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`Molecular
`Molecular
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`Cloning
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`Associate Author
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`Nina Irwin
`
`Managing Editor
`
`Nancy ford
`
`Editor
`
`Chris Nolan
`
`Associate Editor
`
`Michele Ferguson
`
`Rlustrator
`
`Michael Ockler
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`oleeular
`Cloning
`
`A LABORATORY MANUAL
`
`SECOND EDITION
`
`J.Sambrook
`
`UNIVERSITY OF TEXAS SOUTHWESTERN MEDICAL CENTER
`
`E.F. Fritsch
`
`GENETICS INSTITUTE
`
`T. Maniatis
`
`HARVARD UNIVERSITY
`
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`Molecular
`Cloning
`A LABORATORY MANUAL
`SECOND EDITION
`
`All rights rl•st•rn•d
`© 1989 b) ('old Spring Harbor Laboratory Press
`Printc•d in the United States of America
`
`9 8 7 6 5 4
`
`Bovl• and ro1•1>r· design by Emily Harste
`
`Co1>er: The electron micrograph of bacteriophage ;. particles
`stained with uranyl acetate was digitized and assigned false color
`by comput('r. tThomas R. Broker, Louise T. Chow, and J ames 1.
`Garreh;)
`
`Catalog111g 111 Pttbl1cations data
`Sambrook. Joseph
`Molecular cloning . a laboratory manual I E.F.
`Fritsch. T Maniatis-2nd ed.
`cm.
`p.
`Bibliography· p
`Includes index
`ISBN 0-87969-309-6
`l Molecular cloning-baboratory manuals. 2. Eukaryolic cells(cid:173)
`-Laboratory manuals. l. Fritsch. Edward F. II. Maniatis. Thomas
`Ill Title
`QH442.2 M26 1987
`574 .87'3224-dcl9
`
`87-35464
`
`Rescnrchers using the procedures of this manual do so at their own risk. Cold Spring Harbor
`Laboratory makes no representations or warranties with respect to the material set forth in
`this manual and ha1> no liabilil.y in connection with the use of these materials.
`
`Authoriza1ion to photocopy items for internal or personal use, or the internal or personal use of
`specific clients. 1s granted by Cold Spring Harbor Laboratory Press for libraries a nd other
`users registered with the Copyright Clearance Center <CCC I Transactional Reporting Service.
`provided that the base fee of$0.10 per page is paid directly to CCC, 21 Congress St.. Salem MA
`0 1970 [0-87969-309-6/ 89 $00 -.- $0. lOJ This consent docs not extend to other kindb of copying.
`such as copying for general distribution. for advertising or promotional purposes. for creating
`ne'' collectwe works. or for resale.
`
`All ('old Spring Harbor Laboratory Press publications may bt• ordered d1rcc..'lly from Cold
`Spring H:irbor Laborator) Press. JO Skyline Drh•e. Plainview New York 11803. Phone:
`1-800-843-438b In New York 15161 367-8423. FAX: 15161 367-0432.
`
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`Pre-race to First Edition
`
`This manual began as a collection of laboratory protocols that were used
`during the 1980 Cold Spring Harbor course on the Molecular Cloning of
`Eukaryotic Genes. These procedures had been in use in our laboratories at
`that time but were scattered throughout the notebooks of many different
`people.
`In 1981 we decided to produce a more complete and up-to-date
`manual not only for use in the next Cold Spring Harbor course, but also for
`eventual publication. Out of the many permutations of the methods being
`used, we assembled a set of "consensus protocols," which were photocopied
`and widely distributed to many laboratories even as the 1981 course was
`underway. Then in the winter of 1981-1982, the manual was substantially
`rewritten, and new or revised protocols and figures, as well as entirely new
`chapters, were added.
`Even since this last rewriting, however, the field has progressed: New
`methods are constantly being invented and existing techniques are altered in
`response to changing needs. Although we have included in this manual only
`those protocols that have been thoroughly tested and used successfully in our
`laboratories, we make no claim that they are inviolable or perfect. We would
`welcome suggestions for improvements, and we would be grateful to be told
`about any new procedures that are devised.
`The evolution of protocols poses the difficult problem of attribution. We
`have tried to give credit at appropriate places in the text to the people who
`originally developed the procedures presented here, but in many cases
`tracing a particular method to its undisputed roots has proved to be impos(cid:173)
`sible. We therefore wish to apologize-and to express gratitude-to those we
`have been unable to acknowledge for an idea, procedure, or recipe. Our major
`function has been to compile, to ver:ify, and, we hope, to clarify; less frequent(cid:173)
`ly we have introduced modifications, and only in rare instances have we
`devised new protocols. In large part, then, the manual is based on procedures
`developed by others, and it is to them that any credit belongs.
`Because the manual was originally written to serve as a guide to those who
`had little experience in molecular cloning, it contains much basic material.
