrument collects fluorescence data during each cycle of a pre-programmed PCR run. The pre-programming occurs by setting the thermal cycling parameters of a default method.
Page 373 A PCR reaction with a single fluorescent detector per well. See single nucleotide polymorphism. spectral bin A portion of the complete range of the wave length measured by the 7900HT instrument (approximately 500-660 nm). spectral calibration Spectral calibration occurs from pure dye results obtained from a pure dye run. The software stores the spectral information for the pure dye standards within a calibration file located in the SDS directory.
Page 374 The binary representation of the plate document template is persisted in the database, allowing the plate document template to be retrieved and then populated with data and saved as an SDS 7900HT Document. (2) [Chemical] the DNA sample that is being amplified during PCR.
Page 375 B-4, analyzing run data 5-10 5-18 allelic discrimination assay development guidelines DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide Index-1 September 1, 2004 11:38 am, 4351684AIX.fm...
Page 376 4-17 contamination requirements 4-12 decontaminating the sample block 7-14 TaqMan Low Density Array, placing in DRAFT Index-2 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, 4351684AIX.fm...
Page 377 Gene Expression Profile definitions of the T value 6-43 sample bars 6-31 procedure checklist 6-22, 6-40 X-axis 6-31 DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide Index-3 September 1, 2004 11:38 am, 4351684AIX.fm...
Page 378 4-29 adding a hold, cycle set, or step 3-21 replacing the plate adapter 7-12 adding a temperature ramp 3-23 DRAFT Index-4 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, 4351684AIX.fm...
Page 379 7-52 Linear Phase gripper 7-36 Plateau Phase plate stack positions 7-36 SYBR Green Chemistry DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide Index-5 September 1, 2004 11:38 am, 4351684AIX.fm...
Page 380 7-21 sample extracting data 7-25 calibrator 6-20 performing 7-24 sample bars troubleshooting 8-11 about 6-31 when to perform 7-20 DRAFT Index-6 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, 4351684AIX.fm...
Page 381 4-16 about quality control 4-12 example files removing from packaging 4-14 exporting A-17 sealing 4-19 DRAFT Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide Index-7 September 1, 2004 11:38 am, 4351684AIX.fm...
Page 382 Time PCR System upgrading operating system software 7-55 SDS software 7-55 viewing, well information 2-23 views hiding 2-21 maximizing/minimizing 2-22 DRAFT Index-8 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide September 1, 2004 11:38 am, 4351684AIX.fm...
Page 384 Web site at www.appliedbiosystems.com. Applera is committed to providing the world’s leading technology and information for life scientists. Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses. Headquarters 850 Lincoln Centre Drive Foster City, CA 94404 USA Phone: +1 650.638.5800...

Applied Biosystems 7900HT User Manual

Table of Contents

Safety and Emc Compliance Information

1 Product Overview 1

Chapter 1 Product Overview

2 Getting Started 2

Chapter 2 Getting Started

3 Preparing a Run 3

Chapter 3 Preparing a Run

4 Operating the Instrument 4

Chapter 4 Operating the Instrument

5 Analyzing End-Point Data 5

Chapter 5 Analyzing End-Point Data

Section 5.1 Allelic Discrimination

Chapter 6 Analyzing Real-Time Data

Section 6.1 Absolute Quantification

Section 6.3 Dissociation Curve Analysis

7 Maintaining the Instrument 7

Chapter 7 Maintaining the Instrument

8 Troubleshooting 8

Chapter 8 Troubleshooting

Software Reference a

Appendix A Software Reference

Appendix B Designing Taqman Reagent-Based Assays

Kits, Reagents, and Consumables C

Appendix C Kits, Reagents, and Consumables

Appendix D Theory of Operation

Appendix E Limited Warranty Statement

Quick Links

Chapters

Related Manuals for Applied Biosystems 7900HT

Summary of Contents for Applied Biosystems 7900HT