CURRENT
`
`PROTOCOLS
`IN MOLECULAR
`
`BIOLOGY
`
`VOLUME 1
`
`EDITORIAL BOARD
`
`Frederick M. Ausubel
`
`Massachusetts General Hospital & Harvard Medical School
`
`Roger Brent
`Massachusetts General Hospital & Harvard Medical School
`
`Robert E. Kingston
`Massachusetts General Hospital & Harvard Medical School
`
`David D. Moore
`
`Massachusetts General Hospital & Harvard Medical School
`
`J Seidman
`Harvard Medical School
`
`John A. Smith
`University of Alabama
`
`Kevén Struhl
`Harvard Medical School
`
`GUEST EDITORS
`
`Lisa M. Albright
`DNA Sequencing
`
`Donalé M. Coen
`Harvard Medical School
`
`Polymerase Chain Reaction
`
`Ajit Varki
`University of California San Diego
`Glycoproteins
`
`SERIES EDITOR
`
`Virginia Benson Chanda
`
`E?)
`
`John Wiley & Sons, Inc.
`
`CORE 13 (S36)
`
`BEQ 1016
`Page 1
`
`

`
`Copyright © 1994-1997 by John Wiley & Sons, Inc.
`
`Copyright © 1987-1994 by Current Protocols
`
`All rights reserved. Published simultaneously in Canada.
`
`Reproduction or translation of any part of this work beyond that permitted by Section
`107 or 108 of the 1976 United States Copyright Act without the permission of the
`copyright owner is unlawful. Requests for permission or father infonnalion should
`be addressed to the Permissions Department, John Wiley & Sons, Inc.
`
`While the authors, editors, and publisher believe that the specifscatéon and usage of
`reagents, equipment, and dewréces, as set forth in this book, are in accord with current
`recommendations and practice at the time of publication, they accept no legal
`responsibility for any errors or omissions, and make no warranty, express or implied,
`with respect to material contained herein. In view of ongoing research, equipment
`modifications, changes in governmental regulations, and the constant flow of
`information relating to the use of experimental reagents, equipment, and devices, the
`reader is urged to review and evaluate the information provided in the package insert
`or instructions for each chemical, piece of equipment, reagent, or device for, among
`other things, any changes in the instructions or indication of usage and for added
`warnings and precautions. This is particularly important in regard to new or
`infrequently employed chemicals or experimental reagents.
`
`Library of Congress Cataloging in Publication Data:
`
`Current protocols in molecular biology. 3 Vols.
`1. Molecular biology—Technique. 2. Molecular biology—Laboratory
`manuals. I. Ausubel, Frederick M.
`
`QH506.C87 1987
`ISBN 0-471 -50338-X
`
`574.8’8’028
`
`87-21033
`
`Printed in the United States of America
`
`20 19 18 17 16 15 14 13
`
`BEQ 1016
`Page 2
`
`

`
`CURRENT
`PROTOCOLS
`IN MOLECULAR
`BIOLOGY
`
`SUPPLEMENT 38
`
`A brief listing of new topics in Supplement 38 is provided below.
`Please see next page for a full listing of contents and instructions for
`inserting pages.
`
`Overview of Peptide and Protein Analysis by
`Mass Spectrometry
`
`UNIT 10.21
`
`Overview of Baculovirus Expression System
`
`16.9
`
`Maintenance of Insect Cell Cultures and
`
`Generation of Recombinant Baculoviruses
`
`UNIT 16.10
`
`Expression and Purification of Recombinant
`Proteins Using the Baculovirus System
`
`16.11
`
`Inducible Gene Expression Using an
`Autoregulatory, Tetracyline-Controlled System
`
`UNIT 16.21
`
`Also included are a supplement index, The Red Book Bulletin, and a
`pink Reader Response Card.
`
`BEQ 1016
`Page 3
`
`

`
`FULL CONTENTS OF SUPPLEMENT 3 C
`
`Instructions for Adding Pages to Core Manual
`
`Full contents for Supplement 38 are listed below, with instructions for removing old pages and insert-
`ing new pages. Every page included with Supplement 38 is labeled as such beneath the page number.
`
`Remove pages
`
`Insert pages
`
`Subject
`
`Primary change/notes
`
`VOLUME 1
`
`vii-x
`
`VOLUME 2
`
`10.0.1-10.0.4
`
`10.0.5- 10.0.23
`
`16.0.1-16.0.4
`
`16.0.5—16.0.6
`
`16.9.1-16.9.6
`
`vii-x
`
`Main table of contents
`
`Chap. 10: 1-5
`10.0.1—10.0.19
`
`10.21.1—10.21.27
`
`Chapter 10 contents
`
`Chapter 10 introduction
`
`Overview of mass spectrometry
`
`Chap. 16: 1-4
`
`Chapter 16 contents
`
`Reflects new UNITS 10.21 &
`16.21, and revised UNITS 16.9,
`16.10, & 16.11
`
`Reflects new UNIT 10.21
`
`Revision
`
`New UNIT 10.21
`
`Reflects revised UNITS 16.9,
`16.10, & 16.11, and newI6.21
`
`16.0.1—16.0.3
`
`16.9.1-16.9.10
`
`Chapter 16 introduction
`Overview of baculovirus
`
`Revision
`
`Revised UNIT 16.9
`
`16.10.1—16.10.8
`
`16.10.1-16.10.17
`
`16.1 l.1—16.13.7
`
`16.1l.1—16.13.7
`
`(32 pages)
`
`VOLUME 3
`
`(25 pages)
`16.2l.1-16.2l.9
`
`expression system
`Maintenance of insect cell cultures
`
`and generation of recombinant
`baculoviruses
`
`Purification of proteins using
`baculovirus
`
`Revised UNIT 16.10
`
`Revised UNH‘ 16.11
`
`Inducible gene expression
`
`New UNIT 16.21
`
`A.1B.1-A.lC.1
`
`A.1B.1-A.1C.1
`
`Useful measurements and data
`
`Revision
`
`(2 pages)
`A.1C.8—A.1C.1O
`
`(2 pages)
`A.1C.8-A.1C.l2
`
`Supp. 37 index
`£2 Pages)
`
`Supp. 37 & 38 index
`(3 pages)
`
`Characteiéstics of amino acids
`
`New tables in APPENDIX 1C
`
`New entries covering
`Supplements 37 & 38
`
`Supplement 38
`
`Current Protocols in Molecular Biology
`BEQ 1016
`Page 4
`
`

