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Abi 7500 Getting Started Manual

Manual is about: Real-Time PCR System

Summary of 7500

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    Applied biosystems 7500/7500 fast real-time pcr system relative standard curve and comparative c t experiments getting started guide.

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    Applied biosystems 7500/7500 fast real-time pcr system relative standard curve and comparative c t experiments get started design the relative standard curve experiment prepare the relative standard curve reactions run the relative standard curve experiment analyze the relative standard curve experi...

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    © copyright 2007, 2010 applied biosystems. All rights reserved. Information in this document is subject to change without notice. Applied biosystems assumes no responsibility for any errors that may appear in this document. Applied biosystems disclaims all warranties with respect to this document, e...

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    Contents iii applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vii how to use this guide . . . . . . . . . . . . . . . . . ...

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    Applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments iv order materials for the experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 finish the design wizard . . . . . . . . . ....

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    V applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments define the methods and materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 set up the targets . . . . . . . . . . . . . ....

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    Applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments vi appendix a alternate experiment workflows . . . . . . . . . . . . . . . . . . 203 advanced setup workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . ...

  • Page 9: How to Use This Guide

    Vii applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments preface how to use this guide about the system documentation the guides listed below are shipped with the applied biosystems 7500 and 7500 fast real-time pcr s...

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    Preface how to use this guide applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments viii assumptions this guide assumes that you: • are familiar with the microsoft windows ® xp operating system. • are familiar with th...

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    Preface how to obtain more information ix applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments safety alert words safety alert words also appear in user documentation. For more information, see “safety alert words” o...

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    Preface how to obtain more information applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments x documents related to standard curve experiments documents related to the reagent guide note: for more documentation, see “...

  • Page 13: How to Obtain Support

    Preface how to obtain support xi applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments obtaining information from the software help the 7500 software help describes how to use each feature of the touchscreen. Open the...

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    Preface safety conventions used in this document applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments xii safety conventions used in this document safety alert words four safety alert words appear in applied biosyste...

  • Page 15: Symbols On Instruments

    Preface symbols on instruments xiii applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments symbols on instruments electrical symbols on instruments the following table describes the electrical symbols that may be displ...

  • Page 16: Safety Labels On Instruments

    Preface safety labels on instruments applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments xiv environmental symbols on instruments the following symbol applies to all applied biosystems electrical and electronic prod...

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    Preface safety labels on instruments xv applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments locations of warnings the applied biosystems 7500/7500 fast real-time pcr system contains warnings at the locations shown b...

  • Page 18: General Instrument Safety

    Preface general instrument safety applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments xvi general instrument safety physical injury hazard. Use this product only as specified in this document. Using this instrument ...

  • Page 19: Chemical Safety

    Preface chemical safety xvii applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments chemical safety chemical hazard warning chemical hazard. Before handling any chemicals, refer to the material safety data sheet (msds)...

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    Preface chemical safety applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments xviii chemical safety guidelines to minimize the hazards of chemicals: • read and understand the material safety data sheets (msdss) provid...

  • Page 21: Chemical Waste Safety

    Preface chemical waste safety xix applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments chemical waste safety chemical waste hazard hazardous waste. Refer to material safety data sheets and local regulations for handl...

  • Page 22: Electrical Safety

    Preface electrical safety applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments xx electrical safety electrical shock hazard. Severe electrical shock can result from operating the applied biosystems 7500/7500 fast rea...

  • Page 23: Biological Hazard Safety

    Preface biological hazard safety xxi applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments biological hazard safety general biohazard biohazard. Biological samples such as tissues, body fluids, infectious agents, and ...

  • Page 24

    Preface safety and electromagnetic compatibility (emc) standards applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments xxii safety and electromagnetic compatibility (emc) standards this section provides information on...

  • Page 25

    Chapter 1 1 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments get started this chapter covers: ■ about the 7500/7500 fast system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 ■ supp...

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    Chapter 1 get started about the 7500/7500 fast system applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 2 notes about the 7500/7500 fast system the applied biosystems 7500/7500 fast real-time pcr system is a 96-we...

  • Page 27

    Chapter 1 get started about the 7500/7500 fast system 3 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes about the filters the 7500/7500 fast system uses the following filters: for more information for info...

  • Page 28: Supported Consumables

    Chapter 1 get started supported consumables applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 4 notes supported consumables 7500 system the 7500 system supports the consumables listed below. 7 consumable part numb...

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    Chapter 1 get started supported consumables 5 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes 7500 fast system the 7500 fast system supports the consumables listed below. Important! Use only fast consumabl...

  • Page 30

    Chapter 1 get started supported consumables applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 6 notes standard versus fast reaction plates and tubes make sure that you use the correct reaction plate for your syste...

  • Page 31: Experiments

    Chapter 1 get started about relative standard curve and comparative c t experiments 7 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes about relative standard curve and comparative c t experiments real-time...

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    Chapter 1 get started about relative standard curve and comparative c t experiments applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 8 notes • negative controls – wells that contain water or buffer instead of sam...

  • Page 33

    Chapter 1 get started about relative standard curve and comparative c t experiments 9 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes relative standard curve vs. Comparative c t experiments consider the fo...

  • Page 34

    Chapter 1 get started about relative standard curve and comparative c t experiments applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 10 notes pcr options when performing real-time pcr, choose between: • singleple...

