ABI PRISM 7000 Sequence Detection Systems Relative ...

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Chapter 4 Generating Data from RQ Plates Preparing the PCR Master Mix Follow these recommended general PCR practices: • Wear a clean lab coat (not previously worn while handling amplified PCR products or used during sample preparation) and clean gloves when preparing samples for PCR amplification. • Change gloves whenever you suspect that they are contaminated. • Maintain separate areas, dedicated equipment, and supplies for: – Sample preparation and PCR setup – PCR amplification and post-PCR analysis • Never bring amplified PCR products into the PCR setup area. • Open and close all sample tubes carefully. Try not to splash or spray PCR samples. • Quick-spin PCR samples whenever residual sample is present on the inside lid (such as after dropping a tube or when there is condensation on the tube from heating or thawing) • Keep reactions and components capped as much as possible. • Use aerosol-resistant or positive-displacement pipette tips. • Clean lab benches and equipment periodically with freshly diluted 10% chlorine bleach. Preparing the PCR Master Mix The second step in the two-step RT-PCR procedure is amplifying the cDNA. TaqMan ® Universal Master Mix reagents provide a PCR mix that may be used with any appropriately designed primer and probe to detect any DNA or cDNA sequence. The TaqMan Universal PCR Master Mix Protocol (PN 4304449) explains how to use the reagents provided in the kit. The following table lists the universal assay conditions (volume and final concentration) for using the master mix. CHEMICAL HAZARD. TaqMan Universal PCR Master Mix may cause eye and skin irritation. Exposure may cause discomfort if swallowed or inhaled. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Reaction Component Volume (µL) Per Sample Final Concentration TaqMan Universal PCR Master Mix (2✕) 25.0 1✕ Forward primer 5.0 50 to 900 nM Reverse primer 5.0 50 to 900 nM TaqMan probe 5.0 50 to 250 nM cDNA sample 5.0 10 to 100 ng Nuclease-free water 5.0 — Total 50.0 — DRAFT September 26, 2003 3:14 pm, C4_RQPlate.fm Notes 20 Relative Quantification Getting Started Guide for 7000 v1.1
Preparing the PCR Master Mix Preventing Contamination For most TaqMan reagent- or kit-based assays that are designed and run following Applied Biosystems assay development guidelines (refer to the Sequence Detection Systems Chemistry Guide), using a concentration of 900-nM primers and a 250-nM probe provides a highly reproducible and sensitive assay when using cDNA or DNA as a substrate in a singleplex assay. Probes and primers that you design using Primer Express software must be optimized to work with the universal assay conditions, using the volumes listed in the table on page 20. All Assays-by-Design (ABD) and Assays-on-Demand (AOD) products are formulated so that the final concentration of the primers and probes falls within the recommended parameters. But because the primers and probes are supplied as a 20✕ assay mix, the volumes are slightly different from the universal assay conditions, as explained in the following Example. CHEMICAL HAZARD. TaqMan Universal PCR Master Mix may cause eye and skin irritation. Exposure may cause discomfort if swallowed or inhaled. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Example Primers and probes for the sample RQ experiment were obtained from the Assays-on-Demand product line and were provided as a 20✕ Gene Expression Assay Mix. The PCR master mix was prepared as follows (according to guidelines specified in the product insert that comes with all AOD and ABD products): Reaction Component Volume (µL) Per Sample Final Concentration TaqMan Universal PCR Master Mix (2✕) 25.0 1✕ DRAFT September 26, 2003 3:14 pm, C4_RQPlate.fm 20✕ Assays-on-Demand Gene Expression Assay Mix (contains forward and reverse primers and labeled probe) 2.5 1✕ cDNA sample 5.0 50 ng (for the 50-µL reaction) Nuclease-free water 17.5 — Total 50.0 — The reactions were kept on ice until the plate was loaded on the 7000 SDS instrument. 4 Notes Relative Quantification Getting Started Guide for 7000 v1.1 21
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 and 36: 5′ 3′ 5′ cDNA Reverse Primer
Page 37: Primer Extended on mRNA 5′ 3′ 5
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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