`
`Corrections
`
`The following errors have been detected since publication of the last supplement. NOTE: These changes are
`primarily scientific, not typographical, errors; thus it is crucial that they be entered in your manual for optimal
`use of the protocols.
`
`Page
`
`Error
`
`Correction
`
`10.18.4, Additional materials,
`line 2
`
`“150—mm2 flask”
`
`“150-cmz flask”
`
`11.2.19, Reagents and solutions,
`MUP and NPP substrate solutéons,
`line 2
`
`“NaCO3”
`
`14.5.4, Reagents and solutions
`
`“Gelvatol”
`
`“Na2CO3”
`
`“Gelvatol (now called Airvol,
`from Air Products and
`Chemicals)”
`
`15.5.13, step 28
`
`“final DNA concentration is 1
`ug/ml”
`
`“final DNA concentration is 1
`ug/id”
`
`15.5.19, Vent DNA polymerase
`mix, line 2
`
`“Add 0.5 pt] (10 U)”
`
`“Add 0.5 LL] (1 U)”
`
`Cumsnt Protocols in Molecular Biology
`
`5.; plemem 29
`B C? 1016
`Page 5
`
`

`
`COIIIEIHS, Volumes 1, 2, and 3
`
`xiii Foreword by Phillip A. Sharp
`
`xv Preface
`
`xix Contributors
`
`1
`
`Eseizériclzia coli, Plasmids, and Bacteriophages
`I ESCHERICHIA COLI
`
`1.1 Media Preparation and Bacteriological Tools
`1.2
`Growth in Liquid Media
`1.3
`Growth on Solid Media
`
`1.4
`
`Selected Topics from Classical Bacteréal Genetics
`
`II VECTORS DERIVED FROM PLASMIDS
`
`1.5
`
`Introduction to Plasmid Biology
`
`1.6 Minipreps of Plasmid ENA
`
`1.7
`1.8
`
`Large-Scale Preparation of Plasmid DNA
`Introduction of Plasmid DNA into Cells
`
`III VECTORS DERIVED FROM LANIBDA AND RELATED
`BACTERIOPHAGES
`
`1.9
`1.10
`
`1.11
`
`Introduction to Lambda Phages
`Lambda as a Cloning Vector
`
`Plating Lambda Phage to Generate Plaques
`
`1.12 Growing Lambda-Derived Vectors
`
`1.13
`
`Preparing Lambda DNA from Phage Lysates
`
`IV VECTORS DERIVED FROM FILAMENTOUS PHAGES
`
`1.14
`
`1.15
`
`Introduction to Vectors Derived from Filamentous Phages
`
`Preparing and Using M13-Derived Vectors
`
`2 Preparation and Analysis of
`
`A
`
`I PREPARATION OF GENOMZC DNA
`
`2.1
`
`2.2
`
`2.3
`2.4
`
`Purification and Concentration of DNA from Aqueous Solutions
`
`Preparation of Genomic DNA from Mammalian Tissue
`
`Preparation of Genomic DNA from Plant Tissue
`Preparation of Genomic DNA from Bacteria
`
`II RESOLUTION AND RECOVERY OF LARGE DNA FRAGMENTS
`
`2.5A Agarose Gel Electrophoresis
`
`2.5B Pulsed-Field Gel Electrophoresis
`
`2.6
`
`Isolation and Purification of Large DNA Restréction Fragments from
`Agarose Gels
`
`Copyright © 1995 by John Wiley & Sons, Inc.
`
`iii
`
`supplement 30 cpMB
`BEQ 1016
`Page 6
`
`