`However, the current version also deals in detail with almost every labora(cid:173)
`tory task currently used in molecular cloning. We therefore hope that
`newcomers to cloning and veterans alike will find material of value in this
`book.
`Although molecular cloning seems straightforward on paper, it is more
`difficult to put into practice. Most protocols involve a large number of
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`individual steps and a problem with any one of them can lead the experiment(cid:173)
`er into difficulty. It is a good idea to verify the products of each step and to
`include controls to check the efficiency of each reaction. To deal with these
`problems, a well-founded understanding of the principles underlying each
`procedure is essential. We have therefore provided background information
`and references that may be useful should trouble occur.
`This manual could not have been written without the help and advice of
`members of our laboratories and contributions from many others. We
`therefore wish to thank Joan Brooks, John Fiddes, Mary-Jane Gething, Tom
`Gingeras, David Goldberg, Steve Hughes, David Ish-Horowicz, Mike Mathews,
`Patty Reichel, J oe Sorge, Jim Stringer, Richard Treisman, and Nigel Whittle.
`We wish particularly to thank Arg Efstratiadis for his helpful discussions and
`criticisms of Chapter 7; Brian Seed for permission to include a description of
`his unpublished procedure for screening libraries by recombination (Chapter
`10) and many other useful suggestions; Doug Hanahan for advice on trans(cid:173)
`formation (Chapter 8); Bryan Roberts for suggestions on methods of hybrid(cid:173)
`selection and cDNA cloning; Doug Melton for providing a protocol for injec(cid:173)
`tion of Xenopus oocytes; Ronni Greene for suggesting improvements to many
`protocols; Nina Irwin for providing a critical anthology of methods available
`for expressing eukaryotic proteins in bacteria (Chapter 12); Rich Roberts for
`supplying the computer analysis of the sequence of pBR322; Barbara Bach(cid:173)
`mann and Ahmad Bukhari for reviewing and correcting the list of E. coli
`strains; and Tom Broker, Louise Chow, Jeff Engler, and Jim Garrels for
`producing the elegant photographs used for the front and back covers.
`We also thank all those who participated in the Cold Spring Harbor
`Molecular Cloning courses of 1980 and 1981. They were an excellent group of
`students, who struggled through the first two drafts of the manual and made
`many useful suggestions. We also thank Nancy Hopkins, who helped us to
`teach the course the first year and convinced us that producing a manual
`would be a worthwhile task. In 1981 Doug Engel helped teach the course and
`suggested many improvements to the manual. Contributing to the success of
`both courses were the efforts of the teaching assistants, who were Catherine
`O'Connell and Helen Doris Keller in the summer of 1980 and Susan Vande(cid:173)
`Woude, Paul Bates, and Michael Weiss in 1981.
`We wish to thank Patti Barkley and Marilyn Goodwin for their cheerful(cid:173)
`ness and forbearance during the typing of successive revisions of the manu(cid:173)
`script. Our artists, Fran Cefalu and Mike Ockler, worked with great
`dedication and perseverance to produce the drawings for the manual. Joan
`Ebert kept track of the many references added to and deleted from the text
`and assembled the reference list. We are also grateful to Nancy Ford,
`Director of Publications, Cold Spring Harbor Laborato.ry, for her encourage(cid:173)
`ment and support. Finally, without the patience, skill, and diplomacy of
`Doug Owen, who prepared the manuscript for the printer and helped us in
`many other ways, this book would not exist.
`
`Tom Maniatis
`Ed Fritsch
`Joe Sambrook
`
`TI Preface to First Edition
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`Pre£aee to Second Edition
`
`Since the publication of the first edition of this Laboratory Manual in 1982,
`there has been a vast increase both in the number of people who use
`molecular cloning and in the range and power of the techniques used to
`handle recombinant DNA. This remarkable proliferation of cloning methods
`is reflected in the number of gene sequences in the GenBank DNA sequence
`data base. In 1982 there were fewer than 350 gene sequences on file, but in
`less than four years this number grew to almost 5000. Today over 15,000
`sequences are listed. These figures, impressive as they are, do not do justice
`to the increased sophistication with which cloned genes are now analyzed. In
`1982, respectable journals would still accept manuscripts containing little
`more than the partial sequence of a cDNA clone; today, the publication of a
`complete sequence is taken for granted, and papers describing the initial
`cloning of a cDNA will often also contain elegant accounts of the expression of
`the gene product in prokaryotic or eukaryotic hosts. In most cases, this first
`paper is rapidly followed by others that use site-directed mutagenesis to
`explore the relationship between the structure and function of the relevant
`protein. The cloning and analysis of elements that regulate the expression of
`eukaryotic genes has moved at a similar, exciting pace.
`This increase in the range and speed of molecular cloning is reflected in the
`tripling of size of this manual and its consequent division into three volumes.
