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Applied Biosystems 7500 Getting Started

Real-time pcr system
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Getting Started Guide
Applied Biosystems
7500/7500 Fast
Real-Time PCR System
Standard Curve Experiments

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Summary of Contents for Applied Biosystems 7500

  • Page 1 Getting Started Guide Applied Biosystems 7500/7500 Fast Real-Time PCR System Standard Curve Experiments...
  • Page 3 Getting Started Guide Get Started Applied Biosystems 7500/7500 Fast Real-Time PCR System Standard Curve Experiments Design the Standard Curve Experiment Prepare the Reactions Run the Experiment Analyze the Experiment...
  • Page 4 NOTICE TO PURCHASER: Label License The 7500/7500 Fast Real-Time PCR System is covered by US p atents, and corresponding claims in their non-US counterparts, owned by A pplied Biosys- tems . 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.

  • Page 5: Table Of Contents

    About the 7500/7500 Fast System ........
  • Page 6 Export/Import Workflow ..........104 Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve...
  • Page 7 Index ......... 123 Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve...
  • Page 8 Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 9: Preface

    Time PCR System Site Preparation Guide 7500/7500 Fast system. Intended for personnel who schedule, manage, and perform the tasks required to prepare your site for installation of the 7500/7500 Fast system. Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 10 • Know how to handle DNA and/or RNA samples and prepare them for PCR. • Understand data storage, file transfer, and copying and pasting. • Have networking experience, if you plan to integrate the 7500/7500 Fast system into your existing laboratory data flow.

  • Page 11: How To Obtain More Information

    ® Primer Express Software Version 3.0 Getting Started Guide 4362460 ® TaqMan Gene Expression Assays Protocol 4333458 User Bulletin #2: Relative Quantitation of Gene Expression 4303859 Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 12 Using TaqMan Endogenous Control Assays to Select an Endogenous Control 127AP08 for Experimental Studies Application Note For more documentation, see “How to Obtain Support” page Note: Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 13: How To Obtain Support

    Preface How to Obtain Support Obtaining The 7500 Software Help describes how to use each feature of the user interface. Open Information from the Help from within the software by doing one of the following: the Software Help • Press F1.

  • Page 14: Safety Conventions Used In This Document

    Wear appropriate protective eyewear, clothing, and gloves. ELECTRICAL HAZARD. Failure to ground the instrument properly can lead to an electrical shock. Ground the instrument according to the provided instructions. Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 15: Symbols On Instruments

    Indicates the presence of a laser inside the instrument and to proceed with appropriate caution. Indicates the presence of moving parts and to proceed with appropriate caution. xiii Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 16: Safety Labels On Instruments

    WARNING This instrument is designed for AVERTISSEMENT Cet instrument est conçu 12 V, 75 W Halogen lamps only. pour des lampes d'halogène de 12 V et 75 W seulement. Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 17 Locations of The Applied Biosystems 7500/7500 Fast Real-Time PCR System contains warnings at Warnings the locations shown below. Attention Physical hazard Physical hazard Attention Attention Attention Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 18: General Instrument Safety

    Before using a cleaning or decontamination method other than those Decontaminating recommended by the manufacturer, verify with the manufacturer that the proposed the Instrument method will not damage the equipment Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 19: Chemical Safety

    MSDS in your files. Obtaining MSDSs The MSDS for any chemical supplied by Applied Biosystems is available to you free 24 hours a day. To obtain MSDSs: 1. Go to www.appliedbiosystems.com, click Support, then click MSDS Search.
  • Page 20 MSDS. • Comply with all local, state/provincial, or national laws and regulations related to chemical storage, handling, and disposal. xviii Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 21: Chemical Waste Safety

    • Ensure that the instrument waste is stored, transferred, transported, and disposed of according to all local, state/provincial, and/or national regulations. Radioactive or biohazardous materials may require special handling, and IMPORTANT! disposal limitations may apply. Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 22: Electrical Safety

    Moving Parts PHYSICAL INJURY HAZARD. Moving parts can crush and cut. Keep hands clear of moving parts while operating the instrument. Disconnect power before servicing the instrument. Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 23: Biological Hazard Safety

    • Use equipment that comfortably supports you in neutral working positions and allows adequate accessibility to the keyboard, monitor, and mouse. • Position the keyboard, mouse, and monitor to promote relaxed body and head postures. Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 24: Safety And Electromagnetic Compatibility (Emc) Standards

    This instrument has been tested to and complies with standard AS/NZS 2064, “Limits Standards and Methods Measurement of Electromagnetic Disturbance Characteristics of Industrial, Scientific, and Medical (ISM) Radio-frequency Equipment.” xxii Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 25: Get Started

    About the 7500/7500 Fast System ........