`
`III RESOLUTION AND RECOVERY OF SMALL ENA FRAGMENTS
`
`2.7
`
`2.8
`
`Nondenaturing Polyacrylamide Gel Electrophoresis
`
`Sieving Agarose Gel Electrophoresis
`
`IV ANALYSIS OF DNA SEQUENCES BY BLOTTING AND HYBRIDIZATION
`
`2.9A Southern Blotting
`
`2.9B Dot and Slot Blotting of DNA
`
`2.10 Hybridizatioze Analysis of DNA Blots
`
`V SYNTHESIS AND PURIFICATION OF OLIGONUCLEOTIDES
`
`2.11
`
`2.12
`
`Synthesis of Oligonucleotides
`
`Purification of Oligonucleotides Using Iienaturing Polyacrylamide
`Gel Electrophoresis
`
`VI CHROMATOGRAPHY OF NUCLEIC ACIDS
`
`2.33
`
`2.14
`
`Separation of Double- and Single-Stranded Nucleic Acids Using
`Hydroxylapatite Chromatography
`Purification of DNA by Anion-Exchange Chromatography
`
`W. Enzymatic Manipulation of
`
`and RNA
`
`I RESTRICTION ENDONUCLEASES
`
`3.1
`
`Digestion of DNA with Restriction Endonucleases
`
`II RESTRICTION MAPFING
`
`3.2 Mapping by Multiple Endonuclease Digestions
`
`3.3 Mapping by Partial Endonuclease Digestions
`
`III ENZYMES FOR MODIFYING AND RADIOACTIVELY LABELING
`NUCLEIC ACIDS
`
`3.4
`
`3.5
`
`3.6
`
`3.’?
`3.8
`
`3.9
`
`3.10
`3.11
`
`3.12
`3.13
`
`Reagents and Radioisotopes Used to Manipulate Nucleic Acids
`
`DNA-Dependent DNA Polymerases
`
`Template-Independent DNA Polymerases
`
`RNA-Dependent DNA Polymerases
`DNA-Depenéent RNA Polymerases
`
`DNA-Independent RNA Polymerases
`
`Phosphatases and Kinases
`Exonucleases
`
`Endonucleases
`Ribonucleases
`
`3.14 DNA Ligases
`
`3.15
`
`RNA Ligases
`
`IV CONSTRUCTION OF HYBRID DNA MOLECULES
`
`3.16
`
`3.17
`
`Subcloning of DNA Fragments
`
`Constructizzg Recombinant DNA Molecules by the Polymerase
`Chain Reaction
`
`V SPECIALIZED APPLICATIONS
`
`Labeling and Colorimetric Detection of Nonisotopic Probes
`3.18
`3.19 Chemiluminescent Detection of Nonisotopic Probes
`
`iv
`
`Supplement 30
`
`Current Protocols in Molecular Biology
`BEQ 1016
`Page 7
`
`

`
`4 Preparatim: am] Analysis of RNA
`
`I PREPARATION OF RNA FROM EUKARYOTIC AND
`PROKARYOTIC CELLS
`
`4.1
`4.2
`
`4.3
`4.4
`4.5
`
`Preparation of Cytoplasmic RNA from Tissue Culture Cells
`Guaraidine Method for Total RNA Preparation
`
`Phenol/SDS Method for Plant RNA Preparation
`Preparation of Bacterial RNA
`Preparation of Poly(A)+ RNA
`
`II ANALYSIS OF RNA STRUCTURE AND SYNTHESIS
`
`4.6
`4.7
`4.8
`
`4.9
`
`S1 Analysis of Messenger RNA Using Single-Stranded DNA Probes
`Ribonuclease Protection Assay
`Primer Extension
`
`Analysis of RNA by Northern and Slot Blot Hybridization
`
`4.10
`
`Identification of Newly Transcribed RNA
`
`5 Construetion at‘ Recombinant DNA Libraries
`
`I OVERVIEW OF RECOMBINANT DNA LIBRARIES
`
`5.1
`
`5.2
`
`Genomic DNA Libraries
`
`cDNA Libraries
`
`I1 PREPARATION OF INSERT DNA FROM GENOMIC DNA
`
`5.3
`5.4
`
`Size Fractionation Using Sucrose Gradients
`Size Fractionatién Using Agarose Gels
`
`III PREPARATION OF INSERT DNA FROM MESSENGER RNA
`
`5.5
`
`Conversion of mRNA into Double-Stranded cDNA
`
`5.6 Methylation and Addition of Linkers to Double-Stranded cDNA
`
`IV PRODUCTION OF GENOMIC DNA AND cDNA LIBRARIES
`
`5.7
`
`Production of a Genomic DNA Library
`
`5.8A Production of a Complete cDNA Library
`5.8B Prodzzction of a Subtracted cDNA Library
`
`5.9
`
`PCR-Based Subtractive cDNA Cloning
`
`V AMPLIFICATION OF TRANSFORMED OR PACKAGED LIBRARIES
`
`5.10 Amplification of a Bacteriophage Library
`
`5.11 Amplification of Cosmid and Plasmid Libraries
`
`6
`
`Screening of Recomliinant DNA Libraries
`
`I PLATING LIBRARIES AN9 TRANSFER TO FILTER MEMBRANES
`
`6.1
`6.2
`
`Plating and Transferring Bacteriophage Libraries
`Plating and Transferring Cosmid and Plasmid Libraries
`
`II HSFBRIDIZATION WI’i‘H RADIOACTIVE PROBES
`
`6.3
`
`6.4
`
`Using DNA Fragiiients as Probes
`
`Using Synthetic Oligonucleotides as Probes
`
`Iil PURIFICATION OF BACTERIOPHAGE, COSMID, AND PLASMID CLONES
`
`6.5
`6.6
`
`Purification of Bacteriophage Clones
`Purification of Cosmid and Plasmid Clones
`
`Current Protocols in Molecular Biology
`
`V
`
`Supplement 37
`BEQ 1016
`Page 8
`
`