`Techniques that were mentioned only in passing in the first edition, such as
`mutagenesis, expression of cloned genes in mammalian cells, and dideoxy(cid:173)
`mediated sequencing, are now described in depth; new sections have been
`added that deal with recently invented techniques, such as amplification of
`DNA by the polymerase chain reaction; and we have included modern
`variations and embellishments of many of the basic methods that were the
`mainstay of the first edition. We hope that any inconvenience caused by the
`expansion in size of the manual will be compensated by an increase in the
`richness of its content. We hope that this second edition will be a resource for
`the experienced cloner, a starting point for the student, and a guidebook for
`the next generation of investigators in molecular cloning.
`The burgeoning of molecular cloning has also led to its commercialization,
`and we are now able to purchase a wide variety of high quality and
`reasonably priced reagents and enzymes. Although this is a very positive
`development, it has had a few unfortunate side effects. One is the prolifer(cid:173)
`ation of preassembled kits to carry out particular cloning tasks. Although
`these kits reduce the possibility of trivial errors, they also tend to discourage
`experimenters from thinking about what they are doing. It becomes all too
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`easy to follow blindly instructions to add 2 µ.l of solution A without knowing
`what the particular reagent is, why it is necessary, or why it is added at a
`particular point in the protocol. Kits therefore reward the scientifically
`illiterate and inhibit the development of improvements. In an attempt to
`counteract this trend, we have greatly increased the amount of background
`material at the beginning of each chapter of the manual and we have
`provided full references. Users of the manual who read this material should
`have no problem understanding either the general design or the specific
`details of the experimental protocols. We also strongly recommend reading
`the relevant protocols in their entirety before commencing work. This allows
`reagents to be prepared ahead of time and permits the investigator to carry
`out the protocol efficiently.
`This manual could not have been produced without the help and encour(cid:173)
`agement of a large number of people. We are extremely grateful to colleagues
`from the University of Texas Southwestern Medical Center at Dallas, from
`Genetics Institute, from Harvard University, and from many other institu(cid:173)
`tions who have read individual chapters, have submitted protocols, and have
`made many invaluable suggestions that have immeasurably improved the
`manual. We have listed all these individuals with their affiliations in a
`special section at the end of this preface.
`We owe a special debt of thanks to Rick Myers and Alison Cowie of the
`University of California at San Francisco, who read all of the chapters in
`draft, eliminated many embarrassing errors, and made many valuable sug(cid:173)
`gestions for improvements; to Winship Herr of Cold Spring Harbor Labora(cid:173)
`tory and Mary-Jane Gething of Southwestern Medical Center, who drafted
`the chapters on DNA sequencing and expression of cloned genes in mam(cid:173)
`malian cells, respectively; and to Judy Campbell, who contributed signifi(cid:173)
`cantly to the preparation of the chapter on enzymes. We are also grateful to
`Mike Ockler, who provided all of the conceptual illustrations in this edition,
`and to Carolyn Doyle, who assembled the index.
`Nina Irwin has played a special role in the writing of this manual. Among
`her many contributions are the E. coli strain list and the maps of the many
`strains of bacteriophage ,\, plasmids, cosmids, and bacteriophage M13 used in
`this manual. These maps are based on many months of painstaking work to
`reconstruct and verify the genealogy of these host and vector strains. . In
`addition, Nina also drafted the chapter on expression of cloned genes in
`prokaryotic hosts, read and reviewed all of the chapters in manuscript and
`galleys, and searched the scientific literature with great intelligence and skill
`to validate many of the facts that appear.
`We also wish to thank Jim Watson for his continued interest in this
`manual. The support he has provided made this edition possible.
`Finally, we would like to express our deep appreciation of the work of our
`editor Nancy Ford. For over two years, she has worked untiringly to bring
`order and sense to the entire project and to improve and clarify our writing.
`Nancy and her assistant Michele Ferguson have both served as a source of
`encouragement, sympathy, and friendship. We are greatly in their debt.
