Plus/Minus Assay. Getting Started Guide. Applied Biosystems 7900HT Fast Real-Time PCR System. Introduction. Designing a. Plus/Minus Assay.

Transcription

1 Applied Biosystems 7900HT Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide Introduction Designing a Plus/Minus Assay Experiment Preparing the Samples and Reaction Plate Performing a Plus/Minus Assay Pre-Read Run Performing an Amplification Run Performing and Analyzing a Plus/Minus Assay Post-Read Run

2 Copyright 2007, Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. 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. Notice to Purchaser The Applied Biosystems 7900HT Fast Real-Time PCR System is a real-time thermal cycler covered by one or more of US Patents Nos. 6,814,934, 5,038,852, 5,333,675, 5,656,493, 5,475,610, 5,602,756, 6,703,236, 7,238,517, 6,818,437, 7,008,789, 6,563,581, 6,965,105 and 6,719,949 and corresponding claims in their non-us counterparts, owned by Applera Corporation. No right is conveyed expressly, by implication or by estoppel under any other patent claim, such as claims to apparatus, reagents, kits, or methods such as 5 nuclease methods. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. TRADEMARKS: ABI PRISM, Applied Biosystems, MicroAmp, NucPrep, PrepMan, Primer Express and VIC are registered trademarks and AB (Design), Applera, BloodPrep, Celera Genomics, FAM, ROX and TAMRA are trademarks of Applera Corporation or its subsidiaries in the U.S. and/or certain other countries. TaqMan is a registered trademark of Roche Molecular Systems, Inc. All other trademarks are the sole property of their respective owners. Part Number Rev. C 12/2007

3 Contents Preface v How to Use This Guide v How to Obtain More Information vii How to Obtain Support vii Chapter 1 Introduction 1 About the 7900HT Fast System About Plus/Minus Assays Using an IPC About Plus/Minus Assay Experiments Before You Begin Chapter 2 Designing a Plus/Minus Assay Experiment 9 Using TaqMan Probe-Based Chemistry Selecting the Probes and Primers Chapter 3 Preparing the Samples and Reaction Plate 13 Preparing the DNA Samples Preparing the Reaction Mix Preparing the Reaction Plate Chapter 4 Performing a Plus/Minus Assay Pre-Read Run 19 About Plus/Minus Assay Plate Documents Creating a Plus/Minus Assay Plate Document Performing the Pre-Read Run Chapter 5 Performing an Amplification Run 33 About Standard Curve (AQ) Plate Documents Creating a Standard Curve (AQ) Plate Document Performing the Amplification Run Plus/Minus Assay Getting Started Guide for the 7900HT Fast System iii

4 Chapter 6 Performing and Analyzing a Plus/Minus Assay Post-Read Run 47 Performing the Post-Read Run Analyzing the Run and Evaluating the Results Post-Analysis Options Appendix A Sample Plus/Minus Assay Experiment 69 About the Sample Experiment Plus/Minus Assay Experiment Overview Appendix B SDS Automation Controller Software 75 Overview Using the Automation Controller Software Appendix C Analyzing and Viewing Amplification Data 81 Terms Used in Quantitation Analysis Analyzing the Amplification Data Viewing the Amplification Data References 93 Index 95 iv Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

5 Preface How to Use This Guide Purpose of This Guide Audience Assumptions This guide provides procedures for conducting plus/minus assays using the Applied Biosystems 7900HT Fast Real Time PCR System (7900HT Fast System). This guide is intended for principal investigators and laboratory staff who conduct plus/ minus assays using the 7900HT Fast System. This guide assumes that you have: Familiarity with the Microsoft Windows XP operating system. Knowledge of general techniques for handling DNA samples and preparing them for PCR. A general understanding of hard drives and data storage, file transfers, and copying and pasting. Networking experience, if you want to integrate the 7900HT Fast System into your existing laboratory data flow. Text Conventions This guide uses the following conventions: Bold indicates user action. For example: Type 0, then press Enter for each of the remaining fields. Italic text indicates new or important words and is also used for emphasis. For example: Before analyzing, always prepare fresh matrix. A right arrow bracket (>) separates successive commands you select from a dropdown or shortcut menu. For example: Select File > Open. User Attention Words The following user attention words appear in Applied Biosystems user documentation. Each word implies a particular level of observation or action as described below: Note Provides information that may be of interest or help but is not critical to the use of the product. Plus/Minus Assay Getting Started Guide for the 7900HT Fast System v

6 Preface How to Use This Guide IMPORTANT! Provides information that is necessary for proper instrument operation, accurate chemistry kit use, or safe use of a chemical. Indicates a potentially hazardous situation that, if not avoided, may result in minor or moderate injury. It may also be used to alert against unsafe practices. Indicates a potentially hazardous situation that, if not avoided, could result in death or serious injury. Safety Follow specific safety practices when using this instrument. For safety guidelines, refer to the "Safety and EMC Compliance" section in the Applied Biosystems 7900HT Fast Real-Time PCR System Site Preparation Guide (PN ) and the Sequence Detection Systems Software version 2.3 Online Help (SDS Online Help). 1. You can obtain from Applied Biosystems the MSDS for any chemical supplied by Applied Biosystems. This service is free and available 24 hours a day. To obtain MSDSs: 2. Go to click Support, then click MSDS Search. 3. In the Keyword Search field, enter the chemical name, product name, MSDS part number, or other information that appears in the MSDS of interest. Select the language of your choice, then click Search. 4. Find the document of interest, right-click the document title, then select any of the following: Open To view the document Print Target To print the document Save Target As To download a PDF version of the document to a destination that you choose Note: For the MSDSs of chemicals not distributed by Applied Biosystems, contact the chemical manufacturer. 5. To have a copy of a document sent by fax or , select Fax or to the left of the document title in the Search Results page, then click RETRIEVE DOCUMENTS at the end of the document list. 6. After you enter the required information, click View/Deliver Selected Documents Now. For chemicals not manufactured or distributed by Applied Biosystems, contact the chemical manufacturer. vi Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

7 Preface How to Obtain More Information How to Obtain More Information Related Documentation For more information about using the 7900HT Fast System and performing assays, refer to: Applied Biosystems 7900HT Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide (PN ) Applied Biosystems 7900HT Fast Real-Time PCR System Absolute Quantitation using Standard Curve Getting Started Guide (PN ) Applied Biosystems 7900HT Fast Real-Time PCR System Allelic Discrimination Getting Started Guide (PN ) Applied Biosystems 7900HT Fast Real-Time PCR System Relative Quantitation using Comparative C T Getting Started Guide (PN ) Sequence Detection Systems Software version 2.3 Online Help (SDS Online Help) Applied Biosystems 7900HT Fast Real-Time PCR System Maintenance and Troubleshooting Guide (PN ) Applied Biosystems 7900HT Fast Real-Time PCR System Site Preparation Guide (PN ) ABI PRISM 6100 Nucleic Acid PrepStation Users Guide (PN ) DNA Isolation from Fresh and Frozen Blood, Tissue Culture Cells, and Buccal Swabs Protocol (using BloodPrep Chemistry, PN ) NucPrep Chemistry Protocol: Isolation of Genomic DNA from Animal and Plant Tissue (PN ) PrepMan Ultra Sample Preparation Reagent Protocol (PN ) Primer Express Software v3.0 Getting Started Guide (PN ) Real-Time PCR Systems Chemistry Guide (PN ) TaqMan Exogenous Internal Positive Control Reagents Protocol (PN ) TaqMan Low Density Array Getting Started Guide (PN ) TaqMan Universal PCR Master Mix Protocol (PN ) TransPrep Chemistry Protocol: Purification of gdna from Filtrates Obtained After the Isolation of RNA from Homogenized Animal or Plant Tissue Samples (PN ) Send Us Your Comments Applied Biosystems welcomes your comments and suggestions for improving its user documents. You can your comments to: techpubs@appliedbiosystems.com How to Obtain Support To contact Applied Biosystems Technical Support from North America by telephone, call For the latest services and support information for all locations, go to then click the link for Support. Plus/Minus Assay Getting Started Guide for the 7900HT Fast System vii

8 Preface How to Obtain Support At the Support page, you can: Obtain worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities Search through frequently asked questions (FAQs) Submit a question directly to Technical Support Order Applied Biosystems user documents, MSDSs, certificates of analysis, and other related documents Download PDF documents Obtain information about customer training Download software updates and patches viii Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

9 Chapter 1 Introduction 1 Introduction About the 7900HT Fast System See page 2 Designing a Plus/Minus Assay Experiment Preparing the Samples and Reaction Plate About Plus/Minus Assays Using an IPC See page 3 Performing the Plus/Minus Assay Pre-ReadRun About Plus/Minus Assay Experiments See page 4 Performing an Amplification Run Before You Begin See page 7 Performing and Analyzing a Plus/Minus Assay Post-Read Run Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 1

10 Chapter 1 Introduction About the 7900HT Fast System About the 7900HT Fast System System Description The Applied Biosystems 7900HT Fast Real Time PCR System (7900HT Fast System) uses fluorescent-based PCR chemistries to provide: Quantitative detection of nucleic acid sequences using real-time analysis. Qualitative detection of nucleic acid sequences using end-point and dissociationcurve analysis. For more information about the 7900HT Fast System, refer to the Sequence Detection Systems Software version 2.3 Online Help (SDS Online Help) and the Applied Biosystems 7900HT Fast Real-Time PCR System Maintenance and Troubleshooting Guide (PN ). Note: To access the SDS Online Help, select Help > SDS Online Help from the SDS software menu bar. Supported Assay Configurations You can perform several assay types on the 7900HT Fast System using reaction plates in the 96-well, 384-well and TaqMan Low Density Array format.this guide describes only the plus/minus assay type. For information about the other supported assay configurations for the 7900HT Fast System, refer to the following table. Reaction Plate and System Block Options Assay Type Fast 96-well Standard 96-well Standard 384-well TaqMan Low Density Array Standard curve (AQ) Yes Yes Yes No Comparative C T (RQ) Yes Yes Yes Yes Allelic discrimination No Yes Yes No Plus/minus No Yes Yes No Plus/Minus Assay Configuration Plus/minus assays are supported on the 7900HT Fast System using reaction plates in the standard 96-well or standard 384-well format with standard reagents and protocols. IMPORTANT! Plus/minus assays are not supported using Fast reaction plates, reagents and protocols. IMPORTANT! Use standard reaction plates on the 7900HT Fast System with a standard block. Fast 96-well reaction plates do not fit into the standard 96-well block correctly and standard 96-well reaction plates do not fit into the fast 96-well block. 2 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