  • Page 35

    Chapter 1 get started about relative standard curve and comparative c t experiments 11 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes supported reagents taqman ® and sybr ® green reagents applied biosyste...

  • Page 36: How to Use This Guide

    Chapter 1 get started how to use this guide applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 12 notes other reagents if you use fluorescence-based reagents other than taqman ® reagents, you must design your exper...

  • Page 37

    Chapter 1 get started how to use this guide 13 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes using this guide with your own experiments after completing the tutorial exercises in chapters 2 through 9, yo...

  • Page 38

    Chapter 1 get started about the example experiments applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 14 notes about the example experiments about the relative standard curve example experiment the objective of th...

  • Page 39

    Chapter 1 get started about the example experiments 15 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes about the comparative c t ( ∆∆c t ) example experiment the objective of the comparative c t example ex...

  • Page 40

    Chapter 1 get started about the example experiments applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 16 notes about the example experiment data in this getting started guide, you use five files: • in chapter 2, y...

  • Page 41: Example Experiment Workflow

    Chapter 1 get started example experiment workflow 17 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes example experiment workflow this section shows the workflow for the relative standard curve and comparat...

  • Page 42

    Chapter 1 get started example experiment workflow applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 18 notes relative standard curve experiment (continued from page 17) end experiment analyze the experiment (chapt...

  • Page 43

    Chapter 2 19 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes design the relative standard curve experiment this chapter covers: ■ chapter overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...

  • Page 44: Chapter Overview

    Chapter 2 design the relative standard curve experiment chapter overview applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 20 notes chapter overview this chapter explains how to use the design wizard in the 7500 s...

  • Page 45: Create A New Experiment

    Chapter 2 design the relative standard curve experiment create a new experiment 21 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes create a new experiment create a new experiment using the design wizard in...

  • Page 46

    Chapter 2 design the relative standard curve experiment define the experiment properties applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 22 notes define the experiment properties in the experiment properties scr...

  • Page 47

    Chapter 2 design the relative standard curve experiment define the experiment properties 23 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes design guidelines when you design your own relative standard curv...

  • Page 48

    Chapter 2 design the relative standard curve experiment define the methods and materials applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 24 notes define the methods and materials in the methods and materials scr...

  • Page 49

    Chapter 2 design the relative standard curve experiment define the methods and materials 25 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes design guidelines when you design your own relative standard curv...

  • Page 50: Set Up The Targets

    Chapter 2 design the relative standard curve experiment set up the targets applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 26 notes for more information for more information on: • completing the methods and mate...

  • Page 51

    Chapter 2 design the relative standard curve experiment set up the targets 27 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes 3. Set up the target 1 experiment: a. Click the enter target name cell, then en...

  • Page 52

    Chapter 2 design the relative standard curve experiment set up the targets applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 28 notes design guidelines when you design your own relative standard curve experiment: ...

  • Page 53: Set Up The Standards

    Chapter 2 design the relative standard curve experiment set up the standards 29 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes set up the standards in the standards screen, enter the number of points and ...

  • Page 54

    Chapter 2 design the relative standard curve experiment set up the standards applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 30 notes design guidelines when you design your own relative standard curve experiment...

  • Page 55: Set Up The Samples

    Chapter 2 design the relative standard curve experiment set up the samples 31 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes for more information for more information on: • completing the standards screen...

  • Page 56

    Chapter 2 design the relative standard curve experiment set up the samples applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 32 notes 8. In the well count pane, verify that there are: • 12 unknown wells • 30 stand...

  • Page 57

    Chapter 2 design the relative standard curve experiment set up the samples 33 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes • select which targets to test in the samples: – select all sample/target react...

  • Page 58

    Chapter 2 design the relative standard curve experiment set up the relative quantitation settings applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 34 notes set up the relative quantitation settings in the relativ...

  • Page 59: Set Up The Run Method

    Chapter 2 design the relative standard curve experiment set up the run method 35 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes set up the run method in the run method screen, review the reaction volume a...

  • Page 60: Review The Reaction Setup

    Chapter 2 design the relative standard curve experiment review the reaction setup applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 36 notes design guidelines when you design your own relative standard curve exper...

  • Page 61

    Chapter 2 design the relative standard curve experiment review the reaction setup 37 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes complete the reaction setup screen to complete the reaction mix calculat...

  • Page 62

    Chapter 2 design the relative standard curve experiment review the reaction setup applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 38 notes 7. In the standard dilution series for c-myc pane: a. Click the standard...

  • Page 63

    Chapter 2 design the relative standard curve experiment review the reaction setup 39 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes c. Review the components and calculated volumes for the pcr reactions: 7...

  • Page 64

    Chapter 2 design the relative standard curve experiment review the reaction setup applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 40 notes to complete the sample dilution calculations tab: 1. In the reaction set...

  • Page 65

    Chapter 2 design the relative standard curve experiment review the reaction setup 41 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes you can print detailed reaction setup instructions, then save the instru...

  • Page 66

    Chapter 2 design the relative standard curve experiment review the reaction setup applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 42 notes design guidelines when you design your own relative standard curve exper...

  • Page 67

    Chapter 2 design the relative standard curve experiment order materials for the experiment 43 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes order materials for the experiment in the materials list screen...

  • Page 68

    Chapter 2 design the relative standard curve experiment order materials for the experiment applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 44 notes d. In the find assay results dialog box, select the hs99999905_...

