recombinant dna part g

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 from any source or their regulatory regions can be modified at will and reintroduced into a wide variety of cells by transformation. The cells expressing the introduced gene can be measured at the RNA level or protein level. These advantages allow us to solve complex biolog- ical problems, including medical and genetic problems, and to gain deeper understandings at the molecular level. In addition, new recombinant DNA methods are essential tools in the production of novel or better products in the areas of health, agriculture, and industry. The new Volumes 216, 217, and 218 supplement Volumes 153, 154, and 155 of Methods in Enzymology. During the past few years, many new or improved recombinant DNA methods have appeared, and a number of them are included in these new volumes. Volume 216 covers methods related to isolation and detection of DNA and RNA, enzymes for manipu- lating DNA, reporter genes, and new vectors for cloning genes. Volume 217 includes vectors for expressing cloned genes, mutagenesis, identify- ing and mapping genes, and methods for transforming animal and plant cells. Volume 218 includes methods for sequencing DNA, PCR for ampli- fying and manipulating DNA, methods for detecting DNA-protein inter- actions, and other useful methods. Areas or specific topics covered extensively in the following recent volumes of Methods in Enzymology are not included in these three vol- umes: "Guide to Protein Purification," Volume 182, edited by M. P. Deutscher; "Gene Expression Technology," Volume 185, edited by D. V. Goeddel; and "Guide to Yeast Genetics and Molecular Biology," Volume 194, edited by C. Guthrie and G. R. Fink. RAY Wu XV Contributors to Volume 216 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. ROBIN C. ALLSHIRE (51), MRC Human Ge- netics Unit, Western General Hospital, Edinburgh EH4 2XU, Scotland J. ALTENBUC~INER (40), Institute of Indus- trial Genetics, University of Stuttgart, D-7000 Stuttgart 1, Germany MICHELLE A. ALTING-MEES (42), Strate- gene Cloning Systems, La Jolla, Califor- nia 92037 SHR1KANT ANANT (3), Department of Ge- netics, The University of Illinois at Chi- cago, Chicago, Illinois 60612 JANET M. BARSOMIAN (23), New England Biolabs Inc., Beverly, Massachusetts 01915 ROBERT L. BEBEE (4), Corporate Research, GIBCO BRL, Life Technologies Inc., Gaithersburg, Maryland 20898 STEPHAN BECK (15), Imperial Cancer Re- search Fund, London WC2A 3PX, En- gland ASHOK S. BHAGWAT (21), Department of Chemistry, Wayne State University, De- troit, Missouri 48202 ADI D. BHARUCHA (18, 19), Department of Biochemistry, Faculty of Medicine, Laval University, Ste-Foy, Quebec GIK 7P4, Canada WENDY A. BICKMORE (22), MRC Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, Scotland ADRIAN P. BIRD (22), Institute of Cell and Molecular Biology, University of Edin- burgh, Edinburgh EH9 3JR, Scotland H. C. BIRNBOIM (16), Ottawa Regional Cancer Centre, and Departments of Bio- chemistry., Medicine, and Microbiology/ Immunology, University of Ottawa, Ot- tawa, Ontario K1H 8L6, Canada ix KATHRYN J. BOCKHOLD (17), Ddpartment de Biologie Mol~culaire, Institut Pasteur, 75724 Paris Cedex 15, France JOHAN BOTTERMAN (36), Plant Genetics Systems, B-9000 Gent, Belgium ALLAY R. BRASIER (34), Division of Endo- crinology and Hypertension, University of Texas Medical Branch, Galveston, Texas 77555 JORGEN BROSlUS (41), Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, New York 10029 Z. CAI (10), Department of Immunology, Mayo Clinic, Rochester, Minnesota 55905 ALLAN CAPLAN (37), Department of Bacte- riology and Biochemistry, University of Idaho, Moscow, Idaho 83843 C. THOMAS CASKET (7), Howard Hughes Medical Institute, Baylor College of Med- icine, Houston, Texas 77030 FARID F. CHEHAB (14), Department of Lab- oratory Medicine, University of Califor- nia, San Francisco, San Francisco, Cali- fornia 94143 YAWEN L. CHIANG (8), Department