A LABORATORY MANUAL
`
`T. Maniatis
`E. F. Fritsch
`J. Sambrook
`
`Harvard University
`
`Michigan State university
`
`Cold spring Harbor Laboratory
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`y
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`CSH
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`cold Spring Harbor Laboratory
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`Mylan v. Genentech
`IPR2016-00710
`Merck Ex. 1095, Pg. 1
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`Molecular
`Cloning
`A LABORATORY MANUAL
`
`All rights reserved
`© 1982 by Cold Spring Harbor Laboratory
`Printed in the United States of America
`Book and cover design by Emily Harste
`
`Front cover: The electron micrograph of
`bacteriophage k particles stained with uranyl
`acetate was digitized and assigned false color
`by computer. Thomas R. Broker, Louise T.
`Chow, and James I. Garrets
`Back cover: E. coli VL361 with fimbriae was
`negatively stained with phosphotungstic acid
`and the electron micrograph was digitized and
`assigned false color by computer. Jeffrey A.
`Engler, Thomas R. Broker, and James L
`Garrets
`
`Cataloging in Publications data
`Maniatis, T.
`Molecular cloning.
`(A laboratory manual)
`Bibliography: p.
`Includes index.
`1. Molecular cloning.
`2. Eukaryotic
`cells.
`I. Fritsch, Edward F.
`II. Sambrook,
`Joseph.
`III. Title.
`IV. Series.
`QH442.2.F74
`574.87'3224
`ISBN 0-87969-136-0
`
`81-68891
`AACR2
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`i
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`2
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`■ .■*
`/J-
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`£.
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`Users of this manual are cautioned against proceeding" without regard for prudent laboratory
`safety procedures. Cold Spring Harbor Laboratory makes no representations or warranties
`with respect to the material set forth in this manual and will not be liable for any damages
`or
`injury that might result from the use of the procedures set forth herein.
`
`Other manuals available from Cold Spring Harbor Laboratory
`Hybridoma Techniques
`Experiments with Normal and Transformed Cells
`Advanced Bacterial Genetics,
`Experiments in Molecular Genetics
`A Manual for Genetic Engineering
`(Strain Kit available)
`(Strain Kit available)
`Methods in Ye^t Genetics
`
`All Cold Spring Harbor Laboratory publications are available through booksellers or may be ordered
`directly from Cold Spring Harbor Laboratory, Box 100, Cold Spring Harbor, New York.
`SAN 203-6185
`
`Merck Ex. 1095, Pg. 2
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`This manual began as a collection of laboratory protocols that were used
`during the 1980 Cold Spring Harbor course on the Molecular Cloning of
`Eukaryotic Genes. These procedures had been in use in our laboratories at
`that time but were scattered throughout the notebooks of many different
`people. In 1981 we decided to produce a more complete and up-to-date man­
`ual not only for use in the next Cold Spring Harbor course, but also for
`eventual publication. Out of the many permutations of the methods being
`used, we assembled a set of "
`,
`consensus protocols,” which were photocopied
`and widely distributed to many laboratories even as the 1981 course was
`underway. Then in the winter of 1981-1982, the manual was substantially
`rewritten, and new or revised protocols and figures, as well as entirely new
`chapters, were added.
`Even since this last rewriting, however, the field has progressed: New
`methods are constantly being invented and existing techniques are altered in
`response to changing needs. Although we have included in this manual only
`those protocols that have been thoroughly tested and used successfully in oiir
`laboratories, we make no claim that they are inviolable or perfect. We would
`welcome suggestions for improvements, and we would be grateful to be told
`about any new procedures that are devised.
`The evolution of protocols poses the difficult problem of attribution. We
`have tried to give credit at appropriate places in the text to the people who
`originally developed the procedures presented here, but in many cases trac­
`ing a particular method to its undisputed roots has proved to be impossible.
`We therefore wish to apologize—and to express gratitude—to those we have
`been unable to acknowledge for an idea, procedure, or recipe. Our major
`function has been to compile, to verify, and, we hope, to clarify; less fre­
`quently we have introduced modifications, and only in rare instances have
`we devised new protocols. In large part, then, the manual is based on proce­
`dures developed by others, and it is to them that any credit belongs.
`Because the manual was originally written to serve as a guide to those who
`had little experience in molecular cloning, it contains much basic material.
`However, the current version also deals in detail with almost every labora­
`tory task currently used in molecular cloning. We therefore hope that new­
`comers to cloning and veterans alike will find material of value in this book.
