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1 VOLUME1 Molecular CloningA LABORATORY MANUALFOURTH EDITIONThis is a free sample of content from Molecular Cloning: A Laboratory Manual, 4th edition. Click here for more information or to buy the book. 2012 by Cold Spring Harbor Laboratory PressOTHER TITLES FROM CSHL PRESSLABORATORY MANUALSA ntibodies: A Laboratory ManualImaging: A Laboratory ManualLive Cell Imaging: A Laboratory Manual, 2nd EditionManipulating the Mouse Embryo: A Laboratory Manual, 3rd EditionRNA: A Laboratory ManualHANDBOOKSLab Math: A Handbook of Measurements, Calculations, and Other Quantitative Skillsfor Use at the BenchLab Ref, Volume 1: A Handbook of Recipes, Reagents, and Other Reference Tools forUse at the BenchLab Ref, Volume 2: A Handbook of Recipes, Reagents, and Other Reference Tools forUse at the BenchStatistics at the Bench: A Step-by-Step Handbook for BiologistsWEBSITESM olecular Cloning, A Laboratory Manual,4th Edition, Spring Harbor Protocols, is a free sample of content from Molecular Cloning: A Laboratory Manual, 4th edition.

2 Click here for more information or to buy the book. 2012 by Cold Spring Harbor Laboratory PressVOLUME1 Molecular CloningA LABORATORY MANUALFOURTH EDITIONM ichael R. GreenHoward Hughes Medical InstitutePrograms in Gene Function and Expression and in Molecular MedicineUniversity of Massachusetts Medical SchoolJoseph SambrookPeter MacCallum Cancer Centre and thePeter MacCallum Department of OncologyThe University of Melbourne, AustraliaCOLD SPRING HARBOR LABORATORY PRESSCold Spring Harbor, New York is a free sample of content from Molecular Cloning: A Laboratory Manual, 4th edition. Click here for more information or to buy the book. 2012 by Cold Spring Harbor Laboratory PressMolecular CloningA LABORATORY MANUALFOURTH EDITION#2012 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkAll rights reservedPrinted in the United States of AmericaPublisherJohn InglisAcquisition EditorsJohn Inglis, Ann Boyle, Alexander GannDirector of Development, Marketing, & SalesJan ArgentineDevelopmental EditorsJudy Cuddihy, Kaaren Janssen, Michael ZierlerProject ManagerMaryliz DickersonPermissions CoordinatorCarol BrownProduction EditorKathleen BubbeoProduction ManagerDenise WeissSales Account ManagerElizabeth PowersCover art direction and design:Pete Jeffs, 2012 Library of Congress Cataloging-in-Publication DataGreen, Michael R.

3 (Michael Richard), 1954-Molecular cloning : a laboratory manual / Michael R. Green,Joseph Sambrook. 4th ed. of: Molecular cloning : a laboratory manual / JosephSambrook, David W. Russell. bibliographical references and 978-1-936113-41-5 (cloth) ISBN 978-1-936113-42-2( pbk.)1. Molecular cloning Laboratory manuals. I. Sambrook, Sambrook, Joseph. Molecular cloning. III. dc2320120026131098765432 Students and researchers using the procedures in this manual do so at their own risk. Cold Spring Harbor Laboratory makes no repre-sentations or warranties with respect to the material set forth in this manual and has no liability in connection with the use of thesematerials. All registered trademarks, trade names, and brand names mentioned in this book are the property of the respective should please consult individual manufacturers and other resources for current and specific product the exception of those suppliers listed in the text with their addresses, all suppliers mentioned in this manual can be found on theBioSupplyNet Web site at World Wide Web addresses are accurate to the best of our knowledge at the time of for the humane treatment of animals must be observed at all times.

