- Email: [email protected]
Protein–protein recognition
Book reviews / Biochemistry and Molecular Biology Education 29 (2001) 209–218
ManiatisFMolecular Cloning, A Laboratory Manual by Sambrook and Russell. P. Lu Department of Chemistry, University of Pennsylvania, Philadelphia, PA, USA
PII: S 1 4 7 0 - 8 1 7 5 ( 0 1 ) 0 0 0 6 5 - 0
Protein purification applications. A practical approach (2nd edtion) S. Roe (Ed.); Oxford University Press, Oxford, 2001, 184 pp., price $45 paper back, $90 cloth, ISBN 0-19963671-0 When I receive a book for review, I avoid even glancing at the commercial hype on the back cover. Instead, I read the book and then try to imagine I am the editor responsible for writing the back copy. Comparing the two versions, mine and the actual copy, can often be quite revealing and in some cases I am not sure I read the same book. I am happy to report that for this book, Protein Purification Applications, the back cover is right on the mark. This new edition, edited by Simon Roe, fits appropriately into the Practical Approach Series of Oxford University Press. The Preface states that the book contains strategies, detailed procedures, and practical advice for isolating and purifying proteins from common sources. A listing of the chapters confirms this: (1) Fusion protein purification methods; (2) Initial purification of inclusion bodies; (3) Purification for crystallography; (4) Protein purification from mammalian cell culture; (5) Protein purification from microbial cell culture; (6) Protein purification from milk; (7) Protein purification from animal tissue; and (8) Protein purification from plant sources. Most of the topics are of interest to the general practitioner. The main exception is the chapter on the purification of milk proteins, which, although well written, is designed for the specialist. A full array of laboratory procedures is discussed, but throughout the book there is a clear emphasis on chromatographic techniques, as there should be. Separations by chromatography have been the mainstay for past and present work in protein purification, and all indications are that the importance of this technique will continue in the future. Even though each chapter has a different set of authors, the editor has insisted on uniformity, clarity, and balance. Chapter lengths range from 10 pages to over 30 and more important topics are
211
given appropriate space. Mistakes, typographical errors, etc., are rare. This book is written for the lab worker. Theory is dispensed with by a few references to the original literature and authors jump into discussions on procedure. What I found most appealing in the book was the inclusion of about 46 detailed protocols describing actual procedures for protein purification. A bonus for the reader is a ‘‘List of suppliers’’ with contact information for about 50 companies that are primarily in the United Kingdom and the USA. No book is perfect. What are the concerns in this one? In a word, timeliness. The most recent references in any chapter are from 1998 and these are scarce. One would think that with the advent of proteomics and the present and future interest in proteins, a book on this topic and of this small size could have had a shorter publication schedule. But then, this is not a book on proteomics; it is a ‘‘nuts and bolts’’ guide to protein purification, and techniques do tend to have a lasting value. Should you rush a memo to your library to order the book? Probably not. One of the more comprehensive books now available would better serve the purposes of library readers. Should you order a copy for your research lab? If you are in the business of purifying proteins or think you may be in the future, the $45 for a paper copy is a good investment. Rodney Boyer Hope College Chemistry Department, Holland, MI 49423, USA E-mail address: [email protected]
PII: S 1 4 7 0 - 8 1 7 5 ( 0 1 ) 0 0 0 7 5 - 3
Protein–protein recognition Colin Kleanthous (Ed.); Oxford University Press, Oxford, 2000, 314pp, PriceUS$110.00 /d65.00 hardback or US$55.0/d32.50 paperback, ISBN 0-19-9637-61-X hardback and 0-19-9637-60-1 paperback There is the impression that the work on the human genome and other species recently has dominated biochemistry and molecular biology, so in the postgenome era there has been a call from leaders, like Sydney Brenner, for a reversion to biochemistry and, in particular, the study of the interaction of proteins. For some old hands this has led to a wry smile for we recall an immense, and largely successful, effort to elucidate the metabolism of living cells, an area of research almost unknown to many bioinformaticians (I am told that this
212
Book reviews / Biochemistry and Molecular Biology Education 29 (2001) 209–218
