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The enormous advances made in molecular biology have allowed scientists to manipulate DNA with relative ease. This means that molecular biology has become a widely-used tool for answering scientific questions that may be quite unrelated to genetics and cell biology. This new book focuses on the most important techniques needed by undergraduate and post-graduate students when undertaking research in this area. Written in an accessible style and adopting a 'discovery' approach, to encourage the students to explore and experiment for them selves, this book will be invaluable to novices required and interested in learning these techniques.* Students will evaluate the effect of genetic engineering through the practical application of wet biochemistry techniques and prepare mutations that can be evaluated by other classes.* Coverage will include computer tutorials for sequencing and genomics and will possibly include some coverage of molecular modelling too.* Guidelines on searching the primary literature, oral reports, laboratory notebooks record keeping and report writing will either be included within appendices at the back of the book, or on a supplementary web site linked to the book.
Brenda Spangler has over 30 years experience in the field of protein chemistry and protein toxins, specializing in ligand-receptior molecular recognition.
Preface Introduction and Background Part 1 Molecular Biology Experiment 1 Instructions for Pipetman Use Experiment 2 Preparation of Plasmid Template DNA Experiment 3 Estimation of Plasmid DNA ConcentrationPCR Amplification of LTB Gene from Plasmid DNA Experiment 4 Agarose Gel EletrophoresisRecovery of PCR Product Experiment 5 Restriction Digest of LTB InsertPlate Preparation Experiment 6 Purification of Digested Insert and Vector by Agarose Gel EletrophoresisRecovery of Digested Insert and Vector Experiment 7 Determination of DNA ConcentrationsLigation of Insert into Vector Experiment 8 Transformation of Host Cells by Construct Experiment 9 Colony-pick PCR to Check for Presence of InsertStreak Plates with Potential ConstructsInoculate Cultures/Run PCR Experiment 10 Agarose Gel Electrophoresis to Verify Presence of InsertPurification of the Plasmid Construct using a Plasmid Miniprep Kit Experiment 11 DNA Concentration DeterminationEthanol Precipitation of Plasmid DNASequencing Reaction Experiment 12 Purification of Extension Reaction ProductsSequencing Gel Demonstration Experiment 13 Analysis of Sequencing DataVerification of Insert Sequence Experiment 14 Gene ExpressionTransformation into Expression Host Experiment 15 Induction of LTB Gene Expression Part 2 Protein Chemistry Experiment 16 SDS-PAGE of Induction Time CourseTransfer of Protein to Membrane for Western Blot Experiment 17 Visualization of Western Blot Experiment 18 Large Scale Culture Preparation Experiment 19 Isolation of LTB by Affinity Chromatography Experiment 20 SDS-PAGE of Column FractionsImmunoblot to verify Presence of LTB Experiment 21 Protein ConcentrationProtein Crystallization Experiment 22 Setting up an ELISA Microtiter Plate Experiment 23 ELISA of a Panel of Ganglioside Ligands Experiment 24 Protein Structure Determination: X-ray Diffraction Techniques Experiment 25 Characterization of a Protein Crystal Experiment 26 Primer Design