  • Page 69

    Chapter 2 design the relative standard curve experiment order materials for the experiment 45 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes a. Check that the experiment shopping list contains the desired...

  • Page 70: Finish The Design Wizard

    Chapter 2 design the relative standard curve experiment finish the design wizard applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 46 notes design guidelines when you design your own relative standard curve experi...

  • Page 71

    Chapter 2 design the relative standard curve experiment finish the design wizard 47 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes finish the design wizard 1. At the bottom of the 7500 software screen, cl...

  • Page 72

    Chapter 2 design the relative standard curve experiment finish the design wizard applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 48 notes design guidelines when you design your own relative standard curve experi...

  • Page 73

    Chapter 3 49 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes prepare the relative standard curve reactions this chapter covers: ■ chapter overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...

  • Page 74: Chapter Overview

    Chapter 3 prepare the relative standard curve reactions chapter overview applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 50 notes chapter overview this chapter explains how to prepare the pcr reactions for the r...

  • Page 75: Prepare The Template

    Chapter 3 prepare the relative standard curve reactions prepare the template 51 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes prepare the template you prepare the template for the pcr reactions (both sam...

  • Page 76

    Chapter 3 prepare the relative standard curve reactions prepare the template applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 52 notes • for the samples, total rna isolated from liver and kidney tissues • for the...

  • Page 77: Prepare The Sample Dilutions

    Chapter 3 prepare the relative standard curve reactions prepare the sample dilutions 53 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes preparation guidelines when you prepare your own relative standard cu...

  • Page 78

    Chapter 3 prepare the relative standard curve reactions prepare the sample dilutions applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 54 notes required materials • water (to dilute the sample) • microcentrifuge t...

  • Page 79

    Chapter 3 prepare the relative standard curve reactions prepare the standard dilution series 55 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes prepare the standard dilution series you prepare the standard...

  • Page 80

    Chapter 3 prepare the relative standard curve reactions prepare the standard dilution series applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 56 notes 2. Add the required volume of water (diluent) to each empty t...

  • Page 81

    Chapter 3 prepare the relative standard curve reactions prepare the standard dilution series 57 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes 2. Add the required volume of water (diluent) to each empty t...

  • Page 82: Prepare The Reaction Mix

    Chapter 3 prepare the relative standard curve reactions prepare the reaction mix applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 58 notes prepare the reaction mix you prepare the reaction mix using the component...

  • Page 83

    Chapter 3 prepare the relative standard curve reactions prepare the reaction mix 59 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes prepare the reaction mix important! Prepare the reaction mix for each tar...

  • Page 84: Prepare The Reaction Plate

    Chapter 3 prepare the relative standard curve reactions prepare the reaction plate applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 60 notes preparation guidelines when you prepare your own relative standard curv...

  • Page 85

    Chapter 3 prepare the relative standard curve reactions prepare the reaction plate 61 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes required materials • microcentrifuge tubes • pipettors • pipette tips •...

  • Page 86

    Chapter 3 prepare the relative standard curve reactions prepare the reaction plate applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 62 notes 2. For each replicate group, prepare the standard reactions: a. To appr...

  • Page 87

    Chapter 3 prepare the relative standard curve reactions prepare the reaction plate 63 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes 5. Centrifuge the reaction plate briefly to remove air bubbles. 6. Veri...

  • Page 88

    Chapter 3 prepare the relative standard curve reactions prepare the reaction plate applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 64 notes e. While holding the film by the end-tabs, lower the film onto the reac...

  • Page 89

    Chapter 4 65 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes run the relative standard curve experiment this chapter covers: ■ chapter overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....

  • Page 90: Chapter Overview

    Chapter 4 run the relative standard curve experiment chapter overview applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 66 notes chapter overview this chapter explains how to perform a run on the applied biosystem...

  • Page 91: Prepare For The Run

    Chapter 4 run the relative standard curve experiment prepare for the run 67 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes prepare for the run you prepare for the run by opening the example experiment fil...

  • Page 92

    Chapter 4 run the relative standard curve experiment prepare for the run applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 68 notes load the reaction plate into the instrument physical injury hazard. During operat...

  • Page 93

    Chapter 4 run the relative standard curve experiment enable the notification settings (optional) 69 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes • reaction tubes – place the tubes in the pph. Note: micr...

  • Page 94

    Chapter 4 run the relative standard curve experiment enable the notification settings (optional) applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 70 notes set up the notification settings 1. In the 7500 software,...

  • Page 95: Start The Run

    Chapter 4 run the relative standard curve experiment start the run 71 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes run guidelines when you set up the 7500/7500 fast system for automatic notification: • ...

  • Page 96: Monitor The Run

    Chapter 4 run the relative standard curve experiment monitor the run applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 72 notes monitor the run you can view the progress of the run in real time as described below....

  • Page 97

    Chapter 4 run the relative standard curve experiment monitor the run 73 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes the amplification plot screen helps you identify and examine abnormal amplification. ...

  • Page 98: Unload The Instrument

    Chapter 4 run the relative standard curve experiment unload the instrument applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 74 notes if an alert is displayed, click the error for more information, then troublesho...

  • Page 99

    Chapter 5 75 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes analyze the relative standard curve experiment this chapter covers: ■ chapter overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....

