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This is a free sample of content from Molecular Cloning: A Laboratory Manual, 4th edition.Click here for more information or to buy the book.VOLUMEMolecular CloningA LABORATORY MANUALFOURTH EDITION 2012 by Cold Spring Harbor Laboratory Press1
This is a free sample of content from Molecular Cloning: A Laboratory Manual, 4th edition.Click here for more information or to buy the book.OTHER TITLES FROM CSHL PRESSLABORATORY MANUALSAntibodies: 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 BiologistsWEBSITESMolecular Cloning, A Laboratory Manual, 4th Edition, www.molecularcloning.orgCold Spring Harbor Protocols, www.cshprotocols.org 2012 by Cold Spring Harbor Laboratory Press
This is a free sample of content from Molecular Cloning: A Laboratory Manual, 4th edition.Click here for more information or to buy the book.VOLUMEMolecular CloningA LABORATORY MANUALFOURTH EDITIONMichael 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 † www.cshlpress.org 2012 by Cold Spring Harbor Laboratory Press1
This is a free sample of content from Molecular Cloning: A Laboratory Manual, 4th edition.Click here for more information or to buy the book.Molecular CloningA LABORATORY MANUALFOURTH EDITION# 2012 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkAll rights reservedPrinted in the United States of AmericaPublisherAcquisition EditorsDirector of Development, Marketing, & SalesDevelopmental EditorsProject ManagerPermissions CoordinatorProduction EditorProduction ManagerSales Account ManagerJohn InglisJohn Inglis, Ann Boyle, Alexander GannJan ArgentineJudy Cuddihy, Kaaren Janssen, Michael ZierlerMaryliz DickersonCarol BrownKathleen BubbeoDenise WeissElizabeth PowersCover art direction and design: Pete Jeffs, 2012Library of Congress Cataloging-in-Publication DataGreen, Michael R. (Michael Richard), 1954Molecular cloning : a laboratory manual / Michael R. Green,Joseph Sambrook. – 4th ed.p. cm.Rev. ed. of: Molecular cloning : a laboratory manual / JosephSambrook, David W. Russell. 2001.Includes bibliographical references and index.ISBN 978-1-936113-41-5 (cloth) – ISBN 978-1-936113-42-2( pbk.)1. Molecular cloning–Laboratory manuals. I. Sambrook, Joseph.II. Sambrook, Joseph. Molecular cloning. III. Title.QH442.2.S26 2012572.8–dc23201200261310 9 8 7 6 5 4 3 2Students and researchers using the procedures in this manual do so at their own risk. Cold Spring Harbor Laboratory makes no representations 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 owners.Readers should please consult individual manufacturers and other resources for current and specific product information.With 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 www.biosupplynet.com.All World Wide Web addresses are accurate to the best of our knowledge at the time of printing.Procedures for the humane treatment of animals must be observed at all times. Check with the local animal facility for guidelines.Certain 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 so.The materials and methods in this manual may infringe the patent and proprietary rights of other individuals, companies, or organizations. 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 RIGHT.Authorization 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). 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 www.copyright.com.For a complete catalog of all Cold Spring Harbor Laboratory Press Publications, visit our website at www.cshlpress.org. 2012 by Cold Spring Harbor Laboratory Press