`Although molecular cloning seems straightforward on paper, it is more
`difficult to put into practice. Most protocols involve a large number of indi­
`vidual steps and a problem with any one of them can lead the experimenter
`into difficulty. To deal with these problems, a well-founded understanding of
`the principles underlying each procedure is essential. We have therefore
`provided background information and references that may be useful if trou-
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`iv PREFACE
`
`bie should arise. We also suggest that, as a matter of course, the products of
`each step in a protocol be tested to verify that the reaction was successful.
`This manual could not have been written without the help and advice of
`members of our laboratories and contributions from many others. We there­
`fore wish to thank John Fiddes, Mary-Jane Gething, David Goldberg, Steve
`Hughes, David Ish-Horowicz, Mike Mathews, Patty Reichel, Joe Sorge, Jim
`Stringer, Richard Treisman, and Nigel Whittle. We wish particularly to
`thank Arg Efstratiadis for his helpful discussions and criticisms of Chapter
`7; Brian Seed for permission to include a description of his unpublished
`procedure for screening libraries by recombination (Chapter 10) and many
`other useful suggestions; Doug Hanahan for advice on transformation (Chap­
`ter 8); Bryan Roberts for suggestions on methods of hybrid-selection and
`cDNA cloning; Doug Melton for providing a protocol for injection of Xenopus
`ooc3Ttes; Ronni Greene for suggesting improvements to many protocols; Nina
`Irwin for providing a critical anthology of methods available for expressing
`eukaryotic proteins in bacteria (Chapter 12); Rich Roberts for supplying the
`computer analysis of the sequence of pBR322; Barbara Bachmann for
`reviewing and correcting the list of E. coli strains; and Tom Broker, Louise
`Chow, Jeff Engler, and Jim Garrels for producing the elegant photographs
`used.for the front and back covers.
`We also thank all those who participated in the Cold Spring Harbor Molec­
`ular Cloning courses of 1980 and 1981. They were an excellent group of
`students, who struggled through the first two drafts of the manual and made
`many useful suggestions. We also thank Nancy Hopkins, who helped us to
`teach the course the first year and convinced us that producing a manual
`would be a worthwhile task. In 1981 Doug Engel helped teach the course and
`suggested many improvements to the manual. Contributing to the success ofy"
`both courses were the efforts of the teaching assistants, who were Catherine*^^
`O’Connell and Helen Doris Keller in the summer of 1980 and Susan Vande^
`Woude, Paul Bates, and Michael Weiss in 1981.
`We wish to thank Patti Barkley and Marilyn Goodwin for their cheerful­
`ness and forbearance during the typing of successive revisions of the manu­
`script. Our artists, Fran Cefalu and Mike Ockler, worked with great
`dedication and perseverance to produce the drawings for the manual. Joan
`Ebert kept track of the many references added to and deleted from the text
`and assembled the reference list. We are also grateful to Nancy Ford, Direc­
`tor of Publications, Cold Spring Harbor Laboratory, for her encouragement
`and support. Finally, without the patience, skill, and diplomacy of Doug
`Owen, who prepared the manuscript for the printer and helped us in many
`other ways, this book would not exist.
`
`Tom Maniatis
`Ed Fritsch
`Joe Sambrook
`
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`contents
`
`Preface, v .
`
`1 Vector-Host Systems
`Plasmids, 3
`Cloning in Plasmids, 11
`Bacteriophage x, 17
`The Lytic Cycle, 17
`Lysogeny, 22
`Construction of Bacteriophage k Vectors, 22
`Choosing the Appropriate Vector, 24
`Maps of Bacteriophage k Vectors, 25
`Cosmids, 45
`.
`Single-stranded Bacteriophages, 51
`Summary, 52
`
`V
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`2 Propagation and Maintenance of Bacterial Strains
`and Viruses
`strain Verification, 57
`Isolation of Single Colonies, 58
`Growth, Maintenance, and Preservation of Bacterial Strains, 61
`Plaque Purification of Bacteriophage k. 63
`Preparing Stocks of Bacteriophage k from Single Plaques, 65
`Media and Antibiotics, 68
`Liquid Media, 68
`Media Containing Agar or Agarose, 70
`Antibiotics, 71
`
`.