  • Page 100: Chapter Overview

    Chapter 5 analyze the relative standard curve experiment chapter overview applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 76 notes chapter overview the 7500 software analyzes the data using the relative standard...

  • Page 101: Section 5.1

    Chapter 5 analyze the relative standard curve experiment section 5.1 review results 77 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes section 5.1 review results this section covers: ■ analyze the experime...

  • Page 102: Analyze The Experiment

    Chapter 5 analyze the relative standard curve experiment analyze the experiment applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 78 notes analyze the experiment the 7500 software analyzes the experiment and displ...

  • Page 103

    Chapter 5 analyze the relative standard curve experiment analyze the experiment 79 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes 6. See “navigation tips” on page 80 for information on navigating within t...

  • Page 104

    Chapter 5 analyze the relative standard curve experiment analyze the experiment applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 80 notes guidelines when you analyze your own relative standard curve experiment: •...

  • Page 105

    Chapter 5 analyze the relative standard curve experiment analyze the experiment 81 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes how to display multiple plots 1. In the navigation pane, select analysis m...

  • Page 106: View The Standard Curve

    Chapter 5 analyze the relative standard curve experiment view the standard curve applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 82 notes view the standard curve the standard curve screen displays the standard c...

  • Page 107

    Chapter 5 analyze the relative standard curve experiment view the standard curve 83 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes 7. Check that all samples are within the standard curve. In the example e...

  • Page 108

    Chapter 5 analyze the relative standard curve experiment view the standard curve applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 84 notes analysis guidelines when you analyze your own relative standard curve exp...

  • Page 109: View The Amplification Plot

    Chapter 5 analyze the relative standard curve experiment view the amplification plot 85 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes view the amplification plot the amplification plot screen displays am...

  • Page 110

    Chapter 5 analyze the relative standard curve experiment view the amplification plot applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 86 notes view the plot 1. In the navigation pane, select analysis amplificatio...

  • Page 111

    Chapter 5 analyze the relative standard curve experiment view the amplification plot 87 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes 4. View the baseline values (plot settings): a. In the graph type dro...

  • Page 112

    Chapter 5 analyze the relative standard curve experiment view the amplification plot applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 88 notes 5. View the threshold values: a. In plot settings tab, select log in ...

  • Page 113

    Chapter 5 analyze the relative standard curve experiment view the amplification plot 89 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes 6. Locate any outliers: a. In the plot type drop-down list, select c ...

  • Page 114

    Chapter 5 analyze the relative standard curve experiment view the amplification plot applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 90 notes analysis guidelines when you analyze your own relative standard curve...

  • Page 115

    Chapter 5 analyze the relative standard curve experiment view the amplification plot 91 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes threshold examples threshold set correctly the threshold is set in th...

  • Page 116

    Chapter 5 analyze the relative standard curve experiment view the amplification plot applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 92 notes if your experiment does not meet the guidelines above: • omit wells (...

  • Page 117

    Chapter 5 analyze the relative standard curve experiment view the gene expression plot and well table 93 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes view the gene expression plot and well table the gen...

  • Page 118

    Chapter 5 analyze the relative standard curve experiment view the gene expression plot and well table applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 94 notes about the example experiment in the relative standar...

  • Page 119

    Chapter 5 analyze the relative standard curve experiment view the gene expression plot and well table 95 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes 4. Click at the top left of the view replicate resul...

  • Page 120

    Chapter 5 analyze the relative standard curve experiment view the gene expression plot and well table applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 96 notes 5. View the well table: a. In the navigation pane, s...

  • Page 121: Publish The Data

    Chapter 5 analyze the relative standard curve experiment publish the data 97 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes publish the data you can publish the experiment data in several ways: • save the...

  • Page 122

    Chapter 5 analyze the relative standard curve experiment publish the data applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 98 notes.

  • Page 123: Section 5.2

    Chapter 5 analyze the relative standard curve experiment section 5.2 troubleshoot (if needed) 99 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes section 5.2 troubleshoot (if needed) this section covers: ■ ...

  • Page 124: View The Analysis Settings

    Chapter 5 analyze the relative standard curve experiment view the analysis settings applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 100 notes view the analysis settings the analysis settings dialog box displays ...

  • Page 125

    Chapter 5 analyze the relative standard curve experiment view the analysis settings 101 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes analysis guidelines unless you have already determined the optimal se...

  • Page 126

    Chapter 5 analyze the relative standard curve experiment view the analysis settings applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 102 notes 3. In the select multiple endogenous controls dialog box, select the ...

  • Page 127: View The Qc Summary

    Chapter 5 analyze the relative standard curve experiment view the qc summary 103 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes view the qc summary the qc summary screen displays a list of the 7500 softwa...

  • Page 128

    Chapter 5 analyze the relative standard curve experiment view the qc summary applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 104 notes possible flags for standard curve experiments, the flags listed below can be...

  • Page 129: Omit Wells From The Analysis

    Chapter 5 analyze the relative standard curve experiment omit wells from the analysis 105 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes omit wells from the analysis experimental error may cause some well...

  • Page 130

    Chapter 5 analyze the relative standard curve experiment omit wells from the analysis applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 106 notes 5. Click analyze to reanalyze the experiment data with well a9 remo...

  • Page 131: View The Multicomponent Plot

    Chapter 5 analyze the relative standard curve experiment view the multicomponent plot 107 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes view the multicomponent plot the multicomponent plot screen display...