`
`J. Sambrook
`E.F. Fritsch
`T. Maniatis
`
`viil Preface to Second Edition
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`Ed Alderman, Genetics Institute
`Patricia Ashley, University of Texas Southwestern Medical Center
`Margaret Baron, Harvard University
`Frank Baas, Harvard University
`Phil Bird, University of Texas Southwestern Medical Center
`Tom Bittick, University of Texas Southwestern Medical Genter
`Colleen Brewer, University of Texas Southwestern Medical Genter
`Gene Brown, Genetics Institute
`H. Franklin Bunn, Harvard Medical School
`Steve Clark, Genetics Institute
`Preston Dunnmon, University of Texas Southwestern Medical Center
`Anne Ephrussi, Harvard University
`Henry Erlich, Perkin Elmer Cetus
`Chen-Ming Fan, Harvard University
`Ken Ferguson, Cold Spring Harbor Laboratory
`Bill Garrard, University of Texas Southwestern Medical Center
`Bob Gerard, University of Texas Southwestern Medical Center
`Gary Gilliland, Harvard Medical School
`Doug Hanahan, University of California, San Francisco
`Ed Harlow, Cold Spring Harbor Laboratory
`Jean Henneberry, University of Texas Southwestern Medical Center
`Bill Huse, Stratagene
`David Ish-Horowicz, Imperial Cancer Research Fund
`David Israel, Genetics Institute
`Randy Kaufman, Genetics Institute
`Andrew Keller, Harvard University
`John Knopf, Genetics Institute
`Steve Lacey, University of Texas Southwestern Medical Center
`Ray MacDonald, University of Texas Southwestern Medical Center
`Ed Madison, University of Texas Southwestern Medical Center
`Mike Mathews, Cold Spring Harbor Laboratory
`Michael McClelland, University of Chicago
`John McCoy, Genetics Institute
`Steve McKnight, Carnegie Institution of Washington
`~oug Melton, Harvard University
`Alan Michelson, Harvard University
`George Morris, Genetics Institute
`Michael Nelson, University of Chicago
`Robin Reed, Harvard University
`Rich Roberts, Cold Spring Harbor Laboratory
`Laura Roman, University of Texas Southwestern Medical Center
`Susan Rosenberg, University of Utah
`David Russell, University of Texas Southwestern Medical Center
`Brian Seed, Harvard Medical School
`Conrad Seghers, University of Texas Southwestern Medical Center
`Mark Segal, University of Texas Southwestern Medical Center
`Chuck Shoemaker, Harvard School of Public Health
`Huda Shubeita, University of Texas Southwestern Medical Center
`Harinder Singh, University of Chicago
`Beth Smith, Information Center, Genetics Institute
`Joe Sorge, Stratagene
`Lisa Sultzman, Genetics Institute
`Galvin Swifr, University of Texas Southwestern Medical Center
`Patty Temple, Genetics Institute
`Jeff Vieira, Rutgers University
`Geoff Wahl, Salk Institute
`Steve Wasserman, University of Texas Southwestern Medical Center
`Gordon Wong, Genetics Institute
`Rick Young, Massachusetts Institute of Technology
`Jian-Hua Zhang, University of Texas Southwestern Medical Center
`Mark Zoller, Genentech
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`Contents
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`t
`
`BOOK 1
`1
`Plasmid Yeetors
`
`Essential Features of Plasmids 1.3
`
`Replication and Incompatibility 1.3
`Mobilization 1.5
`Selectable Markers 1.5
`
`Plasmid Vectors 1.7
`
`DEVELOPMENT OF PLASMID CLONING VECTORS 1.7
`Plasmid Vectors That Permit Histochemical Identification of Recombinant
`Clones 1.8
`Plasmid Vectors Carrying Origins of Replication Derived from Single-stranded
`Bacteriophages 1.9
`Plasmid Vectors Carrying Bacteriophage Promoters 1.9
`Plasmid Vectors That Allow Direct Selection of Recombinant Clones 1.9
`Plasmid Expression Vectors 1.10
`
`COMMONLY USED PLASMID VECTORS 1.11