`
`V
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`3 Isolation of Bacteriophage x and Plasmid DIMA
`Large-scale Preparation of Bacteriophage k, 76
`Infection, 77
`Induction, 78
`Purification of Bacteriophage k, 80
`Extraction of Bacteriophage k DNA, 85
`Large-scale isolation of Plasmid DNA, 86
`Growth of Bacteria and Amplification-of the Plasmid, 88
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`Vi CONTENTS
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`Harvesting and Lysis of Bacteria, 89
`Purification of Closed Circular DNA by Centrifugation to Equilibrium in Cesium
`Chloride-Ethidium Bromide Gradients, 93
`Removal of RNA from Preparations of Plasmid DNA, 95
`
`97
`
`/
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`149
`
`4 Enzymes used in Molecular Cloning
`Restriction Enzymes. 98
`Isoschizomers, 99
`Methylation, 102
`Digesting DNA with Restriction Endonucleases, 104
`Other Enzymes Used in Molecular Cloning, 107
`Escherichia coli DNA Polymerase I, 108
`Klenow Fragment of E. coH DNA Polymerase !, 113
`T4 DNA Polymerase, 117
`Labeling the 5' Ends of DNA with T4 Polynucleotide Kinase, 122
`RNA*dependent DNA Polymerase, 128
`'
`Dephosphorylation of DNA, 133
`Nuclease 6a/3l, 135
`Nuclease Si, 140
`Mung-bean Nuclease, 141
`■Ribonucleases, 141
`Deoxribonuclease I, 142
`Exonuclease VII, 142
`Exonuclease III, 143
`X Exonuclease, 144
`Poly(A) Polymerase, 145
`T4 DNA Ligase, 146
`■
`T4 RNA Ligase, 147
`fcoRl Methylase, 147
`Terminal Deoxynucleotidyl Transferase, 148
`
`.
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`5 Gel Electrophoresis
`Agarose Gel Electrophoresis, 150
`Cei Electrophoresis Tanks, 153
`■
`Buffers, 156
`■ Preparation of Agarose Gels, 157
`Staining DNA in Agarose Cels, 161
`Photography, 162
`Minigels, 163
`Recovery of DNA from Agarose Cels, 164
`Alkaline Agarose Gels, 171
`Polyacrylamide cel Electrophoresis, 173
`Preparation of Polyacrylamide Gels, 174
`isolation of DNA Fragments from Polyacrylamide Gels, 178
`Strand-separating Gels, 179
`Strand Separation on Polyacrylamide Gels, 180
`Strand Separation on Agarose Gels, 182
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`CONTENTS vn
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`187
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`211
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`Strand Separation of Short Duplex DNA by Electrophoresis through
`Denaturing Polyacrylamide Gels, 184
`
`6 Extraction, Purification, and Analysis of mRNA from
`Eukaryotic Cells
`Isolation of mRNA from Mammalian Ceils, 191
`Isolation of Total Cellular rna, 194
`Selection of Poly(A)'' RNA, 197
`Gel Electrophoresis of RNA, 199
`Nuclease-Si Mapping of RNA, 207
`
`7 Synthesis and Cloning of cDNA
`Synthesis of cDNA, 213
`Synthesis of the First cDNA Strand, 213
`Synthesis of the Second cDNA Strand, 214
`. Cleavage of the Hairpin Loop with Nuclease S1, 216
`Molecular Cloning of Double-stranded cDNA, 217
`Homopoiymeric Tailing, 217
`'
`Synthetic DNA Linkers, 219
`Other Methods of Cloning cDNA, 220
`Strategies for cdna Cloning, 224
`Abundant mRNAs, 224
`Low-abundance mRNAs, 225
`Procedures for cDIMA Cioning, 229
`Synthesis of Double-stranded cDNA, 230
`Digestion with Nuclease S1, 237
`Cioning Double-stranded cDNA, 239
`Annealing Vector and Double-stranded cDNA, 242
`Cloning Double-stranded cDNA by Sequential Addition of Linkers, 243
`
`,
`’
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`8 Introduction of Plasmid and Bacteriophage k DNA into
`Escherichia coli
`Transformation of Escherichia coli by Plasmid DNA, 249
`Transformation by the Calcium Chloride Procedure, 250
`Transformation by the Calcium Chloride/Rubidium Chloride Procedure 252
`Transformation of £. coli xl776, 254
`'
`in Vitro Packaging of Bacteriophage \ DNA. 256
`Maintenance and Testing of Bacteriophage k Lysogens, 258
`Preparation of Extracts—Protocol l, 260
`'
`Packaging in Vitro—Protocol I, 262
`Preparation of Packaging Extracts—Protocol II, 264
`Packaging in Vitro—Protocol il, 268
`
`247
`
`9 Construction of Genomic Libraries
`Construction of Genomic libraries in Bacteriophage k Vectors, 270
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`vlli CONTENTS
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`Preparation of Vector DNA. 275
`Isolation of High-molecular-weight, Eukaryotic DNA from Cells Crown in
`Tissue Culture, 280
`Preparation of 20-kb Fragments of Eukaryotic DNA, 282
`Ligation and Packaging, 286
`Amplification of the Library, 293
`Construction of Genomic Libraries in cosmid vectors, 295
`Cloning in Phosphatase-treated Cosmid Vectors, 296
`Cloning in Cosmid Vectors Digested with Two Restriction Enzymes and
`Treated with Phosphatase, 300
`Amplification. Storage, and Screening of Cosmid Libraries, 304
`
`10 The Identification of Recombinant Clones
`309
`In Situ Hybridization of Bacterial Colonies or Bacteriophage Plaques, 312
`Screening Small Numbers of Bacterial Colonies, 313
`Replicating Colonies onto Nitrocellulose Filters, 316
`Screening Bacteriophage x Plaques by Hybridization, 320
`Screening by Hybridization Following In Situ Amplification of Bacteriophage
`k Plaques, 322
`Hybridization of DNA or RNA immobilized on Filters to Radioactive
`Probes, 324
`Hybridization to Nitrocellulose Filters Containing Replicas of Bacteriophage
`Plaques or Bacteria! Colonies, 326
`Identification of cDNA Clones by Hybridization Selection, 329
`Hybridization Selection Using DNA Bound to Nitrocellulose, 330
`Translation of Hybridization-selected RNA in Reticulocyte Lysates, 344
`Translation of Messenger RNA Injected into Frog Oocytes, 350
`, /
`Screening Bacteriophage k Libraries for Specific DNA sequences by >
`Recombination in E. coli, 353
`The Principle for Selection by Recombination, 353
`Strains Used in ttVX Screening, 356
`■
`.