is the preferred word). Nevertheless, it must be admitted that great progress has been made in methods for the separation and isolation of minute quantities of proteins and the determination of their structure. An added bonus is that it is possible to modify proteins at will by site-directed mutagenesis. Moreover, the bioinformaticians should surely be encouraged in their attempts to understand the rules that determine the tertiary structure of proteins and their interaction with one another. This book is, therefore, very welcome especially as the contributors are experts in their fields. The first chapter by Joel Janin is entitled ‘‘Kinetics and thermodynamics of protein–protein interactions’’. He explains that the aim of physical chemistry of noncovalent protein–protein interactions is to answer some fundamental questions: what is the origin of the affinity which enables biological macromolecules to form stable, complex, self assemblies; what governs the specificity which makes these assemblies unique, which physical features give them functions that their individual components do not have? He concludes that in protein–protein recognition the diversity of the surfaces involved is great and there is no recurrent structural binding motif. Perhaps, in contradiction, Susan Jones and Janet Thornton, in their chapter ‘‘Analysis and classification of protein–protein interactions from a structural perspective’’, conclude that the protein– protein associations do share common characteristics. They say that such sites are hydrophobic, planar and complementary and have the potential to form electrostatic interactions. They start from the 12,000 structures in the Brookhaven Protein Data Bank many of which are protein complexes. They go on to describe a number of methods based on searching for hydrophobic patches on protein surfaces, together with a method of searching for patches for multiple properties matching specific criteria. In the third chapter, Scott Mathews, Grant Mauk and Geoffrey Moore describe protein complexes involving electron transfer proteins. They conclude that such complexes exhibit properties which distinguish them from other complexes. Martin Humphries and Robert Liddington describe the ‘‘Molecular basis of integrin dependent cell adhesion’’. They find that integrins have up to three, but typically two, distinct ligand-binding sites which are intimately linked and allosterically controlled. The simplest allosteric model with which they could try to explain the ligand binding behaviour of integrins is the two-state model formulated by Monod and colleagues. The two states are two distinct quaternary arrangements (T and R) for the ab-hetero dimer, which generates four distinct energetic states : unliganded T (Tu); liganded T (TL); unliganded R (Ru); and liganded R (RL). The interaction of protein antigens and antibodies is considered by Bradford Braden and Robert Poljak in a
chapter on ‘‘Structure and energetics of anti–lysozyme antibodies’’. This is an impressive chapter but they warn that conclusions derived from this particular system may not hold for antibodies in general. They start by stating that the primary paradigm of antibody–antigen interaction is that the three-dimensional structure of the six hypervariable loops, or complementary-determining regions, of an antibody molecule recognize and bind a specific antigenic surface which is also determined by the three dimensional structure of the antigen. Another immunological chapter follows by Tim Dafforn and Arthur Lesk on ‘‘Proteins of the major histocompatibility complex and their interactions with T-cell receptors’’. Mark Hyvonen, Jake Begun and Tom Blundell describe ‘‘Protein–protein interactions in eukaryotic signal transduction’’. They are concerned with two major superfamilies of globular proteins that are widely involved in signal transduction: the G proteins, which occur in smaller G-protein switches like Ras and as part of the heterotrimeric G proteins and the cyclindependent kinases that are involved in the cell cycle. They include small signalling modules recognizing linear epitopes, G proteins binding to various effectors and regulators in a nucleotide-dependent manner and interactions of cyclin-dependent kinases with activators and inhibitors. They point out that such protein–protein interactions are often transient and require careful control–mutations affecting the regulation of the system can have severe consequences, such as the induction of cancer. Michael Laskowski, Jr Qasim and Stephen Lu have a chapter entitled ‘‘Interaction of standard mechanism, canonical protein inhibitors with serine proteinases’’. Proteinases used to have a rather dull image but no more and the authors of this chapter rightly list their major importance in cell biology. They describe the methods used to control proteinase activity by precursor synthesis and the use of specific inhibitors. They make an interesting point that since most precursor proteinases have an additional peptide at the amino terminus; partially synthesized proteins are never active since protein synthesis proceeds from the N-terminus. In respect to inhibitors they make a division between inhibitors devoid of significant class specificity and classspecific inhibitors. The final chapter is on ‘‘Nuclease inhibitors’’ by Colin Kleanthous and Ansgar Pommer. Inhibition of nuclease activity is accomplished either by binding to the active site directly, with the inhibitor mimicking part of the nucleic acid substrate with amino-acid side chains (ribonuclease inhibitor and barstar) or indirectly by binding to an adjacent site and sterically blocking substrate binding (Im9, a colcicin DNase-specific immunity protein). They conclude that, irrespective of the system under study, the kinetics and thermodynamics of complex formation are very similar. In each of
