Applied Biosystems 7300 Getting Started Manual
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Applied Biosystems 7300 Getting Started Manual

Applied Biosystems 7300 Getting Started Manual

Fast real-time pcr system
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See also: Installation and Maintenance Manual
Applied Biosystems 7300/7500/7500 Fast
Real-Time PCR System
Plus/Minus Assay
Getting Started Guide
Introduction
Designing a
Plus/Minus
Experiment
Setting Up the
Reaction Plate
Performing the
Plus/Minus
Pre-Read Run
Generating
Amplification
STANDARD
STANDARD
STANDARD
Data
Performing the
Plus/Minus
Post-Read Run

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  • Page 1 Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Introduction Getting Started Guide Designing a Plus/Minus Experiment Setting Up the Reaction Plate Performing the Plus/Minus Pre-Read Run Generating Amplification STANDARD STANDARD STANDARD Data Performing the Plus/Minus Post-Read Run...
  • Page 2 Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. In no event shall Applied Biosystems be liable for incidental, special, multiple, or consequential damages in connection with or arising from the use of this document.

  • Page 3: Plus/Minus Experiment Workflow

    Plate Document for STANDARD STANDARD STANDARD Amplification Amplification Run Sample Amplification Data Viewing Exporting Performing the Performing the Chapter 6 Plus/Minus Plus/Minus Plus/Minus Post-Read Run Results Plate Data Post-Read Run Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 4 Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 5: Table Of Contents

    About the 7300/7500/7500 Fast System ........
  • Page 6 Viewing the Amplification Data ......... 49 Appendix C Example Plus/Minus Experiment References Index Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 7: Preface

    • A general understanding of hard drives and data storage, file transfers, and copying and pasting. • Networking experience if you want to integrate the 7300/7500/7500 Fast system into your existing laboratory data flow system. This guide uses the following conventions: Text Conventions •...
  • Page 8 Each time you receive a new MSDS packaged with a hazardous chemical, be sure to replace the appropriate MSDS in your files. 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.

  • Page 9: How To Obtain More Information

    Preface How to Obtain More Information How to Obtain More Information For more information about using the 7300/7500/7500 Fast system, refer to the Related Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Online Help or the Documentation documents shown below. Online Help...

  • Page 10: How To Obtain 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 Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 11: Introduction

    About Plus/Minus See page 2 Assays Using an IPC Performing the Plus/Minus Pre-ReadRun Generating STANDARD Amplification Data About See page 4 Plus/Minus Assays Performing the Plus/Minus Post-Read Run Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 12: About The 7300/7500/7500 Fast System

    • Qualitative detection of nucleic acid sequence using end-point and dissociation- curve analysis. The 7300/7500/7500 Fast system allows you to perform several assay types with plates Plus/Minus Assay in the 96-well format. This guide describes the plus/minus assay, which determines whether or not a specific target sequence is present in a sample.
  • Page 13 Nucleotide sequence that you want to identify as present or absent Unknown sample (U) The sample for which you want to determine the presence or absence of a specific target Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 14: About Plus/Minus Assays

    PN 4330708 (50-user license) ® MicroAmp Optical 96-Well Reaction Plates Applied Biosystems (PN 4306757) Optical Adhesive Covers Applied Biosystems (PN 4311971) Reagent tubes with caps, 10-mL Applied Biosystems (PN 4305932) Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 15 Example Experiment boxes appear in subsequent chapters to illustrate workflow details. Refer to Appendix C, “Example Plus/Minus Experiment,” for more information. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 16 Chapter 1 Introduction About Plus/Minus Assays Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 17: Designing A Plus/Minus Experiment

    Setting Up the Reaction Plate Performing the Plus/Minus Pre-Read Run Designing the See page 9 Probe and Primers Generating STANDARD Amplification Data Performing the Plus/Minus Post-Read Run Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 18: Using Taqman ® Probe-Based Reagent Configuration

    The IPC DNA, primers, and probe supplied in these reagents can be used with all Note: ® sample target systems. Refer to the TaqMan Universal PCR Master Mix Protocol (PN 4304449) for instructions on optimizing amplification of your target. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 19: Designing The Probe And Primers

    In the example experiment, we extracted DNA from 84 batches of hamburger meat and tested them for the presence of E. coli using the plus/minus assay on the 7300/7500 Real Time PCR System. Six no IPC/no target template controls, six IPC/no target template controls, and 84 unknown samples were run.
  • Page 20 Chapter 2 Designing a Plus/Minus Experiment Designing the Probe and Primers Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 21: Setting Up The Reaction Plate