4 Check with the local animal facility for experimental procedures in this manual may be the subject of national or local legislation or agency restrictions. Users of thismanual are responsible for obtaining the relevant permissions, certificates, or licenses in these cases. Neither the authors of this manualnor Cold Spring Harbor Laboratory assume any responsibility for failure of a user to do materials and methods in this manual may infringe the patent and proprietary rights of other individuals, companies, or organiza-tions. Users of this manual are responsible for obtaining any licenses necessary to use such materials and to practice such methods. COLDSPRING HARBOR LABORATORY MAKES NO WARRANTY OR REPRESENTATION THAT USE OF THE INFORMATION IN THISMANUAL WILL NOT INFRINGE ANY PATENT OR OTHER PROPRIETARY to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Cold SpringHarbor Laboratory Press, provided that the appropriate fee is paid directly to the Copyright Clearance Center (CCC).

5 Write or call CCCat 222 Rosewood Drive, Danvers, MA 01923 (978-750-8400) for information about fees and regulations. Prior to photocopying items foreducational classroom use, contact CCC at the above address. Additional information on CCC can be obtained at CCC Online at a complete catalog of all Cold Spring Harbor Laboratory Press Publications, visit our website at is a free sample of content from Molecular Cloning: A Laboratory Manual, 4th edition. Click here for more information or to buy the book. 2012 by Cold Spring Harbor Laboratory PressContentsList of TablesxxvPrefacexxxiAcknowledgmentsxxxii iVOLUME 1 PART 1: ESSENTIALSCHAPTER 1 Isolation and Quantification of DNA1 INTRODUCTIONDNA Isolation2 Commercial Kits for Purification of DNA3 Quantifying DNA5 PROTOCOLS1 Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreps112 Preparation of Plasmid DNA by Alkaline Lysis with SDS: Maxipreps153 Isolating DNA from Gram-Negative Bacteria ( , E.)

6 Coli)194 Precipitation of DNA with Ethanol215 Precipitation of DNA with Isopropanol266 Concentrating and Desalting Nucleic Acids with Microconcentrators287 Concentrating Nucleic Acids by Extraction with Butanol308 Preparation of Single-Stranded Bacteriophage M13 DNA by Precipitation withPolyethylene Glycol319 Plating Bacteriophage M133410 Growing Bacteriophage M13 in Liquid Culture3811 Preparation of Double-Stranded (Replicative Form) Bacteriophage M13 DNA4112 Isolation of High-Molecular-Weight DNA Using Organic Solvents to Purify DNA4413 Isolation of High-Molecular-Weight DNA from Mammalian CellsUsing Proteinase K and Phenol47vThis is a free sample of content from Molecular Cloning: A Laboratory Manual, 4th edition. Click here for more information or to buy the book. 2012 by Cold Spring Harbor Laboratory Press14A Single-Step Method for the Simultaneous Preparation of DNA, RNA, and Proteinfrom Cells and Tissues5415 Preparation of Genomic DNA from Mouse Tails and Other Small Samples58 Alternative Protocol: Isolation of DNA from Mouse Tails withoutExtraction by Organic Solvents61 Alternative Protocol.

7 One-Tube Isolation of DNA from Mouse Tails6216 Rapid Isolation of Yeast DNA6417 Using Ethidium Bromide to Estimate the Amount of DNA in Bands afterElectrophoresis through Minigels6618 Estimating the Concentration of DNA by Fluorometry Using Hoechst 332586819 Quantifying DNA in Solution with PicoGreen71 INFORMATION PANELSDNA Extraction from Formaldehyde-Fixed Tissue Embedded in Paraffin Blocks72 Polyethylene Glycol73a-Complementation74X-Gal76 Minimizing Damage to Large DNA Molecules77 Spectrophotometry78 CHAPTER 2 Analysis of DNA81 INTRODUCTIONA garose Gel Electrophoresis82 Analysis of DNA Fragments86 Recovering DNA from Gels87 Southern Blotting88 PROTOCOLS1 Agarose Gel Electrophoresis942 Detection of DNA in Agarose Gels by Staining993 Polyacrylamide Gel Electrophoresis1044 Detection of DNA in Polyacrylamide Gels by Staining1105 Detection of DNA in Polyacrylamide Gels by Autoradiography1126 Alkaline Agarose Gel Electrophoresis114 Additional Protocol: Autoradiography of Alkaline Agarose Gels1177 Imaging: Autoradiography and Phosphorimaging1198 Recovery of DNA from Agarose Gels Using Glass Beads1259 Recovery of DNA from Low-Melting-TemperatureAgarose Gels: Organic Extraction12710 Isolation of DNA Fragments from Polyacrylamide Gels by the Crushand Soak Method13011 Southern Blotting13312 Southern Blotting: Simultaneous Transfer of DNA from an AgaroseGel to Two Membranes141vi/ContentsThis is a free sample of content from Molecular Cloning: A Laboratory Manual, 4th edition.

