Preface Recombinant DNA methods are powerful, revolutionary techniques for at least two reasons. First, they allow the isolation of single genes in large amounts from a pool of thousands or millions of genes. Second, the isolated genes or their regulatory regions can be modified at will and re- introduced into cells for expression at the RNA or protein levels. These attributes allow us to solve complex biological problems and to produce new and better products in the areas of health, agriculture, and industry. Volumes 153, 154, and 155 supplement Volumes 68, 100, and 101 of Methods in Enzyrnology. During the past few years, many new or im- proved recombinant DNA methods have appeared, and a number of them are included in these three new volumes. Volume 153 covers methods related to new vectors for cloning DNA and for expression of cloned genes. Volume 154 includes methods for cloning eDNA, identification of cloned genes and mapping of genes, chemical synthesis and analysis of oligodeoxynucleotides, site-specific mutagenesis, and protein engineer- ing. Volume 155 includes the description of several useful new restriction enzymes, detail of rapid methods for DNA sequence analysis, and a num- ber of other useful methods. RAY Wu LAWRENCE GROSSMAN xiii NATHAN O. KAPLAN June 25, 1917-April 15, 1986 Nathan O. Kaplan In the past half century, knowledge in the natural sciences has pro- gressed at a rate unmatched in previous history. Biochemistry appears closer than ever to the attainment of its ultimate objective: creation of a body of knowledge rationalized in a conceptual structure which provides a solid basis for understanding life processes. In these fabulous times, there have been fabulous people among whom may be included Nathan ("Nate") Kaplan. His many, varied and massive contributions to cru- cially important areas of biochemical research added to his creative activ- ities as an editor, scholar, and academic statesman have left a lasting impression on the history of these exciting times. We are fortunate in having an account of his life philosophy and experiences which he himself provided in "Selected Topics in the History of Biochemistry" (edited by G. Semenga; Vol. 30, p. 255 et seq. ; Elsevier Science Publishers). His potential was manifest early in his career at Berkeley where he collaborated with Barker, Hassid, and Doudoroff in the late 1930s, pro- viding biochemical expertise crucial for the demonstration that in the phosphorolysis of sucrose the phosphate ester formed was glucose 1-phosphate. His first scientific publication on sucrose phosphorylase in- cluded an account of these seminal researches. His full potential was realized when, under the watchful eye of Fritz Lipmann, his great mentor and life-long admirer and friend, he made essential contributions in col- laboration with Lipmann and Dave Novelli to the isolation and character- ization of coenzyme A, work which later formed part of the basis for the Nobel Prize to Lipmann. Nate followed his unerring intuition in continuing his career at the McCollum-Pratt Institute under the aegis of W. D. McElroy. He built a body of research on NAD, NAD analogs, and associated dehydrogenases to earn a leading position as an international authority on the pyridine nucleotide coenzymes. In the course of these investigations he began a life-long collaboration with another "biochemist's biochemist" Sidney Colowick which resulted in the creation of the monumental series Meth- ods in Enzymology, which was to become the definitive source of method- ology in the biochemical sciences. Nate, as he so vividly detailed in the account I have referred to above, stressed the importance of following research wherever it led, even if assured results might not be immediately