oflm- munology, Genetic Therapy Inc., Gaithersburg, Maryland 20878 ING-MING CHIU (44), Departments of later- hal Medicine and Molecular Genetics, and Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210 BRYAN R. CULLEN (31), Howard Hughes Medical Institute, Section of Genetics, Departments of Microbiology and Medi- cine, Duke University Medical Center, Durham, North Carolina 27710 X CONTRIBUTORS TO VOLUME 216 MARC DE BLOCK (36), Plant Genetics Sys- tems, B-9000 Gent, Belgium RUDY DEKEYSER (37), Instituut ter Aan- moediging van het Wetenschappelijk, On- derzoek in Nijverheid en Landbouw, B- 1050 Brussels, Belgium SUSANA DE LA LUNA (33), Centro Nacional de Biotechnologla and Centro de Biologfa Molecular, Universidad Aut6noma de Madrid, Campus de Cantoblanco, 28049 Madrid, Spain Jt3RGEN DENECKE (36), Department of Mo- lecular Genetics, Swedish University of Agricultural Sciences, S-75007 Uppsala, Sweden PETER B. DERVAN (27), Arnold and Mabel Beckman Laboratory of Chemical Syn- thesis, Division of Chemistry and Chemi- cal Engineering, Pasadena, California 91125 JEFFREY R. DE WET (35), Pfizer Central Re- search, Pfizer, Inc., Groton, Connecticut 06340 KATHLEEN D'HALLUIN (36), Plant Genetics Systems, B-9000 Gent, Belgium JAMES EBERWINE (9), Departments of Phar- macology and Psychiatry, University of Pennsylvania Medical School, Philadel- phia, Pennsylvania 19104 GLEN A. EVANS (46), Molecular Genetics Laboratory, The Salk Institute for Biolog- ical Studies, San Diego, California 92138 GEORGE R. FEEHERY (23), New England Biolabs Inc., Beverly, Massachusetts 01915 RICHARD FINNELL (9), Department of Vet- erinary Anatomy~Public Health, Texas A&M University, College Station, Texas 77843 CARL W. FULLER (29), Research and Devel- opment, United States Biochemical Cor- poration, Cleveland, Ohio 44122 GULILAT GEBEYEHU (4), Molecular Biology Research and Development, GIBCO BRL, Life Technologies Inc., Gaithers- burg, Maryland 20898 CHRISTIANE GOBLET (17), D~partement de Biologie Mol~culaire, Institut Pasteur, 75724 Paris Cedex 15, France JEAN GOULD (30), Soil and Crop Sciences Department, Texas A&M University, Col- lege Station, Texas 77843 FRANqOIS GUIDET (1), G1P Prince de Bre- tagne Biotechnologie, Penn Ar Prat, 29250 St. Pol De Ldon, France JOHN D. HARDING (4), Corporate Research, GIBCO BRL, Life Technologies Inc., Gaithersburg, Maryland 20898 GARY G. HERMANSON (46), Molecular Ge- netics Laboratory, The Salk Institute for Biological Studies, San Diego, California 92138 PHILIP HIETER (49), Department of Molecu- lar Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 R. M. HORTON (10), Department of Immu- nology, Mayo Clinic, Rochester, Minne- sota 55905 DENNIS E. HRUBY (32), Center for Gene Re- search and Biotechnology, Department of Microbiology, Oregon State University, Corvallis, Oregon 97331 JAN JANSSENS (36), Plant Genetics Systems, B-9000 Gent, Belgium Y. W. KAN (14), Department of Laboratory Medicine, Howard Hughes Medical Insti- tute, University of California, San Fran- cisco, San Francisco, California 94143 RAJENDRA P. KANDPAL (5), Department of Genetics, Yale University School of Medi- cine, New Haven, Connecticut 06510 DAVID J. KEMP (12), Menzies School of Health Research, Casuarina, Northern Territory 0811, Australia SUN CHANG KIM (26), Department of On- cology, McArdle Laboratory for Cancer Research, University of Wisconsin, Madi- son, Wisconsin 53706 MICHAEL KOOB (2, 28), McArdle Labora- tory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706 CONTRIBUTORS TO VOLUME 216 xi DAVID LANDRY (23), New England Biolabs Inc., Beverly, Massachusetts 01915 PETER LANGRIDGE (1), Centre for Cereal Biotechnology, The WRite Agricultural Research Institute, University of Ade- laide, Glen Osmond, South Australia 5064, Australia CHENG CHI LEE (7), Institute for Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030 JAN LEEMANS (36), Plant Genetics Systems, B-9000 Gent, Belgium KIRSTEN LEHTOMA (44), Department of In- ternal