Book reviews / Biochemistry and Molecular Biology Education 29 (2001) 209–218
the cases mentioned the enzyme is a basic protein bound by an acidic inhibitor. The book is nicely produced with a generous list of references. The distinguished authors have produced scholarly contributions which can be thoroughly recommended for those concerned with this important field. Peter N. Campbell Department of Biochemistry and Molecular Biology University College London, Gower Street, London WC1E 6BT, UK E-mail address: [email protected] PII: S 1 4 7 0 - 8 1 7 5 ( 0 1 ) 0 0 0 6 7 - 4
Unraveling DNAFmolecular biology for the laboratory M.R. Winfrey, M.A. Rott and A.T. Wortman; PrenticeHall, Inc., New York, 1997, 370 pp., price $51.00 Unraveling DNA is a molecular biology lab manual that takes students through all the techniques involved in the isolation, cloning, and characterization of the bioluminescence genes of the lux operon. Unraveling DNA is based on the pedagogical premise that techniques are learned and retained best when presented in an engaging biological context. This lab manual is an excellent example of this pedagogy in practice. The foreward by Prof. Kenneth Nealson provides infectious enthusiasm for the strategy taken by these authors. Indeed, throughout this lab manual the authors’ enthusiasm for exploring bioluminescence draws students into the innate coolness of cloning bioluminescence genes. Each chapter in the text takes the student through a step in the isolation, cloning, mapping, and sequence analysis of the lux operon, each of which can be accomplished in a 3-h lab period. Hence, a two-credit lab course can complete all the chapters that accomplish this feat (22 chapters out of 28). The situation is more problematic for a one-credit lab course, however. Nonetheless, an instructor can streamline the curriculum and successfully clone and analyze these genes meeting just once a week for 3 h. In addition, this text can serve as a lab manual for a more advanced course in molecular biology. For instance, Exercise 3 is an experiment in which students isolate bacteria from natural sources (quite easy to do in landlocked areas it seems). After one isolates bioluminescent bacteria from some ‘‘fishy’’ source, then one can jump to Exercise 23 and use PCR to amplify prospective bioluminescence genes. From here one can carry out the following few exercises, and then go back to earlier ones to clone and
213
characterize these genes. Identification of bacterial clones containing the lux genes is quite funFturn off the lights and look for colonies that light up in the dark! For purists who must expose their students to colony hybridization, Exercise 21 utilizes this technique to identify clones containing one of the lux genes in a subcloning exercise. The last section of the book (Advanced Techniques) includes PCR, pulse-field electrophoresis, DNA sequencing, and DNA sequence analysis using BLAST, all of which can be seamlessly incorporated into the cloning and analysis of the lux genes. The authors are keenly focused on an undergraduate audience. Important steps common to many procedures are highlighted repeatedly; reaction setups are organized into tables that students can use and extend; basic procedures are depicted in excellent diagrams or very useful photographs; and the protocols are very easy to follow. In addition, many of the chapters have (photocopiable) graph paper (in simple and semi-log formats). This lab manual contains valuable reference information for students well beyond their experience in a lab course using this manual. Most of this information is collected in a large set of appendices (B90 pp.) These appendices cover metric system basics; electrophoresis; nucleic acid hybridization; the care and use of enzymes; care and handling of hazardous materials, including radioisotopes; and a nice discussion of ethanol precipitation of nucleic acids Also included in these appendices are maps of cloning vectors, lists of supplies one needs in a molecular biology lab, and the names and addresses of major suppliers. So, while this lab manual is largely a ‘‘cookbook’’ in its basic format, it is also a fine reference text for students who go onto work in a molecular biology laboratory. Unfortunately, its usefulness as a reference text is hindered by the absence of an index. There are no egregious omissions or oversights, but several noteworthy ones caught my attention. In an otherwise careful discussion of the use of micropipettors, the handling of viscous solutions (especially enzymes stored in 50% glycerol) is not discussed. This is a common source of problems in an undergraduate setting. The discussion of the electrophoretic results of the digestion of plasmids with restriction enzymes is incomplete. Here, there is no photograph or discussion of the electrophoretic behavior of uncut, linear, and open circle DNA, which are all commonly seen in these preparations and can easily lead to confusion among neophytes. A step that can thwart any cloning operation is the unability to digest DNA with restriction enzymes. Hence, I am a bit surprised that the authors do not provide more detailed, cautionary information in the removal of culture medium from bacterial pellets and the white precipitate that forms after the addition of