evident. As an example, one notes that his investigations of the pyridine nucleotide cofactors ignited XV xvi NATHAN O. KAPLAN an interest in comparative biochemistry, elaborated in many researches of major significance for biochemical evolution. Nate's intuitive insights into things biochemical also extended to an uncanny ability to assess potential in budding biochemists. His success in finding and recruiting talent was never better shown than in the creation of the Graduate Department of Biochemistry at Brandeis in the late 1950s. Those in the remarkable group he assembled which included W. Jencks, L. Grossman, G. Sato, M. E. Jones, L. Levine, H. Van Vunakis, and J. Lowenstein owed their start in large part to his unstinting guidance and encouragement. He found time to serve on a multitude of policy-making committees and was always available, however hard pressed, to take over editorial chores, however onerous. I recall the many hours he spent helping to organize and edit a Festschrift and symposium celebrating the fact I had survived to age 65. And then there was the salvage and rebuilding opera- tion he so unselfishly initiated to revive the ailing Analytical Biochemistry journal when his old friend, A1 Nason, its Editor-in-Chief, fell seriously ill. No project engaged Nate's attention and devotion more than his la- bors with Colowick to oversee and assure the publication and excellence of the many volumes which make up the Methods in Enzymology series, now numbering more than a hundred, which will stand as a lasting monu- ment to his memory. Certainly nothing could be more appropriate than the present dedication. MARTIN D. KAMEN Contributors to Volume 153 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. GYNI-IEUNG AN (17), Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164 PAUL BATES (6), Department of Microbiol- ogy, University of California, San Fran- cisco, San Francisco, California 94143 CHRISTOPH F. BECK (28), Institut fiir Bi- ologie III, Albert-Ludwigs-Universitgit, D-7800 Freiburg i. Br., Federal Republic of Germany RAMA M. BELAGAJE (25), Department of Molecular Biology, Lilly Research Labo- ratories, A Division of Eli Lilly and Com- pany, Lilly Corporate Center, Indianapo- lis, Indiana 46285 MERVYN J. BmB (9), Department of Ge- netics, John Innes Institute, Norwich NR4 7UH, England GRANT A. BITTER (33), AMGen, Thousand Oaks, California 91320 Jt3RGEN BROSIUS (4), Department of Genet- ics and Development and Center for Neu- robiology and Behavior, Columbia Uni- versity, New York, New York 10032 FRANqOISE BRUNEL (3), Unit of Molecular Biology, International Institute of Cellu- lar and Molecular Pathology, B-1200 Brussels, Belgium JUDY BRUSSLAN (12), Department of Molec- ular Genetics and Cell Biology, The Uni- versity of Chicago, Chicago, Illinois 60637 JuDY CALLIS (21), Horticulture Depart- ment, University of Wisconsin, Madison, Wisconsin 53706 SHING CHANG (32), Microbial Genetics, Ce- tus Corporation, Emeryville, California 94608 KEITH F. CHATER (9), Department of Ge- netics, John Innes Institute, Norwich NR4 7UH, England JOHN DAVlSON (3), Unit of Molecular Biol- ogy, International Institute of Cellular and Molecular Pathology, B-1200 Brus- sels, Belgium R. DEBLAERE (16), Laboratorium voor Genetica, RUksuniversiteit Gent, B-9000 Gent, Belgium HERMAN A. DE BOER (27), Department of Biochemistry of the Gorlaeus Laboratory, University of Leiden, 2300 RA Leiden, The Netherlands GuY O. DUFFAUD (31), Department of Bio- chemistry, State University of New York at Stony Brook, Stony Brook, New York 11794 JAMES E. DUTCnIK (5), Department of Ge- netics, Washington University School of Medicine, St. Louis, Missouri 63110 KEVIN M. EGAN (33), AMGen, Thousand Oaks, California 91320 STEVEN G. ELLIOTT (33), AMGen, Thou- sand Oaks, California 91320 WALTER FlEas (26), Laboratory of Molecu- lar Biology, State University of Ghent, B-9000 Ghent, Belgium R. T. FRALEY (15), Plant Molecular Biology Group, Biological Sciences Department, Corporate Research and Development Staff, Monsanto Company, Chesterfield, Missouri 63198 A. M. FRISCHAUF (8), European Molecular Biology Laboratory, D-6900 Heidelberg, Federal Republic of Germany MICHAEL FROMM (21), United States De- partment of Agriculture, Agricultural Re- search Service, Pacific Basin Area Plant Gene Expression Center, Albany, Califor- nia 94710 JAMES C. GIFFIN (33), AMGen, Thousand Oaks, California 91320 ix X CONTRIBUTORS TO VOLUME 153 SUSAN S. GOLDEN (12), Department of Biol- ogy, Texas A&M University, College Sta- tion, Texas 77843 ROBERT HASELKORN (12), Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois 60637 CYNTHIA HELMS (5), Collaborative Re- search, Inc., Lexington, Massachusetts 02173 J P. HERNALSTEENS (16), Laboratorium Genetische Virologie, Vr~ie Universiteit Brussel, B-1640 Sint-Genesius-Rode, Belgium MICHEL HEUSTERSPREUTE (3), Unit of Mo- lecular Biology, International Institute of Cellular and Molecular Pathology, B-1200 Brussels, Belgium H. HOFTE (16), Plant Genetic Systems, Inc., B-9000 Ghent, Belgium PAUL J. J. HOOYKAAS (18), Department of Plant Molecular Biology, Biochemistry Laboratory, University of Leiden, 2333 AL Leiden, The Netherlands DAVID A. HOPWOOD (9), Department of Ge- netics, John lnnes Institute, Norwich NR4 7UH, England R. B. HORSCH (15), Plant Molecular Biology Group, Biological Sciences Department, Corporate Research and Development Staff, Monsanto Company, Chesterfield, Missouri 63198 HANSEN M. HSIUNG (24), Lilly Research Laboratories, A Division of Eli Lilly and Company, Lilly Corporate Center, Indi- anapolis, Indiana 46285 ANNA HUI (27), Department of Cell Genet- ics, Genentech, Inc., South San Fran- cisco, California 94080 MASAYORI INOUYE (31), Department of Bio- chemistry, University of Medicine and Dentistry of New Jersey at Rutgers, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 PARKASH JHURANI (27), Department of Or- ganic Chemistry, Genentech, Inc., South San Francisco, California 94080 MATTHEW O. JONES (33), AMGen, Thou- sand Oaks, California 91320 TOBIAS KIESER (9), Department of Ge- netics, John Innes Institute, Norwich NR4 7UH, England H. J. KLEE (15), Plant Molecular Biology Group, Biological Sciences Department, Corporate Research and Development Staff, Monsanto Company, Chesterfield, Missouri 63198 RuuD N. H. KONINGS (2), Laboratory of Molecular Biology, Faculty of Science, University of Nijmegen, Toernooiveld, 6525 ED N(jmegen, The Netherlands RAYMOND A. KOSKI (33), AMGen, Thou- sand Oaks, California 91320 C. J. KUHLEMEIER (11), Laboratory of Plant Molecular Biology, The Rockefeller University, New York, New York 10021 S. KUHSTOSS (10), Molecular Genetics Re- search, Lilly Research Laboratories, A Division of Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 CHRISTINE LANG-HINRICHS (22), Institut far Mikrobiologie, lnstitut fiir Giirungsge- werbe und Biotechnologie, D-IO00 Berlin 65, Federal Republic of Germany W. H. R. LANGalDGE (20), Boyce Thomp- son Institute for Plant Research, Cornell University, Ithaca, New York 14853 J. LEEMANS (16), Plant Genetic Systems, Inc., B-9000 Ghent, Belgium H. LEHRACH (8), The Imperial Cancer Re- search Fund, London WC2A 3PX, En- gland B. J. LI (20), Department of Biology, Chungshan University, Kwangchou, K~angdong, People's Republic of China JAMES R. LUPSlCI (4), Department of Pediat- rics and Institute for Molecular Genetics, Baylor College of Medicine, Texas Medi- cal Center, Houston, Texas 77030 WARREN C. MACKELLAR (24), Lilly Re- search Laboratories, A Division of Eli Lilly