Molecular Biology Techniques: An Intensive Laboratory Course

Paperback | May 5, 1993

byWalt Ream, Katharine G. FieldEditorWalt Ream

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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* 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

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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* Provides well-...

From the Jacket

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* Provides we...

Walt Ream, Professor of Microbiology, Oregon State University, received the Richard M. Bressler Senior Faculty Teaching Award for developing the course this book supports. In addition to this work, he has written numerous peer reviewed scholarly papers, review articles, book articles and chapters and has co-authored another book entitl...
Format:PaperbackDimensions:234 pages, 9.25 × 6.5 × 0.68 inPublished:May 5, 1993Publisher:Academic PressLanguage:English

The following ISBNs are associated with this title:

ISBN - 10:0125839901

ISBN - 13:9780125839907

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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 synthesizevirD2flanked 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 ofE. coliwith 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 ofvirD2(in pCS64) and pUC119.
Purification of DNA fragments from agarose.
Ligatation of restriction fragment and vector.
Transformation ofE. coliwith 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 vitroDNA synthesis by primer extension.
Transform synthesis reaction intoE. coliDH5a. Small-scale preparation of plasmid DNA.
Confirmation of mutants by restriction analysis.
Study questions.
DNA Sequencing:
Polyacrylamide sequencing gel electrophoresis.
Didexy sequencing.
Automated Sequencing.
Introduction to databases and gene sequence analysis.
Study questions.
Southern Blot Detection of DNA:
Preparation of genomic DNA fromAgrobacterium tumefaciens.
Restriction digestion of genomic DNA.
Agarose gel electrophoresis of restriction fragments.
Southern blot: denaturation and blotting of DNA gel.
Preparation of probe nick translation.
Hybridization and washing of Southern blots.
Study questions.
Northern Blot Detection of mRNA:
Preparation of RNA from tobacco leaves.
Agarose-formaldehyde gel electrophoresis.
Northern blot: denaturation and blotting of RNA.
Probe preparation. Hybridization and washing of Northern blots.
Study questions.
Protein Interaction Analysis in Yeast:
Yeast transformation.
Filter b-galactosidase assay.
Index.