Medicine and Comprehensive Can- cer Center, The Ohio State University, Columbus, Ohio 43210 GEORGES LI~VESQUE (19), Department of Biochemistry, Faculty of Medicine, Laval University, Ste-Foy, Quebec GIK 7P4, Canada ANDREW M. LEW (13), Walter and Eliza Hall Institute, Melbourne, Victoria 3050, Australia KENNETH R. LUEHRSEN (35), Department of Biological Sciences, Stanford Univer- sity, Stanford, California 94305 SCOTT MACKLER (9), Department of Phar- macology, University of Pennsylvania Medical School, Philadelphia, Pennsylva- nia 19104 MICHAEL H. MALIM (31), Howard Hughes Medical Institute, Departments of Micro- biology and Medicine, University of Pennsylvania School of Medicine, Phila- delphia, Pennsylvania 19104 MICHAEL MCCLELLAND (25), Department of Plant Pathology, University of Ne- braska, Lincoln, Nebraska 68583 KEVIN MIYASHIRO (9), Department of Phar- macology, University of Pennsylvania Medical School, Philadelphia, Pennsylva- nia 19104 DONALD T. MOIR (50), Department of Hu- man Genetics and Molecular Biology, Collaborative Research, Inc., Waltham, Massachusetts 02154 MICHAEL NELSON (25), Department of Plant Pathology, University of Nebraska, Lincoln, Nebraska 68583 JUAN ORTfN (33), Centro Nacional de Biotechnologia and Centro de Biologla Molecular, Universidad Aut6noma de Madrid, Campus de Cantoblanco, 28049 Madrid, Spain MICHAEL PANACCIO (13), Victorian Insti- tute of Animal Science, Attwood, Victoria 3049, Australia WILLIAM J. PAVAN (49), Department of Mo- lecular Biology, Howard Hughes Medical Institute, Princeton University, Prince- ton, New Jersey 08544 L. R. PEASE (10), Department oflmmunol- ogy, Mayo Clinic, Rochester, Minnesota 55905 I. PELLETIER (40), Institute of Industrial Ge- netics, University of Stuttgart, D-7000 Stuttgart 1, Germany SIDNEY PESTKA (20), Department of Molec- Mar Genetics and Microbiology, Univer- sity of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 JAMES C. PIERCE (47), Cancer Therapeutic Program, The Du Pont Merck Pharma- ceutical Company, Wilmington, Dela- ware 19880 ANNA J. PODHAJSKA (26), Department of Microbiology, University of Gdansk, 80-222 Gdansk, Poland MATTHEW L. POULIN (44), Department Molecular Genetics, The Ohio State Uni- versity, Columbus, Ohio 43210 EDOUARD PROST (17), D~partement de Biologie Mol~culaire, Institut Pasteur, 75724 Paris Cedex 15, France J. K. PULLEN (10), Department oflmmunol- ogy, Mayo Clinic, Rochester, Minnesota 55905 PETER J. PUNT (39), Department of Molecu- lar Genetics and Gene Technology, Medi- cal Biological Laboratory, 2280 AA Rijsw(jk, The Netherlands xii CONTRIBUTORS TO VOLUME 216 ROGER H. REEVES (49), Department of Physiology, The Johns Hopkins Univer- sity School of Medicine, Baltimore, Mary- land 21205 ARLETTE REYNAERTS (36), Plant Genetics Systems, B-9000 Gent, Belgium DAVID RON (34), Laboratory of Molecular Endrocrinology, Massachusetts General Hospital, Boston, Massachusetts 02114 J. M. SHORT (42, 43), Strategene Cloning Systems, La Jolla, California 92037 DOUGLAS R. SMITH (50), Department of Human Genetics and Molecular Biology, Collaborative Research, Inc., Waltham, Massachusetts 02154 ADRIENNE P. SMYTH (50), Department of Human Genetics and Molecular Biology, Collaborative Research, Inc., Waltham, Massachusetts 02154 KEN SNIDER (46), Molecular Genetics Lab- oratory, The Salk Institute for Biological Studies, San Diego, California 92138 JAEMOG SOH (20), Department of Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 J. A. SORGE (42, 43), Strategene Cloning Systems, La Jolla, California 92037 CORINNE SPENCER (9), Department of Phar- macology, University of Pennsylvania Medical School, Philadelphia, Pennsylva- nia 19104 NAT L. STERNBERG (47), Cancer Therapeu- tic Program, The Du Pont Merck Phar- maceutical Company, Wilmington, Dela- ware 19880 SCOTT A. STROBEL (27), Arnold and Mabel Beckman Laboratory of Chemical Syn- thesis, Division of Chemistry and Chemi- cal Engineering, Pasadena, California 91125 KIRANUR