ABI PRISM 7000 Sequence Detection Systems Relative ...

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Prim 5′ 3′ Syn Have you selected a PCR method (Single or Multiplex) E T T E E - Endo Ctrl T - Target No page 7 Select a PCR method. Yes Have you designated the targets, calibrator, endogenous control, and replicates for the experiment Target Control Calibrator # of Replicates No page 9 Designate the four essential components of RQ experiments. Yes Have you selected the SDS chemistry (SYBR Green 1 or TaqMan®) and reagent configuration R Q 3′ No page 11 Select the SDS chemistry for your experiment. Yes Have you designed your primers and probes Primer Extended on mRNA 5′ 3′ 5′ cDNA Reverse Primer Oligo d(T) or random hexamer Synthesis of 1st cDNA strand 3′ 5′ cDNA No page 13 Go to chapter 3, Performing Reverse Transcription Choose primers and probes for your experiment. DRAFT September 26, 2003 3:14 pm, C2_Design.fm Chapter 2 Designing an RQ Experiment
Primer Extended on mRNA 5′ 3′ 5′ cDNA Reverse Primer Oligo d(T) or random hexamer Synthesis of 1st cDNA strand 3′ 5′ cDNA Designing an RQ Experiment Workflow Designing an RQ Experiment Performing Reverse Transcription Generating Data from RQ Plates Designing an RQ Experiment • Select the PCR method (multiplex or singleplex). • Designate the targets, calibrator, endogenous control, and replicates. • Select the SDS chemistry and reagent configuration. • Choose the primers and probes. 2 Performing an RQ Study Selecting the PCR Method DRAFT September 26, 2003 3:14 pm, C2_Design.fm Traditional PCR is performed as a singleplex reaction, where a single primer pair is present in the reaction tube or well. Only one target sequence or endogenous control can be amplified per reaction—target sequences and endogenous controls cannot be amplified in the same tube. In multiplex PCR, two or more primer pairs are present in the reaction. Each primer pair amplifies either a target sequence or an endogenous control. The availability of multiple reporter dyes for TaqMan ® probes, each with different emission wavelength maxima, makes multiplex PCR possible. Notes <strong>Relative</strong> Quantification Getting Started Guide for <strong>7000</strong> v1.1 7
Page 1 and 2: 5′ cDNA Reverse Primer Oligo d(T)
Page 3 and 4: Primer Extended on mRNA 5′ 3′ 5
Page 5 and 6: Chapter 5 Performing an RQ Study 33
Page 7 and 8: Preface How to Use This Guide Purpo
Page 9 and 10: Safety and EMC Compliance Informati
Page 11 and 12: Symbols on Instruments Electrical S
Page 13 and 14: Chemical Safety Chemical Hazard War
Page 15 and 16: Physical Hazard Safety Ultraviolet
Page 17 and 18: Safety and Electromagnetic Compatib
Page 19 and 20: Introduction 1 About the 7000 SDS I
Page 21 and 22: About the Sample RQ Experiment Desc
Page 23: Materials and Equipment Description
Page 27 and 28: Specifying the Components of an RQ
Page 29 and 30: Selecting the Sequence Detection Ch
Page 31 and 32: Choosing the Probes and Primers Abo
Page 35 and 36: 5′ 3′ 5′ cDNA Reverse Primer
Page 39 and 40: Preparing the PCR Master Mix Preven
Page 41 and 42: Creating an RQ Plate Document Detec
Page 43 and 44: Specifying Thermal Cycling Conditio
Page 45 and 46: Analyzing and Viewing RQ Plate Data
Page 47 and 48: Analyzing and Viewing RQ Plate Data
Page 49 and 50: Exporting RQ Plate Documents Reanal
Page 53 and 54: Creating an RQ Study Document 5. Co
Page 55 and 56: Analyzing and Viewing the Results o
Page 57 and 58: Analyzing and Viewing the Results o
Page 59 and 60: Omitting Samples from a Study Omitt
Page 61 and 62: Exporting RQ Study Results Exportin
Page 63 and 64: References Kwok, S. and Higuchi, R.
Page 65 and 66: Creating Detectors A Before you can
Page 67 and 68: Upgrading the 7000 Software with RQ
Page 69 and 70: Index SAMPLE DOCUMENT September 26,
Page 71 and 72: Index SAMPLE DOCUMENT September 26,
Page 73 and 74: DRAFT September 26, 2003 3:17 pm, G

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