`Preparation of ttVX, 356 ^
`_ ^
`Transformation of W3110 r m^ (p3), 357
`Plating Bacteriophage k Libraries on W3110 r m"^ (p3) (ttVX), 357
`Genetic Tests for Bacteriophage Containing a Suppressor Gene, 357
`Testing the ttVX System, 358.
`Using the ttVX System, 361
`
`11 Analysis of Recombinant DNA Clones
`Rapid Isolation of Plasmid or Bacteriophage k DNA, 365
`Rapid, Small-scale Isolation of Plasmid DNA, 366
`Rapid, Small-scale Isolation of Bacteriophage k DNA, 371
`Constructing Maps of Sites Cleaved by Restriction Endonucleases, 374
`Mapping by Single and Multiple Digestions, 376
`Sequential Digestion, 377
`Partial Digestion, 379
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`CONTENTS iX
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`Southern Transfer. 382
`Transfer of DNA from Agarose Gels to Nitrocellulose Paper, 383
`Hybridization of Southern Filters, 387
`Subcloning small DNA Fragments into Plasmid Vectors, 390
`Subcloning DNA Fragments with Cohesive Ends, 591
`The Use of Synthetic DNA Linkers in Subcloning, 392
`Creating Restriction Sites by Ligation of Blunt-ended DNA Molecules, 398
`Carrying Out Sequential Cloning Steps Rapidly, 401
`
`403
`
`435
`
`12 vectors That Express Cloned DNA in Escherichia coli
`Promoters, 405
`Vectors Using the pl Promoter of Bacteriophage x, 406
`Other Promoters, 410
`Rfbosome-binding Sites, 411
`Expression of Eukaryotic Genes, 412
`vectors That Express Unfused Eukaryotic Proteins, 412
`. Vectors That Express Fused Eukaryotic Proteins, 422
`Maximizing Expression of Cloned Genes, 431
`Increased Gene Dosage, 432
`Summary, 433
`
`'
`
`Appendices
`Appendix A: Biochemical Techniques, 436
`Glassware and Plasticware, 437
`Preparation of Organic Reagents, 438
`Liquid Media, 440
`Solutions for Working with Bacteriophage k, 443
`Antibiotics, 444
`-
`Preparation of Buffers and Solutions, 445
`Proteolytic Enzymes, 450
`Enzymes, 451
`Buffers for Restriction of Endonuclease Digestion, 453
`Commonly Used Electrophoresis Buffers, 454
`Commonly Used Gel-loading Buffers, 455
`Preparation of Dialysis Tubing, 456
`.
`Drying Down ^^p-iabeled Nucleotides from Mixtures of Ethanol and
`Water, 457
`Purification of Nucleic Acids, 458
`Concentration of Nucleic Acids, 461
`Chromatography through Sephadex G-50, 464
`Quantitation of DNA and RNA, 468
`Autoradiography, 470
`Measurement of Radioactivity in Nucleic Acids, 473
`Preparation of Multimers of Plasmids as Molecular-weight Markers, 474
`Protocol for Sequencing by the Maxam-Cilbert Technique, 475
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`X CONTENTS
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`Appendix B: pBR322, 479
`Nucleotide Sequence of pBR322, 479
`Restriction Sites in pBR322, 488
`Restriction Fragments of pBR322 DNA, 493
`Appendix C: commonly used Bacterial Strains, 504
`
`References, 507
`
`Index, 521
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`Extraction, Purification,
`and Anaiysis of mRNA
`from Eukaryotic Ceiis
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`188 EXTRACTION. PURIFICATION. AND ANALYSIS OF mRNA FROM EUKARYOTIC CELLS
`
`A typical mammalian cell contains about 10“^ jugof RNA, 80-85% of which is
`ribosomal (chiefly, 28S, 18S, and 5S), whereas 10-15% is made up of a variety
`of low-molecular-weight species (transfer RNAs, small nuclear RNAs, etc.).