N. SUBRAMANIAN (3), Depart- ment of Genetics, The University of Illi- nois at Chicago, Chicago, Illinois 60612 WACLAW SZYBALSKI (2, 26), McArdle Lab- oratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706 KENNETH D. TARTOF (48), Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 HIROO TOYODA (ll), Medical Genetics- Birth Defects Center, Department of Medicine and Pediatrics, Cedars-Sinai Medical Center, UCLA School of Medi- cine, Los Angeles, California 90048 LEVY ULANOVSKY (6), Department of Structural Biology, The Weizmann Insti- tute of Science, Rehovot 76100, Israel MARC VAN MONTAGU (37), Laboratorium voor Genetica, Universiteit Gent, B-9000 Gent, Belgium CEES A. M. J. J. VAN DEN HONDEL (39), Department of Molecular Genetics and Gene Technology, Medical Biological Laboratory, 2280 AA Rijswijk, The Neth- erlands M. R. VEN MURTHY (18, 19), Department of Biochemistry, Faculty of Medicine, Laval University, Ste-Foy, Quebec GIK 7P4, Canada P. VIELL (40), Institute of lndustrial Genet- ics, University of Stuttgart, D-7000 Stutt- gart 1, Germany VIRGINIA WALBOT (35), Department of Bio- logical Sciences, Stanford University, Stanford California 94305 JEFF WALL (14), Department of Laboratory Medicine, University of California, San Francisco, San Francisco, California 94143 DAVID C. WARD (5), Department of Genet- ics, Yale University School of Medicine, New Haven, Connecticut 06510 SHERMAN M. WEISSMAN (5), Department of Genetics, Yale University School of Medi- cine, New Haven, Connecticut 06510 ROBERT G. WHALEN (17), D~partement de Biologie Mol~culaire, Institut Pasteur, 75724 Paris Cedex 15, France CONTRIBUTORS TO VOLUME 216 xiii ELIZABETH M. WILSON (32), Center for Gene Research and Biotechnology, De- partment of Microbiology, Oregon State University, Corvallis, Oregon 97331 GEOFFREY G. WILSON (23, 24), New En- gland Biolabs Inc., Beverly, Massachu- setts 01915 MICHAEL WITTY (38), Department of Plant Sciences, University of Cambridge, Cam- bridge CB2 3EA, England ANDREW O. ZELENETZ (45), Division of Hematologic Oncology/Lymphoma of Memorial Hospital Program in Molecular Biology of the Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Cen- ter, New York, New York 10021 [1] MEGABASE DNA FROM PLANTS 3 [1] Megabase DNA Preparation from Plant Tissue By FRANqOIS GUIDET and PETER LANGRIDGE Introduction Traditional DNA extraction methods yield fragments of about 50 to 100 kilobase pairs (kbp) in length. The largest DNA fragments that can be separated by conventional electrophoresis in an agarose gel are 30 to 40 kbp in size. In contrast, the pulsed-field gel electrophoresis (PFGE) technique allows the separation of DNAs of more than 10,000 kbp (10 Mbp). The different principles involved in PFGE are represented by vari- ous acronyms such as FIGE, OFAGE, TAFE, and CHEF. All involve repeated reorientation of the DNA molecules inside the gel matrix due to corresponding changes in electric field parameters (electrode angle, switching time, field inversion, etc.; for a review see Ref. 1). To date the fractionation of DNA molecules has been extended to 12 Mbp, 2 but there does not seem to be any theoretical limit. To take advantage of these dramatic improvements biologists have designed methods to prepare high molecular weight DNA molecules, so- called megabase DNA (Mbp DNA). 3 For various reasons, plant molecular biologists have been slow to develop specific protocols suitable for prepar- ing Mbp DNA. Without exception, the methods used to prepare plant Mbp DNA involved the preparation of protoplasts as a preliminary step, that is, plant cells are freed of their cell wall by digestion with specific en- zymes. 4-8 To circumvent this tedious task, we designed an alternative method that appeared to be both rapid and efficient. 9 The description of an updated version of this method is the subject of the present article. Principle of Method The principle of the method is straightforward. It is based on the assumption that grinding leaf tissue in the presence of liquid nitrogen with t R. Anand, Trends Genet. 