11 Chapter 1 Introduction About Plus/Minus Assays Using an IPC About Plus/Minus Assays Using an IPC Plus/Minus Assay Definition A plus/minus assay is an end-point assay that determines if a specific target sequence is present (plus) or not present (minus) in a sample. In an end-point assay, data are collected at the end of the PCR process. 1 What is an IPC? An internal positive control (IPC; see TaqMan Exogenous Internal Positive Control Reagents kit, PN ) is used in plus/minus assays to monitor the PCR process and to ensure that a negative result is not due to failed PCR in the sample (Kwok and Higuchi, 1989). The IPC consists of a template, a primer set, and a VIC dye-labeled probe that are added to each well of a reaction plate (the IPC is part of the reaction mix; see Preparing the Reaction Mix on page 15). Plus/minus assays with an IPC use fluorogenic 5 nuclease chemistry (also known as TaqMan probe-based chemistry ; see Using TaqMan Probe-Based Chemistry on page 10). During amplification, the sample target and the IPC target generate reporter fluorescence signals such that positive or negative calls can be made on unknown samples (Saiki et al., 1985). Note: Plus/minus assays can also be accomplished without the use of an IPC; however, an IPC ensures that a failed PCR reaction is not mistaken for a negative target result. Note: The SYBR Green I dye-based chemistry and the TaqMan Fast reagent-based chemistry are not supported for plus/minus assays. Terms Used in Plus/Minus Assay Analysis Term Internal positive control (IPC) No amplification control (NAC) No template control (NTC) Nucleic acid target (also called target template or target ) Unknown sample (U; also called sample of interest ) Definition A second TaqMan probe and primer set added to the plate to identify well failure to amplify. Provides a means of determining amplification failure that might give a false negative signal. Wells that contain no target template and blocked IPC (the IPC target template has been blocked by a blocking agent). A sample that does not contain template. Shows background signal and is used as the negative control. Provides a means of measuring contamination that might give a false positive signal. Nucleotide sequence that you want to identify as present or absent. The sample for which you want to determine the presence or absence of a specific target. Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 3

12 Chapter 1 Introduction About Plus/Minus Assay Experiments About Plus/Minus Assay Experiments Plus/Minus Assay Workflow After you design a plus/minus assay experiment and prepare the DNA samples, you perform three runs: A pre-read run using a plus/minus assay plate document to determine the baseline fluorescence associated with primers and probes before amplification. An amplification run using a standard curve (AQ) plate document to generate realtime PCR data, which can be used to analyze and troubleshoot the PCR data for the plus/minus assay, if needed. A post-read run using the original plus/minus assay plate document, which automatically subtracts the baseline fluorescence determined during the pre-read run from the post-read amplified data to calculate the result. The following figure illustrates the complete process. Prepare the Samples Perform a Plus/Minus Pre-Read Run Perform an Amplification Run Perform and Analyze a Plus/Minus Post-Read Run Sample Plus/ Minus Assay Experiment To illustrate how to design, perform, and analyze plus/minus assay experiments, a sample experiment representing a typical plus/minus assay experiment is described in Appendix A on page 69. References to parts of the sample experiment are made in the subsequent chapters (2 to 6) of this guide where applicable. You can use the summarized procedures of the sample plus/minus assay experiment in Appendix A on page 69 to familiarize yourself with the entire plus/minus assay workflow. 4 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

13 Chapter 1 Introduction About Plus/Minus Assay Experiments Required User-Supplied Materials Chemistry/Reagents Item Source 1 DNA isolation and purification chemistry systems: ABI PRISM 6100 Nucleic Acid PrepStation BloodPrep Chemistry (genomic DNA from fresh or frozen blood or cells) NucPrep Chemistry (DNA from animal and plant tissue) PrepMan Ultra Sample Preparation Reagent Kit ABI PRISM TransPrep System (purification of gdna after the isolation of RNA from animal and plant tissue) Labeled primers and probes source: Primer Express Software (custom-designed primers and probes) TaqMan reagents: TaqMan Exogenous Internal Positive Control Reagents (VIC dye-labeled probe) TaqMan Universal PCR Master Mix (2X) Applied Biosystems (PN ) Applied Biosystems (PN ) Applied Biosystems (PN ) Applied Biosystems (PN ) Applied Biosystems web site Contact your Applied Biosystems sales representative. Applied Biosystems (PN ) Applied Biosystems (PN ) Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 5

14 Chapter 1 Introduction About Plus/Minus Assay Experiments Reaction Plates and Covers IMPORTANT! Do not use MicroAmp Caps (domed) or Optical Tubes with the 7900HT Fast System. You can use Optical Caps (PN ) only with standard 96-well plates on the 7900HT Fast System. Item Source Standard 96-well reaction plates: MicroAmp 96-Well Optical Reaction Plate with Barcode (code 128), 20 Plates/Pkg MicroAmp 96-Well Optical Reaction Plate with Barcode (code 128), 500 Plates/Pkg Standard 384-well reaction plates: 384-Well Clear Optical Reaction Plate with Barcode (code 128), 50 Plates/Pkg 384-Well Clear Optical Reaction Plate with Barcode (code 128), 500 Plates/Pkg Optical adhesive covers: ABI PRISM Optical Adhesive Cover Starter Kit Includes 20 ABI PRISM Optical Adhesive Covers, an Applicator, and an ABI PRISM Optical Cover Compression Pad ABI PRISM Optical Adhesive Covers, 100 Covers/Pkg ABI PRISM Optical Adhesive Covers, 25 Covers/Pkg Optical Caps, 8 caps/strip, 2400 caps/300 strips Applied Biosystems (PN ) Applied Biosystems (PN ) Applied Biosystems (PN ) Applied Biosystems (PN ) Applied Biosystems (PN ) Applied Biosystems (PN ) Applied Biosystems (PN ) Applied Biosystems (PN ) Other Consumables and Equipment Item Centrifuge with adapter for 96-well and 384-well plates Gloves Microcentrifuge Microcentrifuge tubes, sterile 1.5-mL Pipette tips, with filter plugs Pipettors, positive-displacement Vortexer Nuclease-free water Tris-EDTA (TE) Buffer, ph 8.0 Source Major Laboratory Supplier (MLS) MLS MLS MLS MLS MLS MLS MLS MLS 6 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

15 Chapter 1 Introduction Before You Begin Before You Begin Calibrating the 7900HT Fast System Check that background and pure-dye runs have been performed regularly for optimal performance of the 7900HT Fast System. For information about calibrating the 7900HT Fast System, refer to the Sequence Detection Systems Software version 2.3 Online Help (SDS Online Help) and the Applied Biosystems 7900HT Fast Real-Time PCR System Maintenance and Troubleshooting Guide (PN ). 1 Accessing the SDS Online Help Automation Options Some steps in this guide refer you to the SDS Online Help for more information. To access the SDS Online Help, select Help > SDS Online Help from the SDS software menu bar. The 7900HT Fast System can run prepared reaction plates individually or in groups using the Automation Accessory with the Zymark Twister Microplate Handler. If you are not using the Automation Accessory, you must run reaction plates individually. For clarity, this guide illustrates only running an individual reaction plate using the SDS Software for the 7900HT Fast System. For information on running multiple reaction plates using the Automation Controller Software, refer to Appendix B on page 75. For information on automated operation of the 7900HT Fast System using the Automation Accessory, see the SDS Online Help. Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 7

16 Chapter 1 Introduction Before You Begin 8 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

17 Chapter 2 Designing a Plus/Minus Assay Experiment Introduction 2 Designing a Plus/Minus Assay Experiment Preparing the Samples and Reaction Plate Using TaqMan Probe-Based Chemistry See page 10 Performing the Plus/Minus Assay Pre-ReadRun Selecting the Probes and Primers See page 11 Performing an Amplification Run Performing and Analyzing a Plus/Minus Assay Post-Read Run Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 9

18 Chapter 2 Designing a Plus/Minus Assay Experiment Using TaqMan Probe-Based Chemistry Using TaqMan Probe-Based Chemistry About the Chemistry Plus/minus assays with an IPC use the fluorogenic 5 nuclease chemistry (also known as TaqMan probe-based chemistry). Note: The SYBR Green I dye-based chemistry and the TaqMan Fast reagent-based chemistry are not supported for plus/minus assays. Description Process TaqMan chemistry TaqMan probe-based chemistry uses a fluorogenic probe to detect a specific PCR product as it accumulates during PCR cycles (Mullis and Faloona, 1987). This process, outlined in the following figures, is the basis for running plus/minus assays on the 7900HT Fast System. 5 3 FORWARD PRIMER R PROBE Q 3 R = REPORTER Q = QUENCHER Step 1: Polymerization A reporter (R) and a quencher (Q) are attached to the 5' and 3' ends of a TaqMan probe. REVERSE PRIMER Step 3: Cleavage During each extension cycle, the Applied Biosystems hot-start DNA polymerase system cleaves the reporter dye from the probe. R Q Step 2: Strand Displacement When both dyes are attached to the probe, reporter dye emission is quenched. R Q Step 4: Polymerization Completed After being separated from the quencher, the reporter dye emits its characteristic fluorescence R Q Two Types of TaqMan Probes Applied Biosystems offers two types of TaqMan probes: TaqMan probes with TAMRA dye as quencher TaqMan MGB (minor groove-binder) probes with non-fluorescent quencher (NFQ) For more information about the TaqMan probe-based chemistry, refer to the Real-Time PCR Systems Chemistry Guide (PN ). 10 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

19 Chapter 2 Designing a Plus/Minus Assay Experiment Selecting the Probes and Primers Chemistry Kits for Plus/Minus Assays The following reagents are available from Applied Biosystems for designing and running plus/minus assays: TaqMan Exogenous Internal Positive Control Reagents with VIC TAMRA dye-labeled probe (PN ) TaqMan Universal PCR Master Mix (2X) (PN ) Note: The IPC DNA, primers, and probe supplied in these reagents can be used with all target samples. Refer to the TaqMan Universal PCR Master Mix Protocol (PN ) for instructions on optimizing amplification of your target. 2 Selecting the Probes and Primers Design and select a probe and primer set for your target sequence(s) using Applied Biosystems Primer Express software. For more information about using this software, refer to the Primer Express Software v3.0 Getting Started Guide (PN ). Sample Experiment For the sample experiment, we extracted DNA from 84 batches of ground beef to test for the presence of E. coli using the plus/minus assay on the 7900HT Fast System. We ran six no IPC/no target template controls, six IPC/no target template controls, and 84 unknown samples. The sample experiment used the TaqMan Exogenous Internal Positive Control Reagents Kit, which is supplied with one 1-mL tube of 10 Exo IPC Mix. This mix contains the IPC primers and VIC dye-labeled probe. We designed the primers/ probe set for E. coli, containing a FAM dye-labeled probe with TAMRA as the quencher, using Applied Biosystems Primer Express Software. Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 11

20 Chapter 2 Designing a Plus/Minus Assay Experiment Selecting the Probes and Primers 12 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

21 Chapter 3 Preparing the Samples and Reaction Plate Introduction Preparing the DNA Samples See page 14 Designing a Plus/Minus Assay Experiment 3 Preparing the Samples and Reaction Plate Preparing the Reaction Mix See page 15 Performing the Plus/Minus Assay Pre-ReadRun Performing an Amplification Run Preparing the Reaction Plate See page 16 Performing and Analyzing a Plus/Minus Assay Post-Read Run Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 13

