Cloning, Gene Expression, and Protein Purification Experimental Procedures and Process Rationale
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The central purpose of this combination lecture/laboratory manual is to present detailed protocols for the multi-step process involved in isolating a gene, cloning and characterizing it, expressing the encoded protein, purifying the protein, and characterizing rudimentary aspects of its basic physical properties. The manual includes 20 experiments designed to train students to prepare, manipulate, and analyse plasmids, to produce fusion proteins in bacteria, and to purify theseproteins based on unique chemical properties or substrate affinities. Electrophoresis, Southern and Western blotting and cominatorial techniques are emphasized. Radioisotopes, fluorophores and solvent effects on protein structure are discussed. (No radioisotopes are required.) The experiments included have been selected because of their high success rate and because they emphasize a project-oriented approach. Sufficient detail and background are provided such that the intended audience includes advanced undergraduate students, beginning graduates, and practising professionals engaged in a broad range of pursuits. Each experiment is accompanied by detailed process rationale. Where appropriate, alternate methods are also discussed. Maximizing its utility for both studetns and instructors, this unique manual precedes each lab experiment with the necessary background theory and principles, which culminate in a detailed process rationale and, ultimately, the experimental techniques themselves. While many lab manuals list background references, this innovative book combines both theory and procedure into one manageable volume. In addition to this theoretical background material, new innovations and insights are providedalong with carefully selected primary source research literature.
List price: $133.95
Copyright year: 2001
Publisher: Oxford University Press, Incorporated
Publication date: 3/1/2001
Size: 8.25" wide x 10.75" long x 0.75" tall
Introductory UnitIntroductory Lecture - Introduction to the Biochemical LaboratoryTheory: Course DescriptionTheory: "Central Dogma of Molecular Biology"Theory: Laboratory SafetyTheory: The Scientific Method: Surviving Recipe MentalityTheory: Proactive TroubleshootingTheory: Introduction to the Biotechnology LaboratoryTheory: Error Analysis and Assay SensitivityTheory: Treatment of Analytical DataTheory: Concentration and Temperature Effects on pKaIntroductory Lab 1 - Basic Biochemical Techniques I: Pipet Calibration and Solution PreparationProcess: PipetsIntroductory Lab 2 - Basic Techniques II: Absorbance Spectroscopy and Protein Concentration DeterminationsProcess: AMP and Tryptophan Absorbance Spectra; Sample CalculationsTheory: Absorption Data for the Nucleoside MonophosphatesProcess: Absorption Spectra Data for the Aromatic Amino Acids at pH 6; UV Absorption Characteristics of the Aromatic Amino Acids. Selected Extinction CoefficientsProcess: BCA Assay Sample DataInnov.: Measurement of Protein in 20 SecondsPart I - Nucleic Acids & CloningUnit 1Lecture 1 - DNA IsolationTheory: Subcloning ProcedureInnov.: The pET Bacterial Plasmid System (Novagen)Lab 1.1 - Media Preparation; Bacterial Growths; Plasmid Minipreps; HindIII Digestion of DNA, Commercial Bacteriophage h DNA BstEII Digest Size StandardsProcess: pUR278 and p2D Restriction MapsProcess: Cloning the myo-3 Gene from C. elegans and Construction of an Expression VectorProcess: C. elegans myo-3 Gene in pUR288Vend. Lit.: Restriction Enzymes HindIII and BstEII; h DNA DigestsProcess: Phage h BstEII DigestLab 1.2 - Agarose Gel ElectrophoresisExercises: Restriction MappingUnit 2Lecuture 2 - Construction of Recombinant PlasmidsInnov.: Protecting and Manipulating Large DNA SubstratesInnov.: Yeast of Burden--Yoking the YACLab 2.1 - Extraction and Cleanup of DNA Bands Cut from Agrose Gels, Quantitation of Yields and Ligation of myo-3 HindIII DNA Insert Fragment into Linearized B-gal Plasmid DNAVend. Lit.: Gibco BRL (TM) T4 DNA LigaseVend. Lit.: DNA Purification Kit (NaI/Glass Bead Method)Alt. App.: The Use of B-Agarase to Recover DNA from Gel SlicesAlt. App.: GELase TMUnit 3Lecture 