`
`IV SCREENING WITH ANTIBODIES
`
`6.7
`
`6.8
`6.9
`
`Immunoscreening of Fusion Proteins Produced in Lambda Plaques
`
`Immunoscreening after Hybrid Selection and Translation
`Yeast Artificial Chromosome Libraries
`
`6.10 Analysis of Isolated YAC Clones
`
`SPECIALIZED STRATEGIES FOR SCREENING LFBRARIES
`
`6.11
`
`Use of Monoclonal Antibodies for Expression Cloning
`
`6.12 Recombination-Based Assay (RBA) for Screening Bacteriophage Lambda Libraries
`
`7
`
`A Seqiiencing
`
`7.1
`
`7.2
`
`DNA Sequencing Strategies
`
`Construction of Nested Deletions for DNA Sequencing
`
`Preparation of Templates for DNA Sequencing
`7.3
`’?.4A DNA Sequencing by the Dideoxy Method
`
`7.4B Dideoxy DNA Sequencing with Ciiemiluminescent Detection
`
`7.5
`
`7.6
`
`7.7
`
`DNA Sequencing by the Chemical Method
`
`Denaturing Gel Electrophoresis for Sequencing
`
`Computer Manipulation of DNA and Protein Sequences
`
`8 Mutagenesis of Clrined DNA
`
`8.1
`
`filigonucleotide-Directed Mutagenesis without Phenotypic Selection
`
`8.2A Mutagenesis with Degenerate Oligonucleotides: Creating Numerous Mutations
`in a Small DNA Sequence
`
`8.2B Gene Synthesis: Assembly of Target Sequences Using Mutually Priming
`Long Oligonucleotides
`
`8.3
`
`8.4
`
`8.5
`
`Region-Specific Mutagenesis
`
`Linker-Scanning Mutagenesis of fiNA
`
`Directed Mutagenesis Using the Polymerase Chain Reaction
`
`9
`
`Introduction of DNA into Mammalian Cells
`
`1 TRANSFECTION OF DNA INTO EUKARYOTIC CELLS
`
`9.1
`
`9.2
`9.3
`
`9.4
`9.5
`
`Calcium Phosphate Transfection
`
`Transfection Using DEAE-Dextran
`'Ii'ansfection by Electroporation
`
`Liposome-Mediated Transfecfion
`Stable Transfer of Genes into Mammalian Cells
`
`II USES OF FUSION GENES IN MAMMALIAN TRANSFECTION
`
`9.6
`
`Overview of Genetic Reporter Systems
`
`9.7A Isotopic Assays for Reporter Gene Activity
`
`9.7B Nonisotopic Assays for Reporter Gene Activity
`
`9.7C Use of the A. Victoria Green Fluorescent Protein to Study Protein Dynamics in Vivo
`9.8
`Direct Analysis of RNA After Transfection
`
`vi
`
`Supplement 37
`
`Cmrent Protocols in Molecular Biology
`BEQ 1016
`Page 9
`
`

`
`III TRANSDUCTION OF GENES USING RETROVIRUS VECTORS
`9.9
`
`Overview of the Retrovinis Transduction System
`
`9.10
`9.11
`
`9.12
`
`9.13
`
`9.14
`
`Preparation of a Specific Retrovirus Producer Cell Line
`
`Transient 'I‘ransfection Methods for Preparation of High-Titer
`Retroviral Supernatants
`
`Large-Scale Preparation and Concerztration of Retrovirus Stocks
`
`Detection of Helper Virus Retrovirus Stocks
`Retrovirus Infection of Cells In Vitro and In Vivo
`
`IV GENE TARGETING BY HOMOLOGOUS RECOMBINATION
`
`9.15
`
`9.16
`
`9.17
`
`Overview of Gene Targeting by Homologous Recombination
`
`Production of a Heterozygous Mutant Cell Line by Homologous Recombination
`(Single Knockout)
`
`Production of a Homozygous Mutant Embryonic Stem Cell Line (Double Knockout)
`
`10 Analysis {sf Pffiteins
`
`I QUANTITATION OF PROTEINS
`
`10.1A Spectrophotometric and Colorimetiéc Deterénination of Protein Concentratior;
`10.1B Quantitative Amino Acid Analysis
`
`ELECTROPHORETIC SEPARATION OF PROTEINS
`
`10.2
`
`10.3
`
`10.4
`10.5
`
`One-{Eimensional Gel Electrophoresis of Proteins
`
`Two-Dimensional Gel Electrophoresis Using the ISO-DALT System
`
`Two-Dimensional Gel Electrophoresis Using the O’Farrell System
`Electroelution of Proteins from Stained Gels
`
`III
`
`DETECTION OF PROTEINS
`
`10.6
`10.7
`
`10.8
`
`Staining Proteins in Gels
`Detection of Proteins on Blot Transfer Membranes
`
`Iinmunoblotting and Immunodetection
`
`PURIFICATION OF PROTEINS BY CONVENTIONAL CHROMATOGRAPHY
`
`1f%.9
`
`Gel-Filtration Chromatography
`
`10.10
`
`Ion-Exchange Chromatography
`
`10.11A Immunoaffinity Chromatography
`
`10.11B Metal-Chelate Affinity Chromatography
`
`PURIFICATION OF PROTEINS BY HIGH-PERFORMANCE LIQUID
`CHROMATOGRAPHY
`
`10.12
`
`10.13
`
`Reversed-Phase High-Performance Liquid Chromatography
`
`Ion-Exchange High-Performance Liquid Chromatography
`
`Size-Exclusion High-Ferformance Liquid Chromatography
`10.1-1
`10.15 High-Performance Chromatofocusing and Hydrophobic-Interaction Chromatography
`
`PURIFICATION OF PROTEINS BY PRECIPITATION
`
`10.16
`
`Immunoprecipitation
`
`VII
`
`SPECIALIZED APPLICATIONS
`
`10.17
`
`10.18
`10.19
`
`10.20
`
`10.21
`
`Synthesizing Proteins In Vitro by Transcription and Translation of Cloned Genes
`
`Biosynthetic Labeling of Proteins
`Isolation of Proteins for Microsequence Analysis
`
`Capillary Electrophoresis of Proteins and Feptides
`
`Overview of Peptide and Protein Analysis by Mass Spectrometry
`
`Current Pzotocols in Molecular Biology
`
`cc
`V1]
`
`Supplement 38
`
`BEQ 1016
`Page 10
`
`