and Company, Lilly Corporate Cen- ter, Indianapolis, Indiana 46285 CONTRIBUTORS TO VOLUME 153 xi PAUL E. MARCH (31), Department of Bio- chemistry, University of Medicine and Dentistry of New Jersey at Rutgers, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 ANNE MARMENOUT (26), Innogenetics, Zwijnaarde, Belgium JOACHIM MESSING (1), Waksman Institute of Microbiology, Rutgers, The State Uni- versity of New Jersey, Piscataway, New Jersey 08855 GREGORY MILMAN (30), Department of Bio- chemistry, The Johns Hopldns University, School of Hygiene and Public Health, Baltimore, Maryland 21205 N. MURRAY (8), Department of Molecular Biology, University of Edinburgh, Edin- burgh EH9 3JR, Scotland KIYOSHI NAGAI (29), Medical Research Council Laboratory of Molecular Biol- ogy, Cambridge CB2 2QH, England SARAN A. NARANG (23), Division of Biologi- cal Sciences, National Research Council of Canada, Ottawa, Ontario, Canada KIA OR6 MAYNARD V. OLSON (5), Department of Ge- netics, Washington University School of Medicine, St. Louis, Missouri 63110 ENZO PAOLETTI (34), Laboratory of Immu- nology, Wadsworth Center for Laborato- ries and Research, New York State De- partment of Health, Albany, New York 12201 BEN P. H. PEETERS (2), Department of Ge- netics, University of Groningen, 9751 NN Haren (GR), The Netherlands MARION E. PERKUS (34), Laboratory of Im- munology, Wadsworth Center for Labo- ratories and Research, New York State Department of Health, Albany, New York 12201 AN'rONIA PICClNI (34), Laboratory of Im- munology, Wadsworth Center for Labo- ratories and Research, New York State Department of Health, Albany, New York 12201 INGO POTRYKUS (19), Institute for Plant Sci- ences, CH-1892 Zurich, Switzerland R. NAGARAJA RAO (10), Molecular Genetics Research, Lilly Research Laboratories, A Division of Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 ERIK REMAUT (26), Laboratory of Molecu- lar Biology, State University of Ghent, B-9000 Ghent, Belgium A. REYNAERTS (16), Plant Genetic Systems, Inc., B-9000 Ghent, Belgium M. A. RICHARDSON (10), Molecular Genet- ics Research, Lilly Research Laborato- ries, A Division of Eli Lilly and Company, Lilly Corporate Center, Indianapolis, In- diana 46285 S. G. ROGERS (15), Plant Molecular Biology Group, Biological Sciences Department, Corporate Research and Development Staff, Monsanto Company, Chesterfield, Missouri 63198 SUSAN M. ROSENBERG (7), Institute of Mo- lecular Biology, University of Oregon, Eugene, Oregon 97403 ROB A. SCHILPEROORT (18), Department of Plant Molecular Biology, Biochemistry Laboratory, University of Leiden, 2333 AL Leiden, The Netherlands KLAUS SCHNEIDER (28), lnstitut fiir Biolo- gic 11I, Albert-Ludwigs-Universit~it, D-7800 Freiburg i. Br., Federal Republic of Germany BR1GITTE E. SCHONER (25), Department of Molecular Genetics, Lilly Research Labo- ratories, A Division of Eli Lilly and Com- pany, Lilly Corporate Center, Indianapo- lis, Indiana 46285 RONALD G. SCHONER (25), Department of Molecular Genetics, Lilly Research Labo- ratories, A Division of Eli Lilly and Com- pany, Lilly Corporate Center, Indianapo- lis, Indiana 46285 RAYMOND D. SHILLITO (19), Biotechnology Research, CIBA-GEIGY Corporation, Research Triangle Park, North Carolina 27709 Guus SIMONS (26), N.I.Z.O., 6710 Ede, The Netherlands ULF STAHL (22), Fachgebiet Mikrobiologie, xii CONTRIBUTORS TO VOLUME 153 Technische Universitdt Berlin, D-IO00 Berlin 65, Federal Republic of Germany WING L. SONG (23), Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A OR6 RICHARD T. SUROSKY (14), Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois 60637 A. A. SZALAY (20), Boyce Thompson Insti- tute for Plant Research, Cornell Univer- sity, Ithaca, New York 14853 LOVEmNE P. TAYLOR (21), Carnagie Insti- tution of