22 Chapter 3 Preparing the Samples and Reaction Plate Preparing the DNA Samples Preparing the DNA Samples Systems and Chemistries for DNA Isolation Applied Biosystems supplies several instrument systems and chemistries for isolating DNA from a variety of starting materials, such as blood, tissue, cell cultures, plant material, and food. System Part Number BloodPrep Chemistry NucPrep Chemistry PrepMan Ultra Sample Preparation Reagent Kit ABI PRISM TransPrep System Applied Biosystems web site ABI PRISM 6100 Nucleic Acid PrepStation For more information, refer to: DNA Isolation from Fresh and Frozen Blood, Tissue Culture Cells, and Buccal Swabs Protocol (using BloodPrep Chemistry, PN ) NucPrep Chemistry Protocol: Isolation of Genomic DNA from Animal and Plant Tissue (PN ) PrepMan Ultra Sample Preparation Reagent Protocol (PN ) TransPrep Chemistry Protocol: Purification of gdna from Filtrates Obtained After the Isolation of RNA from Homogenized Animal or Plant Tissue Samples (PN ) ABI PRISM 6100 Nucleic Acid PrepStation Users Guide (PN ) Quality of DNA Ensure that the DNA you use for a plus/minus assay experiment: Is extracted from the raw material you are testing using an optimized protocol. Does not contain PCR inhibitors. Is intact as visualized by gel electrophoresis. Has not been heated above 60 C, which can cause degradation. Has a A 260/280 ratio >1.7. Sample Experiment For the sample experiment, we froze the meat samples with liquid nitrogen and ground them to a fine powder with a prechilled mortar and pestle. We extracted DNA using the PrepMan Ultra Sample Preparation Reagent Kit (PN ) and protocol (PN ) to obtain a final concentration of 10 ng/µl of DNA for each sample. 14 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

23 Chapter 3 Preparing the Samples and Reaction Plate Preparing the Reaction Mix Note: The reagents, volumes, and final concentrations shown in Preparing the Reaction Mix on page 15 and Preparing the Reaction Plate on page 16 are taken from the TaqMan Exogenous Internal Positive Control Reagents Protocol (PN ). Preparing the Reaction Mix This section describes how to set up both standard 96-well and 384-well reaction plates for a plus/minus assay run with reaction mix. IMPORTANT! Plus/minus assays are not supported using Fast reaction plates, reagents and protocols. Preparing the PCR Reaction Mix Prepare the PCR reaction mix for your selected reaction plate format as shown in the following table. Volume for One Reaction 3 Component Final Concentration Std. 96-well plate Std. 384-well plate TaqMan Universal PCR Master Mix (2 ) 10 Exo IPC Mix (IPC kit) 50 Exo IPC DNA (IPC kit) Target primers, probe, and deionized water 1 25 µl 5 µl 50 to 900 nm 5 µl 1 µl 50 to 900 nm 1 µl 0.2 µl 50 to 250 nm 14 µl 2.8 µl Total Volume 45 µl 9 µl Optimization may be necessary depending on your selected target sequence and primers/probe set. Note: Prepare extra volume to account for pipetting losses. CHEMICAL HAZARD. TaqMan Universal PCR Master Mix (2X) may cause eye and skin irritation. Exposure may cause discomfort if swallowed or inhaled. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 15

24 Chapter 3 Preparing the Samples and Reaction Plate Preparing the Reaction Plate Preparing the Reaction Plate This section describes how to set up both standard 96-well and 384-well reaction plates for a plus/minus assay run with samples and reaction mix. IMPORTANT! Plus/minus assays are not supported using Fast reaction plates, reagents and protocols. Reaction Plate Components Preparing the Reaction Plate A reaction plate for a plus/minus assay contains the following: Internal positive control (IPC) No amplification control (NAC; blocked IPC with no target template) No template control (NTC; IPC with no target template) Unknown DNA sample (U) 1. Pipette the appropriate PCR reaction mix volume into each well of the plate: 45 µl for a standard 96-well reaction plate 9µL for a standard 384-well reaction plate 2. Pipette the control and sample reagent volumes (NAC, NTC and unknowns) into the appropriate wells of your selected reaction plate format, as shown in the following table. Volume Per Well Item Reagent Std. 96-well plate Std. 384-well plate NAC NTC U # 10 Exo IPC Block 5 µl 1 µl 1 TE or H 2 O 5 µl 1 µl sample DNA 5 µl 1 µl No Amplification Control well contains no target template and blocked IPC. No Template Control well contains no target template, only IPC. # Unknown well contains both target template and IPC. TE Tris-EDTA Buffer, ph 8.0. Note: The final reaction volume in each well should be 50 µl for a standard 96- well reaction plate and 10 µl for a standard 384-well reaction plate. 3. Cover the reaction plate with an optical adhesive cover or optical caps. 4. Keep the reactions on ice until the plate is loaded into the 7900HT Fast System. 16 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

25 Chapter 3 Preparing the Samples and Reaction Plate Preparing the Reaction Plate Sample Experiment For the sample experiment, we pipetted the extracted DNA samples into a standard 96-well plate along with negative and positive controls. We added 45 µl of reaction mix and 5 µl of the control and sample reagents as specified in the table and figure below. Wells To prepare Add to each well A1 to A6 NAC 5 µl of 10 Exo IPC Block A7 to A12 NTC 5 µl of 1 TE or H 2 O B1 to H12 U 5 µl of sample DNA Wells A1 to A6 contain blocked IPC and no target template, wells A7 to A12 contain IPC template, but no target template, and wells B1 to H12 contain both IPC and target template. Wells contain blocked IPC and no target template NAC NTC plus IPC Wells contain IPC and no target template 3 Samples GR2363a (Unknown plus IPC) Gemini Plus/Minus 96-wel plat on sample GR2363a Wells contain IPC plus target template Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 17

26 Chapter 3 Preparing the Samples and Reaction Plate Preparing the Reaction Plate 18 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

27 Chapter 4 Performing a Plus/Minus Assay Pre-Read Run Introduction About Plus/Minus Assay Plate Documents See page 20 Designing a Plus/Minus Assay Experiment Preparing the Samples and Reaction Plate Performing the Plus/Minus Assay Pre-ReadRun Creating a Plus/Minus Assay Plate Document See page 21 4 Performing an Amplification Run Performing the Pre-Read Run See page 30 Performing and Analyzing a Plus/Minus Assay Post-Read Run Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 19

28 Chapter 4 Performing a Plus/Minus Assay Pre-Read Run About Plus/Minus Assay Plate Documents About Plus/Minus Assay Plate Documents Plate Document Parameters A plus/minus assay plate document is an SDS software document that stores the data collected from a plus/minus assay run for a single reaction plate. Plus/minus assay plate documents also store other information about the run, including sample names and detectors. When you create a plate document for a plus/minus assay with an IPC, you define for each plus/minus assay reaction plate: Detectors A virtual representation in the SDS software of a TaqMan probe and primer set and an associated fluorescent dye that detects a single target nucleic acid sequence. Detector Tasks A setting that you apply to each well of a plate document. A detector task determines the way the SDS software uses the data collected from the well during analysis. For plus/minus assay plate documents, there are four types of detector tasks: Task Symbol Apply to all detectors of... Unknown IPC IPC + NTC Wells that contain target sequence. Wells that contain IPC. Control wells that contain IPC but no target template. Negative control wells that contain PCR reagents, but no target template and no IPC. Note: Applied Biosystems recommends that you run six replicates of each control (NAC and NTC) to accurately define plus/minus assay thresholds and to obtain plus/minus assay calls with a 99.7% confidence level. Options for Creating a Plate Document Reaction Plate Options This chapter describes how to create a new plate document using the New Plate Wizard. Refer to the SDS Online Help for other methods of creating new plate documents. The plus/minus assay can be run on both standard 96-well and standard 384-well reaction plates. For clarity, this chapter illustrates only the running of a standard 96-well reaction plate. 20 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

29 Chapter 4 Performing a Plus/Minus Assay Pre-Read Run Creating a Plus/Minus Assay Plate Document Creating a Plus/Minus Assay Plate Document To create a new plus/minus assay plate document: 1. If it is not already running, start the SDS software for the 7900HT Fast System: Double-click the SDS v2.3 software icon ( ) on the desktop or Select Start > All Programs > Applied Biosystems > SDS2.3 > SDS Optional. If your System Administrator has enabled the login option, the Login dialog box appears. Enter your User Name and Password, then click OK. Note: For more information, see the SDS Online Help. 3. Click (New Plate Wizard) to open the Create Plate Document Wizard. 4. Select the assay type: a. Select Plus/Minus (+/-). b. Click Next. 4a 4 4b Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 21

30 Chapter 4 Performing a Plus/Minus Assay Pre-Read Run Creating a Plus/Minus Assay Plate Document 5. Enter the plate information: a. Select your Plate Type. b. Optional. In the Barcode field, scan or enter the barcode for the plate. 5a Note: The Automation Accessory must be installed in order to use barcodes. A barcode is also required if you are adding the plate document to the automation queue (see step 5e below). c. Select to Create Document From your preferred source. You can either create a blank plate document or import existing information from a template or setup file. Note: Refer to the SDS Online Help for information about creating plate documents from templates and setup files. 5c 5f d. Optional. Check Save Settings As My Default. Select this option if you want the SDS software to automatically apply the current page settings every time you use the Create Plate Document Wizard. e. Optional. Check Add document to automation queue. The finished plate document will be added the Automation Controller software plate queue. See Appendix B on page 75 for more information. f. Click Next. 22 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

31 Chapter 4 Performing a Plus/Minus Assay Pre-Read Run Creating a Plus/Minus Assay Plate Document 6. Enter the samples to use in the plate: a. Double-click in the Sample field, type a sample name (for example, NAC), then press Enter. The sample row is numbered and another sample row appears. 6a Note: You may also copy sample names from an existing plate document (click Existing Plate) or enter mutiple sample names at once (click Auto name Selected). For more information, see the SDS Online Help. b. Continue entering your remaining samples (repeat step 6a above). Note: To remove sample(s) in the Samples to Use in Plate table, select the sample(s), then click Clear Selected. 6a 6b 7. Enter the detectors to use in the plate: a. Click Create Detector to open the Add Detector dialog box. Note: You may also add detectors from other sources (click Existing Plate, Detector Manager or Assay Information File). For more information, see the SDS Online Help. 4 7a Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 23