  • Page 26: About The 7500/7500 Fast System

    • After the PCR. Regardless of the run type, a data collection point or read on the 7500/7500 Fast instrument consists of three phases: 1. Excitation – The instrument illuminates all wells of the reaction plate within the instrument, exciting the fluorophores in each reaction.
  • Page 27 • Cy3 For More For information on: Information • The 7500/7500 Fast system – Refer to the Applied Biosystems 7500/7500 Fast Real- Time PCR Software Help. Note: To open the Help, select Help 7500 Software Help in the 7500 software.

  • Page 28: Supported Consumables

    Splash Free Support Base MicroAmp Optical Tube without Cap ™ ™ MicroAmp Optical 8-Cap Strip, 0.2-mL MicroAmp Optical Adhesive Film ® MicroAmp Reaction Tube with Cap, 0.2-mL Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 29 Fast Optical 96-Well Reaction Plate, 0.1-mL ® Microamp Splash Free Support Base ™ MicroAmp Optical 8-Cap Strip ™ MicroAmp Fast 8-Tube Strip ™ MicroAmp Optical Adhesive Film Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 30 Standard Reaction Plates (PN 4346906) (PN 4306737) Notch at top-right corner by A12 Notch at top-left corner by A1 30-µL 100-µL maximum maximum reaction reaction volume volume Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 31: About Standard Curve Experiments

    The standard curve method is used to determine the absolute target quantity in samples. Curve With the standard curve method, the 7500 software measures amplification of the target Experiments in samples and in a standard dilution series. Data from the standard dilution series are used to generate the standard curve.
  • Page 32 UNG enzyme can be used to prevent carryover contamination. ® For more information on AmpErase UNG, refer to the Applied Biosystems Real- Note: Time PCR Systems Reagent Guide. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 33 ® ® Applied Biosystems offers TaqMan and SYBR Green reagents for use on the 7500/7500 Fast system. Both reagent types are briefly described in the table below. ® ® If you use TaqMan or SYBR Green reagents, the 7500 software automatically Note: calculates reaction volumes in the Reaction Setup screen.
  • Page 34 98). For More For more information on real-time PCR experiments, PCR options, and reagents, refer to Information the Applied Biosystems Real-Time PCR Systems Reagent Guide. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 35: How To Use This Guide

    This guide functions as both a tutorial and as a guide for performing your own experiments. Using This Guide By using the example experiment data provided with the 7500 software, you can use this as a Tutorial guide as a tutorial for performing a standard curve experiment on a 7500/7500 Fast system.

  • Page 36: About The Example Standard Curve Experiment

    MGB probe is not available as a Note: ® ® TaqMan Gene Expression Assay. It can be ordered as a Custom TaqMan Gene Expression Assay (PN 4331348). Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 37 • In Chapter 5, you will view results in a standard curve example experiment file that contains run data. The data file for the example experiment is installed with the 7500 software. You can find the data file for the example experiment on your computer at: <drive>:\Applied Biosystems\<software name>\experiments\...

  • Page 38: Example Experiment Workflow

    Chapter 1 Get Started Example Experiment Workflow Example Files in the Experiments Folder The experiments folder of the 7500 software contains several example files that you can reference when analyzing your own data. The following example files install with the 7500 software: •...
  • Page 39 1. View the analysis settings; adjust the baseline/threshold. 2. View the quality summary. 3. Omit wells. 4. View the multicomponent plot. 5. View the raw data plot. End Experiment Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 40 Chapter 1 Get Started Example Experiment Workflow Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 41: Design The Standard Curve Experiment

    For more information about any of the topics discussed in this guide, open the Help from within Applied Biosystems 7500/7500 Fast Real-Time PCR Software v2.0 by pressing F1, clicking in the toolbar, or selecting Help 7500 Software Help. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 42: Chapter Overview

    Chapter Overview Chapter Overview This chapter explains how to use the Design Wizard in the 7500 software to set up the standard curve example experiment. The Design Wizard guides you through Applied Biosystems recommended best practices as you enter design parameters for the example experiment.

