Recombinant DNA Laboratory Manual - 1st Edition - ISBN: 9780127844008, 9781483220970

Recombinant DNA Laboratory Manual

1st Edition

Authors: Judith W. Zyskind Sanford I. Bernstein
eBook ISBN: 9781483220970
Imprint: Academic Press
Published Date: 28th January 1989
Page Count: 208
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Description

Recombinant DNA Laboratory Manual is a laboratory manual on the fundamentals of recombinant DNA techniques such as gel electrophoresis, in vivo mutagenesis, restriction mapping, and DNA sequencing. Procedures that are useful for studying either prokaryotes or eukaryotes are discussed, and experiments are included to teach the fundamentals of recombinant DNA technology. Hands-on computer sessions are also included to teach students how to enter and manipulate sequence information.

Comprised of nine chapters, this book begins with an introduction to bacterial growth parameters, how to measure bacterial cell growth, and how to plot cell growth data. The discussion then turns to the isolation and analysis of chromosomal DNA in bacteria and Drosophila; plasmid DNA isolation and agarose gel analysis; and introduction of DNA into cells. Subsequent chapters deal with Tn5 mutagenesis of pBR329; DNA cloning in M13; DNA sequencing; and DNA gel blotting, probe preparation, hybridization, and hybrid detection. The book concludes with an analysis of lambda phage manipulations.

This manual is intended for advanced undergraduate or beginning graduate students and should also be helpful to established investigators who are changing their research focus.

Table of Contents


Preface

Schedule of Laboratory Exercises

Lab I. Bacterial Growth Parameters

Day 1. Measuring Bacterial Cell Growth

Day 2. Plotting Cell Growth Data

Addendum

Lab II. Isolation and Analysis of Bacterial and Drosophila Chromosomal DNA

Day 1. Isolation and Purification of E. Coli Chromosomal DNA

Isolation and Purification of Drosophila DNA

Day 2. Determination of the Concentration and Purity of DNA by UV Spectroscopy

Day 3. Restriction Endonuclease Digestion of Chromosomal DNA

Day 4. Agarose Gel of Chromosomal DNA Restriction Endonuclease Digestions

Day 5. Staining and Photography of Agarose Gel of Chromosomal DNA Restriction Endonuclease Digestions

Lab III. Plasmid DNA Isolation and Agarose Gel Analysis

Day 1. Isolation of Plasmid DNA by the Alkaline-Detergent Method—A Miniprep Procedure

Day 2. Agarose Gel Electrophoresis of Undigested Plasmid DNA

Lab IV. Introduction of DNA into Cells

Day 1. Production of Frozen Competent Cells

Day 2. Transformation of LE392 with pBR329 DNA Isolated from HB101::Tn5

Lab V. Tn5 Mutagenesis of pBR329

Day 1. Marker Screening: Divide Transformants into Tcs and Tcr Classes

Day 2. Purification of Tcs and Tcr Clones

Day 3. Isolation of Plasmid DNA by the Alkaline-Detergent Method and Determination of Recovery by Agarose Gel Electrophoresis

Day 4. Restriction Mapping of the Tn5 Inserts Using PstI and EcoRI

Day 5. Agarose Gel of Plasmid DNA Restriction Endonuclease Digestions

Lab VI. DNA Cloning in M13

Day 1. Isolation of Restriction Fragment from an Agarose Gel

Day 2. Estimation of Recovery of Restriction Fragment and Isolation of M13mp 19 RF DNA

Day 3. Eco RI Digestion of M13mp19 RF DNA and Treatment with Alkaline Phosphatase

Day 4. Removing the Phosphatase and Analysis of the Eco RI Digest of M13mp 19 RF DNA

Day 5. Ligation

Ligation of Eco RI Digested M13mp19 RF DNA and the Purified pRSG 192 Eco RI Fragment

Day 6. Transfection of XL1-Blue with Ligation Mixtures

Day 7. Plaque Purification

Day 8. Isolation of Colorless Plaques that Contain the chb Gene and Growth of Recombinant Bacteriophage

Lab VII. DNA Sequencing

Day 1. Isolation of Recombinant M13mpl9 RF and ss DNA

Day 2. Restriction Digestion and Gel Electrophoresis of Recombinant Phage to Determine Orientation of Insert

Estimating the Recovery of ss DNA Using Agarose Gel Electrophoresis

Day 3. Staining Gel of Pstl Restriction Fragments

Template Annealing and Dideoxy Sequencing

Day 4. Electrophoretic Separation of Sequencing Reactions

Day 5. Developing Autoradiogram and Reading DNA Sequence

Lab VIII. DNA Gel Blotting, Probe Preparation, Hybridization, and Hybrid Detection

Day 0. Agarose Gel Electrophoresis

Day 1. Gel Blotting

Day 2. Baking the Blot, Nick Translation, and Biotinylation of DNA

Day 3. Hybridization

Day 4. Washing the Blot

Day 5. Developing the Film and Developing the Blot

Lab IX. Lambda Phage Manipulations

A. Phage Plating and Plaque Transfer

Day -1 . Preparation of Cells

Day 0. Overnight Culture

Day 1. Plating the Phage

Day 2. Plaque Transfer

B. Bacteriophage Lambda Miniprep

Day 1. Plating the Phage

Day 2. Making a Plate Lysate

Day 3. Phage Isolation

Day 4. Phage DNA Isolation

Day 5. Running Gel, Staining, and Photography

Appendix 1. Use of Computers in a Molecular Biology Laboratory

Appendix 2. Safety in the Recombinant DNA Laboratory

Radioisotopes

Chemical Hazards

Biohazards

Culture Media

Buffers and Other Reagents

Restriction Enzyme Buffers

Appendix 3. Solutions

Appendix 4. Strains and DNA

Appendix 5. Molecular Biology Reagent Suppliers

Appendix 6. Frequently Used Enzymes

Appendix 7. Frequently Used Techniques not Described in Labs

S1 Nuclease Mapping

Primer Extension

RNA Isolation from Bacterial Cells

RNA Isolation from Animal Cells

Electrophoresis of RNA in a Denaturing Formaldehyde-Agarose Gel; RNA Gel Blotting (Northern Blotting)

Labeling DNA at the 3′ End by the Fill-In Reaction with Klenow Fragment

Labeling DNA at the 5′ End with Polynucleotide Kinase

Transduction with P1 Bacteriophage

Large-Scale Plasmid Isolation by the Cleared Lysate Method

Index

Details

No. of pages:
208
Language:
English
Copyright:
© Academic Press 1989
Published:
Imprint:
Academic Press
eBook ISBN:
9781483220970

About the Author

Judith W. Zyskind

Sanford I. Bernstein