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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.
List of contents
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 Concentration
PCR Amplification of LTB Gene from Plasmid DNA
Experiment 4 Agarose Gel Eletrophoresis
Recovery of PCR Product
Experiment 5 Restriction Digest of LTB Insert
Plate Preparation
Experiment 6 Purification of Digested Insert and Vector by Agarose Gel Eletrophoresis
Recovery of Digested Insert and Vector
Experiment 7 Determination of DNA Concentrations
Ligation of Insert into Vector
Experiment 8 Transformation of Host Cells by Construct
Experiment 9 Colony-pick PCR to Check for Presence of Insert
Streak Plates with Potential Constructs
Inoculate Cultures/Run PCR
Experiment 10 Agarose Gel Electrophoresis to Verify Presence of Insert
Purification of the Plasmid Construct using a Plasmid Miniprep Kit
Experiment 11 DNA Concentration Determination
Ethanol Precipitation of Plasmid DNA
Sequencing Reaction
Experiment 12 Purification of Extension Reaction Products
Sequencing Gel Demonstration
Experiment 13 Analysis of Sequencing Data
Verification of Insert Sequence
Experiment 14 Gene Expression
Transformation into Expression Host
Experiment 15 Induction of LTB Gene Expression
Part 2 Protein Chemistry
Experiment 16 SDS-PAGE of Induction Time Course
Transfer 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 Fractions
Immunoblot to verify Presence of LTB
Experiment 21 Protein Concentration
Protein 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
About the author
Brenda Spangler has over 30 years experience in the field of protein chemistry and protein toxins, specializing in ligand-receptior molecular recognition.