3 - The Polymerase Chain ReactionInnov.: Polumerase Chain Reaction Used for Antigen Detection; Immuno-PCR: Very Sensitive Antigen Detection by Means of Specific Antibody-DNA ConjugatesLab 3.1 - Polymerase Chain Reaction Test for myo-3 Gene Insert OrientationUnit 4Lecture 4 - Transcription of Genomic DNA & Analysis of the Resulting mRNAsAlt. App.: Isolation of Total RNA from E. coli CellsAlt. App.: Promega TM PolyATractTM System 1000Alt. App.: Electrophoresis and Northern Blotting of RNAUnit 5Lecture 5 - Transformation and Gene ExpressionInnov.: How Cells Respond to StressLab 5.1 - Preparation of Fresh Transformation - Competent CellsAlt. App.: Ultracomp TM Transformation KitLab 5.2 - Colony Immunoblotting to Screen for TransformantsAlt. App.: The QIAexpressionist, QIAGENTMUnit 6Lecture 6 - Analysis of DNA or RNA by Duplex Hybridization: DNA Isolation, Labeling, and ProbingInnov.: Reduction of Background Problems in Nonradioactive Northern and Southern Blot Analyses Enables Higher Sensitivity than 32P-Based HybridizationLab 6.1 - Labeling of DNA and Probe Construction from Cloned C. elegans myo-3 Gene; Quantitation of DNA ConcentrationVend. Lit.: Digoxigenin Labeling of DNA: Genius TM Nucleic Acid Labeling SystemLab 6.2 - Isolation of C. elegans Genomic DNA, Quantitation of DNA Concentration, and Digestion to Extract the myo-3 GeneLab 6.3 - Southern BlottingPart 2 - Protein PurificationUnit 7Lecture 7 - Protein PurificationTheory: Preparation and Handling of Biological Macromolecules for CrystallizationTheory: Solution STructure of Biomacromolecules in Ionic SolutionsTheory: Solubility as a Function of Protein Structure and Solvent ComponentsTheory: Dominant Forces in Protein FoldingAlt. App.: Hydrophobic Interaction ChromatographyAlt. App.: Centriprep Microconcentrators for Small Volume Concentrations; Centricon-3 and 100Lab 7.1 - The Protein Purifier: A Learning Aid from PharmaciaLab 7.2 - Induction and Purification of B-Galactosidase Fusion Protein from BacteriaLab 7.3 - Gel Filtration of Molecular Weight Standards and Protein FractionationProcess: Gel Filtration and ChromatographyVend. Lit.: Sephadex and SephacrylVend. Lit.: Sigma TM Gel Filtration Molecular Weight MarkersLab 7.4 - Mciroplate B-Galactosidase Assay to Determine Fractions Containing Fusion Protein; MW DeterminationProcess: Time Course Assay of B-GalactosidaseVend. Lit.: B-Galactosidase SubstratesInnov.: Luminescent Reporter Gene Assays for Luciferase and B-Galactosidase Using a Liquid Scintillation CounterLab 7.5 - Ion Exchange Column ChromatographyProcess: Ion Exchange ChromatographyTheory: The Isoelectric Point: Protein Charge Neutrality at a Particular pHAlt. App.: Ion-Pair ChromatographyAlt. App.: HPLC: Ion Exchange and Reverse Phase Methods; Literature SourcesLab 7.6 - Affinity Chromatography and Microplate B-Galactosidase Assays to Determine Fractions Containing Fusion ProteinProcess: Affinity ChromatographyProcess: Affinity Chromatography: One Step Purification of Hybrid Proteins Carrying Fused B-Galactosidase ActivityLab 7.7 - BCA Protein Concentration Assays and B-Galactosidase Assays to Construct an Enzyme Purification TableUnit 8Lecture 8 - Discontinuous Gel Electrophoresis, Protein Mobilities and Apparent Size DeterminationProcess: Discontinuous Gel Electrophoresis & Protein Size DeterminationLab 8.1 - Discontinuous SDS Gel ElectrophoresisUnit 9Lecture 9 - Immunochemical TechniquesInnov.: Immunochemical TechniquesInnov.: The Enzyme Linked Immunosorbent Assay (ELISA)Innov.: How the Immune System Learns About SelfInnov.: Making Monoclonal Antibodies That Won''t Fight BackLab 9.1 - Western BlottingProcess: ImmunoblottingProcess: Western Blots Using Stained Protein GelsUnit 10Lecture 10 - Combinatorial Biochemical TechnologyInnov.: Examples of Combinatorial TechniquesInnov.: Making Antibody Fragments Using Phage Display LibrariesInnov.: Building a Better EnzymeInnov.: The ImmunoZAP Cloning and Expression SystemAppendicesPart 1 Terms ListPart 2 Terms ListLaboratory ReagentsAbbreviations ListCopyright AcknoledgementsSuggested ScheduleSupplies RequiredIndex(Abbreviations:Innov., Innovation/InsightTheory, Theory/PrinciplesProcess, Process RationaleVend. Lit., Vendor LiteratureAlt. App., Alternative Approach