32 Chapter 4 Performing a Plus/Minus Assay Pre-Read Run Creating a Plus/Minus Assay Plate Document b. Enter a name for the detector (for example, IPC). IMPORTANT! The name of the detector must be unique and should reflect the target locus of the assay (for example, IPC or E. coli). Do not use the same name for multiple detectors. 7b 7c 7e 7f 7g c. Accept the Default Group setting. d. Optional. Enter a brief description of the detector (up to 32 characters) in the Description field. e. Leave the AIF Assay ID field blank. Note: For more information on Assay Information Files (AIF), see the SDS Online Help. 7i f. Select the appropriate dyes for the detector from the Reporter and Quencher dye dropdown lists. IMPORTANT! Ensure that the reporter dye for the target is different from the reporter dye for the IPC. The TaqMan Exogenous Internal Positive Control Reagents Kit (PN ) uses an IPC probe labeled with VIC (reporter) and TAMRA (quencher) dyes. Note: Select TAMRA as the quencher for TaqMan probes and None for TaqMan MGB probes. 24 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

33 Chapter 4 Performing a Plus/Minus Assay Pre-Read Run Creating a Plus/Minus Assay Plate Document g. Click the Color box to open the Select Detector Color dialog box, select a color swatch to represent the detector, then click OK. h. Optional. Enter any additional comments for the detector (up to 200 characters) in the field. Step 7g: Choose a color swatch for the detector, then click OK. i. Click OK. The SDS software saves the new detector and displays it in the Detectors to Use in Plate table of the wizard. j. Continue entering your remaining detector(s) (repeat steps 7a through 7i above). Note: To remove detector(s) in the Detectors to Use in Plate table, select the detector(s), then click Clear Selected. k. Click Next. Sample Experiment 7k 4 For the sample plus/minus assay experiment, we created a detector for the E. coli target, and another for the IPC. The E. coli detector was assigned a yellow color and the IPC detector a red color. Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 25

34 Chapter 4 Performing a Plus/Minus Assay Pre-Read Run Creating a Plus/Minus Assay Plate Document 8. Assign samples and detectors to the wells: a. From the Plate Layout drop-down list, select Individual Wells. 8a 8b 8c 8d IMPORTANT! If you change the plate layout after assigning samples or detectors to the wells, all assignments are cleared from the plate grid. b. In the plate grid, select the well(s) containing the first sample. Note: To select more than one well at a time, hold down the Ctrl key or Shift key while selecting the wells. c. In the Detectors in Selected Wells table of the Add Samples and Detectors tab, assign the target detector for the selected well(s). d. Select the appropriate target detector task from the Task drop-down list. e. In the Detectors in Selected Wells table, assign the IPC detector. f. Select the appropriate IPC detector task from the Task drop-down list. 8e 8f 26 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

35 Chapter 4 Performing a Plus/Minus Assay Pre-Read Run Creating a Plus/Minus Assay Plate Document g. In the Samples in Selected Wells table, assign the appropriate sample for the selected well(s). h. Continue assigning samples and detectors for the remaining wells (repeat steps 8b through 8g above). Note: Before you can use a plate document to run a plate, you must create and apply detectors for all samples on the plate. 9. Optional. Verify the chosen sample setup information (sample name, detector, task) for each well. a. Select the Well Inspector tab. b. Select the desired well(s) in the plate grid. Note: To select more than one well at a time, hold down the Ctrl key or Shift key while selecting the wells. 8g 10 c. Verify the information for each well in the Well Inspector tab. 4 Note: If you need to correct the sample setup information for a well, select the well(s) in the Well Inspector table, click Clear Selected, then assign the correct setup information. 10. Click Finish. The Create Plate Document Wizard closes and a new plus/minus assay plate document opens in the main SDS software window. Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 27

36 Chapter 4 Performing a Plus/Minus Assay Pre-Read Run Creating a Plus/Minus Assay Plate Document 11. Select the Setup tab and verify the default setting for the Passive Reference (ROX dye). IMPORTANT! If your experiment does not use all the wells on a plate, do not omit the wells from use at this point. You can omit unused wells after the run is complete. For more information about omitting wells, refer to the SDS Online Help. Note: If necessary, you can change the sample setup information (sample name, detector, task) after a run is complete Save the pre-read plate document: a. Select File > Save As to open the Save As dialog box. b. In the Save in field, navigate to and select a directory to save the plate document. c. In the File name field, enter a file name for the plate document. d. From the Files of type drop-down list, select SDS 7900HT Document (*.sds), then click Save. e. Optional. If you want to use this plate setup again, you can save it as a template. Select File > Save As, type a File name, then select Files of type: (*.sdt). Note: Refer to the SDS Online Help for information about using plate document templates and setup files. 12b 12c 12d 28 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

37 Chapter 4 Performing a Plus/Minus Assay Pre-Read Run Creating a Plus/Minus Assay Plate Document Sample Experiment We created the pre-read plate document with controls and samples as shown below. For each well, two detector tasks were selected, one for the target (E. coli) and one for the IPC. There were three possible combinations (shown below): the NN wells contained the NAC (no target template plus blocked IPC), the NI + wells contained the NTC (no target template plus IPC), and the UI wells contained the unknown sample plus IPC. Well legend: Well color Detector color, task and name Sample name Number of detectors 4 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 29

38 Chapter 4 Performing a Plus/Minus Assay Pre-Read Run Performing the Pre-Read Run Performing the Pre-Read Run About the Pre-Read Run A pre-read run records the background fluorescence of each well of the plus/minus assay plate before performing PCR amplification (see Chapter 5 on page 33). Then, during the post-read run (see Chapter 6 on page 47), the pre-read fluorescence is subtracted from the post-read fluorescence to ensure that fluorescence due only to amplification is recorded. To run an individual plate: 1. Select the Instrument >Plate Read tab. 2. Click Connect to connect the plate document to the instrument Click Open/Close. The instrument tray rotates to the OUT position Load the reaction plate into the instrument tray. IMPORTANT! Ensure that the A1 position is at the top-left side of the instrument. st 6-well plate load in 7900 Well A1 12 Notched corner 30 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

39 Chapter 4 Performing a Plus/Minus Assay Pre-Read Run Performing the Pre-Read Run 5. Click Pre Read. The instrument tray rotates to the IN position. 5 During the pre-read run, the instrument collects one fluorescence scan per well. As the instrument performs the run, it displays status information in the Plate Read tab. After the pre-read run is finished: A message indicates whether or not the run was successful. Click OK to close the dialog box. The instrument tray rotates to the OUT position. The date and time of completion is recorded in the Date Collection Stamp pane and the Pre Read button is disabled. 6. Optional. Review the fluorescence data generated during the pre-read run: a. Click (Hide/Show System Raw Data Pane). 4 b. In the Plate Grid, select the well(s) you want to view. c. Select the Raw Data Plot tab, then select Pre PCR Read from the Spectra drop-down list to view the pre-read run data. Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 31

40 Chapter 4 Performing a Plus/Minus Assay Pre-Read Run Performing the Pre-Read Run 7. Select File > Close. All raw data generated during the pre-read run and any changes to the plate document settings are saved to the file that you specified in step 12 on page Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

41 Chapter 5 Performing an Amplification Run Introduction About Standard Curve (AQ) Plate Documents See page 34 Designing a Plus/Minus Assay Experiment Preparing the Samples and Reaction Plate Creating a Standard Curve (AQ) Plate Document See page 35 Performing the Plus/Minus Assay Pre-ReadRun Performing an Amplification Run 5 Performing the Amplification Run See page 44 Performing and Analyzing a Plus/Minus Assay Post-Read Run Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 33

42 Chapter 5 Performing an Amplification Run About Standard Curve (AQ) Plate Documents About Standard Curve (AQ) Plate Documents Benefits of Real-Time PCR Amplification Using Standard Curve (AQ) Plate Documents for Amplification Because the plus/minus assay is an end-point assay, you can amplify the target sequences offline using any thermal cycler. However, using the 7900HT Fast System to amplify the target sequences provides real-time PCR data. After the plus/minus assay samples are analyzed (see Chapter 6 on page 47), you can study the amplification plots if you observe questionable calls or do not observe data for a well (see Appendix C on page 81). You create and use standard curve (AQ) plate documents to store real-time data for plus/ minus assays. Because the AQ plate document is used only to amplify target sequences (not to quantify the PCR data), you do not need a standard curve for the AQ plate. Note: See the Applied Biosystems 7900HT Fast Real-Time PCR System Absolute Quantitation using Standard Curve Getting Started Guide (PN ) for more information about creating AQ plate documents and performing AQ assays. Detector Tasks For AQ plate documents, there are three types of detector tasks: Task Symbol Apply to detectors of... Unknown Standard NTC Wells that contain target sequences that you are quantifying. Wells that contain samples of known quantities. Negative control wells that contain PCR reagents, but that lack template. Note: For AQ plate documents created for plus/minus assays, the task label Unknown is used for both IPC and the target samples. Options for Creating a Plate Document Reaction Plate Options This chapter describes how to create a new plate document using the New Plate Wizard. Refer to the SDS Online Help for other methods of creating new plate documents. Amplification of the plus/minus assay can be run on both standard 96-well and standard 384-well reaction plates. For clarity, this chapter illustrates only the running of a standard 96-well reaction plate. 34 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

43 Chapter 5 Performing an Amplification Run Creating a Standard Curve (AQ) Plate Document Creating a Standard Curve (AQ) Plate Document To create a new AQ plate document: 1. If it is not already running, start the SDS software for the 7900HT Fast System: Double-click the SDS v2.3 software icon ( ) on the desktop or Select Start > All Programs > Applied Biosystems > SDS2.3 > SDS Optional. If your System Administrator has enabled the login option, the Login dialog box appears. Enter your User Name and Password, then click OK. Note: For more information, see the SDS Online Help. 3. Select (New Plate Wizard) to open the Create Plate Document Wizard. 4. Select the assay type: a. Select Standard Curve (AQ). 4a Note: A standard curve is not necessary for a non-quantitation amplification run. b. Click Next. 5 4b Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 35

44 Chapter 5 Performing an Amplification Run Creating a Standard Curve (AQ) Plate Document 5. Enter the plate information: a. Select your Plate Type. b. Optional. In the Barcode field, scan or enter the barcode for the plate. 5a Note: The Automation Accessory must be installed in order to use barcodes. A barcode is also required if you are adding the plate document to the automation queue (see step 5e below). c. Select to Create Document From your preferred source. You can either create a blank plate document or import existing information from a template or setup file. Note: Refer to the SDS Online Help for information about creating plate documents from templates and setup files. 5c 5f d. Optional. Check Save Settings As My Default. Select this option if you want the SDS software to automatically apply the current page settings every time you use the Create Plate Document Wizard. e. Optional. Check Add document to automation queue. The finished plate document will be added the Automation Controller software plate queue. See Appendix B on page 75 for more information. f. Click Next. 36 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

45 Chapter 5 Performing an Amplification Run Creating a Standard Curve (AQ) Plate Document 6. Enter the samples to use in the plate: a. In the Samples to Use in plate window, click Existing Plate to copy the sample names from the pre-read plate document you created in Chapter 4 on page 19. The Open dialog box opens. 6a Note: You may also enter sample names manually (see step 6 on page 23 for instructions) or enter mutiple sample names at once (click Auto name Selected). For more information, see the SDS Online Help. b. In the Look in field of the Open dialog box, navigate to and select the previously saved pre-read plate document file, then click Open. The SDS Software displays the sample names from the pre-read plate document in the Samples to Use in Plate table of the wizard. Note: To remove sample(s) in the Samples to Use in Plate table, select the sample(s), then click Clear Selected. 6b Sample names appear here 5 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 37