  • Page 43: Create A New Experiment

    Biosystems 7500 Software <software name> where <software name> is the current version of the 7500 software. 2. In the Home screen, click Design Wizard to open the Design Wizard. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 44: Define The Experiment Properties

    In the standard curve example experiment: Example • The experiment is identified as an example. Experiment • The instrument that is selected to run the experiment is the 7500 instrument. ® • A MicroAmp Optical 96-Well Reaction Plate is used.
  • Page 45 Defaults tab (default). In the Default Instrument Type drop-down list, select the appropriate instrument. For More For more information on: Information • Completing the Experiment Properties screen – Open the 7500 Software Help by clicking or pressing F1. • Consumables – See “Supported Consumables” on page •...

  • Page 46: Define The Methods And Materials

    3. Select Standard (~ 2 hours to complete a run) for the ramp speed. 4. Select gDNA (genomic DNA) for the template type. 5. Click Next. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 47 – Select gDNA (genomic DNA) if you have already extracted the gDNA from tissue or sample. You are adding purified genomic DNA to the PCR reactions. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 48: Set Up The Targets

    Set Up the Targets For More For more information on: Information • Completing the Methods & Materials screen – Open the 7500 Software Help by clicking or pressing F1. • Using Advanced Setup – See “Advanced Setup Workflow” on page •...
  • Page 49 ® – SYBR Green Reagents – Select None. For More For more information on completing the Targets screen, open the 7500 Software Help by Information clicking or pressing F1. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve...

  • Page 50: Set Up The Standards

    In the Select Serial Factor drop-down list, select 1:2. 4. Review the Standard Curve Preview pane. The standard curve has the following points: 10000, 5000, 2500, 1250, and 625. 5. Click Next. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 51 2✕, 3✕, and so on. For More For more information on: Information • Completing the Standards screen – Open the 7500 Software Help by clicking pressing F1. ® • Amplification efficiency – Refer to the Amplification Efficiency of TaqMan Gene Expression Assays Application Note.

  • Page 52: Set Up The Samples

    • 3 Negative control wells • 72 Empty wells 9. In the View Plate Layout tab: In the Arrange Plate by drop-down list, select Rows (default). Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 53 (amplification and detection of two or more targets per well), design your experiment using Advanced Setup (see page 98) instead of the Design Wizard. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 54: Set Up The Run Method

    For More For more information on: Information • Completing the Samples screen – Open the 7500 Software Help by clicking pressing F1. • Using Advanced Setup – See “Advanced Setup Workflow” on page Set Up the Run Method In the Run Method screen, review the reaction volume and the thermal profile for the default run method.
  • Page 55 7500 software. For More For more information on: Information • The Run Method library or on completing the Run Method screen – Open the 7500 Software Help by clicking or pressing F1. • Using Advanced Setup – See “Advanced Setup Workflow”...

  • Page 56: Review The Reaction Setup

    6. In the Reactions for RNase P TAMRA pane: Make sure the Master Mix Concentration field displays 2.0✕ (default). Make sure the Assay Mix Concentration field displays 20.0✕ (default). Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 57 7. In the Standard Dilution Series for RNase P TAMRA pane (see page 34): Click the Standard Concentration in Stock field, then enter 20000. Click the units field, then enter copies per µL. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 58 Stock Concentration Sample Volume Diluent Volume Total Volume of Diluted Sample Name (ng/µL) (µL) (µL) (µL) pop1 100.0 1.00 14.15 15.15 pop2 100.0 1.00 14.15 15.15 Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 59 • Detailed Reaction Setup Instructions • Include Plate Layout • Use sample color 3. Click Print to print the reaction setup instructions. 4. Click Next. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 60 • Enter a reaction volume/well. Applied Biosystems recommends a reaction volume of 25 µL for standard curve experiments. The 7500 system supports reaction volumes from 20 to 100 µL. The 7500 Fast system supports reaction volumes from 10 to 30 µL.

  • Page 61: Order Materials For The Experiment

    Applied Biosystems Store. To open the Applied Biosystems Store, you must have an Internet connection. 3. (Optional) Click Print Materials List to print the materials list. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 62 When you are connected to the Applied Biosystems Store, follow the prompts to complete your order. 6. Go to “Finish the Design Wizard” on page Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 63 Make sure that cookies and Java Script are turned on. IMPORTANT! • To find your assay in the Applied Biosystems Store, complete the Find Assay pane: Click the Enter Gene Name field, enter the gene name, then click Find Assay.