This is a free sample of content from Molecular Cloning: A Laboratory Manual, 4th edition.Click here for more information or to buy the book.ContentsList of TablesPrefaceAcknowledgmentsxxvxxxixxxiiiVOLUME 1PART 1: ESSENTIALSCHAPTER 1Isolation and Quantification of DNA1INTRODUCTIONDNA Isolation2Commercial Kits for Purification of DNA3Quantifying DNA5PROTOCOLS1Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreps112Preparation of Plasmid DNA by Alkaline Lysis with SDS: Maxipreps153Isolating DNA from Gram-Negative Bacteria (e.g., E. coli)194Precipitation of DNA with Ethanol215Precipitation of DNA with Isopropanol266Concentrating and Desalting Nucleic Acids with Microconcentrators287Concentrating Nucleic Acids by Extraction with Butanol308Preparation of Single-Stranded Bacteriophage M13 DNA by Precipitation withPolyethylene Glycol31Plating Bacteriophage M1334Growing Bacteriophage M13 in Liquid Culture3891011Preparation of Double-Stranded (Replicative Form) Bacteriophage M13 DNA4112Isolation of High-Molecular-Weight DNA Using Organic Solvents to Purify DNA4413Isolation of High-Molecular-Weight DNA from Mammalian CellsUsing Proteinase K and Phenol47v 2012 by Cold Spring Harbor Laboratory Press
This is a free sample of content from Molecular Cloning: A Laboratory Manual, 4th edition.Click here for more information or to buy the book.vi / Contents14A Single-Step Method for the Simultaneous Preparation of DNA, RNA, and Proteinfrom Cells and Tissues5415Preparation of Genomic DNA from Mouse Tails and Other Small Samples58† Alternative Protocol: Isolation of DNA from Mouse Tails withoutExtraction by Organic Solvents61† Alternative Protocol: One-Tube Isolation of DNA from Mouse Tails6216Rapid Isolation of Yeast DNA6417Using Ethidium Bromide to Estimate the Amount of DNA in Bands afterElectrophoresis through Minigels6618Estimating the Concentration of DNA by Fluorometry Using Hoechst 332586819Quantifying DNA in Solution with PicoGreen71INFORMATION PANELSDNA Extraction from Formaldehyde-Fixed Tissue Embedded in Paraffin Blocks72Polyethylene Glycol73a-Complementation74X-Gal76Minimizing Damage to Large DNA Molecules77Spectrophotometry78CHAPTER 2Analysis of DNA81INTRODUCTIONAgarose Gel Electrophoresis82Analysis of DNA Fragments86Recovering DNA from Gels87Southern Blotting88PROTOCOLS1 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 Extraction12710Isolation of DNA Fragments from Polyacrylamide Gels by the Crushand Soak Method13011Southern Blotting13312Southern Blotting: Simultaneous Transfer of DNA from an AgaroseGel to Two Membranes141 2012 by Cold Spring Harbor Laboratory Press
This is a free sample of content from Molecular Cloning: A Laboratory Manual, 4th edition.Click here for more information or to buy the book.Contents13/ viiSouthern Hybridization of Radiolabeled Probes to Nucleic AcidsImmobilized on Membranes144† Additional Protocol: Stripping Probes from Membranes150INFORMATION PANELSFormamide and Its Uses in Molecular Cloning153Rapid Hybridization Buffers155CHAPTER 3Cloning and Transformation with Plasmid Vectors157INTRODUCTIONPlasmid Vectors158Transformation159PROTOCOLS1The Hanahan Method for Preparation and Transformation of Competent E. coli:High-Efficiency Transformation162The Inoue Method for Preparation and Transformation of Competent E. coli:“Ultracompetent” Cells168Easy Transformation of E. coli: Nanoparticle-Mediated Transformation173† Alternative Protocol: One-Step Preparation of Competent E. coli: Transformationand Storage of Bacterial Cells in the Same Solution1754Transformation of E. coli by Electroporation1775Cloning in Plasmid Vectors: Directional Cloning1836Cloning in Plasmid Vectors: Blunt-End Cloning1867Dephosphorylation of Plasmid DNA1898Attaching Phosphorylated Adaptors/Linkers to Blunt-Ended DNAs1929Cloning PCR Products: Addition of Restriction Sites to the Termini of Amplified DNA19410Cloning PCR Products: Blunt-End Cloning19711Cloning PCR Products: Making T Vectors20012Cloning PCR Products: TA Cloning20313Cloning PCR Products: TOPO TA Cloning20614Screening Bacterial Colonies Using X-Gal and IPTG: a-Complementation21023INFORMATION PANELSCaring for E. coli213The History of Transformation of Bacteria by DNA217A Guide to Cloning the Products of Polymerase Chain Reactions218BioBricks and the Ordered Assembly of DNA Fragments225TOPO Tools: Creating Linear Expression Constructs with Functional tion and Ligases235 2012 by Cold Spring Harbor Laboratory Press