46 Chapter 5 Performing an Amplification Run Creating a Standard Curve (AQ) Plate Document 7. Enter the detectors to use in the plate: a. In the Detectors to Use in plate window, click Existing Plate to copy the detectors from the pre-read plate document you created in Chapter 4 on page 19. The Open dialog box opens. 7a Note: You may also add detectors from other sources (click Create Detector, Detector Manager, or Assay Information File). For more information, see the SDS Online Help. b. In the Look in field of the Open dialog box, navigate to and select the previously saved pre-read plate document file, then click Open. The SDS Software displays the detectors from the pre-read plate document in the Detectors to Use in Plate table of the wizard. Note: To remove detector(s) in the Detectors to Use in Plate table, select the detector(s), then click Clear Selected. 7b Detectors appear here 38 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

47 Chapter 5 Performing an Amplification Run Creating a Standard Curve (AQ) Plate Document c. Click Next. 7c 8. Assign samples and detectors to the wells: a. From the Plate Layout drop-down list, select Individual Wells. 8a 8b 8c 8d IMPORTANT! If you change the plate layout after assigning samples or detectors to the wells, all assignments are cleared from the plate grid. b. In the plate grid, select the well(s) containing the first sample. Note: To select more than one well at a time, hold down the Ctrl key or Shift key while selecting the wells. c. In the Detectors in Selected Wells table of the Add Samples and Detectors tab, assign the target detector for the selected well(s). d. Select the appropriate target detector task from the Task drop-down list. 5 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 39

48 Chapter 5 Performing an Amplification Run Creating a Standard Curve (AQ) Plate Document e. In the Detectors in Selected Wells table, assign the IPC detector. f. Select the appropriate IPC detector task from the Task drop-down list. 8e 8f g. In the Samples in Selected Wells table, assign the appropriate sample for the selected well(s). h. Continue assigning samples and detectors for the remaining wells (repeat steps 8b through 8g above). Note: Before you can use a plate document to run a plate, you must create and apply detectors for all samples on the plate. 8g Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

49 Chapter 5 Performing an Amplification Run Creating a Standard Curve (AQ) Plate Document 9. Optional. Verify the chosen sample setup information (sample name, detector, task) for each well. a. Select the Well Inspector tab. b. Select the desired well(s) in the plate grid. Note: To select more than one well at a time, hold down the Ctrl key or Shift key while selecting the wells. c. Verify the information for each well in the Well Inspector tab. Note: If you need to correct the sample setup information for a well, select the well(s) in the Well Inspector table, click Clear Selected, then assign the correct setup information. 10. Click Finish. The Create Plate Document Wizard closes and a new AQ plate document opens in the main SDS software window. 11. Select the Setup tab and verify the default setting for the Passive Reference (ROX dye). IMPORTANT! If your experiment does not use all the wells on a plate, do not omit the wells from use at this point. You can omit unused wells after the run is complete. For more information about omitting wells, refer to the SDS Online Help. Note: If necessary, you can change the sample setup information (sample name, detector, task) after a run is complete 5 11 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 41

50 Chapter 5 Performing an Amplification Run Creating a Standard Curve (AQ) Plate Document 12. Select the Instrument > Thermal Cycler > Thermal Profile tab. By default, the Standard PCR conditions for the amplification step are displayed IMPORTANT! The 2-min, 50 C step is required for optimal AmpErase UNG activity. Note: For more information about configuring the Thermal Profile, Auto Increment, Ramp Rate and Data Collection tabs, see the SDS Online Help. 13. Accept the default values for: Mode: Standard or 9600 Emulation Note: When Standard mode is selected, the SDS software uses the 9700 thermal cycler ramp rate in the 7900HT Fast System for standard PCR reactions. When the 9600 Emulation mode is selected, the SDS software reduces the 7900HT Fast System ramp rate to match that of the 9600 thermal cycler in the ABI PRISM 7700 Sequence Detection System. Standard mode default thermal profile Standard 96-well reaction plate volume Sample volume: 50 µl for a standard 96- well reaction plate or 20 µl for a standard 384-well reaction plate 14. Save the AQ plate document: a. Select File > Save As to open the Save As dialog box. b. In the Save in field, navigate to and select a directory to save the plate document. c. In the File name field, enter a file name for the plate document. d. From the Files of type drop-down list, select SDS 7900HT Document (*.sds), then click Save. 14b 14c 14d 42 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

51 Chapter 5 Performing an Amplification Run Creating a Standard Curve (AQ) Plate Document e. Optional. If you want to use this plate setup again, you can save it as a template. Select File > Save As, type a File name, then select Files of type: (*.sdt). Note: Refer to the SDS Online Help for information about using plate document templates and setup files. Sample Experiment We created an AQ plate document with controls and samples as shown below. For each well, two detector tasks were selected, one for the target (E. coli) and one for the IPC. There were three possible combinations: the NN wells contained the NAC (no target template, plus blocked IPC), the NU wells contained the NTC (no target template plus IPC), and the UU wells contained the unknown sample plus IPC. Note: For AQ plate documents created for plus/minus assays, the task label Unknown is used for both IPC and the target samples. 5 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 43

52 Chapter 5 Performing an Amplification Run Performing the Amplification Run Performing the Amplification Run To run an individual plate: 1. Select the Instrument >Real-Time tab Click Connect to Instrument to connect the AQ plate document to the instrument Optional. Click Open/Close. The instrument tray rotates to the OUT position. Note: The instrument tray will already be in the OUT position if you are performing the plus/ minus assay runs in sequence (pre-read, amplification, post-read). 4. Load the reaction plate into the instrument tray. IMPORTANT! Ensure that the A1 position is at the top-left side of the instrument. st 6-well plate load in 7900 Well A1 12 Notched corner 5. Click Start Run. The instrument tray rotates to the IN position. During the amplification run, the instrument records the fluorescence resulting from cleavage of TaqMan probes in the presence of the target sequences. As the instrument performs the run, it displays real-time status information in the Real- Time tab. 5 After the amplification run is finished: A message indicates whether or not the run was successful. Click OK to close the dialog box. The instrument tray rotates to the OUT position. 44 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

53 Chapter 5 Performing an Amplification Run Performing the Amplification Run The status values and buttons are grayed-out in the Real-Time tab, and the Analyze button ( ) is enabled. Note: If necessary, the amplification run data can be analyzed later to help troubleshoot the plus/minus assay results (see Appendix C on page 81 for more information). To continue with the plus/minus assay workflow, proceed to step 6 below. 6. Select File > Close. All raw data generated during the amplification run and any changes to the plate document settings are saved to the file that you specified in step 14 on page Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 45

54 Chapter 5 Performing an Amplification Run Performing the Amplification Run 46 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

55 Chapter 6 Performing and Analyzing a Plus/ Minus Assay Post-Read Run Introduction Performing the Post-Read Run See page 48 Designing a Plus/Minus Assay Experiment Preparing the Samples and Reaction Plate Analyzing the Run and Evaluating the Results See page 52 Performing the Plus/Minus Assay Pre-ReadRun Performing an Amplification Run Post Analysis Options See page 65 Performing and Analyzing a Plus/Minus Assay Post-Read Run 6 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 47

56 Chapter 6 Performing and Analyzing a Plus/Minus Assay Post-Read Run Performing the Post-Read Run Performing the Post-Read Run About the Post-Read Run A post-read run records the fluorescence of each well of the plus/minus assay plate after performing PCR amplification (see Chapter 5 on page 33). During the post-read run, the pre-read background fluorescence (see Chapter 4 on page 19) is subtracted from the post-read fluorescence to ensure that fluorescence due only to amplification is recorded. Reaction Plate Options The plus/minus assay can be run on both standard 96- well and standard 384-well reaction plates. For clarity, this chapter only illustrates the running of a standard 96-well reaction plate. To perform the post-read run: 1. Select File > Open, browse to open the previously saved pre-read plate document, then click Open. The plus/minus assay plate document opens in the main SDS software window Optional. Save the current file as a post-read plate document: Note: By default, both the pre-read and postread run data are saved to the same plus/minus assay plate document. Use this option if you want to retain these data sets in two separate plate documents. a. Select File > Save As to open the Save As dialog box. b. In the Save in field, navigate to and select a directory to save the plate document. 48 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

57 Chapter 6 Performing and Analyzing a Plus/Minus Assay Post-Read Run Performing the Post-Read Run c. In the File name field, enter a file name for the plate document. d. From the Files of type drop-down list, select SDS 7900HT Document (*.sds), then click Save. To run an individual plate: 1. Select the Instrument >Plate Read tab. 2. Click Connect to connect the plate document to the instrument Click Open/Close. The instrument tray rotates to the OUT position. Note: The instrument tray will already be in the OUT position if you are performing the plus/ minus assay runs in sequence (pre-read, amplification, post-read) Load the reaction plate into the instrument tray. IMPORTANT! Ensure that the A1 position is at the top-left side of the instrument. st 6-well plate load in 7900 Well A1 12 Notched corner 6 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 49

58 Chapter 6 Performing and Analyzing a Plus/Minus Assay Post-Read Run Performing the Post-Read Run 5. Click Post Read. The instrument tray rotates to the IN position. 5 During the post-read run, the instrument collects one fluorescence scan per well. As the instrument performs the run, it displays status information in the Plate Read tab. After the post-read run is finished: A message indicates whether or not the run was successful. Click OK to close the dialog box. The instrument tray rotates to the OUT position. The date and time of completion is recorded in the Date Collection Stamp pane and the Post Read button is disabled. 6. Optional. Review the raw fluorescence data generated during the post-read run: a. Click (Hide/Show System Raw Data Pane). b. In the Plate Grid, select the well(s) you want to view. c. Select the Raw Data Plot tab, then select Post PCR Read from the Spectra dropdown list to view the post-read run data. 50 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

59 Chapter 6 Performing and Analyzing a Plus/Minus Assay Post-Read Run Performing the Post-Read Run d. To review the subtracted (post-read minus pre-read) raw fluorescence data, select Post PCR-Pre PCR from the Spectra drop-down list. 6 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 51