Washington, Stanford, Califor- nia 94305 TEgESA THIEL (13), Department of Biology, University of Missouri-St. Louis, St. Louis, Missouri 63121 HANS CHRISTIAN THgtGERSEN (29), Bio- struktur Afdeling, Kemisk Institut, /~rhus Universitet, 8200 ]lrhus N, Denmark BIK-KwooN TYE (14), Section of Biochem- istry, Molecular and Cell Biology, Divi- sion of Biological Sciences, CorneU Uni- versity, Ithaca, New York 14853 G. A. VAN ARKEL (11), Department of Mo- lecular Cell Biology, University of Utrecht, 3584 CH Utrecht, The Nether- lands M. VAN MONTAGU (16), Laboratorium Genetische Virologie, Vr~/e Universiteit Brussel, B-1640 Sint-Genesius-Rode, Belgium, and Laboratorium voor Genetica, Rijksuniversiteit Gent, B-9000 Gent, Belgium ELS J. M. VERHOEVEN (2), Department of Biology, Antoni van Leeuwenhoekhuis, 1066 CX Amsterdam, The Netherlands JEFFREY VIEIRA (1), Waksman Institute of Microbiology, Rutgers, The State Univer- sity of New Jersey, Piscataway, New Jer- sey 08855 VIRGINIA WALBOT (21), Department of Bio- logical Sciences, Stanford University, Stanford, California 94305 C. PETER WOLK (13), MSU-DOE Plant Re- search Laboratory, Michigan State Uni- versity, East Lansing, Michigan 48824 FE1-L. YAO (23), Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A OR6 [1] PRODUCTION OF SINGLE-STRANDED PLASMID DNA 3 [1] Production of Single-Stranded Plasmid DNA By JEFFREY VIEIRA and JOACHIM MESSING Introduction In the study of gene structure and function, the techniques of DNA analysis that are efficiently carried out on single-strand (ss) DNA tem- plates, such as DNA sequencing and site-specific in vitro mutagenesis, have been of great importance. Because of this, the vectors developed from the ssDNA bacteriophages M13, fd, or fl, which allow the easy isolation of strand-specific templates, have been widely used. While these vectors are very valuable for the production of ssDNA, they have certain negative aspects in comparison to plasmid vectors (e.g., increased insta- bility of some inserts, the minimum size of phage vectors). Work from the laboratory of N. Zinder showed that a plasmid carrying the intergenic region (IG) of fl could be packaged as ssDNA into a viral particle by a helper phage. 1 This led to the construction of vectors that could combine the advantages of both plasmid and phage vectors. 2 Since that time a number of plasmids carrying the intergenic region of M13 or fl have been constructed with a variety of features) A problem that has been encountered in the use of these plasmid/ phage chimeric vectors (plage) is the significant reduction in the amount of ssDNA that is produced as compared to phage vectors. Phage vectors can have titers of plaque-forming units (pfu) of 1012/ml and give yields of a few micrograms per milliliter of ssDNA. It might then be expected that cells carrying both a plage and helper phage would give titers of 5 × 10H/ ml for each of the two. However, this is not the case due to interference by the plage with the replication of the phage.4 This results in a reduction in the phage copy number and, therefore, reduces the phage gene prod- ucts necessary for production of ssDNA. This interference results in a 10- to 100-fold reduction in the phage titer and a level of ss plasmid DNA particles of about 101° colony forming units (cfu) per milliliter. 1 Phage mutants that show interference resistance have been isolated. 4,5 These mutants can increase the yield of ss plasmid by 10-fold and concurrently G. P. Dotto, V. Enea, and N. D. Zinder, Virology 114, 463 (1981). 2 N. D. Zinder and J. D. Boeke, Gene 19, 1 (1982). 3 D. Mead and B. Kemper, in "Vectors: A Survey of Molecular Cloning Vectors and Their Uses." Butterworth, Massachusetts, 1986. 4 V. Enea and N. D. Zinder, Virology 122, 222 (1982). 