Manipulation and Expression of Recombinant DNA

Manipulation and Expression of Recombinant DNA : A Lab Manual

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This laboratory manual is designed for use in introductory biotechnology courses. Experiments are constructed in a modular fashion, lending this book both to the traditional semester course and the shorter intensive course. Its main objective is to expose students to the basic research protocols used in molecular biology and biochemistry labs. This text covers techniques in detail and teaches students to culture and transform bacteria, grow vector plasmids, purify insert DNA, ligate vector and insert, and ultimately express protein and assay its activity. Comprehensive and complete, this text examines the underlying techniques used in all basic research and biotechnology laboratories.
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Product details

  • Spiral bound | 224 pages
  • 223 x 278 x 15mm | 654g
  • Academic Press Inc
  • San Diego, United States
  • English
  • b&w illustrations, glossary, appendices, index
  • 0125897650
  • 9780125897655

Table of contents

Part 1 Manipulation of DNA: Lab session 1 - culturing E. Coli for plasmid isolation: preparation of solid media, making stabs cultures, streaking out plasmid clones on LB/carb plates, growth curve of E. Coli,; cultures for plasmid preparations; Lab session 2 - growth curve of E. Coli: examination of clones, E. Coli growth curve, preparation of solutions for large scale plasmid DNA preps, inoculation of cultures for large scale plasmid DNA preps; Lab session 3A - large scale preparation of plasmid DNA by alkaline lysis; Lab session 3B - purification of plasmid DNA using columns; Lab session 3C - purification of plasmid DNA using cesium gradients; Lab session 3D - purification of plasmid DNA using polyethylene glycol; Lab session 4 - preparation of vector DNA - ethidium bromide spot test, preparation of Hindill Cut, phosphated vector, gel electrophoresis, electrophoresis of pUR288, shrimp alkaline phosphatase treatment of pUR288, overnight cultures for transformation-competent cells; Lab session 5 - transformation of E. Coli - preparation of frozen, transformation-competent cells, test ligations of pUR288 electrophoresis of ligation products, transformation; Lab session 6 - preparation and ligation of insert DNA: analysis of vector preparation, isolation of myo-3 DNA from agarose, ligations; Lab session 7A - transformation of E. Coli with ligated DNA and labelling a DNA probe: CaCl2-mediated transformation, electrophoration, electrophoresis of ligation reactions, random primed DNA labelling with Dig-11-dUTP; Lab session 7B: labelling DNA with radioactive nucleotides: random primer labelling of DNA using 32p-dCTP; Lab Session 8 - DNA probe preparation, replica plating: estimating the yield of the DIG-labelled probe, making replica plates; Lab session 9 - colony hybrid dilations, DNA probe: colony hybridizations - transfer of colonies to nylon membranes, prehybridization, DNA/DNA hybridization, adding DNA probe; Lab session 10 - restriction of map of plasmid p2D, colony nybridizations. Part 2 Restriction enzyme reactions; colony hybridization with a DNA probe - washing filters probed with Dig-11-dUTP labelled myo-3 fragment; colony hybridization with a DNA probe - washing filters probed with 32p-dCTP labelled myo-3 fragment; gel electrophoresis of restriction digests; Lab session 11 - screening colonies with monoclonal antibodies: colony hybridizations - mAb probe, first day, alkaline lysis minipreps, finish restriction maps; Lab session 12 - isolation and characterization of miniprep DNA from potential transformants. (Part contents).
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Review quote

"This is a clearly written and well thought out laboratory manual that covers many of the basic concepts and laboratory techniques common to molecular biology... The book has many clear and concise diagrams that are a great aid to a student who is not familiar with molecular biology... the protocols distinctly describe how to make solutions used in molecular biology experiments."
--Philip M. Sass, PhD, University of Illinois at Chicago College of Medicine for DOODY'S PUBLISHING REVIEWS
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