ABI PRISM 7000 Sequence Detection Systems Relative ...

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Prim 5′ 3′ Syn Do you have cDNA on mRNA 3 5′ cDNA Reverse Primer Oligo d(T) or random hexamer DNA strand 5′ cDNA No Chapter 3 Reverse transcribe total RNA to cDNA. Yes Have you prepared the PCR master mix PCR Master Mix No page 20 Prepare the PCR master mix as directed in the TaqMan ® Universal PCR Master Mix Protocol. Yes Have you created an RQ plate document No page 22 Create a new RQ plate document. Yes Have the detectors for the experiment been added to the software No page 23 Create detectors. Yes Are the default thermal cycling conditions for PCR set No page 25 Program the default PCR conditions or enter the thermal cycling conditions for one-step RT-PCR Have you saved the data from the PCR run Have you viewed the RQ plate data Yes Yes Start the run. Yes No No page 26 page 28 Go to chapter 5, Performing an RQ Study Save the data in the RQ plate document. View the RQ plate data to confirm that the run was successful. DRAFT September 26, 2003 3:14 pm, C4_RQPlate.fm Chapter 4 Generating Data in RQ Plates
Primer Extended on mRNA 5′ 3′ 5′ cDNA Reverse Primer Oligo d(T) or random hexamer Synthesis of 1st cDNA strand 3′ 5′ cDNA Generating Data from RQ Plates Workflow Designing an RQ Experiment Performing Reverse Transcription Generating Data from RQ Plates Generating Data from RQ Plates 1. Prepare the PCR Master Mix using TaqMan Universal PCR Master Mix. 2. Create an RQ Plate document. 3. Create detectors. 3. Set thermal cycling parameters. 4. Save and run the RQ plate. Performing an RQ Study 5. Analyze and view the results. DRAFT September 26, 2003 3:14 pm, C4_RQPlate.fm Before You Begin Calibrating the 7000 SDS instrument Preventing Contamination Check that background and pure-dye runs have been performed regularly to ensure optimal performance of the 7000 SDS instrument. For more information about calibrating the 7000 SDS instrument, refer to the Online Help for the 7000 SDS instrument. PCR techniques require special laboratory practices to avoid false positive amplifications (Kwok and Higuchi, 1989). The high throughput and repetition of these techniques can lead to amplification of a single DNA molecule (Saiki et al., 1985; Mullis and Faloona, 1987). 4 Notes Relative Quantification Getting Started Guide for 7000 v1.1 19
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 and 24: 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: 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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