    Setting Up the Reaction Plate Performing the Plus/Minus Pre-Read Run Setting Up See page 13 the Reaction Plate Generating STANDARD Amplification Data Performing the Plus/Minus Post-Read Run Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 22: Preparing Dna

    PrepMan Ultra Sample Preparation Reagent Kit (PN 4322547) and protocol (PN 4318925) to obtain a final concentration of 10 ng/µL of DNA for each sample. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 23: Setting Up The Reaction Plate

    Contains blocked IPC and no target NTC plus IPC Contains IPC and no target Samples (Unknown plus IPC) GR2363a Contains IPC plus target Gemini Plus/Minus 96-well plate one sample Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 24 2 µL of 1✕ TE or H § ‡ No Amplification Control – well contains no target template and blocked IPC. § No Template Control – well contains no target template, only IPC. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 25 4306737) Notch at top-right corner by A12 Notch at top-left corner GR2386 by A1 7900 HS96 well plate comparison 30-µL 100-µL maximum maximum reaction reaction volume volume Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 26 A7 to A12 5 µL of sample DNA 2 µL of sample DNA ‡ B1 to H12 ‡ Unknown – well contains both target template and IPC. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 27: Performing The Plus/Minus Pre-Read Run

    Creating a See page 18 Plus/Minus Plus/Minus Pre-Read Run Plate Document Generating STANDARD Amplification Data Performing the See page 24 Pre-Read Run Performing the Plus/Minus Post-Read Run Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 28: The Pre-Read Run

    Applied Biosystems recommends you run six replicates of each control (NAC and Note: NTC) to accurately define plus/minus thresholds and obtain plus/minus calls with a 99.7% confidence level. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 29 All detectors of control wells that contain IPC but no target template. All detectors of negative control wells that contain PCR reagents, but no target template and no IPC. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 30 Plus/Minus assay. Accept the default settings for the Container and Template fields (96-Well Clear and Blank Document). In the Plate Name field, type Plus/Minus Pre- Read. Click Next>. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 31 Select Task > NTC. An N appears in the well. Select the IPC detector by checking the Use box next to it. Select Task > NTC. A second N appears in the well. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 32 Click Finish. Enter sample names for each well. Click or select View > Well Inspector from the menu. Click-drag to select replicate wells. Type the sample name. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 33 NN box is the NAC (no target template, plus blocked IPC), the NI box seen below is the NTC (no target template plus IPC), and the UI box is the unknown sample plus IPC. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 34: Performing The Pre-Read Run

    (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 Save As type: (*.sdt). Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 35: Performing The Pre-Read Run

    Select File > Close. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 36 Chapter 4 Performing the Plus/Minus Pre-Read Run Performing the Pre-Read Run Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 37: Generating Amplification Data

    Setting Up the Reaction Plate Performing the Plus/Minus Pre-Read Run Performing the See page 32 Amplification Run Generating STANDARD STANDARD STANDARD Amplification Data Performing the Plus/Minus Post-Read Run Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 38: Creating A Plate Document For Sample Amplification

    Because the plus/minus assay is an end-point assay, you can amplify the target sequences Benefits of offline using any thermal cycler. However, using the 7300/7500/7500 Fast system to Real-Time amplify the target sequences provides real-time PCR data. After the plus/minus samples...
  • Page 39 Positive Control Reagents kit uses an IPC ™ VIC-labeled probe with TAMRA dye-labeled quencher. To remove a detector in the Detectors in Note: Document window, select the detector, then click Remove. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 40 Select Task > Unknown. A U appears in the boxes. Select the IPC detector by checking the Use box next to it. Select Task > Unknown. Another U appears next to the target U. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 41 You can omit unused wells after the run. For information about omitting unused wells, refer to the Online Help. Verify the information about each well in the Setup tab. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 42: Performing The Amplification Run

    • Sample volume – 50 µL (20 µL for the Fast system). • 9600 Emulation – selected. The 9600 Emulation feature is not Note: available for the 7300 instrument. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 43 (next to A1). You can change the sample setup Note: information (sample name, detector, task) after a run is complete. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 44 Chapter 5 Generating Amplification Data STANDARD STANDARD STANDARD Performing the Amplification Run Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 45 Viewing See page 39 Plus/Minus Results Performing the Plus/Minus Pre-Read Run Generating STANDARD Amplification Data Exporting See page 43 Plus/Minus Plate Data Performing the Plus/Minus Post-Read Run Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 46: Chapter 6 Performing The Plus/Minus Post-Read Run

    Select the Instrument tab. Accept the default value for sample volume. Select File > Save As, type the name Plus/Minus Post-Read for the plus/minus plate document, then click Save. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 47: Performing The Post-Read Run

