Description

This manual is designed as an intensive introduction to the various tools of molecular biology. It introduces all the basic methods of molecular biology including cloning, PCR, Southern (DNA) blotting, Northern (RNA) blotting, Western blotting, DNA sequencing, oligo-directed mutagenesis, and protein expression.

Key Features

@introbul:Key Features @bul:* Provides well-tested experimental protocols for each technique * Lists the reagents and preparation of each experiment separately * Contains a complete schedule of experiments and the preparation required * Includes study questions at the end of each chapter

Readership

Beginning graduate students in molecular biology, biochemistry, genetics, microbiology, and cell biology. Also researchers and technicians in molecular biology, biotechnology, cell biology, microbiology, and biochemistry.

Table of Contents

Preface. Course Synopsis: Introduction. Safety Precautions. Daily Schedule. Acknowledgements. Exercises: I. DNA Preparation, Polymerase Chain Reaction, and Molecular Cloning: Cesium chloride-ethidium bromide density gradient centrifugation. PCR to synthesize virD2 flanked with restriction sites. Restriction digests of plasmid pGEX2 and PCR products. Purification of DNA fragments from agarose. Ligation of PCR product to pGEX2 vector. Transformation of E. coli with the ligated plasmid. Small-scale preparation of plasmid DNA by the alkaline lysis method. Restriction analysis. Study questions. Protein Expression, Purification, and Analysis: Expression and purification of a fusion protein. SDS-polyacrylamide gel electrophoresis. Silver stain detection of proteins. Study questions. Oligonucleotide-Directed Mutagenesis: Restriction digests of virD2 (in pCS64) and pUC119. Purification of DNA fragments from agarose. Ligatation of restriction fragment and vector. Transformation of E. coli with the ligated plasmid and recovery clones. Small-scale preparation of plasmid DNA from broth cultures. Restriction digest of DNA: examination to confirm insert. Preparation of single-stranded DNA template. Phosphorylation of oligonucleotide. Annealing mutant oligonuleotide to template. In vitro DNA synthesis by primer extension. Transform synthesis reaction into E. coli DH5a. Small-scale preparation of plasmid DNA. Confirmation of mutants by restriction analys

Details

No. of pages:
234
Language:
English
Copyright:
© 1999
Published:
Imprint:
Academic Press
Print ISBN:
9780125839907
Electronic ISBN:
9780080536828