This is a free sample of content from Molecular Cloning: A Laboratory Manual, 4th edition.Click here for more information or to buy the book.viii / ContentsCondensing and Crowding Reagents240The Discovery of Restriction Enzymes241Restriction Enzymes242Chloramphenicol245The ccdB Gene247Bacteriophage l248Bacteriophage M13249Plasmids251Cosmids258CHAPTER 4Gateway Recombinational Cloning261John S. Reece-Hoyes and Albertha J.M. WalhoutINTRODUCTIONBasic Principles and Applications of Gateway Cloning262Disadvantages of Gateway Cloning and Alternative Cloning Systems264PROTOCOLS1Propagating Gateway Vectors2Generating an ORF Entry Clone and Destination Clone2703Using Multisite LR Cloning to Generate a Destination Clone277267INFORMATION PANELGenerating Gateway-Compatible Vectors280CHAPTER 5Working with Bacterial Artificial Chromosomes and Other High-Capacity Vectors281Nathaniel Heintz and Shiaoching GongINTRODUCTIONDevelopment of High-Capacity Vectors: Advantages and Disadvantages282Working with Bacterial Artificial Chromosomes286PROTOCOLS1 Small-Scale Isolation of BAC DNA and Verification by PCR2942 Large-Scale Preparation and Linearization of BAC DNA2973 Examination of BAC DNA Quality and Quantity by Pulsed-Field GelElectrophoresis3014 Two-Step BAC Engineering: Preparation of Shuttle Vector DNA3035 Preparation of the A Homology Arm (A-Box) and B Homology Arm (B-Box)3066 Cloning of the A and B Homology Arms into the Shuttle Vector3097 Preparation and Verification of the Recombinant Shuttle Vector312 2012 by Cold Spring Harbor Laboratory Press
This is a free sample of content from Molecular Cloning: A Laboratory Manual, 4th edition.Click here for more information or to buy the book.Contents/ix8Electroporation of Competent BAC Host Cells with the RecombinantShuttle Vector3159Verification of Cointegrates and Selection of ResolvedBAC Clones31710One-Step BAC Modification: Preparation of Plasmids32111Preparation of the A Homology Arm (A-Box)32412Cloning of the A Homology Arm into Reporter-Shuttle Vector32613Transformation of the BAC Host with the RecA Vector32914Transfer of the Reporter Vector into BAC/RecA Cells and Selectionof Cointegrates33115Growth of S. cerevisiae and Preparation of DNA33416Small-Scale Preparations of Yeast DNA336INFORMATION PANELSCRE–loxP338Using EGFP as a Reporter342Primer Design for Homology Arms, Cointegration, and Resolution343Yeast Media344CHAPTER 6Extraction, Purification, and Analysis of RNA from Eukaryotic Cells345INTRODUCTIONPROTOCOLSIntroduction to the Isolation of Total RNA Using Monophasic Lysis Reagents1348Purification of Total RNA from Mammalian Cells and Tissues351† Alternative Protocol: Preparing RNA from Smaller Samples3542Isolation of Total RNA from Zebrafish Embryos and Adults3553Total RNA Isolation from Drosophila melanogaster3574Total RNA Extraction from Caenorhabditis elegans3595Total RNA Extraction from Saccharomyces cerevisiae Using HotAcid Phenol3626Quantifying and Storing RNA3657Precipitation of RNA with Ethanol3728Removing DNA Contamination from RNA Samples by Treatment withRNase-Free DNase I3759þIsolation of Poly(A) Messenger RNA Using Magnetic Oligo(dT) BeadsIntroduction to Hybridization of RNA by Northern Transfer101112377381Separation of RNA according to Size: Electrophoresis of RNA through Agarose GelsContaining Formaldehyde388Separation of RNA according to Size: Electrophoresis of RNA through Denaturing UreaPolyacrylamide Gels393Transfer and Fixation of Denatured RNA in Agarose Gels to Membranes401† Alternative Protocol: Capillary Transfer by Downward Flow406 2012 by Cold Spring Harbor Laboratory Press