    Click the green analysis button ( ) to start analysis. All data generated during the run are saved to the plus/minus plate document that you specified in step Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 48 Post-read results for the presence of E. coli are displayed in the Results > Plate tab. For an explanation of results see “Viewing Plus/Minus Results” on page Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 49 When the post-read is complete, select the Plate tab on the Results page to display the The Plate Tab plus/minus calls for the presence or absence of the target sequence for each well. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 50: Viewing Plus/Minus Results

    IPC and no target template. This plot demonstrates that there is no amplification for IPC or the E. coli target sequence. Well A1 No E. coli present Blocked IPC, no amplification Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 51 E. coli target sequence is less than the target threshold, while the IPC sample signal is above the calculated IPC threshold defined by the SDS software. Well G2 No E. coli present Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 52 (?) 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. Well B9 E. coli Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 53 • Calibration Data (*.csv) • Spectra (*.csv) • Component (*.csv) • Rn (*.csv) • Results (*.csv) Refer to the Online Help for information about the export file types. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 54: Exporting Plus/Minus Plate Data

    Type a file name for the export file. The name of the dialog box depends on Note: the type of data you selected to export. Click Save. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 55 (such as IPC or E. coli). Do not use the same name for multiple detectors. Optionally, click the Description field, then enter a brief description of the detector. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 56: Appendix A Creating Detectors

    When creating detectors, you use the reporter dye and quencher information (and Note: optionally, the gene name or symbol for the sample name). You can view the contents in ® a spreadsheet program, such as Microsoft Excel. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 57 For more information about system calibration Online Help files, refer to the Online Help. Click OK & Reanalyze. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 58: Appendix B Viewing Amplification Data

    To analyze the amplification data (AQ plate), click or select Analysis > Analyze. Starting the The software generates several types of result views, as described in the following Analysis section. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 59 • A calculated value – quantity (default; displays are not determined for runs without standard curves), ∆Rn, or C . Select Analysis > Display to select the value to display. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 60: Viewing The Amplification Data

    Component Tab the duration of the PCR run. Only the first selected well is shown at one time. Double-clicking the y-axis displays the Graph Settings dialog box. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 61 This plot displays the Rn dye fluorescence as a function of cycle. You can use this plot to identify and examine irregular amplification and to manually set the threshold and baseline parameters for the run. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 62 , and Std Dev C You can format the display of the report and how the report is printed. Refer to the Online Help for more information. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 63 Graph Settings dialog box, which allows you to adjust the plot settings. Settings The adjustable settings depend on which plot you are viewing. Refer to the Online Help for more information. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 64 Go to the plate Results tab to view the plus and minus calls for the presence or absence Overview of of the target sequence for each well. For further determination of the results, check the Result Calls plots for each well. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 65 • A post-read run using the original plus/minus 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. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 66: Appendix C Example Plus/Minus Experiment

    Mix (IPC kit) gloves. 50✕ Exo IPC 50 to 900 nM DNA (IPC kit) Target 14.0 50 to 250 nM primers, probe, and deionized water Total 45.0 18.0 — Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 67 ‡ No Amplification Control – Well contains no target template and no IPC. § No Template Control – Well contains no target template, only IPC. # Unknown – Well contains both target template and IPC. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 68 PCR conditions are displayed. Select File > Save As, type a name for the plus/minus plate document, then click Save. Load the reaction plate into the instrument. Click Pre-Read. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 69 By default, the standard PCR conditions for the PCR step are displayed. After the run, a message indicates if the run is successful, or if errors were encountered. Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 70 Analysis > Analyze. Click the Results tab to view results for each well. If you need to troubleshoot the plus/minus results, see “Viewing the Amplification Data” on page Notes Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 71 Methods Enzymol. 155:335–350. 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 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 72: References

    References Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 73 47, plus/minus plate document bold text, when to use templates dyes calibration, background and pure dye exporting plate data CAUTION, description Color box Component tab Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...

  • Page 74: Index

    36, OK & Reanalyze button PrepMan Ultra Sample Preparation Reagent Kit 12, omitting unused wells Pre-Read button 25, online help pre-read run 25, creating plate document performing Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 75 TaqMan Exogenous Internal Positive Control Reagents Kit 8, TaqMan probe-based chemistry TaqMan Universal PCR Master Mix 8, 13, 55, protocol target template 3, task Technical Support, contacting template documents text conventions threshold Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 76 Index Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide...
  • Page 78 Web site at www.appliedbiosystems.com. Applera is committed to providing the world’s leading technology and information for life scientists. Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses. Headquarters 850 Lincoln Centre Drive Foster City, CA 94404 USA Phone: +1 650.638.5800...

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