  • Page 64: Finish The Design Wizard

    Finish the Design Wizard To finish the Design Wizard, review the plate layout, then select an exit option. About the The 7500 software automatically selects locations for the wells in the reaction plate. In Example the standard curve example experiment: Experiment •...
  • Page 65 Chapter 2 Design the Standard Curve Experiment Finish the Design Wizard Finish the 1. At the bottom of the 7500 software screen, click Finish Designing Experiment. Design Wizard 2. In the Review Plate Layout for Experiment window, review the plate layout. Make sure there are: •...
  • Page 66 In the Data Folder field, browse to then select the desired location. For More For more information on using Advanced Setup, see “Advanced Setup Workflow” on Information page Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 67: Prepare The Reactions

    Note: Help from within Applied Biosystems 7500/7500 Fast Real-Time PCR Software v2.0 by pressing F1, clicking in the toolbar, or selecting Help 7500 Software Help. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 68: Chapter Overview

    3. Prepare the reaction mix for each target assay. 4. Prepare the reaction plate. Run the Experiment (Chapter 4) Analyze the Experiment (Chapter 5) End Experiment Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 69: Prepare The Sample Dilutions

    • Sample dilutions are necessary because the sample volume is limited to 10% of the Experiment total reaction volume in the 7500 software. Because the total reaction volume is 25 µL/reaction, the sample volume is 2.5 µL/reaction. • The stock concentration is 100 ng/µL. After diluting the sample according to the Sample Dilutions Calculations table, the sample has a concentration of 6.6 ng/µL.
  • Page 70 Guidelines • Sample dilutions may be necessary because the sample volume is limited to 10% of the total reaction volume in the 7500 software. You must perform the sample dilutions before adding the samples to the final reaction mix. ®...

  • Page 71: Prepare The Standard Dilution Series

    Chapter 3 Prepare the Reactions Prepare the Standard Dilution Series Prepare the Standard Dilution Series Prepare the standard dilution series using the volumes that were calculated by the 7500 software (“Complete the Reaction Mix Calculations Tab (see page 33)” on page 32).
  • Page 72 • The quality of pipettors and tips and the care used in measuring and mixing dilutions affect accuracy. • Use TE buffer or water to dilute the standards. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 73: Prepare The Reaction Mix

    Exposure may cause discomfort if swallowed or inhaled. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 74 Information the reagents you are using in the PCR reactions: ® • TaqMan Gene Expression Assays Protocol ® • Custom TaqMan Gene Expression Assays Protocol Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 75: Prepare The Reaction Plate

    – 15 Standard wells – 3 Negative control wells – 72 Empty wells • The plate layout that the 7500 software automatically generates is used: Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 76 Mix the reactions by gently pipetting up and down, then cap the tubes. Centrifuge the tubes briefly to remove air bubbles. Add 25 µL of the standard reaction to the appropriate wells in the reaction plate. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 77 7500 software. You can either: – Accept the plate layout automatically generated by the software. – Use Advanced Setup to change the plate layout in the software. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 78 – Custom TaqMan Gene Expression Assays Protocol • Consumables – See “Supported Consumables” on page • Using Advanced Setup to change the plate layout – See page Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 79: Run The Experiment

    For more information about any of the topics discussed in this guide, open the Help from within Applied Biosystems 7500/7500 Fast Real-Time PCR Software v2.0 by pressing F1, clicking in the toolbar, or selecting Help 7500 Software Help. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 80: Chapter Overview

    Chapter 4 Run the Experiment Chapter Overview Chapter Overview This chapter explains how to perform a run on the Applied Biosystems 7500/7500 Fast Real-Time PCR System. Example Start Experiment Experiment Workflow Design the Experiment (Chapter 2) Prepare the Experiment (Chapter 3) Run the Experiment (Chapter 4) 1.

  • Page 81: Prepare For The Run

    A12 position at notched A1 position at the GR2477 7300/7500 the top-right of the tray. top-left of the tray. barcode on plate GR2475 7500 keyed corner Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 82 • Place tube strips with samples in the PPH vertically, starting in columns 6 and 7 and moving outward. • A maximum of 6 tube strips can be used in the 7500 Fast instrument. Leave columns 2, 3, 10, and 11 empty.