`
`11
`
`Iiilmunology
`I
`IMMUNOASSAYS
`11.1
`
`Conjugation of Enzymes Antibodies
`
`11.2
`
`11.3
`
`Enzyme-Linked Immunosorbent Assay (ELISA)
`Isotype Determination of Antéléodies
`
`II PREPARATION OF MONOCLONAL ANTIBODIES
`11.4
`
`Immunization of Mice
`
`11.5
`
`11.6
`
`11.7
`
`11.8
`
`11.9
`11.10
`11.11
`
`Preparation of Myeloma Cells
`
`Preparation of Mouse Feeder Cells for Fusion and Cloning
`Fusion of Myeloma Cells with Immune Spleen Cells
`
`Cloning of Hybridoma Cell Lines by Limiting Dilution
`Freezing and Recovery of Hybridoma Cell Lines
`
`Peoduction of Monoclonal Antibody Supernatant and Ascites Fliiids
`Purification of Monoclonal Antibodies
`
`III PREPARATION OF POLYCLONAL ANTISERA
`11.12
`
`Production of Polyclonal Antisera
`
`11.13
`
`Puréficatéon of Immunoglobulin G Fraction from Antiserum, Ascites Fluid, or
`Hybridoma Superisatants
`
`IV PREPARATION OF ANTIPEPTIDE ANTIBODIES
`
`11.14
`
`11.15
`
`Selection of an Immunogenic Peptide
`
`Production of Antipeptide Antibodies
`
`V DETERMINATION OF SPECIFIC ANTIBODY TITER AND ISOTYPE
`11.16 Determination of the Specific Antibody Titer
`
`12 DNAaProtein Interaetians
`
`12.1
`
`12.2
`
`12.3
`
`12.4
`
`12.5
`
`12.6
`12.7
`
`12.8
`
`12.9
`
`12.10
`
`12.11
`
`Preparation of Nuclear and Cytoplasmic Extracts from Mammalian Cells
`
`Mobility Shift DNA-Binding Assay Using Gel Electrophoresis
`Methylation and Uracil Interference Assays for Analysis of
`Protein-DNA Interactions
`
`DNase I Footprint Analysis of Protein-DNA Binding
`UV Crosslinking of Proteins to Nucleic Acids
`
`Purification of DNA-Binding Proteins Using Biotin/Streptavétfin Affinity Systems
`
`Detection, Purificaéon, and Characterization of cDNA Clones Encoding
`DNA-Bénding Proteins
`
`Rapid Separation of Protein-Bouné DNA from Free DNA Using
`Nitrocellulose Filters
`
`Analysis of DNA-Protein Interactions Using Proteins Synthesized In Vitro
`from Cloned Genes
`
`Purification of Seql:encesSpecif%c DNA-Binding Proteins by
`Affinity Chromatography
`
`Determination of Protein-DNA Sequence Specificity by PCR-Assisted
`Binding-Site Selectéon
`
`viii
`
`Supplement 38
`
`Current Protocols in Molecular Biology
`BEQ 1016
`Page 11
`
`