8 Click here for more information or to buy the book. 2012 by Cold Spring Harbor Laboratory Press13 Southern Hybridization of Radiolabeled Probes to Nucleic AcidsImmobilized on Membranes144 Additional Protocol: Stripping Probes from Membranes150 INFORMATION PANELSF ormamide and Its Uses in Molecular Cloning153 Rapid Hybridization Buffers155 CHAPTER 3 Cloning and Transformation with Plasmid Vectors157 INTRODUCTIONP lasmid Vectors158 Transformation159 PROTOCOLS1 The Hanahan Method for Preparation and Transformation of CompetentE. coli:High-Efficiency Transformation1622 The Inoue Method for Preparation and Transformation of CompetentE. coli: Ultracompetent Cells1683 Easy Transformation ofE. coli:Nanoparticle-Mediated Transformation173 Alternative Protocol: One-Step Preparation of CompetentE. coli: Transformationand Storage of Bacterial Cells in the Same Solution1754 Transformation ofE.

9 Coliby Electroporation1775 Cloning in Plasmid Vectors: Directional Cloning1836 Cloning in Plasmid Vectors: Blunt-End Cloning1867 Dephosphorylation of Plasmid DNA1898 Attaching Phosphorylated Adaptors/Linkers to Blunt-Ended DNAs1929 Cloning PCR Products: Addition of Restriction Sites to the Termini of Amplified DNA19410 Cloning PCR Products: Blunt-End Cloning19711 Cloning PCR Products: Making T Vectors20012 Cloning PCR Products: TA Cloning20313 Cloning PCR Products: TOPO TA Cloning20614 Screening Bacterial Colonies Using X-Gal and IPTG:a-Complementation210 INFORMATION PANELSC aring forE. coli213 The History of Transformation of Bacteria by DNA217A Guide to Cloning the Products of Polymerase Chain Reactions218 BioBricks and the Ordered Assembly of DNA Fragments225 TOPO Tools: Creating Linear Expression Constructs with Functional Elements227 Antibiotics229 Adaptors232 Linkers234 Ligation and Ligases235 Contents/viiThis is a free sample of content from Molecular Cloning: A Laboratory Manual, 4th edition.

10 Click here for more information or to buy the book. 2012 by Cold Spring Harbor Laboratory PressCondensing and Crowding Reagents240 The Discovery of Restriction Enzymes241 Restriction Enzymes242 Chloramphenicol245 TheccdBGene247 Bacteriophagel248 Bacteriophage M13249 Plasmids251 Cosmids258 CHAPTER 4 Gateway Recombinational Cloning261 John S. Reece-Hoyes and Albertha WalhoutINTRODUCTIONB asic Principles and Applications of Gateway Cloning262 Disadvantages of Gateway Cloning and Alternative Cloning Systems264 PROTOCOLS1 Propagating Gateway Vectors2672 Generating an ORF Entry Clone and Destination Clone2703 Using Multisite LR Cloning to Generate a Destination Clone277 INFORMATION PANELG enerating Gateway-Compatible Vectors280 CHAPTER 5 Working with Bacterial Artificial Chromosomes and Other High-Capacity Vectors281 Nathaniel Heintz and Shiaoching GongINTRODUCTIOND evelopment of High-Capacity Vectors.