  • Page 132

    Chapter 5 analyze the relative standard curve experiment view the multicomponent plot applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 108 notes 6. View the rox dye signal. In the example experiment, the rox dye ...

  • Page 133: View The Raw Data Plot

    Chapter 5 analyze the relative standard curve experiment view the raw data plot 109 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes analysis guidelines when you analyze your own relative standard curve exp...

  • Page 134

    Chapter 5 analyze the relative standard curve experiment view the raw data plot applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 110 notes 4. Drag the show cycle slider from cycle 1 to cycle 40. In the example ex...

  • Page 135

    Chapter 6 111 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes design the comparative c t experiment this chapter covers: ■ chapter overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....

  • Page 136: Chapter Overview

    Chapter 6 design the comparative c t experiment chapter overview applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 112 notes chapter overview this chapter explains how to use the design wizard in the 7500 software...

  • Page 137: Create A New Experiment

    Chapter 6 design the comparative c t experiment create a new experiment 113 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes create a new experiment create a new experiment using the design wizard in the 75...

  • Page 138

    Chapter 6 design the comparative c t experiment define the experiment properties applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 114 notes define the experiment properties in the experiment properties screen, en...

  • Page 139

    Chapter 6 design the comparative c t experiment define the experiment properties 115 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes design guidelines when you design your own comparative c t experiment: •...

  • Page 140

    Chapter 6 design the comparative c t experiment define the methods and materials applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 116 notes define the methods and materials in the methods and materials screen, se...

  • Page 141

    Chapter 6 design the comparative c t experiment define the methods and materials 117 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes design guidelines when you design your own comparative c t experiment: •...

  • Page 142

    Chapter 6 design the comparative c t experiment define the methods and materials applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 118 notes – select rna if you are performing 1-step rt-pcr. You are adding total r...

  • Page 143: Set Up The Targets

    Chapter 6 design the comparative c t experiment set up the targets 119 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes set up the targets in the targets screen, enter the number of targets you want to quan...

  • Page 144

    Chapter 6 design the comparative c t experiment set up the targets applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 120 notes design guidelines when you design your own comparative c t experiment: • identify each...

  • Page 145: Set Up The Samples

    Chapter 6 design the comparative c t experiment set up the samples 121 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes set up the samples in the samples screen, enter the number of samples, replicates, and...

  • Page 146

    Chapter 6 design the comparative c t experiment set up the samples applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 122 notes 8. In the well count pane, verify that there are: • 96 unknown wells • 0 negative cont...

  • Page 147

    Chapter 6 design the comparative c t experiment set up the samples 123 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes – select specify sample/target reactions to specify the targets to test in each sample...

  • Page 148

    Chapter 6 design the comparative c t experiment set up the relative quantitation settings applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 124 notes set up the relative quantitation settings in the relative quant...

  • Page 149: Set Up The Run Method

    Chapter 6 design the comparative c t experiment set up the run method 125 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes set up the run method in the run method screen, review the reaction volume and the ...

  • Page 150

    Chapter 6 design the comparative c t experiment set up the run method applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 126 notes design guidelines when you design your own comparative c t experiment: • enter a nu...

  • Page 151: Review The Reaction Setup

    Chapter 6 design the comparative c t experiment review the reaction setup 127 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes review the reaction setup in the reaction setup screen, select the assay type (...

  • Page 152

    Chapter 6 design the comparative c t experiment review the reaction setup applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 128 notes c. Review the components and calculated volumes for the pcr reactions: 7. Repea...

  • Page 153

    Chapter 6 design the comparative c t experiment review the reaction setup 129 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes 4. Review the calculated volumes for the sample dilutions: print reaction setup...

  • Page 154

    Chapter 6 design the comparative c t experiment review the reaction setup applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 130 notes design guidelines when you design your own comparative c t experiment: • if you...

  • Page 155

    Chapter 6 design the comparative c t experiment order materials for the experiment 131 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes order materials for the experiment in the materials list screen, revie...

  • Page 156

    Chapter 6 design the comparative c t experiment order materials for the experiment applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 132 notes 3. In the display drop-down list, select all items (default), then rev...

  • Page 157

    Chapter 6 design the comparative c t experiment order materials for the experiment 133 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes a. Check that the experiment shopping list contains the desired materi...

  • Page 158: Finish The Design Wizard

    Chapter 6 design the comparative c t experiment finish the design wizard applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 134 notes design guidelines when you design your own comparative c t experiment: • select ...

  • Page 159

    Chapter 6 design the comparative c t experiment finish the design wizard 135 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes • the experiment is saved as is and closed. Note: for the example experiment, do...

  • Page 160

    Chapter 6 design the comparative c t experiment finish the design wizard applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 136 notes 4. In the save experiment dialog box, enter comparative ct example setup.Eds in ...

  • Page 161

    Chapter 7 137 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes prepare the comparative c t reactions this chapter covers: ■ chapter overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....

  • Page 162: Chapter Overview

    Chapter 7 prepare the comparative c t reactions chapter overview applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 138 notes chapter overview this chapter explains how to prepare the pcr reactions for the comparat...

  • Page 163: Prepare The Template

    Chapter 7 prepare the comparative c t reactions prepare the template 139 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes prepare the template prepare the template for the pcr reactions using the high-capac...

  • Page 164

    Chapter 7 prepare the comparative c t reactions prepare the template applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 140 notes • total rna isolated from liver, kidney, brain, and universal tissues • one of appli...