2, 278 (1986). : M. J. Orbach, D. Vollrath, R. W. Davis, and C. Yanofsky, Mol. Cell. Biol. 8, 1469 (1988). 3 D. C. Schwartz and C. R. Cantor, Cell (Cambridge, Mass.) 37, 76 0984). 4 p. Guzman and J. R. Ecker, Nucleic Acids Res. 16, ll091 (1988). 5 M. W. Ganal, N. D. Young, and S. D. Tanksley, Mol. Gen. Genet. 215, 395 (1989). 6 C. Jung, M. Kleine, F. Fischer, and R. G. Herrmann, Theor. Appl. Genet. 79, 663 0990). 7 R. A. J. van Daelen, J. J. Jonkers, and P. Zabel, Plant Mol. Biol. 12, 341 (1989). 8 W. Y. Cheung and M. D. Gale, Plant Mol. Biol. 14, 881 (1990). 9 F. Guidet, P. Rogowsky, and P. Langridge, Nucleic Acids Res. 18, 4955 (1990). Copyright © 1992 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 216 All rights of reproduction in any form reserved. 4 ISOLATION, SYNTHESIS, DETECTION OF DNA AND RNA [1] a mortar and pestle allows the preparation of plant cells, either isolated or in small aggregates. These plant cells, surrounded by a more or less damaged cell wall, contain intact organelles and their membranes are amenable to digestion by the combined action of a detergent (sarkosyl) and a proteolytic enzyme (proteinase K). The integrity of the DNA molecules is maintained by the addition of a chelating agent (ethylenediaminetetraacetic acid; EDTA), which helps protect the DNA from nucleases. Most impor- tant, the plant material (powder) is embedded in agarose prior to digestion, thus avoiding any mechanical shearing during subsequent treatments. Once the various cell membranes have been dissolved, the DNA molecules are liberated from their associated proteins by the proteinase K. The entire treatment is done at 53-55 °, which is still within the optimal temperature range of action for the proteinase K but well out of the active range for most plant nucleases. The DNA remains in the cavities created inside the agarose plugs by the original plant cells while solutes and small products of cell wall degradation diffuse out of the plugs. The DNA is still accessible to DNA-modifying enzymes such as restriction endonucleases and can be subjected to molecular biological manipulation. Materials and Reagents The plant materials used are either green leaves of 10-day-old seedlings, seeds, or commercial flour. Wheat-rye recombinant plants have been described in Rogowsky et al.l° and are obtained from Ken W. Shepherd (Waite Institute, South Australia). Seeds from alfalfa, lentils, and soybeans are from a local shop. Rye (cv. 'South Australian') flour is from W. Thomas Company (Port Adelaide, South Australia). Low melting temperature (LMP) and LE agarose are both from FMC BioProducts (Rockland, ME), proteinase K and restriction enzymes are from Boehringer (Mannheim, Germany), and radiolabeled dCTP and the transfer membrane HyBond N + are from Amersham (Arlington Heights, IL). The PFGE system used was a CHEF DR II from Bio-Rad Labora- tories (Richmond, CA). Solutions used to treat or store the plugs include a lysis solution (10 mM Tris-HC1, pH 8.0,500 mM EDTA, 1% (v/v) sarkosyl, 1 mg/ml protein- ase K), 1 × ET (1 mM Tris-HCl, pH 8.0, 50 mM EDTA), and TE (10 mM Tris-HC1, pH 8.0, l mM EDTA). After the electrophoretic runs the gels are stained with ethidium bro- mide (1/zg/ml) for 45 min and destained extensively to optimize the signal- i0 p. Rogowsky, F. Guidet, P. Langridge, K. W. Shepherd, and R. M. D. Koebner, Theor. Appl. Genet. 82, 537 (1991). [1] MEGABASE DNA FROM PLANTS 5 to-background ratio. Destaining of up to 20 hr does not visibly affect the sharpness of the DNA bands. The gels are then photographed and irradi- ated for 1 min with 254-nm UV light, depurinated in 0.25 M HC1 for 15 min, treated with alkali (1.5 M NaC1, 0.5 M NaOH) twice for 15 min, and equilibrated in the alkali transfer solution (I .5 M NaC1, 0.25 M NaOH) for 15 min prior to setting up the capillary transfer system. The transfer lasts for 24 hr, The hybridization conditions have been reported in Rogowsky et al. 1° The probe pAWI73 detects a moderately repeated rye-specific element. 