Molecular Cloning, A Laboratory Manual: Set of Volumes 1,2, and 3, Third Edition / Edition 3

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The first two editions of this manual have been mainstays of molecular biology for nearly twenty years, with an unrivalled reputation for reliability, accuracy, and clarity.

In this new edition, authors Joe Sambrook and David Russell have completely updated the book, revising every protocol and adding a mass of new material, to broaden its scope and maintain its unbeatable value for studies in genetics, molecular cell biology, developmental biology, microbiology, neuroscience, and immunology.

Handsomely redesigned and presented in new bindings of proven durability, this three-volume work is essential for everyone using today’s biomolecular techniques.

The opening chapters describe essential techniques, some well-established, some new, that are used every day in the best laboratories for isolating, analyzing and cloning DNA molecules, both large and small.

These are followed by chapters on cDNA cloning and exon trapping, amplification of DNA, generation and use of nucleic acid probes, mutagenesis, and DNA sequencing.

The concluding chapters deal with methods to screen expression libraries, express cloned genes in both prokaryotes and eukaryotic cells, analyze transcripts and proteins, and detect protein-protein interactions.

The Appendix is a compendium of reagents, vectors, media, technical suppliers, kits, electronic resources and other essential information.

As in earlier editions, this is the only manual that explains how to achieve success in cloning and provides a wealth of information about why techniques work, how they were first developed, and how they have evolved

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Editorial Reviews

Designed to be used in tandem with the Web site, this three-volume set provides complete descriptions of 250 laboratory protocols in DNA science<-->35 percent of which were created especially for this edition. Coverage includes techniques for isolating, analyzing and cloning both large and small DNA molecules; cDNA cloning and exon trapping, amplification of DNA, generation and use of nucleic acids probes, mutagenesis, and DNA sequencing; and methods to screen expression libraries, express cloned genes in both pro- and eukaryotic cells, analyze transcripts and proteins, and detect protein-protein interactions. In addition to annotations within protocols, 115 information panels spread throughout the volumes provide insight into the reasons why methods are carried out in a certain manner and how techniques have evolved. The appendices are a compendium of reagents, vectors, media, techniques, suppliers, kits, and electronic resources. Annotation c. Book News, Inc., Portland, OR (
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Product Details

  • ISBN-13: 9780879695774
  • Publisher: Cold Spring Harbor Laboratory Press
  • Publication date: 12/5/2000
  • Edition description: 3 Book Set
  • Edition number: 3
  • Pages: 2368
  • Product dimensions: 9.50 (w) x 13.60 (h) x 4.00 (d)

Meet the Author

Joe Sambrook, Peter MacCallum Cancer Institute, Melbourne, Australia

David Russell, University of Texas Southwestern Medical Center, Dallas

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Read an Excerpt

Nucleic acids do not enter bacteria under their own power, but require assistance traversing the outer and inner cell membranes and in reaching an intracellular site where they can be expressed and replicated. The methods that have been devised to achieve these goals fall into two classes: chemical and physical.

Chemical Methods

E. coli cells washed in cocktails of simple salt solutions achieve a state of competence during which DNA molecules may be admitted to the cell. Most of the chemical methods currently used for bacterial transformation are based on the observations of Mandel and Higa (1970), who showed that bacteria treated with ice-cold solutions of CaC12 and then briefly heated to 37°C or 42°C could be transfected with bacteriophage T, DNA. The same method was subsequently used to transform bacteria with plasmid DNA (Cohen et al. 1972) and E. coli chromosomal DNA (Oishi and Cosloy 1972).

This simple and robust procedure regularly generates between 105 and 106 transformed colonies of E. coli per pg of supercoiled plasmid DNA. This is more than enough for routine tasks such as propagating a plasmid or transferring a plasmid from one strain of E. coli to another. However, higher efficiencies of transformation are required when recovery of every possible clone is of paramount importance, for example, when constructing cDNA libraries or when only minute amounts of foreign DNA are available. Starting in the 1970s and continuing to this day, many variations on the basic technique have been described in the literature, all directed toward optimizing the efficiency of transformation of different bacterial strains by plasmids. The variations include using complex cocktails of divalent cations in different buffers, treating cells with reducing agents, adjusting the ingredients of the cocktail to the genetic constitution of particular strains of E. coh, harvesting cells at specific stages of the growth cycle, altering the temperature of growth of the culture before exposure to chemicals, optimizing the extent and temperature of heat shock, freezing and thawing cells, and exposing cells to organic solvents after washing in divalent cations. By all these treatments and more, it is now possible on a routine basis to achieve transformation frequencies ranging from 106 to 109 transformants/pg of superhelical plasmid DNA (for reviews, please see Hanahan 1987; Hanahan et al. 1995; Hanahan and Bloom, 1996; Hengen 1996).