  • Page 165

    Chapter 7 prepare the comparative c t reactions prepare the template 141 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes preparation guidelines when you prepare your own comparative c t ( ∆∆c t ) experimen...

  • Page 166: Prepare The Sample Dilutions

    Chapter 7 prepare the comparative c t reactions prepare the sample dilutions applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 142 notes prepare the sample dilutions perform sample dilutions before adding the samp...

  • Page 167

    Chapter 7 prepare the comparative c t reactions prepare the sample dilutions 143 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes prepare the sample dilutions 1. Label a separate microcentrifuge tube for ea...

  • Page 168: Prepare The Reaction Mix

    Chapter 7 prepare the comparative c t reactions prepare the reaction mix applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 144 notes prepare the reaction mix prepare the reaction mix using the components and volum...

  • Page 169

    Chapter 7 prepare the comparative c t reactions prepare the reaction mix 145 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes prepare the reaction mix important! Prepare the reaction mix for each target ass...

  • Page 170: Prepare The Reaction Plate

    Chapter 7 prepare the comparative c t reactions prepare the reaction plate applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 146 notes for more information for more information on preparing the reaction mix, refer...

  • Page 171

    Chapter 7 prepare the comparative c t reactions prepare the reaction plate 147 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes prepare the reaction plate 1. For each replicate group, prepare the reactions ...

  • Page 172

    Chapter 7 prepare the comparative c t reactions prepare the reaction plate applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 148 notes 2. Seal the reaction plate with optical adhesive film. 3. Centrifuge the react...

  • Page 173

    Chapter 7 prepare the comparative c t reactions prepare the reaction plate 149 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes e. While holding the film by the end-tabs, lower the film onto the reaction pl...

  • Page 174

    Chapter 7 prepare the comparative c t reactions prepare the reaction plate applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 150 notes.

  • Page 175

    Chapter 8 151 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes run the comparative c t experiment this chapter covers: ■ chapter overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...

  • Page 176: Chapter Overview

    Chapter 8 run the comparative c t experiment chapter overview applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 152 notes chapter overview this chapter explains how to perform a run on the applied biosystems 7500/...

  • Page 177: Prepare For The Run

    Chapter 8 run the comparative c t experiment prepare for the run 153 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes prepare for the run prepare for the run by opening the example experiment file you creat...

  • Page 178: Run The Experiment

    Chapter 8 run the comparative c t experiment run the experiment applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 154 notes run the experiment perform the following procedures in chapter 4 for the comparative c t ...

  • Page 179

    Chapter 9 155 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes analyze the comparative c t experiment this chapter covers: ■ chapter overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...

  • Page 180: Chapter Overview

    Chapter 9 analyze the comparative c t experiment chapter overview applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 156 notes chapter overview the 7500 software analyzes the data using the comparative c t ( ∆∆c t ...

  • Page 181: Section 9.1

    Chapter 9 analyze the comparative c t experiment section 9.1 review results 157 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes section 9.1 review results this section covers: ■ analyze the experiment . . ...

  • Page 182: Analyze The Experiment

    Chapter 9 analyze the comparative c t experiment analyze the experiment applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 158 notes analyze the experiment the 7500 software analyzes the experiment and displays res...

  • Page 183

    Chapter 9 analyze the comparative c t experiment analyze the experiment 159 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes 5. Click analyze. The software analyzes the data using the default analysis setti...

  • Page 184

    Chapter 9 analyze the comparative c t experiment analyze the experiment applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 160 notes navigation tips how to select wells to display specific wells in the analysis scr...

  • Page 185

    Chapter 9 analyze the comparative c t experiment analyze the experiment 161 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes how to display multiple plots use the multiple plots view to display up to four p...

  • Page 186

    Chapter 9 analyze the comparative c t experiment view the gene expression plot and well table applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 162 notes view the gene expression plot and well table displays the r...

  • Page 187

    Chapter 9 analyze the comparative c t experiment view the gene expression plot and well table 163 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes view the plot and well table 1. In the navigation pane, sel...

  • Page 188

    Chapter 9 analyze the comparative c t experiment view the gene expression plot and well table applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 164 notes 4. Click at the top left of the view technical replicates t...

  • Page 189

    Chapter 9 analyze the comparative c t experiment view the gene expression plot and well table 165 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes 5. View the well table: a. In the navigation pane, select a...

  • Page 190: View The Amplification Plot

    Chapter 9 analyze the comparative c t experiment view the amplification plot applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 166 notes view the amplification plot the amplification plot screen displays amplifica...

  • Page 191

    Chapter 9 analyze the comparative c t experiment view the amplification plot 167 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes view the plot 1. In the navigation pane, select analysis amplification plot....

  • Page 192

    Chapter 9 analyze the comparative c t experiment view the amplification plot applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 168 notes 4. View the baseline values (plot settings tab): a. In the graph type drop-d...

  • Page 193

    Chapter 9 analyze the comparative c t experiment view the amplification plot 169 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes 5. View the threshold values: a. In plot settings tab, select log in the gra...

  • Page 194

    Chapter 9 analyze the comparative c t experiment view the amplification plot applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 170 notes 6. Locate any outliers: a. In plot settings tab, select c t vs well in the p...

  • Page 195

    Chapter 9 analyze the comparative c t experiment view the amplification plot 171 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes analysis guidelines when you analyze your own comparative c t experiment, lo...

