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

ISBN-13: 9780470843604 / Angielski / Miękka / 2002 / 220 str.

Brenda D. Spangler

Opis

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.

Spis treści

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

Blurb

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.