5 A. Levinson, D. Silver, and B. Seed, J. Mol. Appl. Genet. 2, 507 (1984). Copyright © 1987 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 153 All rights of reproduction in any form reserved. 4 VECTORS FOR CLONING DNA [1] increase the level of phage by a similar amount. Whether wild-type (wt) phage or an interference-resistant mutant is used as helper the yield of plasmid ssDNA is usually about equal to that of the phage, 3 and as the plasmid size increases the ratio shifts to favor the phage. 5 In order to increase both the quantitative and qualitative yield of the plasmid ssDNA, a helper phage, M13KO7, has been constructed that preferentially pack- ages plasmid DNA over phage DNA. In this chapter, M13KO7 will be described and its uses discussed. M13 Biology Certain aspects of M13 biology and M13 mutants play an important role in the functioning of M13KO7, so a short review of its biology is appropriate. 6,7 M13 is a phage that contains a circular ssDNA molecule of 6407 bases packaged in a filamentous virion which is extruded from the cell without lysis. It can infect only cells having an F pili, to which it binds for entering the cell. The phage genome consists of 9 genes encoding 10 proteins and contains an intergenic region of 508 bases. The proteins expressed by the phage are involved in the following processes: I and IV are involved in phage morphogenesis, III, VI, VII, VIII, and IX are virion proteins, V is an ssDNA binding protein, X is probably involved in repli- cation, and II creates a site-specific (+) strand nick within the IG region of the double-stranded replicative form (RF) of the phage DNA molecule at which DNA synthesis is initiated. Phage replication consists of three phases: (1) ss-ds, (2) ds-ds, and (3) ds-ss. The ss-ds phase is carried out entirely by host enzymes. For phases 2 and 3, gene II, which encodes both proteins II and X, is required for initiating DNA synthesis; all other functions necessary for synthesis are supplied by the host. The DNA synthesis initiated by the action of the gene II protein (glIp) leads to both the replication of the ds molecule and the production of the ssDNA that is to be packaged in the mature virion. The phage is replicated by a rolling circle mechanism that is terminated by glIp cleaving the displaced (+) strand at the same site and resealing it to create a circular ssDNA molecule. Early in the phage life cycle this ssDNA molecule is converted to the ds RF but later in the phage life cycle gVp binds to the (+) strand, preventing it from being converted to dsDNA and resulting in it being packaged into viral particles. The assembly of the virion occurs in the cell membrane where the gVp is replaced by the 6 D. T. Denhardt, D. Dressier, and D. S. Ray (eds.), "The Single-Stranded DNA Phages." Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1978. 7 N. D. Zinder and K. Horiuchi, Microbiol. Rev. 49, 101 (1985). [...]... vectors, i.e., the pKUN plasmids These plasmids allow the separate biosynthesis of both DNA strands of a recombinant plasmid in an ss form and thus overcome the drawbacks of the filamentous phage vectors described above 2°,2°a 2t D Denhardt, D Dressier, and D S Ray (eds.), "The Single-Stranded DNA Phages." Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1978 22 N D Zinder and K Horiuchi, Microbiol... Ff, different primers must be used for sequence analysis, hybridization studies, or site-directed mutagenesis of the DNA inserts The ssDNA packaged under the direction of IKe should be sequenced with the aid of the master primer; on the other hand, for sequence analysis of the ssDNA packaged under the direction of Ff, the reverse primer should be used (Fig 2B) (see Materials, Reagents, and Procedures)... dry it can be resuspended in TE and used in the same manner as has been previously described for the use of M13 ssDNA templates 17 2 For the screening of plasmid for inserts a colony selected from a plate is added to 2-3 ml of medium containing M13KO7 (-107/ml) and grown at 37° for a few hours Kanamycin is then added and the cultures are incubated for 14-18 hr at 37° The cells are then pelleted and... 55 G P Dotto, V Enea, and N D Zinder, Proc Natl Acad Sci U.S.A 78, 5421 (1981) 56 G P Dotto, K Horiuchi, and N D Zinder, Proc Natl Acad Sci U.S.A 79, 7122 (1982) 57 G P Dotto, K Horiuchi, K S Jakes, and N D Zinder, J Mol Biol 162, 335 (1982) 18 VECTORSFOR CLONINGDNA [2] into mature filamentous particles (RF to SS) 21,22,58,59Gene V protein is a phage-encoded ssDNA binding protein that, by binding to... Mazur and N D Zinder, Virology 68, 490 (1975) 60 S Johnston and D S Ray, J Mol Biol 177, 685 (1984) 61 M H Kim, J C Hines, and D S Ray, Proc Natl Acad Sci U.S.A 78, 6784 (1981) 62 G P Dotto and N D Zinder, Nature (London) 311, 279 (1984) 63 G P Dotto and N D Zinder, J Mol Biol 172, 507 (1984) 64 j M Cleary and D S Ray, Proc Natl Acad Sci U.S.A 77, 4638 (1980) [2] PLASMIDS FOR THE PRODUCTION OF s s D N... electrophoresis (data not shown) A large variation in the yield of ss plasmid DNA has been seen between different bacterial strains MV 1184 (derived from JM 83) and MV 1190 (derived from JM 101) have given satisfactory yields MV 1304 (derived from JM 105) gives much reduced yields and JM 109 undergoes significant lysis when it contains both plasmid and phage Acknowledgments We would like to thank B... Boehringer, Pharmacia, New England BioLabs, or Bethesda Research Laboratories, and should be used as recommended by the supplier Media, Nutritional Supplements, and Buffers Use distilled water for media and buffers (dH20) and double-distilled water (ddH20) for enzyme reactions All biochemicals can be purchased from Merck (Darmstadt, FRG) unless stated otherwise Minimal glucose plates: Mix after autoclaving... mixed with 6/~1 of loading buffer and electrophoresed on a 1% agarose gel, stained with ethidium bromide, and viewed with UV illumination Discussion The use of M13KO7 for the production of ss plasmid DNA normally gives titers ofcfu of 1011-5 × 10Wml and phage titers 10- to 100-fold lower ~7j Messing, this series, Vol 101, p 20 [1] PRODUCTION OF SINGLE-STRANDED PLASMID D N A 11 (Fig 4A) Plasmids containing... products for asymmetric DNA synthesis, as well as the proteins required for phage assembly and extnision, both filamentous phages and filamentous particles containing ss plasmid DNA will bud from the cell These particles can easily be concentrated and purified (see Materials, Reagents, and Procedures) and used, for example, for sequence analysis, l,l°,13 mutagenesis, 2,4,1° DNA recombination studies,... alkaline lysis method of Birnboim and Doly 76 as described by Maniatis et al., 77 with the exception that lysozyme is omitted from solution I After isolation, the plasmid DNA is further purified by cesium chloride centrifugation To 8 ml of plasmid DNA in TE buffer add 8.2 g of CsCI in a Ti50 centrifuge tube, mix gently to dissolve the CsCI, and subsequently add 0.5 ml of ethidium bromide Overlay the solution . and thus overcome the drawbacks of the filamentous phage vectors described above. 2°,2°a 2t D. Denhardt, D. Dressier, and D. S. Ray (eds.), "The Single-Stranded DNA Phages." Cold. of Canada, Ottawa, Ontario, Canada K1A OR6 [1] PRODUCTION OF SINGLE-STRANDED PLASMID DNA 3 [1] Production of Single-Stranded Plasmid DNA By JEFFREY VIEIRA and JOACHIM MESSING Introduction. viral particles. The assembly of the virion occurs in the cell membrane where the gVp is replaced by the 6 D. T. Denhardt, D. Dressier, and D. S. Ray (eds.), "The Single-Stranded DNA Phages."