1 Method About 0.4 g of young green leaves are ground to a fine powder in liquid nitrogen using a pestle and mortar. The powder is transferred to a crucible preheated to 50 °, mixed with 2 ml of 0.7% (w/v) LMP agarose in 1 x ET, and gently stirred with a sterile spatula to obtain a homogeneous mixture (alternatively the powder can be mixed with I ml of I x ET and then added to 1 ml of 1.4% agarose solution). The mixture is then poured directly into the mold (Bio-Rad CHEF DR II mold), shaking it gently while pouring to maintain the homogeneity of the mixture. It is then allowed to set at 4 ° for 20 min in a lying position to avoid a deposit of debris at the bottom of each agarose plug. The plugs are transferred into petri dishes and incubated in the lysis solution; we use 10 ml of lysis solution per l0 plugs (each plug is about 250/A agarose mixture). The incubation is done at 53-55 ° on a rocking platform in an oven or by floating the petri dishes in a water bath (to be on the safe side it is wise to float the petri dishes inside a plastic box with a minimum of water, the box itself floating in the water bath). At the end of the treatment the plugs are stored at 4 ° in 1 x ET. We have extended the method and used flour or crushed seeds instead of the leaf material. 12 The seeds are crushed in a mortar and pestle without liquid nitrogen. Most of the results presented here have been obtained by using crushed seeds or flour. Results Source of Material The present method of direct Mbp DNA isolation was developed be- cause high yields were obtained rapidly and without the elaborate tech- It F. Guidet, P. Rogowsky, C. Taylor, W. Song, and P. Langridge, Genome 34, 81 (1991). 12 F. Guider and P. Langridge, C.R. Acad. Sci. Paris, Ser. 3 314, 7 (1992). 6 ISOLATION, SYNTHESIS, DETECTION OF DNA AND RNA [1] SIZE (Kbp) 2500 1600 1125 1020 945 85O 8OO 770 7OO 63O 580 460 370 290 245 A M 0 1 2 4 7 2448 Fi6. 1. Release and restriction digestion of DNA from rye flour embedded in agarose. Two series of plugs were incubated with lysis solution at 55 ° for the periods indicated at the top of each lane (time is in hours). One series (A) was electrophoresed directly (run condi- tions: time ramp 50 to 90 sec at 200 V, in a 1% agarose gel in 0.5 × TBE buffer for 24 hr). The other series was incubated with HindIII and subsequently electrophoresed (B) (run conditions: time ramp 1 to 6 sec at 125 V, in a 1.5% agarose gel in 0.5 × TBE buffer for 18 hr). The arrow in (B) indicates relic DNA that hybridizes with an rDNA probe. The gel from (B) was transferred and probed with pAW 173 (C). (A) and (B) are ethidium bromide-stained gels; (C) is an autoradiogram. niques involved in the preparation of nuclei or protoplasts. Like other authors 6,7 we were not able to isolate Mbp DNA from nuclei, although we obtained good-quality DNA from protoplasts prepared from young leaves or suspension cultures. However, the yield of leaf protoplasts, especially in the case of cereals, is very low; that is, only a small fraction of the leaf cells can be turned into protoplasts and are amenable to lysis. Cell [...]... txg of plasmid 16 Gentle tapping of the tube dissolves the yellowish pellet containing plasmid DNA, leaving the white coating at the top (which consists mainly of protein) floating intact in the solution Vortexing, on the other hand, disperses this white material into the DNA solution; the dispersed particles hinder the migration of the DNA into an agarose gel in the preliminary identification of recombinant. .. authorswouldliketo thankDr PeterRogowskyfor criticalreadingof the manuscript and friendlysuggestions.This workhas been supportedby the AustralianResearchCouncil [2] PREPARING AND USING AGAROSE MICROBEADS 13 [2] P r e p a r i n g a n d U s i n g Agarose Microbeads B y MICHAEL KOOB and WACLAW SZYBALSKI