60 Chapter 6 Performing and Analyzing a Plus/Minus Assay Post-Read Run Analyzing the Run and Evaluating the Results Analyzing the Run and Evaluating the Results After completing the plus/minus assay post-read run, the SDS software compares the relationship between the spectral changes in the unknown samples and the control reactions (NAC and NTC) defined previously (see About Plus/Minus Assays Using an IPC on page 3). The software first calculates an IPC threshold from the NAC control reactions and a target threshold from the NTC control reactions. The software then uses the target threshold to determine amplification of the unknown sample signal for each well and the IPC threshold to determine amplification of the IPC signal in each unknown sample well. Criteria for Calling Unknowns To call the unknown samples, the SDS software initially compares the: Normalized reporter dye signal of each unknown sample to the target threshold IPC sample signal to the IPC threshold The results for the unknown sample are then determined as follows: If the unknown sample signal is above the target threshold, then the call for the unknown sample is plus (+). If the unknown sample signal is below the target threshold, the SDS software compares the IPC sample signal to the IPC threshold. If the IPC sample signal is: Above the IPC threshold, then the call for the unknown sample is minus ( ). Below the IPC threshold, then the call for the unknown sample is an unconfirmed minus (?). If neither the target threshold nor the IPC threshold can be calculated from the NAC and NTC control reactions (either due to missing or failed control reactions), then both the target and IPC thresholds are set at zero and the call for the unknown sample is undetermined ( ). Note: Plus/minus assays can also be accomplished without the use of an IPC; however, an IPC ensures that a failed PCR reaction is not mistaken for a negative target result. Analyzing the Post-Read Run 1. In the plus/minus assay plate document, click (or select Analysis > Analysis Settings) to open the Analysis Settings dialog box. 2. Set the auto-analysis settings for the plus/minus assay plate document: a. Select All Detectors (default) from the Detector drop-down list. Note: You can also choose to select an individual detector from the Detector drop-down list. b. Select a percentage value (default 99.0) from the Quality Value drop-down list to apply as the quality interval for auto-calling samples (the greater the value, the more stringent the calls). 52 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

61 Chapter 6 Performing and Analyzing a Plus/Minus Assay Post-Read Run Analyzing the Run and Evaluating the Results c. Click OK to save your settings and close the Analysis Settings dialog box. 2a 2b 2c Note: For more information on assigning threshold quality values, see the SDS Online Help. 3. Click (or select Analysis > Analyze). The SDS software analyzes the post-read run data and displays the plus/minus assay results in the Results tab. After analysis is complete, you can: Evaluate the analysis results. See Viewing the Results on page 54 for more information. Export and/or print the analysis results. See Post-Analysis Options on page 65 for more information. or Select File > Close. All raw data generated during the post-read run and any changes to the plate document settings are saved to the file that you specified in step 2 on page 48. IMPORTANT! Analysis results are not saved to the plate document. You must re-analyze a plus/minus assay plate document containing both pre- and postread run data each time you open it in the SDS software. 6 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 53

62 Chapter 6 Performing and Analyzing a Plus/Minus Assay Post-Read Run Analyzing the Run and Evaluating the Results Tip: Reanalyzing Data Once you click the Analyze button, it becomes disabled. To reanalyze the data, you can: Reenable the Analyze button by changing the setup information in the plate document (for example, removing a well or omitting a detector assignment), then click (or select Analysis > Analyze) again. Change analysis settings in the Analysis Settings dialog box, then click OK. The SDS software automatically reanalyzes the data when you click OK. (For information on using the Analysis Settings dialog box, see Analyzing the Post-Read Run on page 52.) Note: In order for the SDS software to automatically reanalyze the data via the Analysis Settings dialog box, you must have already analyzed the data once by clicking the Analyze button. Viewing the Results When the post-read analysis is complete, the plate document displays the plus/minus assay calls for the presence or absence of the target sequence for each well. You can view the plus/minus assay results using the following display options: Results table (see Using the Results Table on page 56) Results view of the plate grid (see Using the Plate Grid on page 57) Plus Minus Results Inspector plot (see Using the Plus Minus Results Inspector Plot on page 59) 54 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

63 Chapter 6 Performing and Analyzing a Plus/Minus Assay Post-Read Run Analyzing the Run and Evaluating the Results The figure below illustrates the plus/minus assay plate document default views after the post-read analysis is complete. Plate Grid Plus Minus Results Inspector Plot Results Table The results displays are actively synchronized. In the example shown below, selecting a well in the plate grid also selects the corresponding data in the Results table and Plus Minus Results Inspector plot. For more information on viewing plus/minus assay data, see the SDS Online Help. Well G11 selected Results for well G11 6 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 55

64 Chapter 6 Performing and Analyzing a Plus/Minus Assay Post-Read Run Analyzing the Run and Evaluating the Results Using the Results Table The Results table displays the assay-specific setup and analysis properties for the plate document in a table format. Once the plus/minus assay post-read analysis is complete, the Results table displays the following default parameters: Well position Sample name Detector name Signal (as R n value) for the detector Threshold R n value for the IPC or target detector (depending on the task assigned to the well) Mean of the R n value for the detector Standard deviation of the R n value for the NTC detector task (if applicable) Detector task: Unknown, NTC, IPC, IPC + Plus/minus assay calls for each well. The call for NAC and NTC wells is designated as Control. The IPC call for unknown wells is designated as either IPC Failed or IPC Succeeded. The sample call for unknown wells is designated as plus (+), minus ( ), unconfirmed minus (?) or undetermined ( ). Note: See Appendix C on page 81 for an explanation of terms used in quantitation analysis. Note: You can configure the Results table entries either by double-clicking on a column header, by selecting a user-defined Table Settings profile from the drop-down list, or by clicking (Table Settings) to open the Table Settings dialog box. For more information, see the SDS Online Help. 56 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

65 . Chapter 6 Performing and Analyzing a Plus/Minus Assay Post-Read Run Analyzing the Run and Evaluating the Results The figure below shows the Results table default parameters. Select a Table Settings profile to filter the data Double-click on a column header to sort the data Using the Plate Grid The plate grid displays the assay-specific setup and analysis properties for the plate document in a well format corresponding to the type of reaction plate used for the run. As shown in the following figure, the default Setup view displays the following default parameters for each unknown sample well in the plate document: Well color Detector task: Unknown (U), NTC (N), IPC (I), IPC + (I + ) Detector name Signal (as R n value) for the detector Sample name Plus/minus assay call: plus (+), minus ( ), unconfirmed minus (?) or undetermined ( ) Number of detectors 6 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 57

66 Chapter 6 Performing and Analyzing a Plus/Minus Assay Post-Read Run Analyzing the Run and Evaluating the Results Well color Detector color, task, name, call and R n Sample call Sample name Number of detectors Note: No calls are displayed for the NAC and NTC wells. Once the plus/minus assay post-read analysis is complete, the plate grid switches to the Results view. You can also right-click in the plate grid and select Setup View or Results View from the pop-up menu. By default, the Results view of the plate grid displays the plus/minus assay call for the unknown sample wells in the plate document, as shown in the figure below. Control wells (no calls shown) Sample calls Note: You can configure the display of well information in the plate grid by selecting View > Display Setting and then selecting Results Grid in the Display Settings dialog box. For more information, see the SDS Online Help. 58 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

67 Chapter 6 Performing and Analyzing a Plus/Minus Assay Post-Read Run Analyzing the Run and Evaluating the Results Sample Experiment In the sample plus/minus assay experiment using an IPC, the SDS software subtracted the pre-read run fluorescence signal from the post-read run fluorescence signal to account for background fluorescence. Post-read results for the presence of E. coli are displayed in the plate grid Setup view shown below. For an explanation of results see Criteria for Calling Unknowns on page 52. Using the Plus Minus Results Inspector Plot Once the plus/minus assay post-read analysis is complete, the Results tab displays the Plus Minus Results Inspector plot, which by default plots the signal intensity (as R n value) of the target (unknown) detector against each well position in the plate document, as shown in the figure below. Note: Only one target detector can be displayed at a time. 6 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 59

68 Chapter 6 Performing and Analyzing a Plus/Minus Assay Post-Read Run Analyzing the Run and Evaluating the Results As illustrated in the following figure, you can choose to view the results for all of the call categories (default) or for individual call categories from the Show Calls drop-down list. Refer to Criteria for Calling Unknowns on page 52 for more information about the plus/minus assay call categories. By default, the results for the NTC controls are displayed while the IPC controls are not shown. The IPC threshold (calculated from the NAC wells) and the target threshold (calculated from the NTC wells) are also represented in the plot as indicated in the figure below. Call category selection Detector selection Controls selection Data viewing tools Call icon legend Call icons Detector R n value IPC threshold Target threshold Well number Note: You can configure the display of information in the Plus Minus Results Inspector plot by selecting View > Display Setting and then selecting Plus/Minus Result Inspector in the Display Settings dialog box. For more information about Display Settings or data viewing tools for the Plus Minus Results Inspector plot, see the SDS Online Help. 60 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

69 Chapter 6 Performing and Analyzing a Plus/Minus Assay Post-Read Run Analyzing the Run and Evaluating the Results You can use the Plus Minus Results Inspector plot R n values from the plus/minus assay plate document to identify any unconfirmed minus (?) or undetermined ( ) calls needing futher data review using the results from the amplification run you performed in Chapter 5 on page 33. See Using the Amplification Plots on page 64 for more information. The following Sample Experiment boxes show the Plus Minus Results Inspector plots of each type of result: NAC, NTC, plus (+), minus ( ) and unconfirmed minus (?). Note: You will only encounter an undetermined ( ) result if neither the target threshold nor the IPC threshold can be calculated by the SDS software. Undetermined ( ) results are not displayed for the unknown samples comprising the Sample Experiment. Sample Experiment The Results Inspector plot below is for unknown sample well A5, a no amplification control (NAC), which contains blocked IPC and no target template. This plot shows no amplification for IPC or the E. coli target sequence. Plate Grid Well A5 Blocked IPC, no amplification No target (E. coli) present 6 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 61

70 Chapter 6 Performing and Analyzing a Plus/Minus Assay Post-Read Run Analyzing the Run and Evaluating the Results Sample Experiment The Results Inspector plot below is for well A10, a no template control (NTC), which contains IPC but no target template. This plot shows no amplification for the E. coli target sequence. Plate Grid Well A10 IPC present No target (E. coli) present The Results Inspector plot below is for unknown sample well F3, which displays a minus ( ) result because the amplification for the E. coli target sequence is less than the target threshold, but the IPC sample signal is above the calculated IPC threshold defined by the SDS software. Plate Grid Well F3 IPC present Target (E. coli) below threshold 62 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

71 Chapter 6 Performing and Analyzing a Plus/Minus Assay Post-Read Run Analyzing the Run and Evaluating the Results Sample Experiment The Results Inspector plot below is for unknown sample well C3, which displays a plus (+) result (for the E. coli target sequence) because it has amplification above the target threshold. Plate Grid Well C3 Target (E. coli) present IPC present The Results Inspector plot below is for unknown sample well G11, which displays an unconfirmed minus (?) result because the unknown sample signal is below the target threshold, and the IPC sample signal is below the calculated IPC threshold defined by the SDS software. Plate Grid Well G11 IPC below threshold Target (E. coli) below threshold 6 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 63