`pBR322 1.12
`pUC18, pUC19 1.13
`pUC118, pUC119 1.14
`pSP64, pSP65, pGEM-3, pGEM-3Z, pGEM-3Zf{-), pGEM-4, pGEM-4Z 1.15
`1TAN13 1.19
`BLUESCRIPT M13 + , M13 -
`
`1.20
`
`Extraction and Purification of Plasmid DNA 1.21
`
`Growth of the Bacterial Culture 1.21
`Harvesting and Lysis of the Bacteria 1.22
`Purification of Plasmid DNA 1.23
`
`SMALL-SCALE PREPARATIONS OF PLASMID DNA 1.25
`Harvesting and Lysis of Bacteria 1.25
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`HARVESTING 1.25
`L YSIS BY ALKALI 1.25
`L YSIS BY BOILING 1.29
`Solving Problems That Arise with Minipreparations of Plasmid DNA 1.31
`Rapid Disruption of Bacterial Colonies to Test the Size of Plasmids 1.32
`LARGE-SCALE PREPARATIONS OF PLASMID DNA 1.33
`Amplification of Plasmids in Rich Medium 1.33
`Harvesting and Lysis of Bacteria 1.34
`HARVESTING 1.34
`LYSIS BY BOILING 1.34
`LYSIS BY SODIUM DODECYL SULFATE 1.36
`L YSIS BY ALKALI 1.38
`
`PURIFICATION OF PLASMID DNA 1.40
`Purification of Plasmid DNA by Precipitation with Polyethylene Glycol 1.40
`Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl(cid:173)
`Ethidium Bromide Gradients 1.42
`CONTINUOUS GRADIENTS 1.42
`DISCONTINUOUS GRADIENTS 1.44
`Removal of Ethidium Bromide from DNAs Purified by Equilibrium
`Centrifugation in CsCl-Ethidium Bromide Gradients 1.46
`METHOD 1: EXTRACTION WITH ORGANIC SOLVENTS 1.46
`METHOD 2: ION-EXCHANGE CHROMATOGRAPHY 1.47
`Decontamination of Ethidium Bromide Solutions 1.49
`DECONTAMINATION OF CONCENTRATED SOLUTIONS OF ETHIDIUM BROMIDE 1.49
`DECONTAMINATION OF DILUTE SOLUTIONS OF ETHIDIUM BROMIDE 1.50
`Removal of RNA from Preparations of Plasmid DNA 1.51
`CENTRIFUGATION THROUGH 1 M NaCl 1.51
`CHROMATOGRAPHY THROUGH BIO-GEL A-150m OR SEPHAROSE CL-4B 1.52
`
`Strategies for Cuming in Plasmid Vectors 1.53
`STRATEGIES FOR LIGATION 1.53
`The Nature of the Ends of the Foreign DNA Fragment 1.53
`FRAGMENTS CARRYING NONCOMPLEMENTARY PROTRUDING TERMINI 1.53
`FRAGMENTS CARRYING IDENTICAL TERMIN1 (BLUNT-ENDED OR PROTRUDING) 1.56
`FRAGMENTS CARRYING BLUNT ENDS
`l.56
`The Nature of the Restriction Sites in the Plasmid Vector and the Foreign
`DNA 1.59
`DEPHOSPHORYLATION OF LINEARIZED PLASMID DNA 1.60
`Test Ligations and Transformations 1.62
`LIGATION REACTIONS 1.63
`Setting Up Ligation Reactions Between Foreign DNA and Plasmid Vectors 1.63
`Ligation of Cohesive Termini 1.68
`Ligation of Blunt-ended DNA 1. 70
`CONDENSING AGENTS 1.70
`Rapid Cloning in Plasmid Vectors 1. 72
`
`Preparation and Transformation of Competent E. coli 1. 74
`Transformation of E. coli by High-voltage Electroporation
`(Electrotransformation) 1. 75
`Protocol I: Preparation of Fresh or Frozen Competent E. coli 1. 76
`Protocol II: Fresh Competent E. coli Prepared Using Calcium Chloride 1.82
`
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`Identification of Bacterial Colonies That Contain Recombinant
`Plasmids 1.85
`RESTRICTION ANALYSIS OF SMALL-SCALE PREPARATIONS OF PLASMID
`DNA 1.85
`
`a-COMPLEMENTATION 1.85
`Testing Bacteria for a-Complementation 1.86
`INSERTIONAL INACTNATION 1.87
`
`SCREENING BY HYBRIDIZATION 1.90
`Transferring Small Numbers of Colonies to Nitrocellulose Filters 1.92
`Replicating Colonies onto Nitrocellulose Filters 1.93
`METHOD l 1.93
`METHOD 2 1.96
`Lysis of Colonies and Binding of DNA to Nitrocellulose Filters 1.98
`METHOD 1 1.98
`METHOD 2 1.100
`Hybridization to Nitrocellulose Filters Containing Replicas of Bacterial
`Colonies 1.101
`
`References 1.105
`
`2
`Bacteriophage A Yectors
`
`Molecular Biology of Bacteriophage A 2.3
`THE LYTIC CYCLE 2.3
`Adsorption 2.3
`Immediate Early Transcription 2.5