The improvements in transformation frequency are a tribute to the power of empirical experimentation. How these combinations of chemical agents and physical treatments induce a state of competence remains as obscure today as in Mandel and Higa's time, as does the mechanism by which plasmid DNA enters and establishes itself in competent E. coh. Nevertheless, the improvements made since the late 1970s have eliminated the efficiency of transformation as a potential limiting factor in molecular cloning.

There are two ways to obtain stocks of chemically induced competent E. coli. The first option is to purchase frozen competent bacteria from a commercial source. These products are very reliable and generally yield transformants at frequencies >108 colonies/pg of supercoiled plasmid DNA. However, they are many times more expensive than competent cells prepared in the laboratory. Commercially produced competent cells are nevertheless an excellent yardstick to measure the efficiency of locally generated stocks of competent cells - and they are a godsend to investigators who carry out transformations so infrequently that it is not economical for them to expend the effort required to produce their own competent cultures. In addition, several companies sell competent stocks of strains of E. coli that carry specific genetic markers and are used for particular purposes in molecular cloning. Examples of these include (1) SURE strains, which carry disabling mutations in DNA-repair pathways responsible for the high rate of rearrangement of certain eukaryotic genomic sequences, and (2) strains deficient in methylases such as Dam and Dcm. Plasmids propagated in these strains can be cleaved by restriction enzymes whose activity is normally blocked by methylation of overlapping Dam or Dcm sites. It is cost-effective and far less aggravating to purchase competent stocks of strains such as these, which are tricky to grow and difficult to transform.

For laboratories using standard strains of E. coli, it makes sense to prepare stocks of competent bacteria in-house. The procedure for high-efficiency transformation (Hanahan 1983), described in Protocol 23, works well with K-12 strains of E. coli such as DH 1, DH5, and MM294 and yields competent cultures that can be either used immediately or stored in small aliquots at -70°C until required. If prepared carefully, these competent bacteria can yield up to 109 transformed colonies/pg of supercoiled plasmid DNA. Similar efficiencies can be achieved with the method using "ultra-competent" bacteria (Inoue et al. 1990), described in Protocol 24, in which the bacterial culture is grown at room temperature. However, as discussed above, such high frequencies of transformation are required only rarely; for most routine cloning tasks, competent bacteria prepared by simpler procedures are more than adequate. As a general rule, the more sophisticated the method used to prepare competent cells, the more inconsistent the results. The final method in the series of transformation protocols (Protocol 25) (Cohen et al. 1972) is both robust and durable and yields competent cells that generate 106 to 10' transformed colonies/wg of supercoiled plasmid DNA.

Physical Methods

Exposure to an electrical charge destabilizes the membranes of E. coli and induces the formation of transient membrane pores through which DNA molecules can pass (Neumann and Rosenheck 1972; for reviews, please see Zimmerman 1982; Tsong 1991; Weaver 1993). This method, which is known as electroporation, was originally developed to introduce DNA into eukaryotic cells (Neumann et al. 1982) and was subsequently adapted for transformation of E. coli (Dower et al. 1988; Taketo 1988) and other bacteria by plasmids (Chassy and Flickinger 1987; Fiedler and Wirth 1988; Miller et al. 1988). It is the easiest, fastest, most efficient, and most reproducible method for transformation of bacterial cells with DNA.

Transformation efficiencies in excess of 101° transformants/pg of DNA have been achieved by optimizing various parameters, including the strength of the electrical field, the length of the electrical pulse, the concentration of DNA, and the composition of the electroporation buffer (Dower et al. 1988; Tung and Chow 1995).

More than 80% of the cells in a culture can be transformed to ampicillin resistance by electroporation, and efficiencies approaching the theoretical maximum of one transformant per molecule of plasmid DNA have been reported (Smith et al. 1990).

Plasmids ranging in size from 2.6 kb to 85 kb can be introduced with efficiencies ranging from 6 x 101° transformants/gg of DNA to 1 x 101 transformants/gg DNA, respectively. This is 10-20 times higher than can be achieved with competent cells prepared by chemical methods. Transformation frequencies of this magnitude are especially useful when constructing large and highly complex cDNA libraries (please see Chapter 11).

Electroporation works well with most commonly used laboratory strains of E. coli (Dower et al. 1988; Tung and Chow 1995).