`
`13
`
`Saccharomyces cerevisiae
`I BASIC TECHNIQUES OF YEAST GENETICS
`
`13.1
`13.2
`13.3
`
`Preparation of Yeast Media
`Growth and Manipulation of Yeast
`Mutagenesis of Yeast Cells
`
`II YEAST VECTORS
`
`13.4
`13.6
`
`Yeast Cloning Vectors and Genes
`Yeast Vectors for Expression of Cloned Genes
`
`III MANIPULATION OF YEAST GENES
`
`13.7
`
`13.8
`
`13.9
`
`Introduction of DNA into Yeast Cells
`
`Cloning Yeast Genes by Complementation
`
`Segregation of Plasmids from Yeast Cells
`
`13.10 Manipulation of Cloned Yeast DNA
`
`IV PREPARATION OF YEAST DNA, RNA, AND PROTEINS
`
`13.11
`13.12
`
`13.13
`
`Preparation of Yeast DNA
`Preparation of Yeast RNA
`
`Preparation sf Frotein Extracts from Yeast
`
`14
`
`In situ Hybridization and Imniumiliistochenfistry
`
`14.1
`14.2
`
`14.3
`14.4
`
`14.5
`
`14.6
`
`14.7
`14.8
`
`14.9
`
`Fixation, Embedding, and Sectioning of Tissues, Embryos, and Single Cells
`Cryosectioning
`
`In situ Hybridization to Cellular RNA
`Detection of Hybridized Probe
`
`Counterstaining and Mounting of Autoradiographed In situ Hybridization Slides
`
`Immunohistochemistry
`
`In situ Hybridization and Detection Using Nonisotopic Probes
`In situ Polymerase Chain Reaction and Hybridization to Detect Low-Abundance
`Nucleic Acid Targets
`Whole-Mount in situ Hybridization and Detection of RNAs in Vertebrate Embryos
`and Isolated Organs
`
`15 The Pelymerase Chain Reaction
`
`15.1
`
`15.2
`
`15.3
`
`15.4
`15.5
`
`15.6
`
`15.7
`15.8
`
`Enzymatic Amplification of DNA by the Polymerase Chain Reaction:
`Standard Procedures and Optimization
`
`Direct DNA Sequencing of Polymerase Chain Reaction Products
`
`Quantitation of Rare DNAs by the Polymerase Chain Reaction
`
`Enzymatic Amplification of RNA by the Polymerase Chain Reaction
`Ligation-Mediated PCR for Genomic Sequencing and Footprinting
`
`cDNA Amplification Using One-Sided (Anchored) PCR
`
`Molecular Cloning of FCR Products
`Differential Display of mRNA by PCR
`
`Current Pratosols in Molecular Biology
`
`ix
`
`Supplement 38
`
`BEQ 1016
`Page 12
`
`

`
`16 Protein Expression
`I EXPRESSION OF PROTEINS El ESCHERICHIA COLI
`
`16.1
`
`16.2
`
`Overview of Protein Expression in E. coli
`
`Expression Using the T7 RNA Polymerase/Promoter System
`
`Expression Using Vectors with Phage A. Regulatory Sequences
`16.3
`16.4A Introduction to Expression by Fusion Protein Vectors
`
`16.4B
`
`Enzymatic and Chemical Cleavage of Fusion Proteins
`
`16.5
`16.6
`16.7
`
`16.8
`
`Expression and Purification of (ml and trpE Fméon Proteim
`Expression and Purification of Maltose-Binding Protein Fusions
`Expression and Purification of Glutathione—S-Tlansfelase Fusion Pmteins
`
`Expression and Purification of Tiiioredoxin Fusion Proteins
`
`II EXPRESSION OF PROTEINS IN INSECT CELLS USING
`BACULOVIRUS VECTORS
`
`Overview of the Baculovirus Expression System
`16.9
`16.10 Maintenance of Insect Cell Cultures and Generation of Recombinant Baculovéruses
`
`16.11
`
`Expression and Purification of Recombinant Proteins Using the Baculovirus System
`
`III EXPRESSION OF PROTEINS IN MAMMALIAN CELLS
`
`16.12
`
`16.13
`
`16.14
`
`Overview of Protein Expression in Mammalian Cells
`
`Transient Expression of Proteins Using COS Cells
`
`Amplification Using CHO Cell Expression Vectors
`
`IV EXPRESSION OF PROTEINS H31 MAMMALIAN CELLS USING VACCINIA
`
`16.15
`
`Overeiiew of the Vaccinia Virus Expression System
`
`Preparation of Cell Cultures and Vaccinia Virus Stocks
`16.16
`16.17 Generation of Recombinant Vaccinia Viruses
`16.18
`Characterization of Recombinant Vaccinia Viruses and Their Products
`
`16.19 Gene Expression Using the VacciniaI'I‘7 RNA Polymerase Hybrid System
`
`16.20
`
`Expression of Proteins Using Semliki Forest Virus Vectors
`
`V SPECIALIZED EXPRESSION SYSTEMS
`
`16.21
`
`Inducible Gene Expression Using an Autoregulatory, Tetracyline-Controlled System
`
`17 Preparation and Analysis of Glycoconjugates
`
`I SPECIAL CONSIDERATIONS OF GLYCOCONJUGATES AND THEIR PURIFICATION
`17.1
`17.2
`
`Special Considerations for Glycoproteins and Their Purification
`
`Special Considerations for Proteoglycans and Giycosaminoglycans
`and T”;1eir Purification
`
`17.3
`
`Specéal Considerations for Glycolipids and Their Purification
`
`II DETECTION OF SACCHARIDES ON GLYCOCONJUGATES
`17.4
`
`Metabrslic Radiolabeling of Animal Cell Glycoconjugates
`Chemical Labeling of Carbohydrates by Oxidation and Sodium Borohydride Reduction
`
`17.5
`
`17.6
`17.7
`
`17.8
`17.9
`
`17.10A
`
`17.101!
`17.11
`
`X
`
`Supplement 38
`
`Analysis of Saccharéde Structure and Function Using Glycosyltsransferases
`Lectin Analysis of Proteins Blotted onto Filters
`
`Detection of Glycophospholipid Anchors on Proteins
`
`Direct Chemical Analysis of Glycoconjugates for Carbohydrates
`
`Inhibition of N-Linked Glycosylafion
`
`Inhibition of Glycolipid Biosynthesis
`
`Synthetic Glycosides as Primers of Oligosaccharide Biosynthesis and Inhibitors of
`Glycoprotein and Proteoglycan Assembly
`
`Cument Protocols in Molecular Biology
`BEQ 1016
`Page 13
`
`