ManiatisFMolecular Cloning, A Laboratory Manual by Sambrook and Russell. P. Lu Department of Chemistry, University of Pennsylvania, Philadelphia, PA, USA
PII: S 1 4 7 0 - 8 1 7 5 ( 0 1 ) 0 0 0 6 5 - 0
Protein purification applications. A practical approach (2nd edtion) S. Roe (Ed.); Oxford University Press, Oxford, 2001, 184 pp., price $45 paper back, $90 cloth, ISBN 0-19963671-0 When I receive a book for review, I avoid even glancing at the commercial hype on the back cover. Instead, I read the book and then try to imagine I am the editor responsible for writing the back copy. Comparing the two versions, mine and the actual copy, can often be quite revealing and in some cases I am not sure I read the same book. I am happy to report that for this book, Protein Purification Applications, the back cover is right on the mark. This new edition, edited by Simon Roe, fits appropriately into the Practical Approach Series of Oxford University Press. The Preface states that the book contains strategies, detailed procedures, and practical advice for isolating and purifying proteins from common sources. A listing of the chapters confirms this: (1) Fusion protein purification methods; (2) Initial purification of inclusion bodies; (3) Purification for crystallography; (4) Protein purification from mammalian cell culture; (5) Protein purification from microbial cell culture; (6) Protein purification from milk; (7) Protein purification from animal tissue; and (8) Protein purification from plant sources. Most of the topics are of interest to the general practitioner. The main exception is the chapter on the purification of milk proteins, which, although well written, is designed for the specialist. A full array of laboratory procedures is discussed, but throughout the book there is a clear emphasis on chromatographic techniques, as there should be. Separations by chromatography have been the mainstay for past and present work in protein purification, and all indications are that the importance of this technique will continue in the future. Even though each chapter has a different set of authors, the editor has insisted on uniformity, clarity, and balance. Chapter lengths range from 10 pages to over 30 and more important topics are
211
given appropriate space. Mistakes, typographical errors, etc., are rare. This book is written for the lab worker. Theory is dispensed with by a few references to the original literature and authors jump into discussions on procedure. What I found most appealing in the book was the inclusion of about 46 detailed protocols describing actual procedures for protein purification. A bonus for the reader is a ‘‘List of suppliers’’ with contact information for about 50 companies that are primarily in the United Kingdom and the USA. No book is perfect. What are the concerns in this one? In a word, timeliness. The most recent references in any chapter are from 1998 and these are scarce. One would think that with the advent of proteomics and the present and future interest in proteins, a book on this topic and of this small size could have had a shorter publication schedule. But then, this is not a book on proteomics; it is a ‘‘nuts and bolts’’ guide to protein purification, and techniques do tend to have a lasting value. Should you rush a memo to your library to order the book? Probably not. One of the more comprehensive books now available would better serve the purposes of library readers. Should you order a copy for your research lab? If you are in the business of purifying proteins or think you may be in the future, the $45 for a paper copy is a good investment. Rodney Boyer Hope College Chemistry Department, Holland, MI 49423, USA E-mail address: [email protected]
PII: S 1 4 7 0 - 8 1 7 5 ( 0 1 ) 0 0 0 7 5 - 3
Protein–protein recognition Colin Kleanthous (Ed.); Oxford University Press, Oxford, 2000, 314pp, PriceUS$110.00 /d65.00 hardback or US$55.0/d32.50 paperback, ISBN 0-19-9637-61-X hardback and 0-19-9637-60-1 paperback There is the impression that the work on the human genome and other species recently has dominated biochemistry and molecular biology, so in the postgenome era there has been a call from leaders, like Sydney Brenner, for a reversion to biochemistry and, in particular, the study of the interaction of proteins. For some old hands this has led to a wry smile for we recall an immense, and largely successful, effort to elucidate the metabolism of living cells, an area of research almost unknown to many bioinformaticians (I am told that this
212
Book reviews / Biochemistry and Molecular Biology Education 29 (2001) 209–218