72 Chapter 6 Performing and Analyzing a Plus/Minus Assay Post-Read Run Analyzing the Run and Evaluating the Results Using the Amplification Plots Amplification plots are obtained from the standard curve (AQ) plate document that you created in Chapter 5 on page 33 to amplify the samples in a standard 96-well plate. After the plus/minus assay samples are analyzed in the plus/minus assay plate document, you can study the amplification plots from the AQ plate document if you observe questionable calls or do not observe data for a well. Note: For more information on analyzing the plus/minus assay amplification data, see Appendix C on page Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

73 Chapter 6 Performing and Analyzing a Plus/Minus Assay Post-Read Run Post-Analysis Options Post-Analysis Options The following options are available after the analysis: Exporting Data as Text Files (below) Exporting Data as Graphic Files (page 66) Printing a Data Report (page 67) Exporting Data as Text Files You can export raw or analyzed data from plate documents into tab-delimited text files (*.txt). The text files can then be imported into spreadsheet software, such as Microsoft Excel software. 1. Open the plate document from which you wish to export data. 2. Click or select File > Export to open the Export dialog box. 3. In the Look in field, navigate to and select a directory for the new file From the Export drop-down list, select the data type to export. 4 Note: The data type options shown depends on the plate document you selected. For more information about the data type options, see the SDS Online Help. 5. Select to export data from All Wells or select group of wells (Selected Wells). Note: To use the Selected Wells option, you must select a subset of wells in the plate document before opening the Export dialog box From the Format options, select the appropriate software version. 10 Note: Accept the default SDS v2.3 file format when exporting data from plus/minus assay plate documents Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 65

74 Chapter 6 Performing and Analyzing a Plus/Minus Assay Post-Read Run Post-Analysis Options 7. Optional. Check Save Settings As My Default. Check this option if you want the SDS software to automatically apply the current settings every time you export data as a text file. 8. In the File name field, type a name for the new file. 9. From the Files of type drop-down list, select Tab-delimited Text (*.txt). 10. Click Export. The software exports the new file to the specified directory. Exporting Data as Graphic Files You can export the plate grid and plot images from plate documents as Joint Photographic Experts Group (JPEG) graphic files (*.jpeg). The JPEG files can then be viewed in most common word processing, spreadsheet, and HTML-based software. 1. Open the plate document from which you wish to export data. 2. To export the: Plate grid, right-click in the plate grid, then select Save Grid to Image File. The Save dialog box appears. 66 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

75 Chapter 6 Performing and Analyzing a Plus/Minus Assay Post-Read Run Post-Analysis Options Plot, right-click in the plot, then select Save Plot to Image File. The Save dialog box appears. Note: If desired, adjust the plot dimensions (length and width) as you want them to appear in the exported file. The exported file retains the dimensions of the original screen element. 3. Complete the Save dialog box: a. In the Save in field, navigate to and select a directory for the new file. b. In the File name field, type a name for the new file. c. From the Files of type drop-down list, select JPEG File. d. Click Save. The software saves the new file to the specified directory. 3a 3d 3b 3c Printing a Data Report You can use the SDS software to print a report of the analyzed data containing individual or multiple elements of the plate document. Note: For more information on printing reports, see the SDS Online Help. 1. Open the plate document from which you wish to print a report. 2. Click (or select File > Print Report) to open the Print Report dialog box. 6 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 67

76 Chapter 6 Performing and Analyzing a Plus/Minus Assay Post-Read Run Post-Analysis Options 3. In the Include Data window, select the plate document element(s) to print. Note: By default, the selected data from all wells of the plate grid will be included in the printed report. Select data to print 4. Optional. Click Page Setup to format the display of the report and how the report is printed. 5. Optional. Check Save Settings As My Default. Check this option if you want the SDS software to automatically apply the current settings every time you print a report Optional. Click Preview to view the report before printing it. 7. Click Print to open the Print dialog box and print the report. 8. When the report has finished printing, click Done to close the Print Report dialog box. 68 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

77 Appendix A Sample Plus/Minus Assay Experiment A About the Sample Experiment Overview Description To illustrate how to design, perform, and analyze plus/minus assay experiments, this section provides a sample experiment. The sample experiment represents a typical plus/minus assay experiment that you can use to familiarize yourself with the plus/minus assay workflow. Details about the plus/minus assay workflow, including Sample Experiment boxes to illustrate workflow details, appear in the previous chapters of this guide (2 to 6). The objective of the sample plus/minus assay experiment is to determine if an E. coli target sequence is present or not present in each batch of ground beef for analysis. The experiment uses duplex PCR, where a set of primers and a VIC dye-labeled probe for the Internal Positive Control (IPC) plus a set of primers and a FAM dye-labeled probe for the target E. coli sequence are run together in each PCR reaction. The probe/primers set for detecting the target E. coli sequence are custom designed using the Applied Biosystems Primer Express software. Note: Plus/minus assays can also be accomplished without the use of an IPC; however, an IPC ensures that a failed PCR reaction is not mistaken for a negative target result. Reactions are set up for PCR in standard 96-well reaction plates using the TaqMan Universal PCR Master Mix (2X) and appropriate primers and probes. The sample plus/minus assay experiment data and results are generated using a 7900HT Fast System by performing: A pre-read run using a plus/minus assay plate document to determine the baseline fluorescence associated with primers and probes before amplification. An amplification run using a standard curve (AQ) plate document to generate realtime PCR data, which can be used to analyze and troubleshoot the PCR data for the plus/minus assay, if needed. A post-read run using the original plus/minus assay plate document, which automatically subtracts the pre-read baseline fluorescence determined during the pre-read run, then assigns positive or negative calls using the amplified data. Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 69

78 Appendix A Sample Plus/Minus Assay Experiment Plus/Minus Assay Experiment Overview Plus/Minus Assay Experiment Overview 1. Design the experiment as explained in Chapter 2 on page 9. a. Order the TaqMan Exogenous Internal Positive Control Reagents kit and the TaqMan Universal PCR Master Mix (2X). b. Design the primers and FAM dye-labeled probe set for E. coli detection with Applied Biosystems Primer Express software. 2. Extract DNA from samples (see Preparing the DNA Samples on page 14) using the PrepMan Ultra Sample Preparation Reagent Kit (PN ) and protocol (PN ) to obtain a final concentration of 10 ng/µl of DNA for each sample. 3. Prepare sufficient reaction mix (see Preparing the PCR Reaction Mix on page 15) for a standard 96-well plate using the volumes listed in the example table to the right.. CHEMICAL HAZARD. TaqMan Universal PCR Master Mix (2X) may cause eye and skin irritation. Exposure may cause discomfort if swallowed or inhaled. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Note: Prepare extra volume to account for pipetting losses. Component TaqMan Universal PCR Master Mix (2 ) Final Concentration Volume for One Reaction Std. 96-well plate 1 25 µl 10 Exo IPC Mix (IPC kit) 50 to 900 nm 5 µl 50 Exo IPC DNA (IPC kit) 50 to 900 nm 1 µl Target primers, probe, and deionized water 50 to 250 nm 14 µl Total Volume 45 µl Optimization may be necessary depending on your selected target sequence and primers/probe set. 70 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

79 Appendix A Sample Plus/Minus Assay Experiment Plus/Minus Assay Experiment Overview 4. Prepare the reaction plate (see Preparing the Reaction Plate on page 16). a. Pipette 45 µl of the reaction mixture into each well of a standard 96-well reaction plate. b. Pipette 5 µl of IPC block, TE or water, or unknown sample into the designated wells of a standard 96-well plate such as the example indicated in the table to the right. Note: The final reaction volume in each well is 50 µl. NAC NTC plu us IPC GR2363 A c. Keep the reaction plate on ice until you are ready to load it into the 7900HT Fast System. 5. Create a plus/minus assay pre-read plate document. For more detail, see Chapter 4 on page 19. a. Add detectors and samples to the plate document. b. Specify the detectors and tasks for each well. IMPORTANT! If your experiment does not use all the wells on a plate, do not omit the wells from use at this point. You can omit unused wells after the run is completed. For more information about omitting wells, refer to the SDS Online Help. Wells If preparing... Add A1 to A6 NAC A7 to A12 NTC B1 to H12 U # 5 µl of 10 Exo IPC Block 5 µl of 1 TE or H 2 O 5 µl of sample being tested for E. coli No Amplification Control well contains no target template and blocked IPC. No Template Control well contains no target template, only IPC. # Unknown well contains both target template and IPC. TE Tris-EDTA Buffer, ph 8.0. The figure on the right shows a completed plus/ minus assay pre-read plate setup with detectors, tasks and sample names. Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 71

80 Appendix A Sample Plus/Minus Assay Experiment Plus/Minus Assay Experiment Overview 6. Perform the plus/minus assay pre-read run. a. Select the Instrument > Plate Read tab, then click Connect. b. Select File > Save As, enter a name for the plus/minus assay pre-read plate document, then click Save. c. Load the reaction plate into the instrument. d. Click Pre Read. 7. Create a standard curve (AQ) plate document. For more detail, see Chapter 5 on page 33. a. Add detectors and samples to the plate document. b. Specify the detectors and tasks for each well. IMPORTANT! If your experiment does not use all the wells on a plate, do not omit the wells from use at this point. You can omit unused wells after the run is completed. For more information about omitting wells, refer to the SDS Online Help. The figure on the right shows a completed standard curve (AQ) plate setup with detectors, tasks and sample names. Note: A standard curve is not necessary for a non-quantitation amplification run. 72 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

81 Appendix A Sample Plus/Minus Assay Experiment Plus/Minus Assay Experiment Overview 8. Perform the plus/minus amplification run. a. Select the Instrument > Thermal Cycler > Thermal Profile tab. By default, the Standard PCR conditions for the PCR step are displayed. A b. Select File > Save As, type a name for the AQ Plate document, then click Save. c. Load the reaction plate into the instrument, then click Start Run. After the run, a message indicates if the run is successful, or if errors were encountered. 9. Perform the plus/minus assay post-read run. : a. Open the plus/minus assay pre-read plate document, then use it to perform the postread run. For more detail, see Chapter 6 on page 47. b. Select the Instrument > Plate Read tab, then click Connect. c. Select File > Save As, enter a name for the plus/minus assay post-read plate document, then click Save. d. Load the reaction plate into the instrument. e. Click Post Read. After the run, a message indicates if the run is successful, or if errors were encountered. Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 73

82 Appendix A Sample Plus/Minus Assay Experiment Plus/Minus Assay Experiment Overview 10. Analyze the plus/minus assay data, as explained in Chapter 6 on page 47. a. Click or select Analysis > Analyze. b. Click the Results tab to view results for each well in the plate grid, Results table and Plus/Minus Results Inspector Plot. c. If you need to troubleshoot the plus/minus assay results, see Appendix C on page Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

