Biotechnology

A Laboratory Course

1st Edition - March 28, 1990
Author: Jeffery M. Becker
Language: English
eBook ISBN:
9 7 8 - 0 - 3 2 3 - 1 5 9 1 3 - 5

Biotechnology: A Laboratory Course is a series of laboratory exercises demonstrating the in-depth experience and understanding of selected methods, techniques, and instrumentation… Read more

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Biotechnology: A Laboratory Course is a series of laboratory exercises demonstrating the in-depth experience and understanding of selected methods, techniques, and instrumentation used in biotechnology. This manual is an outgrowth of an introductory laboratory course for senior undergraduate and first year graduate students in the biological sciences at The University of Tennessee. This book is composed of 19 chapters and begins with some introductory notes on record keeping and safety rules. The first exercises include pH measurement, the use of micropipettors and spectrophotometers, the concept of aseptic technique, and preparation of culture media. The subsequent exercises involve the application of the growth curve, the isolation, purification, and concentration of plasmid DNA from Escherichia coli, and the process of agarose gel electrophoresis. Other exercises include the preparation, purification, and hybridization of probe, the transformation of Saccharomyces cerevisiae, the transformation of E. coli by plasmid DNA, and the principles and applications of protein assays. The final exercises explore the ?-galactosidase assay and the purification and determination of ?-galactosidase in permeabilized yeast cells. This book is of great value to undergraduate biotechnology and molecular biology students.

Preface

Acknowledgments

Suggested Schedule for Exercises

Introductory Notes

Record Keeping and Safety Rules

Format of Student Laboratory Records

Ten Dos and Don'ts of Record Keeping

Criteria for Grading the Laboratory Notebook

Safety Rules in the Laboratory

Exercise 1

Measurement of pH

Exercise 2

Use of Micropipettors and Spectrophotometers

Exercise 3

Aseptic Technique: Transferring a Culture

Exercise 4

Establishing a Pure Culture: The Streak Plate

Exercise 5

Preparation of Culture Media

Exercise 6

The Growth Curve

Exercise 7

Isolation of Plasmid DNA from Escherichia Coli: The Mini-Prep

Exercise 8

Purification, Concentration, and Quantitation of DNA

Exercise 9

Isolation of Plasmid DNA: The Maxi-Prep

Exercise 10

Restriction Digestion and Agarose Gel Electrophoresis

Exercise 11

Southern Transfer

Exercise 12

Preparation, Purification, and Hybridization of Probe

Exercise 13

Transformation of Saccharomyces cerevisiae

Exercise 14

Transformation of Escherichia coli by Plasmid DNA

Exercise 15

Protein Assays

Exercise 16

β-Galactosidase Assay

Exercise 17

Determination of β-Galactosidase in Permeabilized Yeast Cells

Exercise 18

Assay of β-Galactosidase in Cell Extracts

Exercise 19

β-Galactosidase Purification

Part A Gel Filtration Chromatography: Column Calibration

Part Β Ammonium Sulfate Salting-Out of β-Galactosidase and Column Chromatography

Part C Sodium Dodecyl Sulfate—Polyacrylamide Gel Electrophoresis

Appendix 1

Alternative Protocols and Experiments

Exercise 6A Isolation and Characterization of Auxotrophic Yeast Mutants

Exercise 9A Large-Scale Isolation of Plasmid DNA by Column Chromatography

Exercise 12A Colony Hybridization

Appendix 2

Buffer Solutions

Appendix 3

Preparation of Buffers and Solutions

Appendix 4

Properties of Some Common Concentrated Acids and Bases

Appendix 5

Use of Micropipettors

Appendix 6

Safe Handling of Microorganisms

Appendix 7

List of Cultures

Appendix 8

Storage of Cultures

Appendix 9

Sterilization Methods

Appendix 10

Preparation of Stock Solutions for Culture Media

Appendix 11

Growth in Liquid Medium

Appendix 12

Determination of Viable Cells

Appendix 13

Determination of Cell Mass

Appendix 14

Determination of Cell Number

Appendix 15

Nomenclature of Strains

Appendix 16

Glassware and Plasticware

Appendix 17

Preparation of Tris and EDTA

Appendix 18

Basic Rules for Handling Enzymes

Appendix 19

Manufacturers' and Distributors' Addresses

Glossary

Index