Unlike chemical transformation, the number of transformants generated by electroporation is marker-dependent. For example, when pBR322, which carries genes conferring resistance to two antibiotics (ampicillin and tetracycline), is introduced into E. coli by electroporation, the number of tetracycline-resistant transformants is - 100-fold less than the number of ampicillinresistant transformants (Steele et al. 1994). This effect is not seen when the plasmid is introduced into the bacteria by chemical transformation. A likely explanation is that damage or depolarization caused by the pulse of electrical current prevents or delays insertion into the inner cell membrane of the antiporter protein responsible for tetracycline resistance.

Of course, the ease and efficiency of electroporation come at a price. Electroporation is an expensive business, requiring costly electrical equipment and highly priced specially designed cuvettes. Nevertheless, for many investigators, electroporation, because of its reproducibility and lack of mumbo-jumbo, is the preferred option. For a method for the electroporation of bacterial cells, see Protocol 26. For more details, please see the information panel on ELECTROPORATION.....

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Table of Contents

Chapter 1: Plasmids and Their Usefulness in Molecular Cloning
Chapter 2: Bacteriophage lambda and Its Vectors
Chapter 3: Working with Bacteriophage M13 Vectors
Chapter 4: Working with High-capacity Vectors
Chapter 5: Gel Electrophoresis of DNA and Pulsed-field Agarose Gel Electrophoresis
Chapter 6: Preparation and Analysis of Eukaryotic Genomic DNA
Chapter 7: Extraction, Purification, and Analysis of mRNA from Eukaryotic Cells
Chapter 8: In vitro Amplification of DNA by the Polymerase Chain Reaction
Chapter 9: Preparation of Radiolabeled DNA and RNA Probes
Chapter 10: Working with Synthetic Oligonucleotide Probes
Chapter 11: Preparation of cDNA Libraries and Gene Identification
Chapter 12: DNA Sequencing
Chapter 13: Mutagenesis
Chapter 14: Screening Expression Libraries
Chapter 15: Expression of Cloned Genes in Escherichia coli
Chapter 16. Introducing Cloned Genes into Cultured Mammalian Cells
Chapter 17: Analysis of Gene Expression in Cultured Mammalian Cells
Chapter 18: Protein Interaction Technologies
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The First Edition of This Manual was written some 20 years ago, when basic methods in mol- ecular cloning were far from robust and were established in only a few laboratories. The appearance of that book did much to change the picture. The manual's didactic character gave readers confidence to use techniques that still seemed magical during the late 1970s and early 1980s. The second edition, published just 7 years after the first, was smoother in style and far richer in reliable content. By then, individual methods had become more durable and portable, but it was still difficult to string them successfully together into multistep procedures. This new edition, almost certainly the last to appear in book form, reflects a mature discipline working at full power and with high reliability. During the 1990s, many of the drearier, more repetitive techniques of molecular biology have been automated; demanding, multistep procedures have been converted into kits; high-quality genomic and cDNA libraries are now available from the shelves of commercial manufacturers; and all manipulations involving nucleic acids have benefited greatly from improvements in the quality of reagents and enzymes. As a consequence of these and other advances, competent laboratory workers can now easily avoid experimental problems that beset even the best investigators just a few years ago. This is not to say that everything works perfectly all of the time or that no further improvement is possible. However, difficulties now can largely be avoided by careful planning and application of existing knowledge rather than by experimental trial and error.

A major goal of all three editions of Molecular Cloning has been to provide researchers with up-to-date protocols that work reproducibly. Users of the previous editions will recognize many of the organizational features in the experimental sections of this book. Nevertheless, the revision of the text has been extensive and detailed. Ancient protocols have been modernized, while new protocols have been added to reflect the continuing penetration of molecular cloning into almost all areas of biomedical research. Of equal importance has been our desire to explain how and why particular methods work, and with reasoned arguments for choosing between alternative procedures. This edition therefore contains not only annotations at crucial points in the protocols, but also an abundance of material in the form of Information Panels, which are placed at the end of the chapters as well as in Appendix 9. We hope that these 115 panels spread throughout the three volumes of the book will provide clear insights into the reasons why methods are carried out in a certain manner and how techniques have progressively evolved. Finally, we have provided extensive references to the scientific literature so that curious readers can trace methods and ideas to their roots. Few will read this book from beginning to end. But we hope that the community of cloners will find in these pages much to stimulate the mind and to facilitate the work of their hands.