  • Page 196

    Chapter 9 analyze the comparative c t experiment view the amplification plot applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 172 notes threshold examples threshold set correctly the threshold is set in the expon...

  • Page 197

    Chapter 9 analyze the comparative c t experiment view the amplification plot 173 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes if your experiment does not meet the guidelines above, troubleshoot as follo...

  • Page 198: Publish The Data

    Chapter 9 analyze the comparative c t experiment publish the data applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 174 notes publish the data you can publish the experiment data in several ways: • save the plot a...

  • Page 199: Section 9.2

    Chapter 9 analyze the comparative c t experiment section 9.2 troubleshoot (if needed) 175 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes section 9.2 troubleshoot (if needed) this section covers: ■ view th...

  • Page 200: View The Analysis Settings

    Chapter 9 analyze the comparative c t experiment view the analysis settings applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 176 notes view the analysis settings the analysis settings dialog box displays the anal...

  • Page 201

    Chapter 9 analyze the comparative c t experiment view the analysis settings 177 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes analysis guidelines unless you have already determined the optimal settings f...

  • Page 202

    Chapter 9 analyze the comparative c t experiment view the analysis settings applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 178 notes 3. In the select multiple endogenous controls dialog box, select the targets ...

  • Page 203: View The Qc Summary

    Chapter 9 analyze the comparative c t experiment view the qc summary 179 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes view the qc summary the qc summary screen displays a list of the 7500 software flags...

  • Page 204

    Chapter 9 analyze the comparative c t experiment view the qc summary applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 180 notes possible flags for comparative c t experiments, the flags listed below may be genera...

  • Page 205: Omit Wells From The Analysis

    Chapter 9 analyze the comparative c t experiment omit wells from the analysis 181 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes omit wells from the analysis experimental error may cause some wells to be ...

  • Page 206: View The Multicomponent Plot

    Chapter 9 analyze the comparative c t experiment view the multicomponent plot applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 182 notes analysis guidelines when you analyze your own comparative c t experiment, c...

  • Page 207

    Chapter 9 analyze the comparative c t experiment view the multicomponent plot 183 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes view the multicomponent plot 1. In the navigation pane, select analysis mul...

  • Page 208

    Chapter 9 analyze the comparative c t experiment view the multicomponent plot applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 184 notes analysis guidelines when you analyze your own comparative c t experiment, l...

  • Page 209: View The Raw Data Plot

    Chapter 9 analyze the comparative c t experiment view the raw data plot 185 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes view the raw data plot the raw data plot screen displays the raw fluorescence (no...

  • Page 210

    Chapter 9 analyze the comparative c t experiment view the raw data plot applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 186 notes 4. Drag the show cycle slider from cycle 1 to cycle 40. In the example experiment...

  • Page 211: Section 9.3

    Chapter 9 analyze the comparative c t experiment section 9.3 perform a study of multiple experiments 187 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes section 9.3 perform a study of multiple experiments ...

  • Page 212: Section Overview

    Chapter 9 analyze the comparative c t experiment section overview applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 188 notes section overview the 7500 software can combine the analysis of experiments that use the...

  • Page 213: Design A Study

    Chapter 9 analyze the comparative c t experiment design a study 189 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes design a study this section explains how to use the 7500 software to set up a comparative...

  • Page 214

    Chapter 9 analyze the comparative c t experiment design a study applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 190 notes define study properties in the experiment properties screen, enter identifying informatio...

  • Page 215

    Chapter 9 analyze the comparative c t experiment design a study 191 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes design guidelines when you design your own comparative c t study: • enter an study name t...

  • Page 216

    Chapter 9 analyze the comparative c t experiment design a study applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 192 notes how to simplify data lists using the filter query to narrow your search for an experiment...

  • Page 217

    Chapter 9 analyze the comparative c t experiment design a study 193 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes e. In the plate layout, select wells d6 and d7, then click >> to add the technical replic...

  • Page 218

    Chapter 9 analyze the comparative c t experiment design a study applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 194 notes design guidelines when you add a biological replicate group: • enter a biological replica...

  • Page 219: Analyze The Study

    Chapter 9 analyze the comparative c t experiment analyze the study 195 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes analyze the study this section explains how to use the 7500 software to analyze the co...

  • Page 220

    Chapter 9 analyze the comparative c t experiment analyze the study applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 196 notes view the gene expression plot, replicate results table, and well table the gene expres...

  • Page 221

    Chapter 9 analyze the comparative c t experiment analyze the study 197 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes the well results data group displays data for each well in the reaction plates that ar...

  • Page 222

    Chapter 9 analyze the comparative c t experiment analyze the study applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 198 notes 4. Click at the top left of the replicate results data group. The figure below shows t...

  • Page 223

    Chapter 9 analyze the comparative c t experiment analyze the study 199 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes analysis guidelines when you review the gene expression plot of a comparative c t stud...

  • Page 224

    Chapter 9 analyze the comparative c t experiment analyze the study applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 200 notes view the amplification plot the amplification plot displays post-run amplification of ...

  • Page 225

    Chapter 9 analyze the comparative c t experiment analyze the study 201 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes view multiple plots in the study menu, select multiple plots view. The operation of th...

  • Page 226: Publish The Data

    Chapter 9 analyze the comparative c t experiment publish the data applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 202 notes compare analysis settings the compare settings screen allows you to perform a side-by-s...