`Delayed Early Transcription 2.5
`DNA Replication 2.5
`Late Transcription 2.5
`Assembly 2. 7
`Lysis 2.8
`
`LYSOGENY 2.8
`
`Bacteriophage A Vectors 2.9
`CONSTRUCTION OF BACTERIOPHAGE A VECTORS: A BRIEF HISTORY 2.9
`CHOOSING THE APPROPRIATE BACTERIOPHAGE A VECTOR 2.11
`MAPS OF BACTERIOPHAGE A VECTORS 2.15
`A'
`2.17
`CHARON 4A 2.18
`CHARON 21A 2.20
`CHARON 32 2.22
`CHARON 33 2.24
`
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`CHARON 34 2.26
`CHARON 35 2.28
`CHARON 40 2.30
`EMBL3A 2.32
`,\2001 2.34
`ADASH 2.36
`AFIX 2.38
`AgtlO 2.40
`Agtll 2.42
`Agtl8 2.44
`Agt20 2.46
`Agt22 2.48
`AORF8 2.50
`AZAP/R 2.52
`
`CHOOSING A BACTERIAL HOST FOR BACTERIOPHAGE A VECTORS 2.55
`Restriction and Modification 2.55
`Amber Suppressors 2.55
`Recombination Systems 2.55
`
`Bacteriophage A Growth, Purification, and DNA Extraction 2.60
`PLAQUE PURIFICATION OF BACTERIOPHAGE A 2.60
`Preparation of Plating Bacteria 2.60
`Plating Bacteriophage A 2.61
`Picking Bacteriophage A Plaques 2.63
`
`PREPARING STOCKS OF BACTERIOPHAGE A FROM SINGLE PLAQUES 2.64
`Plate Lysate Stocks 2.64
`PREPARATION OF PLATE LYSATE STOCKS: PROTOCOL I 2.65
`PREPARATION OF PLATE LYSATE STOCKS: PROTOCOL II 2.66
`Small-scale Liquid Cultures 2.67
`Long-term Storage of Bacteriophage A Stocks 2.68
`LARGE-SCALE PREPARATION OF BACTERIOPHAGE A 2.69
`Infection at Low Multiplicity 2. 70
`Infection at High Multiplicity 2. 72
`
`PURIFICATION OF BACTERIOPHAGE A 2. 73
`Standard Method for Purification of Bacteriophage A 2. 73
`Alternative Methods for Purification of Bacteriophage A 2. 77
`PELLETING BACTERIOPHAGE PARTICLES 2.77
`GLYCEROL STEP GRADIENT 2.78
`EQUILIBRIUM CENTRIFUGATION IN CESIUM CHLORIDE 2.79
`
`EXTRACTION OF BACTERIOPHAGE A DNA 2.80
`
`Cloning in Bacteriophage A 2.82
`PREPARATION OF VECTOR DNA 2.82
`Digestion of Bacteriophage A DNA with Restriction Enzymes 2.83
`Purification of Bacteriophage A Arms 2.85
`CENTRIFUGATION THROUGH SUCROSE DENSITY GRADIENTS 2.85
`CENTRIFUGATION THROUGH SODIUM CHLORIDE GRADIENTS 2.88
`Preparation of Vectors Treated with Alkaline Phosphatase 2.90
`Digestion of Bacteriophage A Vectors with Two Restriction Enzymes 2.92
`Ligation of Bacteriophage A Arms to Fragments of Foreign DNA 2.94
`
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`PACKAGING OF BACTERIOPHAGE A DNA IN VITRO 2.95
`Maintenance and Testing of Lysogens of Bacteriophage A 2.96
`Preparation of Packaging Extracts and Packaging of Bacteriophage A DNA In
`Vitro 2.98
`PROTOCOL I: PREPARATION OF PACKAGING EXTRACTS FROM TWO LYSOGENS 2.100
`PROTOCOL I: PACKAGING IN VITRO USING TWO EXTRACTS 2.104
`PROTOCOL II: PREPARATION OF PACKAGING EXTRACTS FROM ONE LYSOGEN 2.105
`PROTOCOL II: PACKAGING IN VITRO USING ONE EXTRACT 2.107
`
`Identification and Analysis of Recombinants 2.108
`IN SITU HYBRIDIZATION OF BACTERIOPHAGE A PLAQUES 2.108
`Immobilization of Bacteriophage A Plaques on Nitrocellulose Filters or Nylon
`Membranes 2.109
`Immobilization of Bacteriophage A Plaques on Nitrocellulose Filters Following In
`Situ Amplification 2.112
`Hybridization to Nitrocellulose Filters Containing Replicas of Bacteriophage A
`Plaques 2.114
`
`RAPID ANALYSIS OF BACTERIOPHAGE A ISOLATES 2.118
`Plate Lysate Method 2.118
`Liquid Culture Method 2.121
`
`References 2.122
`
`3
`Cosmid 'Vectors
`
`Cloning in Cosmid Vectors 3.5
`
`Cosmid Vectors 3. 7
`
`COSMID VECTORS FOR PROPAGATION OF EUKAilYOTIC DNA IN
`BACTERIA 3.9
`pJBS 3.9
`c2RB 3.13
`pcoslEMBL 3.17
`
`COSMID VECTORS FOR TRANSFECTION OF MAMMALIAN CELLS 3.18
`pHC79-2cos/tk 3.19
`pCV103, pCV107, pCV108 3.19
`pTM, pMCS, pNNL 3.19
`pHSG274 3.19
`cos202, cos203 3.19
`pWE15, pWE16 3.21