As might be imagined of a book that has been long in the making, scores of individuals have provided material. We particularly thank Erica Golemis and her colleagues for Chapter 18 on "Protein Interaction Techniques," a large and rapidly changing field that neither of us felt comfortable covering. The people listed on the facing page have all made valuable contributions some verbal, some written, and some corrective - that we gratefully acknowledge. Other colleagues have provided, sometimes unwittingly, critical insights into problems of both style and substance. In addition, the chocolate-loving editorial and production staff at Cold Spring Harbor Laboratory Press have spent thousands of hours meticulously checking references, facts, and grammar and producing, we think, a book of harmonious and elegant design. Without the enduring efforts, diligence, and cheerful dedication of Maryliz Dickerson, Inez Sialiano, Joan Ebert, Mary Cozza, Dorothy Brown, Susan Schaefer, Danny deBruin, Nora McInerny, and Denise Weiss, this book, if it existed at all, would be an embarrassment. The manual could not have been completed without the patient understanding and speedy responses of the librarians at Cold Spring Harbor Laboratory, The University of Texas Southwestern Medical Center at Dallas, and the Peter MacCallum Cancer Institute, Melbourne.

We owe deep debts to our Associate Authors, Kaaren Janssen and Nina Irwin, who have given us unstinting support, expert work, clarifying ideas, and dedicated and unflagging optimism. Sian Curtis and Michael Zierler ironed out scientific problems in the protocols, and Sian also assembled the appendices. Mark Curtis converted rough drafts drawn on scraps of paper into elegant and intelligent illustrations. All of these people came up with many good suggestions. Foolishly, perhaps, we did not accept them all, so any remaining errors of fact or interpretation are ours alone. Personal debts can never be adequately acknowledged. Jan Argentine, our Managing Editor, has given us support in more ways than we can list here. She has given ungrudgingly of her time and has brought common sense, order, civility, and timeliness to a process that sometimes threatened to fall out of control. No writers could have received greater help and friendship. We owe special thanks to Daphne Davis, who cheerfully provided answers to many questions concerning experimental details. We have also benefited from the encouragement and sustaining enthusiasm of many others - in particular, John Inglis and Jim Watson at Cold Spring Harbor, Nancy Ford, who worked with us during the early stages of this project, and Rose Williams in Melbourne. Kate Simpson, a person of rare charm and intelligence, worked on the manual for a few months but did not live long enough to see the project completed. We hope that some of her lively grace shines through these pages. Finally, we owe an unquantifiable debt to our families, who have seen these three volumes built sentence by sentence and whose encouragement has never flagged.

Joe Sambrook

David Russell

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  • Anonymous

    Posted September 21, 2001

    Essential Reference Book

    A classic reference work in Molecular Biology, newly and completely updated. It has extensive coverage of nearly all core molecular biology techniques and often presents several different means of accomplishing the same goal. The website is easy to access and the protocols are clearly written. The website makes it easy to print out protocols. In addition each protocol contains references to the primary literature supporting its claims. In the internet version of the protocol the references are hotlinked to PubMed, greatly simplifying access to the underlying references. I definitely recommend this book to all labs using molecular biology. Make sure to check out the website!

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  • Anonymous

    Posted September 29, 2001

    The molecular biology bible

    Any lab serious about molecular biology has this book. A lab staple for over 20 years the new updated version is even more comprehensive reflecting the continuing development of molecular biology. Most importantly the series still retains the historical significance and the background or reference material for many of the techniques. A must have!

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  • Anonymous

    Posted August 14, 2001

    Molecular cloning-reprinted and ready to go!

    Molecular cloning has been a lab staple for years. Now reprinted so you can update the old lab copy worn out by years of student use! Its a must have for any lab serious about molecular biology. Its also useful for student training. Many times there are simple explanations for the lab techniques we have adopted as dogma, but are unsure why. Molecular cloning has the answers and is a great resource. I highly recommend this book for its depth and breadth of protocols and guidance in the complicated realm of cloning!

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  • Anonymous

    Posted August 15, 2001

    a laboratory bible!

    Sambrook and Russell answer every question you could think of, and then some. This book is a goldmine of information, packed with protocols, but also filled with the extra information that transforms a simple set of instructions into an amazingly helpful how-to manual. The approach taken is that of an experimentor (ie. 'How to win the battle with RNase'--it really is a battle!), with hints and suggestions usually learned by watching an old pro. The information is well-organized, and very well illustrated to give a clear view of how an experiment is performed and especially the logic behind it. The manual also satisfies the insatiable curiosity of a scientist rather than a technician: how do pharmacological agents work? What are their structures? And the troubleshooting sections provide an invaluable resource. All in all, Sambrook and Russell have created an essential weapon in any scientist's artillery.

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