Molecular Biology Techniques An Intensive Laboratory Course

ISBN-10: 0125839901
ISBN-13: 9780125839907
Edition: 1999
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Book details

List price: $89.95
Copyright year: 1999
Publisher: Elsevier Science & Technology Books
Publication date: 11/12/1998
Binding: Paperback
Pages: 234
Size: 6.50" wide x 9.50" long x 0.75" tall
Weight: 0.946
Language: English

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 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 from Agrobacterium 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

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