This is a free sample of content from Molecular Cloning: A Laboratory Manual, 4th edition.Click here for more information or to buy the book.x / Contents13Transfer and Fixation of Denatured RNA in Polyacrylamide Gels to Membranes byElectrophoretic Transfer40814Northern Hybridization41015Dot and Slot Hybridization of Purified RNA415Introduction to Mapping RNA42016Mapping RNA with Nuclease S143017Ribonuclease Protection: Mapping RNA with Ribonuclease and RadiolabeledRNA Probes437Analysis of RNA by Primer Extension44518INFORMATION PANELSHow to Win the Battle with RNase450Inhibitors of RNases451Diethylpyrocarbonate452Nuclease S1454CHAPTER 7Polymerase Chain Reaction455INTRODUCTIONThe Basic Polymerase Chain Reaction456Design of Oligonucleotide Primers for Basic PCR462Detecting, Analyzing, and Quantifying mRNAs464Contamination in PCR466PROTOCOLS1 The Basic Polymerase Chain Reaction4702 Hot Start PCR4773 Touchdown PCR4814 PCR Amplification of GC-Rich Templates4845 Long and Accurate PCR (LA PCR)4906 Inverse PCR4947 Nested PCR4998 Amplification of cDNA Generated by Reverse Transcription of mRNA: Two-StepRT-PCR50300515009 Rapid Amplification of Sequences from the 5 Ends of mRNAs: 5 -RACE10Rapid Amplification of Sequences from the 3 Ends of mRNAs: 3 -RACE52311Screening Colonies by PCR531INFORMATION PANELSTaq DNA Polymerase533PCR in Theory537Ribonuclease H538The dut and ung Genes of E. coli539Terminal Transferase540 2012 by Cold Spring Harbor Laboratory Press
This is a free sample of content from Molecular Cloning: A Laboratory Manual, 4th edition.Click here for more information or to buy the book.Contents/xiCHAPTER 8Bioinformatics541Jui-Hung Hung and Zhiping WengINTRODUCTIONPROTOCOLS1Visualizing Genomic Annotations with the UCSC Genome Browser544Introduction to Sequence Alignment and Homology Search5542Sequence Alignment and Homology Search with BLAST and ClustalW5573Designing PCR Primers Using Primer3Plus564Introduction to Expression Profiling by Microarray and RNA-seq5714577Expression Profiling by Microarray and RNA-seqIntroduction to Mapping Billions of Short Reads to a Reference Genome5885591Mapping Billions of Short Reads to a Reference GenomeIntroduction to Peak-Finding Algorithms5986604Identifying Regions Enriched in a ChIP-seq Data Set (Peak Finding)Introduction to Motif Finding6127617Discovering cis-Regulatory MotifsINFORMATION PANELSData Formats625Algorithms, Portals, and Methods628VOLUME 2PART 2: ANALYSIS AND MANIPULATION OF DNA AND RNACHAPTER 9Quantification of DNA and RNA by Real-Time Polymerase Chain Reaction631INTRODUCTIONReal-Time PCR Chemistries632Instruments for Real-Time PCR639Extracting Data from a Real-Time PCR Experiment: Data Analysisand Normalization Methods641Designing Primers and Probes and Optimizing Conditions for Real-Time PCR643Constructing a Standard Curve648Performing Real-Time PCR650Performing Real-Time RT-PCR650MIQE Guidelines654Real-Time PCR Protocols654 2012 by Cold Spring Harbor Laboratory Press
This is a free sample of content from Molecular Cloning: A Laboratory Manual, 4th edition.Click here for more information or to buy the book.xii / ContentsPROTOCOLS1Optimizing Primer and Probe Concentrations for Use in Real-Time PCR6582Constructing a Standard Curve6633Quantification of DNA by Real-Time PCR6674Quantification of RNA by Real-Time RT-PCR6705Analysis and Normalization of Real-Time PCR Experimental Data674INFORMATION PANELSMultiplex PCR680SNP Genotyping681CHAPTER 10Nucleic Acid Platform Technologies683Oliver RandoINTRODUCTIONMicroarray Applications685Performing Microarray Experiments688PROTOCOLS1 Printing Microarrays6942 Round A/Round B Amplification of DNA6983 T7 Linear Amplification of DNA (TLAD) for Nucleosomaland Other DNA , 500 bp7024 Amplification of