  • Page 83: Enable The Notification Settings (Optional)

    Enable the Notification Settings (Optional) Enable the Notification Settings (Optional) Enable the notification settings so that the 7500 software alerts you by e-mail when the 7500/7500 Fast instrument begins and completes the run, or if an error occurs during the run.
  • Page 84 Run Guidelines When you set up the 7500/7500 Fast system for automatic notification: • Your system must be set up for network use. Refer to the Applied Biosystems 7500/7500 Fast Real-Time PCR Systems Maintenance Guide. • Select the events for which you want to receive e-mail notifications. Select: –...

  • Page 85: Start The Run

    2. Click START RUN Monitor the Run You can view the progress of the run in real time as described below. During the run, periodically view all three available plots from the 7500 software for potential problems. To… Action Stop the run 1.
  • Page 86 If you notice abnormal amplification or a complete absence of florescence, troubleshoot the error as explained in the 7500 Software Help (click or press F1). Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 87 If an alert appears, click the error for more information, then troubleshoot the problem as explained in the 7500 Software Help (click or press F1). Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 88: Unload The Instrument

    1. Push the tray door to open it. 2. Remove the reaction plate. 3. Push the tray door to close it. GR2477 7300/7500 barcode on plate GR2471 7500 tray top view Push Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 89: Analyze The Experiment

    For more information about any of the topics discussed in this guide, open the Help from within Applied Biosystems 7500/7500 Fast Real-Time PCR Software v2.0 by pressing F1, clicking in the toolbar, or selecting Help 7500 Software Help. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 90: Chapter Overview

    1. View the analysis settings; adjust the baseline/threshold. 2. View the quality summary. 3. Omit wells. 4. View the multicomponent plot. 5. View the raw data plot. End Experiment Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 91: Section 5.1: Review Results

    Publish the Data ........... 83 Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 92: Analyze The Experiment

    (for example, the Amplification Plot screen, QC Summary screen, and so on). About the For the standard curve example experiment, use the data file that is installed with the Example 7500 software. The data file was created with the same design parameters that are Experiment provided in Chapter 2, then run and analyzed on a 7500/7500 Fast instrument.
  • Page 93 Analyze the Experiment Guidelines When you analyze your own standard curve experiment: • Immediately after a run, the 7500 software automatically analyzes the data using the default analysis settings, then displays the Amplification Plot screen on your computer. • To reanalyze the data, select all the wells in the plate layout, then click Analyze.
  • Page 94 4. To display two plots in columns, click (Show plots in two columns). 5. To display a specific plot, select the plot in the drop-down list above each plot display. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 95: View The Standard Curve

    View the Standard Curve View the Standard Curve The Standard Curve screen displays the standard curve for samples designated as standards. The 7500 software calculates the quantity of an unknown target from the standard curve. About the In the standard curve example experiment, you review the Standard Curve screen for the...
  • Page 96 In the Group By drop-down list, select Replicate. Observe the values in the C column. In the example experiment, the C values are within the expected range (>8 and <35). Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 97 90). • Rerun the experiment. For More For more information on: Information • The Standard Curve screen – Open the 7500 Software Help by clicking pressing F1. ® • Amplification efficiency – Refer to the Amplification Efficiency of TaqMan Gene Expression Assays Application Note.

  • Page 98: View The Amplification Plot

    3. In the Amplification Plot screen: In the Plot Type drop-down list, select ∆Rn vs Cycle (default). In the Plot Color drop-down list, select Well (default). Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 99 Verify that the baseline is set correctly: The end cycle should be set a few cycles before the cycle number where significant fluorescence is detected. In the example experiment, the baseline is set correctly. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 100 Select the Threshold check box to show the threshold. Verify that the threshold is set correctly. In the example experiment, the threshold is in the exponential phase. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 101 In the Plot Type drop-down list, select C vs Well. Look for outliers in the amplification plot. In the example experiment, there are no outliers for RNase P. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 102 • Correct baseline and threshold values – See the threshold examples on page 79 the baseline examples on page Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 103 The standard deviation is significantly higher than that for a plot where the threshold is set correctly. Drag the threshold bar down into the exponential phase of the curve. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 104 • Manually adjust the baseline and/or threshold (see “View the Analysis Settings” on page 86). For More For more information on the Amplification Plot screen, open the 7500 Software Help by Information clicking or pressing F1. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve...

