The Nucleic Acid Protocols Handbook

The Nucleic Acid Protocols Handbook is today's most comprehensive up-to-date treasury of all the key molecular biology methods-ranging from DNA extraction to gene localization in situ-needed to function effectively in the modern laboratory. Each of the 120 highly successful techniques follows the format of the much acclaimed Methods in Molecular Biology(TM) series, providing an introduction to the scientific basis of each technique, a complete listing of all the materials and reagents needed, and clear step-by-step instructions to permit error-free execution. Included for each technique are notes about pitfalls to avoid, troubleshooting tips, alternate methods, and explanations of the reasons for certain steps-all key elements contributing significantly to success in the lab. The authord are well-experienced masters of the techniques described, often having perfected and optimized them in their own laboratories. In The Nucleic Acid Protocols Handbook, Ralph Rapley has brought together thewidest-ranging, one-volume collection of classic and cutting-edge techniques for the successful isolation, analysis, and manipulation of nucleic acids. Eminently practical and reproducible, these proven methods will help both experienced researchers and those entering the field attain the laboratory mastery needed-not only to deepen understanding of biological processes at the molecular level, but also to begin realizing the biotechnological bounty so clearly in prospect.


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Part I. Nucleic Acid Extraction. Isolation of High-Molecular-Weight DNA from Animal Cells, Ian Garner. Isolation of mRNA by Affinity Chromatography, Sian Bryant and David L. Manning. Isolation and Purification of DNA from Plants, Justin Stacey and Peter G. Isaac. Purification of Uncontaminated, Intact Plant RNA, Shu-Hua Cheng, Brandon D. Moore, and Jeffrey R. Seemann. An Improved Method to Isolate Mitochondrial RNA from Green Plant Tissue, Fei Ye and Ralf Reski. Isolating Chromosomal DNA from Bacteria, Elisabeth Chachaty and Patrick Saulnier. Bacterial DNA Extraction for Polymerase Chain Reaction and Pulsed-Field Gel Electrophoresis, Elisabeth Chachaty and Patrick Saulnier. Isolation of Fungal Nucleic Acids, Surapareddy Sreenivasaprasad. Total RNA Isolation from Bacteria, John Heptinstall. Simultaneous RNA and DNA Extraction from Biopsy Material, Culture Cells, Plants, and Bacteria, Udo Döbbeling. Spectrophotometric Analysis of Nucleic Acids, John Heptinstall and Ralph Rapley. Part II. Basic Separation and Analysis of DNA. Restriction Endonuclease Digestion of DNA, Duncan R. Smith. Agarose Gel Electrophoresis of Nucleic Acids, D. Ross Williams and Ralph Rapley. Preparation of RNA Dot Blots, Rachel Hodge. Native Polyacrylamide Gel Electrophoresis, Adrian J. Harwood. Southern Blotting of Agarose Gels by Capillary Transfer, Ralph Rapley and Jane Davenport -Jones. Pulsed-Field Gel Electrophoresis, John Maule. HPLC of DNA and PCR Products, Elena D. Katz. Part III. Probe Design, Synthesis, and Labeling. End-Labeling of DNA Fragments, Adrian J. Harwood. Nick Translation and Random Hexamer Labeling of DNA, Jane Davenport-Jones. Generation of Labeled Probes by Polymerase Chain Reaction, Y. M. Dennis Lo and Shu F. An. Nonradioactive Oligonucleotide Probe Labeling, Sue Fowler and Ian Durrant. Preparation of Direct, Enzyme-Labeled DNA Probes, Ian Durrant and Timothy Stone. Random Prime Labeling of DNA Probes with Fluorescein-Tagged Nucleotides, Bronwen M. Harvey, Claire B.Wheeler, and Martin W. Cunningham. Hybridization and Detection of Fluorescein-Labeled DNA Probes Using Chemiluminescence, Claire B. Wheeler, Bronwen M. Harvey, and Martin W. Cunningham. Hybridization of Enzyme-Labeled Probes and Detection by Chemiluminescence, Timothy Stone and Ian Durrant. Hybridization and Competition Hybridization of Southern Blots, Rosemary Kelsell. Autoradiography and Fluorography, Eric Quéméneur. Part IV. RNA Analysis Techniques. Formaldehyde Gel Electrophoresis of Total RNA, Sian Bryant and David L. Manning. RNA Probes for the Analysis of Gene Expression, Dominique Belin. Primer Extension Analysis of mRNA, Maggie Walmsley, Mark Leonard, and Roger Patient. S1 Mapping Using Single-Stranded DNA Probes, Stéphane Viville and Roberto Mantovani. Measurements of Rate of Transcription in Isolated Nuclei by Nuclear 'Run-Off' Assay, Rai Ajit K. Srivastava and Gustav Schonfeld. One-Tube RT-PCR with Sequence-Specific RT Primers, Ulrich Pfeffer and Paola Ferro. Characterization of RNA Using Continuous RT-PCR Coupled with ELOSA, François Mallet. Quantitative Analysis of RNA Species by Polymerase Chain Reaction and Solid-Phase Minisequencing, Anu Suomalainen and Ann-Christine Syvänen. Nonradioactive Northern Blotting of RNA, Rainer Löw. Analysis of RNA by Northern Blotting Using Riboprobes, Rai Ajit K. Srivastava. Part V. Gene Library Construction and Screening. Production of Double-Stranded cDNA for Gene Library Synthesis, Jane Kirk and Steve Mayall. Using Rapid Amplification of cDNA Ends (RACE) to Obtain Full-Length cDNAs, Yue Zhang and Michael A. Frohman. cDNA Library Construction Using Streptavidin-Paramagnetic Beads and PCR, Kris N. Lambert and Valerie M. Williamson. Rapid (Ligase-Free) Subcloning of Polymerase Chain Reaction Products, Alan R. Shuldiner and Keith Tanner. Subtraction Hybridization cDNA Libraries, Clifford W. Schweinfest, Peter S. Nelson, Michael W. Graber, Rita I. Demopoulos, and Takis S. Papas. Cloning Polymerase Chain Reaction

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