RNA7095 Direct Cyanine-dUTP Labeling of RNA7156 Indirect Aminoallyl-dUTP Labeling of RNA7187 Cyanine-dCTP Labeling of DNA Using Klenow7218 Indirect Labeling of DNA7249 Blocking Polylysines on Homemade Microarrays72610Hybridization to Homemade Microarrays729CHAPTER 11DNA Sequencing735Elaine Mardis and W. Richard McCombieINTRODUCTIONHistory of Sanger/Dideoxy DNA Sequencing736Next-Generation Sequencing742Overview of Next-Generation Sequencing Instruments752Sanger Sequencing versus Next-Generation Sequencing: When to Do What?760Introduction to Protocols761 2012 by Cold Spring Harbor Laboratory Press
This is a free sample of content from Molecular Cloning: A Laboratory Manual, 4th edition.Click here for more information or to buy the book.Contents/xiiiSECTION I. LIBRARY PREPARATIONS FOR VARIOUS PLATFORMSCapillary Sequencing1Preparing Plasmid Subclones for Capillary Sequencing7642Preparing PCR Products for Capillary Sequencing7703Cycling Sequencing Reactions773Illumina4Whole Genome: Manual Library Preparation7765Whole Genome: Automated, Nonindexed Library Preparation783† Additional Protocol: Automated Library Preparation7896Whole Genome: Automated, Indexed Library Preparation7927Preparation of a 3-kb Mate-Pair Library for Illumina Sequencing7998Preparation of an 8-kb Mate-Pair Library for Illumina Sequencing808† Additional Protocol: AMPure Bead Calibration821RNA-Seq: RNA Conversion to cDNA and Amplification824† Additional Protocol: RNAClean XP Bead Cleanup (before RNA-Seq)830Solution-Phase Exome Capture831† Additional Protocol: AMPure XP Bead Cleanup840† Additional Protocol: Agarose Gel Size Selection84211Automated Size Selection84412Library Quantification Using SYBR Green-qPCR84813Library Quantification Using PicoGreen Fluorometry85214Library Quantification: Fluorometric Quantitation of Double-Stranded orSingle-Stranded DNA Samples Using the Qubit System857910Pyrosequencing15Preparation of Small-Fragment Libraries for 454 Sequencing16sstDNA Library Capture and emPCR86617Roche/454 Sequencer: Executing a Sequencing Run874859SECTION II. POSTSEQUENCING ANALYSES18Validation88319Quality Assessment of Sequence Data88520Data Analysis887INFORMATION PANELSBiotin888Magnetic Beads890Fragmenting of DNA892Analysis of DNA Methylation in Mammalian Cells893CHAPTER 12Paul M. Lizardi, Qin Yan, and Narendra WajapeyeeINTRODUCTIONDNA Methylation Affects and Reveals Biological Phenomena894Experimental Approaches for Analysis of DNA Methylation895 2012 by Cold Spring Harbor Laboratory Press
This is a free sample of content from Molecular Cloning: A Laboratory Manual, 4th edition.Click here for more information or to buy the book.xiv / ContentsAdvantages and Limitations of Different Approaches for Analyzing DNA Methylation898Future Perspectives899PROTOCOLS1DNA Bisulfite Sequencing for Single-Nucleotide-Resolution DNA MethylationDetection9002Methylation-Specific PCR for Gene-Specific DNA Methylation Detection9073Methyl-Cytosine-Based Immunoprecipitation for DNA Methylation Analysis9114High-Throughput Deep Sequencing for Mapping Mammalian DNA Methylation9155Roche 454 Clonal Sequencing of Bisulfite-Converted DNA Libraries9276Illumina Sequencing of Bisulfite-Converted DNA Libraries932INFORMATION PANELSPublic Domain Software for Identifying CpG Islands in Promoter and Coding Regionsof Mammalian Genes937Designing Primers for the Amplification of Bisulfite-Converted Product to PerformBisulfite Sequencing and MS-PCR939Postsequence Processing of High-Throughput Bisulfite Deep-Sequencing Data940CHAPTER 13Preparation of Labeled DNA, RNA, and Oligonucleotide Probes943INTRODUCTIONRadioactive versus Nonradioactive Labeling of Nucleic Acids944Types of Nonradioactive Detection Systems948Designing Oligonucleotides for Use as Probes953PROTOCOLS1 Random Priming: Labeling of Purified DNA Fragments by Extension of