`All of these RNAs are of defined size and sequence and can be isolated in
`virtually pure form by gel electrophoresis, density gradient centrifugation,
`or ion-exchange chromatography. By contrast, mRNA, which makes up
`between 1% and 5% of the total cellular RNA, is heterogeneous both in size
`(from a couple of hundred bases to several kilobases in length) and sequence.
`However, virtually all mammalian mRNAs carry at their 3' ends a poly(A)
`tract that is generally long enough to allow mRNAs to be purified by affinity
`chromatography on oligo(dT) cellulose. The resulting, physically heterogene­
`ous population of molecules collectively encodes all of the polypeptides syn­
`thesized by the cell.
`The keys to obtaining good preparations of eukaryotic mRNA are to min­
`imize ribonuclease activity during the initial stages of extraction and to
`avoid the accidental introduction of trace amounts of ribonuclease from the
`glassware and solutions. The following four elements are therefore impor­
`tant in the successful isolation of mRNA from mammalian cells.
`
`1. Use of Exogenous inhibitors of RNases
`Two types of inhibitors of RNase are currently in widespread use: vanadyl-
`ribonucleoside complexes and RNasin.
`
`Vanadyl-^ibonucleoside complexes. The complexes formed between tho^'
`oxovanadium ion and any of the four ribonucleosides are transition-st£d;e
`analogs that bind to many RNases and inhibit their activity almost com­
`pletely (Berger and Birkenmeier 1979). The four vanadyl-ribonucleoside
`complexes are added to intact cells and used at a concentration of 10 mM
`during all stages of RNA extraction and purification. The resulting mRNA
`is isolated in high yield and in a form that can be translated efficiently in
`cell-free, protein-synthesizing systems. If necessary, the vanadyl-ribonucleo-
`side complexes can be removed from the RNA preparation by multiple
`extractions with phenol containing 0.1% 8-hydroxyquinoline.
`Vanadyl-ribonucleoside complexes are prepared as follows (Berger and
`Birkenmeier 1979):
`
`1. Dissolve 0.5 mmole of each of the four ribonucleosides in 8 ml of water in a
`boiling-water bath.
`
`2. Purge the solution with nitrogen gas, while adding 1 ml of 2 M vanadyl
`sulfate.
`
`3. Adjust the pH to
`
`6 by dropwise addition of 10 M NaOH.
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`ISOLATION OFmRNA FROM MAMMALIAN CELLS 189
`
`4. Adjust the pH carefully to 7.0 by dropwise addition of 1 M NaOH under
`nitrogen gas in the water bath. As the complexes form, the solution
`changes color from blue to green-black.
`
`5. Adjust the volume to 10 ml; divide the solution into small aliquots and
`store at -20°C under nitrogen gas. The concentration of the complex is
`200 mM. Dilute 1:20 for use.
`
`Vanadyl-ribonucleoside complexes are commercially available from Bethes-
`da Research Laboratories, P.O. Box 577, Gaithersburg, MD 20760,
`
`RNasin. RNasin is a protein (Mr —40,000), isolated from rat liver and
`human placenta, that is a potent inhibitor of RNase. RNasin is effective
`during cell-free translation (Scheel and Blackburn 1979) and reverse tran­
`scription of mRNA (de Martynoff et al. 1980). Like vanadyl-ribonucleoside
`complexes, RNasin can be included in enzymatic reactions. Its advantage
`over vanadyl-ribonucleoside complexes is that it can be easily extracted with
`phenol.
`RNasin is commercially available from Biotec, Inc., 2800 Fish Hatchery
`Rd., Madison, WI 53711.
`
`2. Methods That Disrupt Cells and inactivate Nucleases Simultaneously
`Proteins readily dissolve in solutions of potent chaotropic agents such as
`guanidinium chloride and guanidinium isothiocyanate (Cox 1968). Cellular
`structures disintegrate and nucleoproteins dissociate rapidly from nucleic
`acids as ordered secondary structure is lost. Even RNase, an enzyme that is
`resistant to many forms of physical abuse (such as boiling), is inactive in the
`presence of 4 M guanidinium isothiocyanate and reducing agents like
`/3-mercaptoethanol (Sela et al. 1957). This combination of reagents can there­
`fore be used to isolate intact RNA from tissues, such as the pancreas, that are
`rich in RNase (Chirgwin et al. 1979).
