Recombinant DNA Laboratory Manual
- 1st Edition - January 28, 1989
- Imprint: Academic Press
- Authors: Judith W. Zyskind, Sanford I. Bernstein
- Language: English
- Paperback ISBN:9 7 8 - 1 - 4 8 3 2 - 0 7 5 4 - 4
- eBook ISBN:9 7 8 - 1 - 4 8 3 2 - 2 0 9 7 - 0
Recombinant DNA Laboratory Manual is a laboratory manual on the fundamentals of recombinant DNA techniques such as gel electrophoresis, in vivo mutagenesis, restriction mapping,… Read more
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Request a sales quoteRecombinant DNA Laboratory Manual is a laboratory manual on the fundamentals of recombinant DNA techniques such as gel electrophoresis, in vivo mutagenesis, restriction mapping, and DNA sequencing. Procedures that are useful for studying either prokaryotes or eukaryotes are discussed, and experiments are included to teach the fundamentals of recombinant DNA technology. Hands-on computer sessions are also included to teach students how to enter and manipulate sequence information. Comprised of nine chapters, this book begins with an introduction to bacterial growth parameters, how to measure bacterial cell growth, and how to plot cell growth data. The discussion then turns to the isolation and analysis of chromosomal DNA in bacteria and Drosophila; plasmid DNA isolation and agarose gel analysis; and introduction of DNA into cells. Subsequent chapters deal with Tn5 mutagenesis of pBR329; DNA cloning in M13; DNA sequencing; and DNA gel blotting, probe preparation, hybridization, and hybrid detection. The book concludes with an analysis of lambda phage manipulations. This manual is intended for advanced undergraduate or beginning graduate students and should also be helpful to established investigators who are changing their research focus.
PrefaceSchedule of Laboratory ExercisesLab I. Bacterial Growth Parameters Day 1. Measuring Bacterial Cell Growth Day 2. Plotting Cell Growth Data AddendumLab II. Isolation and Analysis of Bacterial and Drosophila Chromosomal DNA Day 1. Isolation and Purification of E. Coli Chromosomal DNA Isolation and Purification of Drosophila DNA Day 2. Determination of the Concentration and Purity of DNA by UV Spectroscopy Day 3. Restriction Endonuclease Digestion of Chromosomal DNA Day 4. Agarose Gel of Chromosomal DNA Restriction Endonuclease Digestions Day 5. Staining and Photography of Agarose Gel of Chromosomal DNA Restriction Endonuclease DigestionsLab III. Plasmid DNA Isolation and Agarose Gel Analysis Day 1. Isolation of Plasmid DNA by the Alkaline-Detergent Method—A Miniprep Procedure Day 2. Agarose Gel Electrophoresis of Undigested Plasmid DNALab IV. Introduction of DNA into Cells Day 1. Production of Frozen Competent Cells Day 2. Transformation of LE392 with pBR329 DNA Isolated from HB101::Tn5Lab V. Tn5 Mutagenesis of pBR329 Day 1. Marker Screening: Divide Transformants into Tcs and Tcr Classes Day 2. Purification of Tcs and Tcr Clones Day 3. Isolation of Plasmid DNA by the Alkaline-Detergent Method and Determination of Recovery by Agarose Gel Electrophoresis Day 4. Restriction Mapping of the Tn5 Inserts Using PstI and EcoRI Day 5. Agarose Gel of Plasmid DNA Restriction Endonuclease DigestionsLab VI. DNA Cloning in M13 Day 1. Isolation of Restriction Fragment from an Agarose Gel Day 2. Estimation of Recovery of Restriction Fragment and Isolation of M13mp 19 RF DNA Day 3. Eco RI Digestion of M13mp19 RF DNA and Treatment with Alkaline Phosphatase Day 4. Removing the Phosphatase and Analysis of the Eco RI Digest of M13mp 19 RF DNA Day 5. Ligation Ligation of Eco RI Digested M13mp19 RF DNA and the Purified pRSG 192 Eco RI Fragment Day 6. Transfection of XL1-Blue with Ligation Mixtures Day 7. Plaque Purification Day 8. Isolation of Colorless Plaques that Contain the chb Gene and Growth of Recombinant BacteriophageLab VII. DNA Sequencing Day 1. Isolation of Recombinant M13mpl9 RF and ss DNA Day 2. Restriction Digestion and Gel Electrophoresis of Recombinant Phage to Determine Orientation of Insert Estimating the Recovery of ss DNA Using Agarose Gel Electrophoresis Day 3. Staining Gel of Pstl Restriction Fragments Template Annealing and Dideoxy Sequencing Day 4. Electrophoretic Separation of Sequencing Reactions Day 5. Developing Autoradiogram and Reading DNA SequenceLab VIII. DNA Gel Blotting, Probe Preparation, Hybridization, and Hybrid Detection Day 0. Agarose Gel Electrophoresis Day 1. Gel Blotting Day 2. Baking the Blot, Nick Translation, and Biotinylation of DNA Day 3. Hybridization Day 4. Washing the Blot Day 5. Developing the Film and Developing the BlotLab IX. Lambda Phage Manipulations A. Phage Plating and Plaque Transfer Day -1 . Preparation of Cells Day 0. Overnight Culture Day 1. Plating the Phage Day 2. Plaque Transfer B. Bacteriophage Lambda Miniprep Day 1. Plating the Phage Day 2. Making a Plate Lysate Day 3. Phage Isolation Day 4. Phage DNA Isolation Day 5. Running Gel, Staining, and PhotographyAppendix 1. Use of Computers in a Molecular Biology LaboratoryAppendix 2. Safety in the Recombinant DNA Laboratory Radioisotopes Chemical Hazards Biohazards Culture Media Buffers and Other Reagents Restriction Enzyme BuffersAppendix 3. SolutionsAppendix 4. Strains and DNAAppendix 5. Molecular Biology Reagent SuppliersAppendix 6. Frequently Used EnzymesAppendix 7. Frequently Used Techniques not Described in Labs S1 Nuclease Mapping Primer Extension RNA Isolation from Bacterial Cells RNA Isolation from Animal Cells Electrophoresis of RNA in a Denaturing Formaldehyde-Agarose Gel; RNA Gel Blotting (Northern Blotting) Labeling DNA at the 3′ End by the Fill-In Reaction with Klenow Fragment Labeling DNA at the 5′ End with Polynucleotide Kinase Transduction with P1 Bacteriophage Large-Scale Plasmid Isolation by the Cleared Lysate MethodIndex
- Edition: 1
- Published: January 28, 1989
- No. of pages (eBook): 208
- Imprint: Academic Press
- Language: English
- Paperback ISBN: 9781483207544
- eBook ISBN: 9781483220970
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