RandomOligonucleotides9652 Random Priming: Labeling of DNA by Extension of Random Oligonucleotides in thePresence of Melted Agarose9713 Labeling of DNA Probes by Nick Translation9744 Labeling of DNA Probes by Polymerase Chain Reaction978† Additional Protocol: Asymmetric Probes5 Synthesis of Single-Stranded RNA Probes by In Vitro Transcription† Additional Protocol: Using PCR to Add Promoters for Bacteriophage-EncodedRNA Polymerases to Fragments of DNA9829849916 Synthesis of cDNA Probes from mRNA Using Random Oligonucleotide Primers9947 Radiolabeling of Subtracted cDNA Probes by Random Oligonucleotide Extension9978 Labeling 30 Termini of Double-Stranded DNA Using the Klenow Fragment ofE. coli DNA Polymerase I10039 Dephosphorylation of DNA Fragments with Alkaline Phosphatase100910Phosphorylation of DNA Molecules with Protruding 50 -Hydroxyl Termini 2012 by Cold Spring Harbor Laboratory Press1012
This is a free sample of content from Molecular Cloning: A Laboratory Manual, 4th edition.Click here for more information or to buy the book.Contents/ xv11Phosphorylation of DNA Molecules with Dephosphorylated Blunt Ends orRecessed 50 Termini101512Phosphorylating the 50 Termini of Oligonucleotides Using T4 Polynucleotide Kinase101813Labeling the 30 Termini of Oligonucleotides Using Terminal DeoxynucleotidylTransferase1021† Alternative Protocol: Synthesizing Nonradiolabeled Probes Using TdT1023† Additional Protocol: Tailing Reaction1024† Additional Protocol: Modifications for Synthesizing Nonradiolabeled Probes1026Labeling of Synthetic Oligonucleotides Using the Klenow Fragment of E. coli DNAPolymerase I102815Purification of Labeled Oligonucleotides by Precipitation with Ethanol103216Purification of Labeled Oligonucleotides by Size-Exclusion Chromatography103417Purification of Labeled Oligonucleotides by Chromatography on a Sep-Pak C18 Column103718Hybridization of Oligonucleotide Probes in Aqueous Solutions: Washing in BuffersContaining Quaternary Ammonium Salts103914INFORMATION PANELSPreparation of Stock Solutions of dNTPs1043E. coli DNA Polymerase I and the Klenow Fragment1044In Vitro Transcription Systems1050Alkaline Phosphatase1053Melting Temperatures1055Methods for In Vitro Mutagenesis1059CHAPTER 14Matteo Forloni, Alex Y. Liu, and Narendra WajapeyeeINTRODUCTIONMutagenesis Approaches1064Research Goals1065Commercial Kits1065PROTOCOLS1Random Mutagenesis Using Error-Prone DNA Polymerases10682Creating Insertions or Deletions Using Overlap Extension PCR Mutagenesis10803In Vitro Mutagenesis Using Double-Stranded DNA Templates: Selectionof Mutants with DpnI10874Altered b-Lactamase Selection Approach for Site-Directed Mutagenesis10955Oligonucleotide-Directed Mutagenesis by Elimination of a Unique Restriction Site(USE Mutagenesis)11026Saturation Mutagenesis by Codon Cassette Insertion11087Random Scanning Mutagenesis11158Multisite-Directed Mutagenesis11209Megaprimer PCR-Based Mutagenesis1124 2012 by Cold Spring Harbor Laboratory Press
This is a free sample of content from Molecular Cloning: A Laboratory Manual, 4th edition.Click here for more information or to buy the book.xvi / ContentsINFORMATION PANELSDomain Mutagenesis1127High-Throughput Site-Directed Mutagenesis of Plasmid DNA11286N -Methyladenine, Dam Methylase, and Methylation-SensitiveRestriction Enzymes1129PART 3: INTRODUCING GENES INTO CELLSCHAPTER 15Introducing Genes into Cultured Mammalian Cells1131Priti Kumar, Arvindhan Nagarajan, and Pradeep D. UchilINTRODUCTIONTransient Versus Stable Transfection1133Transfection Methods1133Transfection Controls1133Optimization and Special Considerations1136Assessing Cell Viability in Transfected Cell Lines1137PROTOCOLS1DNA Transfection Mediated by Cationic Lipid Reagents1139† Alternative Protocol: Transfection Using DOTMA and DOGS1145† Additional Protocol: Histochemical Staining of Cell