`
`III RELEASE OF SACCHARIDES FROM GLYCOCONJUGATES
`17.12
`Sialidases
`
`17.l3A
`17.13B
`
`17.l4A
`17.14B
`
`17.l5A
`17.15B
`
`17.16
`
`17.17A
`17.17B
`
`Endoglycosidase and Glycoamidase Release of N-Linked Oligosaccharides
`Analysis of Glycosaminoglycans with Polysaccharide Lyases
`
`Preparation of Glycopeptides
`
`Detection of Individual Glycosylation Sites on Glycoproteins
`[3-Elimination for Release of 0-Linked Glycosaminoglycans from Proteoglycans
`B-Elimination for Release of 0-GalNAc-Linked Oligosaccharides from
`Glycoproteins and Glycopeptidos
`
`Acid Hydrolysis for Release of Monosaccharides
`
`Enzymatic Release of Oligosaccharides from Glycolipids
`Endo-[3-Galactosidases and Keratanase
`
`IV ANALYSIS OF SACCHARIDES RELEASED FROM GLYCOCONIUGATES
`17.18
`
`Analysis of Monosaccharides
`
`17.19A
`
`17.193
`
`17.20
`17.21A
`
`l7.21B
`17.22A
`
`17.22B
`17.23
`
`Total Compositional Analysis by High-Performance Liquid Chromatography or
`Gas-Liquid Chromatography
`
`Composition of Labeled Monosaccharides from Glycosaminoglycans
`Analysis of Oligosaccharide Negative Charge by Anion-Exchange Chromatography
`HPLC Methods for the Fractionation and Analysis of Negatively Charged
`Oligosaccharides and Gangliosides
`
`Fractionation and Analysis of Neutral Oligosaccharides By HPLC
`Nitrous Acid Degradation of Glycosaminoglycans
`
`Analysis of Disaccharides and Tetrasaccharides Released from Glycosaminoglycans
`
`Analysis of Sulfate Esters by Solvolysis or Hydrolysis
`
`18 Analysis (‘sf Protein Phosphorylation
`18.1
`
`Overview of Protein Phosphorylation
`Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for
`Immunoprecipitation
`
`18.2
`
`18.3
`18.4
`
`18.5
`18.6
`
`Phosphoamino Acid Analysis
`Analysis of Phosphorylation of Unlabeled Proteins
`
`Detection of Phosphorylation by Enzymatic Techniques
`
`Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides
`
`19
`
`Informatics for Molecular Biologists
`19.1
`
`Internet Basics for Biologists
`
`19.2
`
`19.3
`
`Sequence Databases: Information Retrieval and Data Submission
`Sequence Similarity Searching Using the BLAST Family of Programs
`
`Analysis of Protein Interactions
`20.1
`
`Interaction Trap/Two-Hybrid System to Identify Interacting Proteins
`
`20.2
`
`Affinity Purification of Proteins Binding to GST Fusion Proteins
`
`Current Protocols in Molecular Biology
`
`xi
`
`Supplement 35
`
`BEQ 1016
`Page 14
`
`

`
`Appendices
`A.1
`
`Standard Measurements, Data, and Abbreviations
`
`A.2
`
`A.3
`
`A.4
`
`A.5
`
`Commonly Used Reagents and Equipment
`
`Commonly Used Techniques in Biochemistry and Molecular Biology
`Selected Suppliers of Reagents and Equipment
`Vectors
`
`Index
`
`xii
`
`Supplement 3 5
`
`Current Protocols in Molecular Biology
`
`BEQ 1016
`Page 15
`
`

`
`FOREWORD
`
`7 he breadth of knowledge required for modern research in molecular biology is truly
`staggering. A single series of experiments can encompass genetic manipulation of
`specific bacteiéal strains and their phage, the appropriate use of several highly specific
`enzymes, and the culturing and genetic engineering of mammalian cells. For individuals
`who have followed the evolution of research in molecular biology, it was surprising to
`watch the mammalian 7». immunoglobulin gene being cloned in the prokaryotic R. phage.
`Neither immunologists nor bacterial geneticists expected the ultimate union when the
`phage and gene were named. Now a single individual must master both fields to be
`successful. Research that depends upon such breadth of knowledge needs protocol books
`that explain why certain methods are used and provide references for further reading. This
`manual fulfills that need.
`
`All scientists realize that they figuratively stand on the shoulders of their predecessors.
`Nothing more concretely reflects this dependence than a laboratory protocol book. It is a
`monument to the hours of labor that students, technicians and other professionals have
`expended in developing methods that work every time. Good methods have another
`characteristic: they have been honed to the minimum of time and effort required to achieve
`the desired results. Most investigators collect personal protocol books of “tried and true”
`recipes. This new manual with its continuously growing and evolving set of protocols is
`an excellent basis from which to begin such a collection.
`
`two unique features of this manual that are in tune with the rapid advances in
`There
`molecular biology. First, the manual evolves continuously with the addition of new
`protocols in emerging areas of research. These new protocols are added as quarterly
`supplements to the core manual. Second, the manual evolves through a “network” of the
`users. Readers are encouraged to contribute corrections and improvements on techniques
`in each supplement. Tinus, the book is designed to grow and change as biology grows and
`changes.
`
`Molecular biology has always been an intensely experimental science. Young people,
`more commonly, have the energy and intensity to push the field forward. The excellent
`group of young scientists who have contributed to this book are representatives of this
`tradition.
`
`Phillip A. Sharp
`Cambridge, Massachusetts
`
`February I 993
`
`Copyright © 1995 by John Wiley & Sons, Inc.
`
`Current Protocols
`in Molecular
`Biology
`
`xiii
`
`Supplement 30 CPMB
`BEQ 1016
`Page 16
`
`