  • Page 227

    Appendix a 203 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes alternate experiment workflows this appendix covers: ■ advanced setup workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....

  • Page 228: Advanced Setup Workflow

    Appendix a alternate experiment workflows advanced setup workflow applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 204 notes advanced setup workflow when you create an experiment using advanced setup in the 7500 ...

  • Page 229

    Appendix a alternate experiment workflows advanced setup workflow 205 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes 4. Prepare the pcr reactions: 5. Run the experiment: important! While the 7500/7500 fas...

  • Page 230: Quickstart Workflow

    Appendix a alternate experiment workflows quickstart workflow applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 206 notes quickstart workflow when you create an experiment using quickstart, you can run the reactio...

  • Page 231

    Appendix a alternate experiment workflows quickstart workflow 207 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes 4. In the 7500 software, complete the reaction plate setup: 5. Analyze the data: a. Open th...

  • Page 232: Template Workflow

    Appendix a alternate experiment workflows template workflow applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 208 notes template workflow you can use a template to create a new experiment. Templates allow you to c...

  • Page 233

    Appendix a alternate experiment workflows template workflow 209 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes 5. Prepare the pcr reactions: 6. Run the experiment: important! While the 7500/7500 fast inst...

  • Page 234: Export/import Workflow

    Appendix a alternate experiment workflows export/import workflow applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 210 notes export/import workflow use the export/import workflow to set up a new experiment using s...

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    Appendix a alternate experiment workflows export/import workflow 211 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments notes c. In the home screen, click advanced setup. Note: if you do not see the advanced setup ...

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    Appendix a alternate experiment workflows export/import workflow applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 212 notes 5. Analyze the data: a. Open the experiment in the 7500 software. B. In the navigation p...

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    213 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments bibliography kwok, s. And higuchi, r. 1989. Avoiding false positives with pcr. Nature 339:237–238. Saiki, r.K., scharf, s., faloona, f., et al. 1985. Enzymatic...

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    Bibliography applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 214.

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    215 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments glossary advanced setup in the 7500 software, a feature that allows you to set up your experiment according to your experiment design. Advanced setup provides ...

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    Glossary applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 216 amplification stage part of the instrument run in which pcr produces amplification of the target. The amplification stage is called a cycling stage in...

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    Glossary 217 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments baseline-corrected normalized reporter ( ∆ rn) the magnitude of normalized fluorescence generated by the reporter: 1. In experiments that contain data...

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    Glossary applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 218 data collection a process during the instrument run in which an instrument component detects fluorescence data from each well of the reaction plate. T...

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    Glossary 219 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments experiment name entered during experiment setup, the name that is used to identify the experiment. Experiment names cannot exceed 100 characters and c...

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    Glossary applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 220 melt curve a plot of data collected during the melt curve stage. Peaks in the melt curve can indicate the melting temperature (tm) of the target or ca...

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    Glossary 221 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments passive reference a dye that produces fluorescence. Because the passive reference signal should be consistent across all wells, it is used to normaliz...

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    Glossary applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 222 quickstart a feature in 7500/7500 fast systems that allows you to run an experiment without entering plate setup information. R 2 value regression coe...

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    Glossary 223 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments regression coefficients values calculated from the regression line in standard curves, including the r 2 value, slope, and y-intercept. You can use th...

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    Glossary applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 224 sample dna (10 ✕) in the 7500 software, a reaction component displayed on the reaction mix calculations tab of the reaction setup screen. The software...

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    Glossary 225 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments standard sample that contains known standard quantities. Standard reactions are used in quantitation experiments to generate standard curves. See also...

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    Glossary applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 226 system dye dye supplied by applied biosystems and precalibrated on the 7500/7500 fast system. Before you use system dyes in your experiments, make sur...

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    Glossary 227 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments template in the design wizard of the 7500 software (and in quickstart for quantitation experiments), the type of nucleic acid to add to the pcr reacti...

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    Glossary applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 228.

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    229 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments index numerics 1-step rt-pcr 10 , 25 , 26 , 36 , 42 , 118 , 126 , 130 2-step rt-pcr 10 , 14 , 15 , 25 , 26 , 117 , 118 7500/7500 fast system consumables 4 data...

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    Index applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 230 ∆c t mean 164 , 198 ∆c t sd 164 ∆c t se 198 ∆∆c t 198 ∆∆c t sd 164 flag 198 normalized qty mean 95 normalized qty std err 95 omit 95 , 164 , 198 rq 95 , ...

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    Index 231 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments expfail flag 104 , 180 export/import 13 , 210 f fast reaction plates, about 6 flag column 198 flags analysis settings 101 , 177 in comparative ct experim...

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    Index applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 232 q qc summary screen 103 , 179 , 201 quickstart 13 , 206 r radioactive waste, handling xix ramp speed 24 , 25 , 116 , 117 raw data plot screen 109 , 185 r...

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    Index 233 applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments analyze 195 create 189 design 189 , 190 , 192 properties 190 workflow 18 , 156 study properties screen 190 sybr green reagents 3 , 11 , 25 , 26 , 28 , 42...

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    Index applied biosystems 7500/7500 fast real-time pcr system getting started guide for relative standard curve and comparative c t experiments 234.

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    Www.Appliedbiosystems.Com 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 re...