Introduction Protocols for preparing and manipulating large DNA molecules without breakage are required for the... electrophoresis on a 2% agarose gel and visualization of the DNA bands by staining with ethidium bromide to prevent masking of DNA bands smaller than 500 bp in length by the diffuse RNA band 20 RNA should be removed prior to sequencing because fragments of RNA could interfere with the sequencing reaction by annealing with the template and causing incorrect priming 21 E Y Chen and P H Seeburg, D N A 4, 165 (1985)... volume of 2 ml (NotI-digested DNA from microbeads made with 1 × 109 cells will give fine, light DNA bands on PFGE, and that from microbeads made with 2 × 10 9 cells will give intense, heavy bands.) Saccharomyces cerevisiae Grow cells by inoculating 10 ml YPD (10 g yeast extract, 20 g peptone, 20 g glucose, distilled water to 1 liter) with a fresh culture and shake overnight at 30° Wash the cells once... alcohol-precipitated DNA One bench-top clinical centrifuge (e .g. , IEC model HNS-II; International Equipment Co., Needham Heights, MA): Equipped with a standard horizontal rotor and swinging buckets for eighteen 15-ml capped plastic tubes This centrifuge is used for the low-speed centrifugation of animal cells One refrigerated superspeed centrifuge (e .g. , Sorvall RC-5B; Du Pont Co., Wilmington, DE): Equipped... as giant DNA- carrying "cells," thus converting DNA into a "solid state" and allowing its easy and rapid transfer to various solutions by sedimenting, washing, and resuspending the microbeads In addition to E coli and S cerevisiae, we have successfully applied these microbead protocols to Pseudomonas aeruginosa, Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Plasmodium species, and Magnaporthe grisea... banding pattern within the smeary background of the digested DNA (marked with arrowheads in Fig 3) is a good indication of precise restriction digestion Southern Blot Hybridization Southern blot hybridization on pulsed-field gels is more difficult to perform than on normal gels The reasons are not clear, but factors such as DNA quality, electrophoretic separation, and gel treatment may have a big influence... It seems of great importance to depurinate the large DNA fragments for successful transfer An example of a Southern blot is shown in Fig 4 As pAW173 hybridizes to a repetitive DNA sequence in rye, individual bands are difficult to visualize in the lane smears (some discrete bands are indicated by arrows in Fig 4) Concluding Remarks The procedure described for producing very large DNA fragments from... described in step 8 The resulting DNA preparation is suitable for doublestranded DNA sequencing by the Chen and Seeburg procedure, 21 using oligodeoxynucleotide primers flanking the MCS region (available commercially) Method H: The Phenol-Chloroform Lysis-Extraction Method for Isolation of Plasmids from Bacteria 1 Grow bacteria overnight and pellet down in a !.5-ml microfuge tube as described in method... vibrating nub, causing the liquid to froth violently To entrap larger cells, such as those from human tissue culture, less vigorous mixing should be used to produce slightly larger microbeads c Attempting to embed cells at unusually high concentrations will result in large numbers of cells trapped on the microbead surface and free in solution This in turn will lead to severe clumping and, following cell . detection of DNA and RNA, enzymes for manipu- lating DNA, reporter genes, and new vectors for cloning genes. Volume 217 includes vectors for expressing cloned genes, mutagenesis, identify- ing and. and mapping genes, and methods for transforming animal and plant cells. Volume 218 includes methods for sequencing DNA, PCR for ampli- fying and manipulating DNA, methods for detecting DNA- protein. YAWEN L. CHIANG (8), Department oflm- munology, Genetic Therapy Inc., Gaithersburg, Maryland 20878 ING-MING CHIU (44), Departments of later- hal Medicine and Molecular Genetics, and