  • Page 105: View The Well Table

    In the example experiment, note that the quantity values within each replicate group are similar. Note: To move a column, drag the column heading. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 106 . The software groups the wells by value: low, medium, high, and undetermined. In the example experiment, the C values are within the expected range (>8 and <35). Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 107: Publish The Data

    This indicates tight precision. • Flag – The software groups the flagged and unflagged wells. A flag indicates that the software found an error in the flagged well. For a description of the 7500 software flags, see “View the QC Summary”...
  • Page 108 Chapter 5 Analyze the Experiment Publish the Data Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 109: Section 5.2: Troubleshoot (If Needed)

    View the Raw Data Plot ..........94 Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 110: View The Analysis Settings

    The Analysis Settings dialog box displays the analysis settings for the threshold cycle ), flags, and advanced options. If the default analysis settings in the 7500 software are not suitable for your experiment, you can change the settings in the Analysis Settings dialog box, then reanalyze your experiment.
  • Page 111 Unless you have already determined the optimal settings for your experiment, use the Guidelines default analysis settings in the 7500 software. If the default settings are not suitable for your experiment, you can change the settings. Use the: • C Settings tab to manually set the threshold and baseline.

  • Page 112: View The Qc Summary

    Chapter 5 Analyze the Experiment View the QC Summary View the QC Summary The QC Summary screen displays a list of the 7500 software flags, and includes the flag frequency and location for the open experiment. About the In the standard curve example experiment, you review the QC Summary screen for any Example flags generated by the experiment data.
  • Page 113 – Adjust the sensitivity so that more wells or fewer wells are flagged. – Change the flags that are applied by the 7500 software. For More For more information on the QC Summary screen or on flag settings, open the 7500 Information Software Help by clicking or pressing F1.

  • Page 114: Omit Wells From The Analysis

    Look for any outliers in the replicate group (be sure they are flagged). In the example experiment, the 7500 software flagged well B12 as a potential outlier. 5. Select the Omit check box next to well B12. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 115 Guidelines groups for outliers. If needed, remove outliers manually using the Well Table. For More For more information on omitting wells from the analysis, open the 7500 Software Help Information by clicking or pressing F1. Within the Help, search for the omit well topics: 1.

  • Page 116: View The Multicomponent Plot

    Hide the plot legend. 5. Observe the ROX dye signal. In the example experiment, the ROX dye signal remains constant throughout the PCR process, which indicates typical data. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 117 7. Select the negative control wells one at a time and check for amplification. In the example experiment, there is no amplification in the negative control wells. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 118: View The Raw Data Plot

    • Negative control wells – There should be no amplification in the negative control wells. For More For more information on the Multicomponent Plot screen, open the 7500 Software Help Information by clicking or pressing F1. View the Raw Data Plot The Raw Data Plot screen displays the raw fluorescence (not normalized) for each optical filter for the selected wells during each cycle of the real-time PCR.
  • Page 119 Guidelines filter: • Characteristic signal growth • No abrupt changes or dips For More For more information on the Raw Data Plot screen, open the 7500 Software Help by Information clicking or pressing F1. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve...
  • Page 120 Chapter 5 Analyze the Experiment View the Raw Data Plot Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 121: Appendix A Alternate Experiment Workflows

    Note: Help from within Applied Biosystems 7500/7500 Fast Real-Time PCR Software v2.0 by pressing F1, clicking in the toolbar, or selecting Help 7500 Software Help. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 122: Advanced Setup Workflow

    Appendix A Alternate Experiment Workflows Advanced Setup Workflow Advanced Setup Workflow When you create an experiment using Advanced Setup in the 7500 software, you can set up the experiment according to your own design. 1. Double-click (7500 software) or select Start All Programs Applied Biosystems 7500 Software <software name>...
  • Page 123 In the navigation pane, select an analysis screen to view the data (for example, select QC Summary to view a quality summary of the data). Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 124: Quickstart Workflow

    Load the reaction plate into the instrument. Start the run (Optional) Monitor the run. Unload the reaction plate from the instrument. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 125 In the navigation pane, select an analysis screen to view the data (for example, select QC Summary to view a quality summary of the data). Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 126: Template Workflow

    3. (Optional) If you want to modify the experiment, use Advanced Setup (see page 98). 4. Click Save, enter a file name, then click Save to save the experiment. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 127 In the navigation pane, select an analysis screen to view the data (for example, select QC Summary to view a quality summary of the data). Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 128: Export/Import Workflow

    Excel software), open an exported text file. Replace the parameters of the text file as needed. When finished, save the file as a tab-delimited text file. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 129 Load the reaction plate into the instrument. Start the run (Optional) Monitor the run. Unload the reaction plate from the instrument. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 130 In the navigation pane, select an analysis screen to view the data (for example, select QC Summary to view a quality summary of the data). Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 131: Bibliography