`■
`Guanidinium isothiocyanate is made up according to the following steps.
`
`1. To a 100-g bottle of guanidinium isothiocyanate (Eastman Laboratory and
`Specialty Chemicals or Fluka), add 100 ml of deionized H2O, 10.6 ml of
`1 M Tris ■ Cl (pH 7.6), and 10.6 ml of 0.2 M EDTA. Stir overnight at room
`temperature.
`
`2. Warm the solution while stirring to 60-70°C for 10 minutes to assist
`dissolution. Often there is a residue of insoluble material that is removed
`by centrifugation at 3000p for 10 minutes at 20°C.
`
`3. Add 21.2 ml of 20% Sarkosyl (sodium lauryl sarkosinate) and 2.1 ml of
`^-mercaptoethanol to the supernatant and bring the volume to 212 ml
`with sterile H2O.
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`190 EXTRACTION, PURIFICATION, AND ANALYSIS OF mRNA FROM EUKARYOTIC CELLS
`
`4. Filter through a disposable Nalgene filter and store at 4°C in a tightly
`sealed, brown glass bottle.
`.
`
`3. Use of RNase-free Glassware and Plasticware
`Sterile, disposable plasticware is essentially free of RNase and can be used
`for the preparation and storage of RNA without pretreatment. General
`laboratory glassware, however, is often a source of RNase contamination and
`should be treated by baking at 250°C for 4 or more hours. In addition, some
`workers treat glassware for 12 hours at 37®C with a solution of 0.1% diethyl-
`pyrocarbonate, which is a strong but not absolute inhibitor of RNase
`(Fedorcsak and Ehrenberg 1966). Before using glassware treated in this
`way, it is important to remove traces of diethylpyrocarbonate by heating to
`lOO^C for 15 minutes (Kumar and Lindberg 1972) or by autoclaving. Other­
`wise, there is a danger that the remaining traces of diethylpyrocarbonate
`will inactivate the RNA by carboxymethylation.
`'
`It is a good idea to set aside items of glassware and batches of plasticware
`that are to be used only for experiments with RNA, to mark them distinc­
`tively, and to store them in a designated place.
`A potentially major source of contamination with RNase is the hands of
`investigators. There is little use in going to great lengths to rid glassware of
`contamination if no care is taken to keep one’s fingers out of harm’s way.
`Gloves should therefore be worn at all stages during the preparation of
`materials and solutions used for the isolation and analysis of RNA and dur­
`ing all manipulations involving RNA.
`
`4. Careful Preparation of Solutions
`All solutions should be prepared using baked glassware, glass-distilled au6»-
`claved water, and dry chemicals that are reserved for work with RNA and
`that are handled with baked spatulas. Wherever possible, the solutions
`should be treated with 0.1% diethylpyrocarbonate for at least 12 hours and
`autoclaved (15 minutes, liquid cycle). Note that diethylpyrocarbonate cannot
`be used to treat solutions containing Tris. It is highly unstable in the pres­
`ence of Tris buffers and decomposes rapidly into ethanol and carbon dioxide.
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`ISOLATION OFmRNA FROM MAMMALIAN CELLS 191
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`ISOLATION OF mRNA FROM MAMMALIAN CELLS
`The method given below is a modification of that of Favaloro et al. (1980) for
`the isolation of mRNA from monolayers of mammalian cells grown in tissue
`culture. However, it can also be used to isolate mRNA from mammalian cells
`grown in suspension or from any mammalian tissue that can be dispersed
`into single cells.
`
`1. Remove the medium from the cells and wash the monolayers three or
`four times with ice-cold, phosphate-buffered saline (PBS). Add 2 ml of
`ice-cold PBS to each of the plates and stand them on a bed of ice.
`
`2. Scrape off the cell sheet from each plate in turn by using a rubber
`policeman. With a wide-mouthed pipette, transfer the cell suspension
`into a Corex centrifuge tube and store on ice until the cells have been
`removed from all of the plates.
`
`3. Centrifuge at 2000fir for 5 minutes at 4°C,
`
`/
`
`4. Remove the supernatant by aspiration and resuspend the cell pellet in
`ice-cold lysis buffer (0.25 ml per 85-mm dish).
`
`Lysis buffer
`0.14 M NaCl
`1.5 mM MgCh
`10 mM Tris • Cl (pH 8.6)
`0.5% NP-40
`1000 units/ml RNasin
`10 mM vanadyl-ribonucleoside complexes
`or
`
`•
`
`.
`
`5. Vortex for 10 seconds and then underlay the cell suspension with an
`equal volume of lysis buffer containing sucrose (24% w/v) and NP-40 (1%).
`Store on ice for 5 minutes.