is the preferred word). Nevertheless, it must be admitted that great progress has been made in methods for the separation and isolation of minute quantities of proteins and the determination of their structure. An added bonus is that it is possible to modify proteins at will by site-directed mutagenesis. Moreover, the bioinformaticians should surely be encouraged in their attempts to understand the rules that determine the tertiary structure of proteins and their interaction with one another. This book is, therefore, very welcome especially as the contributors are experts in their fields. The first chapter by Joel Janin is entitled ‘‘Kinetics and thermodynamics of protein–protein interactions’’. He explains that the aim of physical chemistry of noncovalent protein–protein interactions is to answer some fundamental questions: what is the origin of the affinity which enables biological macromolecules to form stable, complex, self assemblies; what governs the specificity which makes these assemblies unique, which physical features give them functions that their individual components do not have? He concludes that in protein–protein recognition the diversity of the surfaces involved is great and there is no recurrent structural binding motif. Perhaps, in contradiction, Susan Jones and Janet Thornton, in their chapter ‘‘Analysis and classification of protein–protein interactions from a structural perspective’’, conclude that the protein– protein associations do share common characteristics. They say that such sites are hydrophobic, planar and complementary and have the potential to form electrostatic interactions. They start from the 12,000 structures in the Brookhaven Protein Data Bank many of which are protein complexes. They go on to describe a number of methods based on searching for hydrophobic patches on protein surfaces, together with a method of searching for patches for multiple properties matching specific criteria. In the third chapter, Scott Mathews, Grant Mauk and Geoffrey Moore describe protein complexes involving electron transfer proteins. They conclude that such complexes exhibit properties which distinguish them from other complexes. Martin Humphries and Robert Liddington describe the ‘‘Molecular basis of integrin dependent cell adhesion’’. They find that integrins have up to three, but typically two, distinct ligand-binding sites which are intimately linked and allosterically controlled. The simplest allosteric model with which they could try to explain the ligand binding behaviour of integrins is the two-state model formulated by Monod and colleagues. The two states are two distinct quaternary arrangements (T and R) for the ab-hetero dimer, which generates four distinct energetic states : unliganded T (Tu); liganded T (TL); unliganded R (Ru); and liganded R (RL). The interaction of protein antigens and antibodies is considered by Bradford Braden and Robert Poljak in a
chapter on ‘‘Structure and energetics of anti–lysozyme antibodies’’. This is an impressive chapter but they warn that conclusions derived from this particular system may not hold for antibodies in general. They start by stating that the primary paradigm of antibody–antigen interaction is that the three-dimensional structure of the six hypervariable loops, or complementary-determining regions, of an antibody molecule recognize and bind a specific antigenic surface which is also determined by the three dimensional structure of the antigen. Another immunological chapter follows by Tim Dafforn and Arthur Lesk on ‘‘Proteins of the major histocompatibility complex and their interactions with T-cell receptors’’. Mark Hyvonen, Jake Begun and Tom Blundell describe ‘‘Protein–protein interactions in eukaryotic signal transduction’’. They are concerned with two major superfamilies of globular proteins that are widely involved in signal transduction: the G proteins, which occur in smaller G-protein switches like Ras and as part of the heterotrimeric G proteins and the cyclindependent kinases that are involved in the cell cycle. They include small signalling modules recognizing linear epitopes, G proteins binding to various effectors and regulators in a nucleotide-dependent manner and interactions of cyclin-dependent kinases with activators and inhibitors. They point out that such protein–protein interactions are often transient and require careful control–mutations affecting the regulation of the system can have severe consequences, such as the induction of cancer. Michael Laskowski, Jr Qasim and Stephen Lu have a chapter entitled ‘‘Interaction of standard mechanism, canonical protein inhibitors with serine proteinases’’. Proteinases used to have a rather dull image but no more and the authors of this chapter rightly list their major importance in cell biology. They describe the methods used to control proteinase activity by precursor synthesis and the use of specific inhibitors. They make an interesting point that since most precursor proteinases have an additional peptide at the amino terminus; partially synthesized proteins are never active since protein synthesis proceeds from the N-terminus. In respect to inhibitors they make a division between inhibitors devoid of significant class specificity and classspecific inhibitors. The final chapter is on ‘‘Nuclease inhibitors’’ by Colin Kleanthous and Ansgar Pommer. Inhibition of nuclease activity is accomplished either by binding to the active site directly, with the inhibitor mimicking part of the nucleic acid substrate with amino-acid side chains (ribonuclease inhibitor and barstar) or indirectly by binding to an adjacent site and sterically blocking substrate binding (Im9, a colcicin DNase-specific immunity protein). They conclude that, irrespective of the system under study, the kinetics and thermodynamics of complex formation are very similar. In each of