`
`PREFACE
`
`lthough mastery of the techniques in this manual will enable the reader to pursue
`research in molecular genetics, the manual is not intended to be a substitute for
`graduate-level courses in molecular biology or a comprehensive textbook in the field.
`Introductory texts that we recommend include: Molecular Biology of the Gene (4th ed.),
`by J .D. Watson, N.H. Hopkins, J.W. Roberts, J.A. Steitz, and A.M. Weiner; Molecular
`Genetics: An Introductory Narrative, by G.S. Stent and R. Calendar; From Genes to
`Clones, by E.-L. Winnacker; and Genetics and Molecular Biology, by R. Schleif. In
`addition, An Introduction to GeneticAnalysis, by D.T. Suzuki, A.J.F. Griffiths, J .H. Miller,
`and RC. Lewontin, is a good place to learn classical genetics, and The Molecular Biology
`of the Cell, by B. Alberts, D. Bray, J. Lewis, M. Raff, K. Roberts, and J.D. Watson is a
`compendium of useful information about many aspects of cellular and molecular biology.
`Finally, we strongly recommend that readers gain first-hand experience in basic tech-
`niques and safety procedures by working in a molecular biology laboratory.
`
`An inevitable hazard of manual writing is that protocols become obsolete or that new
`techniques are developed—usually just as the pages are going to press. This problem is
`particularly severe in a fast-moving field such as molecular genetics. To safeguard our
`manual from inexorable obsolescence, and as a means of correcting errors in a timely
`manner, we provide a quarterly updating service. The looseleaf format will facilitate the
`replacement of pages that contain errors or have become obsolete and will accommodate
`the addition of new protocols to the appropriate section of the manual. The publisher can
`provide further details about subscribing to the quarterly supplements.
`
`HOW TO USE '§HIS MANUAL
`
`Format and firganization
`
`This publication is available in both looseleaf and CD-ROM format. The material covered
`in the two versions is identical.
`
`For looseleaf purchasers, two binders are provided to accommodate the growth of the
`manual via the quarterly update service. The first volume contains Chapters 1 through 9,
`and the second volume holds Chapter 10 through the appendices, including any new
`chapters to be added with the quarterly supplements. A full index and table of contents
`are included with both volumes. The looseleaf format of the binders allows easy insertion
`of new pages, units, and chapters that are added. The index and table of contents are
`updated with each supplement.
`
`CD-ROM purchasers receive a completely new disc containing the entire manual every
`quarter. The CD-ROM User’s Guide describes in detail the many features designed for
`accessing information presented in the CD. Topics of interest can be located primarily by
`using either the search index (by typing in the desired word or string of words) or by
`scanning through the expandable table of contents and selecting the desired units. In
`addition, hyperlinks jump between related units when cross-references are selected and
`from solutions and reagents listed in the materials list to their corresponding recipes.
`
`Subjects in this manual are organized by chapters and sections, and protocols are
`contained in units. Units generally describe a method and include one or more protocols
`with listings of materials, the protocol steps and annotations, recipes for unique reagents
`and solutions, and commentaries on the “hows” and “whys” of the method. Overview
`units contain theoretical discussions that lay the foundation for subsequent protocols. Page
`numbering in the looseleaf version reflects this modular arrangement; for example, page
`
`Copyright © 1995 by John Wiley & Sons, Inc.
`
`Current Protocols
`in Molecular
`Biology
`
`XV
`
`Supplement 30 CPMB
`BEQ 1016
`Page 17
`
`

`
`7.4.2 refers to Chapter 7 (DNA sequencing), Unit 4 (the dideoxy method), page 2 of that
`particular unit.
`
`Many reagents and procedures are employed repeatedly throughout the manual. Rather
`than duplicate this information, cross-references among units are used extensively. Early
`chapters (and appendices) describe these commonly used techniques (basic microbiology
`and basic manipulations of enzymes, DNA, and RNA), while later chapters describe their
`application (constructing libraries, DNA sequencing, mutagenesis, transfection, and
`protein analysis). Thus, whenever a particular enzyme is used in a protocol, the appropriate
`unit in Chapter 3——describing reaction conditions for that enzyme—is cross-referenced
`(e.g., UNIT3.7 for reverse transcriptase). Similarly, throughout the book readers are referred
`to UNIT 1.3 for spreading or streaking a plate, to UNIT 2.1 for phenol extraction/alcohol
`precipitation, to UNIT 2.5 for agarose gel electrophoresis, and so on. By tumiieg to these
`units, the reader will find instructions for the techniques, recipes for relevant reagents,
`and commentary. As a result, protocols in the later chapters of the book—which can be
`lengthy and complex—are not overburdened with steps describing auxiliary procedures
`required to prepare, purify, and analyze the sample or molecule of interest.
`
`Introductory and Explanatory Information
`
`Because this publication is first and foremost a compilation of molecular biology tech-
`niques we have not provided extensive instructive material. We have, however, included
`explanatory infornatéon where required to help readers gain an intuitive grasp of the
`procedures.