    Saiki, R.K., Scharf, S., Faloona, F., et al. 1985. Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350–1354. Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 132 Bibliography Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 133: Glossary

    Glossary Advanced Setup In the 7500/7500 Fast system software, a feature that allows you to set up your experiment according to your experiment design. Advanced Setup provides you with maximum flexibility in the design and setup of your experiment. assay information file (AIF).
  • Page 134 See also cycling stage. assay In the 7500/7500 Fast system, a PCR reaction mix that contains primers to amplify a target and a reagent to detect the amplified target. ® Assay ID Identifier assigned by Applied Biosystems to TaqMan Gene Expression Assays and ®...
  • Page 135 Dye that is not supplied by Applied Biosystems. Custom dyes may be adapted for use in experiments on the 7500/7500 Fast system. When using custom dyes, the custom dye should be added to the Dye Library and a custom dye calibration performed.
  • Page 136 The instrument transforms the signal to electronic data, and the data are saved in the experiment file. In the 7500/7500 Fast system software, a data collection point is indicated by an icon in the thermal profile: •...
  • Page 137 In presence/absence experiments, abbreviation for internal positive control (IPC). In the 7500/7500 Fast system software, the task for the IPC target in wells that contain the IPC and do not contain IPC blocking agent. See also internal positive control (IPC).
  • Page 138 PCR run. negative control In the 7500/7500 Fast system software, the task for targets or SNP assays in wells that (NC) contain water or buffer instead of sample. No amplification of the target should occur in negative control wells.
  • Page 139 7500/7500 Fast system, the grid contains 8 rows and 12 columns. In the 7500/7500 Fast system software, you can use the plate layout as a selection tool to assign well contents, to view well assignments, and to view results. The plate layout can be printed, included in a report, exported, and saved as a slide for a presentation.
  • Page 140 Glossary QuickStart A feature in 7500/7500 Fast systems that allows you to run an experiment without entering plate setup information. value Regression coefficient calculated from the regression line in the standard curve. The R value indicates the closeness of fit between the standard curve regression line and the individual C data points from the standard reactions.
  • Page 141 Method for determining relative target quantity in samples. With the relative standard curve curve method method, the 7500/7500 Fast system software measures amplification of the target and of the endogenous control in samples, in a reference sample, and in a standard dilution series.
  • Page 142 SNP Assay Library In the 7500/7500 Fast system software, a collection of SNP assays to add to genotyping experiments. The SNP assays in the library contain the SNP assay name, SNP assay color, and for each allele, the allele name or base(s), reporter, quencher, and allele colors.
  • Page 143 For example, the standard stock is used to prepare the first dilution point, the first dilution point is used to prepare the second dilution point, and so on. In the 7500/7500 Fast system software, the volumes needed to prepare a standard dilution series are calculated by the number of dilution points, the number of standard replicates, the starting quantity, the serial factor, and the standard concentration in the stock.
  • Page 144 The nucleic acid sequence that you want to amplify and detect. target color In the 7500/7500 Fast system software, a color assigned to a target to identify the target in the plate layout and analysis plots. Target Library In the 7500/7500 Fast system software, a collection of targets to add to experiments. The targets in the library contain the target name, reporter, quencher, and target color.
  • Page 145 (Tm). unknown In the 7500/7500 Fast system software, the task for the target or SNP assay in wells that contain the sample you are testing: • In quantitation experiments, the task for the target in wells that contain a sample with unknown target quantities.
  • Page 146 Glossary Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...

  • Page 147: Index

    QC Summary screen create new view Raw Data Plot screen define experiment properties view Standard Curve screen define methods and materials view Well Table finish Design Wizard workflow Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 148 NOAMP flag guidelines NOISE flag analysis NOSIGNAL flag chemical safety xviii notification settings chemical waste disposal chemical waste safety design preparation OFFSCALE flag Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 149 Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 150 WARNING, description waste disposal, guidelines Well Table wells negative control omit selecting standard unknown workflows Advanced Setup example experiment Export/Import QuickStart Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments...
  • Page 152 Worldwide Sales and Support Applied Biosystems vast distribution and service network, composed of highly trained support and applications personnel, reaches 150 countries on six continents. For sales office locations and technical support, please call our local office or refer to our Web site at www.appliedbiosystems.com.

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