`
`6. Centrifuge at 10,000p for 20 minutes at 4°C in a swing-out rotor.
`
`7. Recover the turbid, upper (cytoplasmic) layer and add an equal volume
`of 2 X PK buffer.
`
`2 X PK buffer
`0.2 M Tris-Cl (pH 7.5)
`25 mM EDTA
`0.3 M NaCl
`2% w/v SDS
`Add proteinase K to a final concentration of 200 iiglml. Mix and incu­
`bate at 37°C for 30 minutes.
`
`Merck Ex. 1095, Pg. 15
`
`

`

`192 EXTRACTION, PURIFICATION, AND ANALYSIS OF mRNA FROM EUKARYOTIC CELLS
`
`Note, If nuclear RNA is also to be prepared, discard the clear sucrose
`phase and resuspend the nuclear pellet at the bottom of the centrifuge
`tube in lysis buffer (0.25 ml per 85-mm dish). Add an equal volume of 2 x
`PK buffer and proteinase K to a final concentration of 200 ixg/ml. Dis­
`rupt the nuclei and shear the liberated DNA by repeatedly squirting the
`viscous solution through a sterile 19-gauge hypodermic needle. Incubate
`at 37°C for 30 minutes.
`
`8. Remove the proteins by extracting once with phenol/chioroform.
`
`9. Recover the aqueous phase, add 2.5 volumes of ethanol, and store at
`-20°C for at least 2 hours.
`
`10. Centrifuge for 10 minutes at 5000^' at 0°C. Discard the supernatant and
`wash the pellet with 75% ethanol containing 0.1 M sodium acetate (pH
`5.2).
`
`11. Redissolve nucleic acids in a small volume (50 m1 per 85-mm dish) of:
`50 mM Tris • Cl (pH 7.5)
`1 mM EDTA
`
`Then add MgCh to 10 mM and RNasin or vanadyl-ribonucleoside com­
`plexes to final concentrations of 2000 units/ml or 2 mM, respectively.
`• Add pancreatic DNase I (Worthington RNase-free DPFF) from which
`RNase has been removed either by chromatography on agarose-5'-(4-
`aminophenyl-phosphoryl) uridine 2'(3') phosphate (Miles) as described
`by Maxwell et ai. (1977) (see page 451) or by adsorption to macaloid
`(Schaffner 1982) (see page 452). Incubate for 30 minutes at 37°C. '
`12. Add EDTA and SDS to final concentrations of 10 mM and 0.2%,
`respectively.
`
`13. Extract the solution once with phenol/chloroform.
`
`14. Add sodium acetate (pH 5.2) to 0.3 M and precipitate the nucleic acids
`with 2 volumes of ethanol. Store the RNA in 70% ethanol at -70°C.
`Cytoplasmic RNA may be further purified and freed from any con­
`taminating oligodeoxyribonucleotides by chromatography on oligo(dT)-
`cellulose (see page 197).
`
`Note
`i. Oligodeoxyribonucleotides contaminating preparations of nuclear RNA
`may be removed as follows:
`
`Merck Ex. 1095, Pg. 16
`
`

`

`ISOUTION OF mRNA FROM MAMMALIAN CELLS 193
`
`a. Resuspend the pellet in 20% sodium acetate by repeated pipetting up
`and down.
`
`b. Centrifuge for 10 minutes in an Eppendorf centrifuge.
`
`c. Discard the supernatant and redissolve the pellet in TE. Precipitate
`with ethanol.
`
`ii. Cytoplasmic RNA can usually be purified by oligo(dT) chromatography
`without DNase treatment.
`
`M-I
`
`Merck Ex. 1095, Pg. 17
`
`

`

`194 EXTRACTION, PURIFICATION. AND ANALYSIS OF mRNA FROM EUKARYOTIC CELLS
`
`ISOLATION OF TOTAL CELLULAR RNA
`
`The two methods given below are used to isolate RNA from cells that cannot
`be fractionated easily into cytoplasm and nuclei (e.g., frozen fragments of
`tissue) or from cells that are particularly rich in RNase (e.g., pancreatic
`cells).
`
`Guanidinium/Hot Phenol Method^
`1. Add 4 M guanidinium isothiocyanate mixture (prepared as described on
`page 189) to a tissue fragment or washed cell pellet in a plastic, disposa­
`ble centrifuge tube. Use 1 ml of guanidinium isothiocyanate mixture for
`10’ cells or 5 ml for every gram of tissue.
`Note. Tissue fragments may require disruption by homogenization in an
`omnimixer or polytron mixer.
`,
`
`2. Bring the mixture to 60°C and, while maintaining this temperature,
`draw the slimy suspension into a syringe fitted with an 18-gauge needle.