83 Appendix B SDS Automation Controller Software Overview The Sequence Detection Systems Automation Controller Software v2.3 provides an interface between the Zymark Twister Microplate Handler, the 7900HT Fast System, the fixed-position bar code reader, and the plate documents created in the main Sequence Detection Systems Software v2.3 (SDS Software v2.3). This Automation Controller software controls and coordinates the action of the 7900HT Fast instrument and the automation module. It initiates and controls the sequence detection run and acquires data during the run. This option allows for plates to be run as part of a group or batch allowing for highthroughput unattended operation. For more information about the Automation Controller, refer to the SDS Online Help. B Using the Automation Controller Software Note: Before launching the Automation Controller software, you must close the main SDS Software v2.3 application. Failure to do so will result in an instrument connection failure. 1. Double-click (the Automation Controller shortcut icon). An initialization popup will appear displaying the status of instrument connection and other verifications. If you encounter an error, refer to the SDS Online Help. Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 75

84 Appendix B Using the Automation Controller Software 2. If you have already created and saved the desired plates (sent them to the queue) in the main SDS application, then the plates will appear in the Plate Queue tab. For more information about creating plate documents and sending them to the queue, refer to Chapter You can add plates by clicking Add Plates or (or click ). a. At the Open dialog box, navigate to the location where the plate files are located. b. Use the barcode reader to scan the Plate IDs. Note that you can automatically scan the next plate without having to select Add Plates again. You can also save the list of queued plates, by clicking Save List. Note: To remove a plate, click on the plate name then click Remove. To remove all plates, click Remove All. 76 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

85 Appendix B Using the Automation Controller Software 4. Place the plates in the input stack of the Zymark Twister Microplate Handler. The order of the plates may vary from the order they were scanned in (the Handler s fixed barcode reader will verify the location of each plate). The software matches the plate document and the method before it starts the run. Orient the plates inside the stacks so that the well A1 of each plate corresponds to the locations shown in the figure below. X Zymark Twister Microplate Handler (top view) B GR2104 Bar code Well A1 5. Click into the boxes next to each stack location where the plates are located. 6. Select Tools > Batch Settings to specify the desired output settings. a. In the Batch Settings dialog box, at the Auto-Analysis Workflow tab, click into a box to select that export option. You can also change the export directory by clicking the Browse button (located in the Export Destination box) and specifying a new location for the file data to be exported to. Note that exported data will be saved as *.txt files. 6a b. Click on Change Format to assign attributes to be included in the export file name. 6b Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 77

86 Appendix B Using the Automation Controller Software c. At the Filename dialog box, click into a field box (under the Include column) to deselect an attribute. 6c d. Click OK. e. Back at the Batch Settings dialog box, click on the Notifications tab to specify desired notification options. For more information on settings, see your network administrator. 6d 6e f. Click OK. 7. Verify that the plate adapter and the output stack of the Handler are empty. 8. Click Start Batch. 6f 78 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

87 Appendix B Using the Automation Controller Software B Note: Note that if the plate type does not match the plate block installed on the instrument, the Automation Controller will not start. You can assign plate types by selecting Tools > Options and selecting the desired plate format. The handler loads the plates and the instrument starts the run. 8 Note: To stop the run, click Stop Batch. You will see a warning message asking you to verify stopping the run. 9. Click on the Run Status tab to view the plate run status. Note that once a plate is being processed, it will not appear in the Processed Plates tab or be listed in the Queued Plates section of the Batch Status box. The plate information will be available under the Currently Running Plate section. Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 79

88 Appendix B Using the Automation Controller Software 9 10 Note: For standard curve (AQ) and ddct (RQ) plates, a view of the amplification and temperature plot will be available and can be selected by clicking on the radial button. 10. Click on Processed Plates to view information about the processed plates. 80 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

89 Appendix C Analyzing and Viewing Amplification Data Terms Used in Quantitation Analysis The following are terms commonly used in quantitation analysis. Term Baseline Threshold cycle (C T ) Passive reference Reporter dye Normalized reporter (R n ) Delta R n ( R n ) Definition A line fit to fluorescence intensity values during the initial cycles of PCR, in which there is little change in fluorescence signal. The fractional cycle number at which the fluorescence intensity exceeds the threshold intensity. A dye that provides an internal fluorescence reference to which the reporter dye signal can be normalized during data analysis. Normalization is necessary to correct for fluorescence fluctuations caused by changes in concentration or volume. The dye attached to the 5 end of a TaqMan probe. The dye provides a signal that indicates specific amplification. The ratio of the fluorescence intensity of the reporter dye signal to the fluorescence intensity of the passive reference dye signal. The magnitude of the signal generated by a set of PCR conditions. ( R n = R n baseline) C The figure below is a representative DNA amplification plot and includes some of the terms defined above. Sample R n Rn Threshold Baseline C T No Template Control Cycle Number Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 81

90 Appendix C Analyzing and Viewing Amplification Data Analyzing the Amplification Data Analyzing the Amplification Data Before you can analyze the amplification data from the standard curve (AQ) plate document you generated in Chapter 5 on page 33, you must specify parameter values (baseline and threshold settings or C T ) for the analysis. Unless you have already determined the optimal baseline and threshold settings (C T ) for your experiment, use the automatic baseline and threshold feature of the SDS software (Automatic C T ) to analyze the amplification run. If the baseline and threshold are called correctly for each well, you can proceed to view the results (see Reviewing the Automatic CT Results on page 83). Otherwise, you must manually set the baseline and threshold. For more information about determining C T, refer to the Real-Time PCR Systems Chemistry Guide (PN ). For more information about manually adjusting C T, refer to the SDS Online Help. Starting the Analysis 1. Open the AQ plate document you want to analyze. 2. Click or select Analysis > Analysis Settings. The Analysis Settings dialog box opens In the Detector drop-down list of the Detector tab, select All Detectors. 4. In the Ct Analysis window, select Automatic Ct. The SDS software will automatically generate baseline values for each well and threshold values for each detector. 5. Click OK to apply any changes and close the Analysis Settings dialog box. 6. Click or select Analysis > Analyze. The Results and QC Summary tabs are available after analysis completes Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

91 Appendix C Analyzing and Viewing Amplification Data Analyzing the Amplification Data Reviewing the Automatic C T Results Click the Results tab to view results of the amplification run for each well. The SDS software Automatic C T analysis process calculates baseline and threshold values for a detector based on the assumption that the data exhibits a typical amplification curve. A typical amplification curve has a: Plateau phase (a) Linear phase (b) Exponential (geometric) phase (c) Background (d) Baseline (e) Threshold a b c R n d C Cycle e IMPORTANT! Experimental error (such as contamination, pipetting errors, etc.) can produce atypical amplification curves that can result in incorrect baseline and threshold value calculations by the SDS software. Therefore, Applied Biosystems recommends you examine the amplification plot and review the assigned baseline and threshold parameter values for each well after analysis completes, and if necessary manually adjust the baseline and threshold. For more information, see the SDS Online Help. Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 83

92 Appendix C Analyzing and Viewing Amplification Data Viewing the Amplification Data Viewing the Amplification Data The following options are available for further determination of the analysis results: Using the Plate Grid Results View (below) Using the Raw Data Plot (page 85) Using the Multicomponent Data Plot (page 85) Using the Amplification Plot Views (page 86) Using the Plate Grid Results View Before analysis, the plate grid displays the setup information for each well, including: detector and sample information. After analysis, the plate grid displays the category and number of data flags generated for each well of the plate, as illustrated below. Two data flags are assigned to well C12 (flagged well) Three data flags are assigned to well F12 (omitted well) Note: A list of the data flags generated for the plate document is also displayed in both the QC Summary tab and Results Table. The list of data flags includes: HMD (the well has missing data, FOS (fluorescence is off-scale), LME (large mean-squared error), EW (the well is empty) and BPR (bad passive reference). See the SDS Online Help for more information about viewing and configuring the plate data flags. 84 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

93 Appendix C Analyzing and Viewing Amplification Data Viewing the Amplification Data Using the Raw Data Plot The Raw Data Plot (click ) displays the fluorescence spectra and temperature profile of selected wells. The vertical slider in the Temperature plot allows you to see the spectra for each run cycle by dragging it with the pointer. Click and drag the slider to view temporal changes in the raw data profile C Using the Multicomponent Data Plot The Multicomponent Data Plot (click ) displays the complete spectral contribution of each dye in a selected well over the duration of the PCR run. Note: Only one well can be displayed at a time. Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 85

94 Appendix C Analyzing and Viewing Amplification Data Viewing the Amplification Data Using the Amplification Plot Views The Amplification Plot views allow you to review the post-run amplification results for selected wells from the plate grid. Several different views are available from the Plot drop-down list: R n vs. Cycle (below) R n vs.cycle (page 87) C T vs. Well Position (page 88) Note: For more information on using the Amplification Plot views to evaluate amplification results, see the SDS Online Help. R n vs. Cycle (linear scale) This plot displays normalized reporter dye fluorescence signal (as R n ) in linear scale as a function of run cycle. You can use this plot to identify and examine irregular amplification. Note: For more information about R n, refer to the Real-Time PCR Systems Chemistry Guide (PN ). 86 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

95 Appendix C Analyzing and Viewing Amplification Data Viewing the Amplification Data R n vs.cycle (log scale) This plot displays the reporter dye fluorescence signal (as R n ) in log scale as a function of run cycle, and is the default view after analysis completes. You can use this plot to identify and examine irregular amplification and to manually set the threshold and baseline parameters for the run. C Threshold IMPORTANT! Applied Biosystems recommends verifying that the baseline and threshold were called correctly for each well. If necessary, adjust the values manually as described in the SDS Online Help. Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 87

96 Appendix C Analyzing and Viewing Amplification Data Viewing the Amplification Data Ct vs. Well Position Plot This plot displays threshold cycle (C T ) as a function of well position. You can use this plot to locate outliers from detector data sets. See Omitting Samples below. Omitting Samples Experimental error (such as contamination, pipetting errors, etc.) can produce atypical amplification data for some wells. These wells typically produce C T values that differ significantly from the average. If included in the baseline and threshold value calculations, these outlying data (outliers) can result in erroneous threshold cycle (C T ) measurements. To ensure precision, carefully review your data set for outliers. You can remove outliers manually using the Ct vs. Well Position plot and the plate grid. For more information about omitting wells, refer to the SDS Online Help. Note: You will need to re-analyze the data each time you remove samples from the data set. 88 Plus/Minus Assay Getting Started Guide for the 7900HT Fast System

97 Appendix C Analyzing and Viewing Amplification Data Viewing the Amplification Data Adjusting Plot Settings Click or select View > Display Setting to open the Display Settings dialog box and adjust the selected plot settings. Example Amplification Plots Sample Experiment Note: The adjustable settings depend on which plot you are viewing. Refer to the SDS Online Help for more information. The following Sample Experiment boxes show the amplification plots of the NAC, NTC, plus (+), minus ( ), and unconfirmed minus (?) results. C The amplification plot below is for well A5, a no amplification control (NAC), which contains blocked IPC and no target template. This plot shows no amplification for IPC or the E. coli target sequence. Plate Grid Well A5 No target (E. coli) present Blocked IPC, no amplification Plus/Minus Assay Getting Started Guide for the 7900HT Fast System 89