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Methods in Molecular Biology and Protein Chemistry. Cloning and Characterization of an Enterotoxin Subunit

  • ID: 2174261
  • Book
  • April 2002
  • 220 Pages
  • John Wiley and Sons Ltd
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Methods in Molecular Biology is a discovery–based guide that will enable students to master current molecular biology and biochemistry techniques as an integrated part of a research project. Written in an accessible style, this book adopts a practical hands–on approach to encourage the reader to explore and experiment independently. The book is designed to support a one semester laboratory course in which students participate in an ongoing research project, give a research presentation on a topic related to the project, and write a research article suitable for publication.

Methods in Molecular Biology features:

∗ a carefully structured sequence of experiments that enables students to develop key laboratory skills whilst contributing towards a live, research project.

∗ a mid–term mock exercise that encourages students to test test their undertanding half way through the book, assess what they have learnt, utilize the primary literature and propose additional research applications and methods.

∗ a supplementary Website, that includes:

guidelines on searching the primary literature using internet and library search tools

corrections,additions, and troubleshooting suggestions

advice on; how to prepare laboratory notebooks, present oral reports, record keeping and report writing

suggestions for future projects based on modified text protocols

This book will be invaluable for undergraduate students of biology, biochemistry microbiology and biomedical sciences. It will also be useful for those students taking chemistry, chemical engineering and animal and plant science courses looking to improve their practical experience in the lab.
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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
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Brenda D. Spangler
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