`CHAROMID 9 VECTORS 3.25
`
`Construction of Genomic DNA Libraries in Cosmid Vectors 3.27
`CLONING IN PHOSPHATASE-TREATED COSMID VECTORS 3.28
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`Preparaticn of Vector DNA 3.28
`TEST LIGATION 3.29
`Partial Digestion of Eukaryotic DNA with MboI or Sau3AI 3.32
`Ligation and Packaging 3.33
`
`CLONING IN COSMID VECTORS DIGESTED WITH TWO RESTRICTION
`ENZYMES AND TREATED WITH PHOSPHATASE 3.35
`Preparation of Vector DNA 3.36
`Treatment of Eukaryotic DNA with Alkaline Phosphatase 3.38
`Ligation and Packaging 3.40
`
`PREPARATION OF VECTORS CONTAINING TWO cos SITES 3.42
`
`Amplification and Storage of Cosmid Libraries 3.44
`Replica Filters 3.46
`Amplification of Cosmid Libraries in Liquid Culture 3.50
`Preparation of a Transducing Lysate of Packaged Cosmids 3.52
`
`Problems Commonly Encountered When Using Cosmids 3.54
`
`References 3.56
`
`4
`Single-stranded, Filamentous
`Bacteriophage J'eetors
`
`The Biology of Filamentous Bacteriophages 4.3
`
`Filamentous Bacteriophages as Vectors 4. 7
`BACTERIOPHAGE M13 VECTORS 4. 7
`BACTERIAL HOSTS FOR BACTERIOPHAGE M13 VECTORS 4.12
`PROBLEMS COMMONLY ENCOUNTERED WHEN CLONING IN
`FILAMENTOUS BACTERIOPHAGE VECTORS 4.16
`PHAGEMIDS: PLASMIDS CONTAINING AN ORIGIN OF REPLICATION
`DERIVED FROM A FILAMENTOUS BACTERIOPHAGE 4.17
`THE USES OF SINGLE-STRANDED DNA GENERATED BY RECOMBINANTS
`CONSTRUCTED IN PHAGEMID AND FILAMENTOUS BACTERIOPHAGE
`VECTORS 4.20
`
`Propagation of Bacteriophage M13 and Preparation of DNA 4.21
`PLAQUE PURIFICATION OF BACTERIOPHAGE M13 4.21
`Preparation of Plating Bacteria 4.21
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`Plating Bacteri.ophage M13 4.22
`Picking Bacteri.ophage M13 Plaques 4.24
`
`PREPARING STOCKS OF BACTERIOPHAGE M13 FROM SINGLE
`PLAQUES 4.25
`Liquid Cultures 4.25
`
`PREPARATION OF BACTERIOPHAGE M13 DNA 4.26
`Small-scale Preparations of the Replicative Form of Bacteri.ophage M13
`DNA 4.26
`Small-scale Preparati.ons of Single-stranded Bacteri.ophage M13 DNA 4.29
`Large-scale Preparati.ons of the Replicatiue Form of Bacteri.ophage M13
`DNA 4.31
`Large-scale Preparati.ons of Single-stranded Bacteri.ophage M13 DNA 4.32
`
`Cloning into Bacteriophage M13 Vectors and Transfection of
`Competent Bacteria 4.33
`Cloning DNA Fragments into Bacteri.ophage M13 Vectors 4.35
`Preparati.on of Competent Bacteria 4.36
`Transfecti.on of Competent Bacteria with Bacteri.ophage M13 DNA 4.37
`
`Identification and Analysis of Recombinants 4.39
`Direct Gel Electrophoresis 4.39
`In Situ Hybridization of Bacteriophage M13 Plaques 4.41
`Analysis of Small-scale Preparations of the Replicatiue Form of Bacteriophage
`M13 DNA 4.41
`Testing the Orientation of DNA Cloned in Bacteriophage M13 Vectors 4.42
`
`Cloning in Phagemids 4.44
`Growth of M13K07 4.47
`Production of Single-stranded Phagemid DNA 4.48
`Screening Colonies by Superinfecti.on 4.49
`
`References 4.51
`
`E~mes llsed in Molecular Cloning
`
`Restriction and DNA Methylation Enzymes 5.3
`LIGATION OF TERMINI CREATED BY RESTRICTION ENZ¥MES 5.10
`Compatible Cohesive Termini 5.10
`Blunt Ends 5.11
`Incompatible Cohesiue Termini 5.11
`
`ISOSCHIZOMERS 5.14
`
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`DNA METHYLATION 5.15
`Methylation by Commonly Used Strains of E. coli 5.15
`dam METHYLASE 5.15
`dcm METHYLASE 5.15
`METHYLATION-DEPENDENT RESTRICTION SYSTEMS IN E. coli 5.15
`M.EcoRI METHYLASE 5.16
`Modification of Restriction Sites by DNA Methylation 5.16
`Influence of Methylation on DNA Mapping 5.26
`
`DIGESTING DNA WITH RESTRICTION ENZYMES 5.28
`Setting Up Digestions with Restriction Enzymes 5.31
`