`Forcefully eject the suspension back into the tube. Repeat until the vis­
`cosity of the suspension is reduced by shearing of the liberated chromo­
`somal DNA.
`
`3. Add an equal volume of phenol preheated to 60°C and continue to pass
`the emulsion through the syringe.
`
`4. Add 0.5 volume of:
`
`0.1 M sodium acetate (pH 5.2)
`10 mM Tris Cl (pH 7.4)
`1 mM EDTA
`
`5. Add an equal volume of a 24:1 solution of chloroform and isoamyl alcohol
`and shake vigorously for 10-15 minutes while maintaining the tempera­
`ture at 60°C.
`
`6. Cool on ice and centrifuge at 20009^ for 10 minutes at 4°C.
`
`7. Recover the aqueous phase and reextract with phenol/chloroform.
`
`8. Centrifuge and recover the aqueous phase and reextract twice with
`chloroform.
`
`9. Add 2 volumes of ethanol. Store at -20°C for 1-2 hours.
`
`10. Recover the RNA by centrifugation at 12,000gf for 20 minutes at 4°C.
`'Feramisco et al. (1982)
`
`Merck Ex. 1095, Pg. 18
`
`

`

`ISOLATION OF TOTAL CELLULAR RNA 195
`
`11. Dissolve the pellet in the original starting volume (step 1) of:
`
`0.1 M Tris-Cl (pH 7.4)
`50 mM NaCi
`10 mM EDTA
`0.2% SDS
`Add proteinase K to a final concentration of 200 M^/ml. Incubate for 1-2
`hours at 37°C.
`12. Heat to 60°C. Add 0.5 volume of phenol preheated to 60°C and mix. Add
`0.5 volume of chloroform and mix vigorously at 60°C for 10 minutes.
`
`13. Cool in ice and centrifuge at 2000gr for 10 minutes at 4°C.
`
`14. Extract once more with phenol/chloroform at 60°C and then extract
`twice with chloroform at room temperature.
`15. Precipitate the nucleic acids with ethanol by centrifugation and rinse
`the pellet in 70% ethanol. Store the RNA in 70% ethanol at -70°C.
`
`16. Dissolve the RNA in sterile water. 5 x 10^ cells should yield approxi­
`mately 5-10 mg of RNA.
`
`/
`
`a
`
`n
`
`m
`
`Merck Ex. 1095, Pg. 19
`
`

`

`196 EXTRACTION, PURIFICATION, AND ANALYSIS OF mRNA FROM EUKARYOTIC CELLS
`
`Guanldinium/Ceslum Chloride Method^
`1. To a fragment of tissue or a cell pellet, add 5 volumes of:
`6 M guanidinium isothiocyanate
`5 mM sodium citrate (pH 7.0)
`0.1 M ^-mercaptoethanol
`0.5% Sarkosyl
`
`.
`
`Disperse the tissue by homogenization or the cell pellet by vortexing.
`
`2. Add 1 g of cesium chloride to each 2.5 ml of homogenate.
`
`3. Layer the homogenate onto a 1.2-ml cushion of 5.7 M CsCl in 0.1 M EDTA
`(pH 7.5) in a Beckman SW50.1 polyallomer tube (or its equivalent). (Other
`types of centrifuge tubes can be used; see Chirgwin et al. [1979]).
`
`4. Centrifuge at 35,000 rpm for 12 hours at 20°C. This procedure takes
`advantage of the fact that the buoyant density of RNA in cesium chloride
`is much greater than that of other cellular macromolecules. During ..
`cen-
`trifugation, the RNA forms a pellet on the bottom of the tube while most
`of the DNA and protein floats upward in the cesium chloride solution.
`
`5. Discard the supernatant, dry the walls of the centrifuge tubes thoroughly,
`and dissolve the pellet of RNA in:
`
`10 mM Tris-Cl (pH 7.4)
`5 mM EDTA
`1% SDS
`
`6. Extract once with a 4:1 mixture of chloroform and 1-butanol and transfer
`the aqueous phase to a fresh tube. Reextract the organic phase with an
`equal volume of:
`
`10 mM Tris • Cl (pH 7.4)
`5 mM EDTA
`1% SDS
`
`Combine the two aqueous phases.
`
`.
`
`7. Add 0.1 volume of 3 M sodium acetate (pH 5.2) and 2.2 volumes of ethanol.
`Store at — 20°C for at least 2 hours. Recover the RNA by centrifugation.
`
`8. Dissolve the pellet in 1 ml of H2O and reprecipitate with ethanol. Store the
`RNA in 70% ethanol at ~70°C.
`
`'Glisin et al. (1974); Ullrich et al. (1977).
`
`Merck Ex. 1095,