Book reviews / Biochemistry and Molecular Biology Education 29 (2001) 209–218
the cases mentioned the enzyme is a basic protein bound by an acidic inhibitor. The book is nicely produced with a generous list of references. The distinguished authors have produced scholarly contributions which can be thoroughly recommended for those concerned with this important field. Peter N. Campbell Department of Biochemistry and Molecular Biology University College London, Gower Street, London WC1E 6BT, UK E-mail address: [email protected] PII: S 1 4 7 0 - 8 1 7 5 ( 0 1 ) 0 0 0 6 7 - 4
Unraveling DNAFmolecular biology for the laboratory M.R. Winfrey, M.A. Rott and A.T. Wortman; PrenticeHall, Inc., New York, 1997, 370 pp., price $51.00 Unraveling DNA is a molecular biology lab manual that takes students through all the techniques involved in the isolation, cloning, and characterization of the bioluminescence genes of the lux operon. Unraveling DNA is based on the pedagogical premise that techniques are learned and retained best when presented in an engaging biological context. This lab manual is an excellent example of this pedagogy in practice. The foreward by Prof. Kenneth Nealson provides infectious enthusiasm for the strategy taken by these authors. Indeed, throughout this lab manual the authors’ enthusiasm for exploring bioluminescence draws students into the innate coolness of cloning bioluminescence genes. Each chapter in the text takes the student through a step in the isolation, cloning, mapping, and sequence analysis of the lux operon, each of which can be accomplished in a 3-h lab period. Hence, a two-credit lab course can complete all the chapters that accomplish this feat (22 chapters out of 28). The situation is more problematic for a one-credit lab course, however. Nonetheless, an instructor can streamline the curriculum and successfully clone and analyze these genes meeting just once a week for 3 h. In addition, this text can serve as a lab manual for a more advanced course in molecular biology. For instance, Exercise 3 is an experiment in which students isolate bacteria from natural sources (quite easy to do in landlocked areas it seems). After one isolates bioluminescent bacteria from some ‘‘fishy’’ source, then one can jump to Exercise 23 and use PCR to amplify prospective bioluminescence genes. From here one can carry out the following few exercises, and then go back to earlier ones to clone and
213
characterize these genes. Identification of bacterial clones containing the lux genes is quite funFturn off the lights and look for colonies that light up in the dark! For purists who must expose their students to colony hybridization, Exercise 21 utilizes this technique to identify clones containing one of the lux genes in a subcloning exercise. The last section of the book (Advanced Techniques) includes PCR, pulse-field electrophoresis, DNA sequencing, and DNA sequence analysis using BLAST, all of which can be seamlessly incorporated into the cloning and analysis of the lux genes. The authors are keenly focused on an undergraduate audience. Important steps common to many procedures are highlighted repeatedly; reaction setups are organized into tables that students can use and extend; basic procedures are depicted in excellent diagrams or very useful photographs; and the protocols are very easy to follow. In addition, many of the chapters have (photocopiable) graph paper (in simple and semi-log formats). This lab manual contains valuable reference information for students well beyond their experience in a lab course using this manual. Most of this information is collected in a large set of appendices (B90 pp.) These appendices cover metric system basics; electrophoresis; nucleic acid hybridization; the care and use of enzymes; care and handling of hazardous materials, including radioisotopes; and a nice discussion of ethanol precipitation of nucleic acids Also included in these appendices are maps of cloning vectors, lists of supplies one needs in a molecular biology lab, and the names and addresses of major suppliers. So, while this lab manual is largely a ‘‘cookbook’’ in its basic format, it is also a fine reference text for students who go onto work in a molecular biology laboratory. Unfortunately, its usefulness as a reference text is hindered by the absence of an index. There are no egregious omissions or oversights, but several noteworthy ones caught my attention. In an otherwise careful discussion of the use of micropipettors, the handling of viscous solutions (especially enzymes stored in 50% glycerol) is not discussed. This is a common source of problems in an undergraduate setting. The discussion of the electrophoretic results of the digestion of plasmids with restriction enzymes is incomplete. Here, there is no photograph or discussion of the electrophoretic behavior of uncut, linear, and open circle DNA, which are all commonly seen in these preparations and can easily lead to confusion among neophytes. A step that can thwart any cloning operation is the unability to digest DNA with restriction enzymes. Hence, I am a bit surprised that the authors do not provide more detailed, cautionary information in the removal of culture medium from bacterial pellets and the white precipitate that forms after the addition of