`Other Enzymes Used in Molecular Cloning 5.33
`DNA POLYMERASES 5.35
`DNA Polymerase I (Holoenzyme) 5.36
`Large Fragment of DNA Polymerase I (Klenow Fragment) 5.40
`Bacteriophage T4 DNA Polymerase 5.44
`Bacteriophage T7 DNA Polymerase 5.48
`Modified Bacteriophage T7 DNA Polymerase (Sequenase™) 5.49
`Taq DNA Polymerase and AmpliTaq TM 5.50
`Reuerse Transcriptase (RNA-dependent DNA Polymerase) 5.52
`Terminal Transferase (Terminal Deoxynucleotidyl Transferase) 5.56
`
`DNA-DEPENDENT RNA POLYMERASES 5.58
`Bacteriophage SP6 and Bacteriophages T7 and T3 RNA
`Polymerases 5.58
`
`LIGASES, KINASES, AND PHOSPHATASES 5.61
`Bacteriophage T4 DNA L igase 5.62
`E. coli DNA Ligase 5.64
`Bacteriophage T4 RNA Ligase 5.66
`Bacteriophage T4 Polynucleotide Kinase 5.68
`Alkaline Phosphatases 5. 72
`
`NUCLEASES 5. 73
`Nuclease BAL 31 5.73
`N,uclease SJ 5. 78
`Mung-bean Nuclease 5.80
`Ribonuclease A 5.81
`Ribonuclease Tl 5.82
`Deoxyribonuclease I 5.83
`Exonuclease III 5.84
`Bacteriophage A Exonuclease 5.86
`
`DNA-binding Proteins 5.87
`
`Single-stranded DNA-binding Protein (SSB) 5.87
`RecA Protein 5.88
`Topoisomerase I 5.89
`
`References 5.90
`
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`6
`Gel Electrophoresis 0£ DNA
`
`Agarose Gel Electrophoresis 6.3
`
`Factors Affecting the Rate of DNA Migration in Agarose Gels 6.4
`MOLECULAR SIZE OF THE DNA 6.4
`AGAROSE CONCENTRATION 6.4
`CONFORMATION OF THE DNA 6.5
`APPLIED VOLTAGE 6.6
`DIRECTION OF THE ELECTRIC FIELD 6.6
`BASE COMPOSITION AND TEMPERATURE 6.6
`PRESENCE OF INTERCALATING DYES 6.6
`COMPOSITION OF THE ELECTROPHORESIS BUFFER 6.6
`Apparatuses Used for Agarose Gel Electrophoresis 6.8
`
`PREPARATION AND EXAMINATION OF AGAROSE GELS 6.9
`Preparation of an Agarose Gel 6.9
`Minigels 6.14
`Staining DNA in Agarose Gels 6.15
`Decontamination of Ethidium Bromide Solutions 6.16
`DECONTAMINATION OF CONCENTRATED SOLUTIONS OF ETHIDIUM BROMIDE 6.16
`DECONTAMINATION OF DILUTE SOLUTIONS OF ETHIDIUM BROMIDE 6.17
`Photography 6.19
`Alkaline Agarose Gels 6.20
`
`RECOVERY AND PURIFICATION OF DNA FRACTIONATED ON AGAROSE
`GELS 6.22
`Electrophoresis onto DEAE-cellulose Membrane 6.24
`Electroelution into Dialysis Bags 6.28
`Recovery of DNA from Low-melting-temperature Agarose Gels 6.30
`Purification of DNA Recovered from Agarose Gels 6.32
`PASSAGE THROUGH DEAE-SEPHACEL 6.32
`EXTRACTION WITH ORGANIC SOLVENTS 6.34
`
`Polyacrylamide Gel. Electrophoresis 6.36
`
`PREPARATION OF NONDENATURING POLYACRYLAMIDE GELS 6.39
`
`DETECTION OF DNA IN POLYACRYLAMIDE GELS 6.44
`Staining with Ethidium Bromide 6.44
`Autoradiography 6.45
`UNFIXED, WET GELS 6.45
`FIXED, DRIED GELS 6.45
`
`ISOLATION OF DNA FRAGMENTS FROM POLYACRYLAMIDE GELS 6.46
`"Crush and Soak" Method 6.46
`
`Other Types of Gels 6.49
`
`STRAND SEPARATING GELS 6.49
`
`DENATURING GRADIENT POLYACRYLAMIDE GELS 6.49
`
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`PULSED-FIELD GEL ELECTROPHORESIS 6.50
`Design of the Apparatus 6.51
`Staining DNA Separated by Pulsed-field Gel Electrophoresis 6.52
`Preparation of DNA for Pulsed-field Gel Electrophoresis 6.53
`ISOLATION OF INTACT DNA FROM MAMMALIAN CELLS 6.53
`ISOLATION OF INTACT DNA FROM YEAST 6.55
`RESTRICTION ENZ¥ME DIGESTION OF DNA IN AGAROSE BLOCKS 6.57
`Markers for Pulsed-field Gel Electrophoresis 6.58
`
`References 6.60
`
`7
`.Extraction, Purification, and Analysis
`of' Messenger .RNA f'rorn Eukaryotic Cells
`
`Extraction and Purification of RNA 7.3
`
`CONTROLLING RIBONUCLEASE ACTNITY 7.3
`Laboratory Procedures 7.3
`Inhibitors of Ribonucleases 7.4
`Methods That Disrupt Cells and Inactivate Ribonucleases Simultaneously 7.5
`
`ISOLATION OF RNAs 7.6