Monolayers for b-Galactosidase1148Calcium-Phosphate-Mediated Transfection of Eukaryotic Cells with Plasmid DNAs1150† Alternative Protocol: High-Efficiency Calcium-Phosphate-Mediated Transfection ofEukaryotic Cells with Plasmid DNAs1156Calcium-Phosphate-Mediated Transfection of Cells with High-Molecular-WeightGenomic DNA1160† Alternative Protocol: Calcium-Phosphate-Mediated Transfection of Adherent Cells1163† Alternative Protocol: Calcium-Phosphate-Mediated Transfection of Cells Growingin Suspension1165Transfection Mediated by DEAE-Dextran: High-Efficiency Method1167† Alternative Protocol: Transfection Mediated by DEAE-Dextran: IncreasedCell Viability11705DNA Transfection by Electroporation11736Analysis of Cell Viability by the alamarBlue Assay11777Analysis of Cell Viability by the Lactate Dehydrogenase Assay11808Analysis of Cell Viability by the MTT Assay1183234INFORMATION PANELSOptical Transfection1186Cotransformation1188Selective Agents for Stable Transformation1190Lipofection1194 2012 by Cold Spring Harbor Laboratory Press
This is a free sample of content from Molecular Cloning: A Laboratory Manual, 4th edition.Click here for more information or to buy the book.Contents/ xviiLinearizing Plasmids before Transfection1197Transfection of Mammalian Cells with Calcium Phosphate –DNA Coprecipitates1198Chloroquine Diphosphate1200DEAE-Dextran Transfection1201Electroporation1203CHAPTER 16Introducing Genes into Mammalian Cells: Viral Vectors1209Guangping Gao and Miguel Sena-EstevesINTRODUCTIONFactors to Consider When Choosing a Viral Vector1211The Major Types of Viruses Currently Used as Vectors1212In Vivo Expression1221Adenovirus Vectors1221Adeno-Associated Virus Vectors1224Retrovirus and Lentivirus Vectors1227PROTOCOLS1Construction of Recombinant Adenovirus Genomes by Direct Cloning12332Release of the Cloned Recombinant Adenovirus Genome for Rescue and Expansion12383Purification of the Recombinant Adenovirus by Cesium Chloride GradientCentrifugation12444Characterization of the Purified Recombinant Adenovirus Vector for Viral GenomeStructure by Restriction Enzyme Digestions12495Measuring the Infectious Titer of Recombinant Adenovirus Vectors UsingTCID50 End-Point Dilution and qPCR1253† Additional Protocol: Preparation of a DNA Standard for qPCR12626Detection Assay for Replication-Competent Adenovirus by ConcentrationPassage and Real-Time qPCR12647Production of rAAVs by Transient Transfection12748Purification of rAAVs by Cesium Chloride Gradient Sedimentation12789Purification of rAAVs by Iodixanol Gradient Centrifugation128310Purification of rAAV2s by Heparin Column Affinity Chromatography128711Enrichment of Fully Packaged Virions in Column-Purified rAAV Preparationsby Iodixanol Gradient Centrifugation Followed by Anion-Exchange ColumnChromatography129012Titration of rAAV Genome Copy Number Using Real-Time qPCR129413Sensitive Determination of Infectious Titer of rAAVs Using TCID50End-Point Dilution and qPCR129814Analysis of rAAV Sample Morphology Using Negative Staining andHigh-Resolution Electron Microscopy130115Analysis of rAAV Purity Using Silver-Stained SDS-PAGE130416Production of High-Titer Retrovirus and Lentivirus Vectors1307 2012 by Cold Spring Harbor Laboratory Press
This is a free sample of content from Molecular Cloning: A Laboratory Manual, 4th edition.Click here for more information or to buy the book.xviii / Contents17Titration of Lentivirus Vectors131418Monitoring Lentivirus Vector Stocks for Replication-Competent Viruses1319INFORMATION PANELSAdenovirus Vectors1322AAV Vectors1323Lentivirus Vectors1324Basic Elements in Viral Vectors1326Assays Done in Transduced Cells1328Transgene Expression Cassettes1330VOLUME 3PART 4: GENE EXPRESSIONCHAPTER 17Analysis of Gene Regulation Using Reporter Systems1335Pradeep D. Uchil, Arvindhan Nagarajan, and Priti KumarINTRODUCTIONIntroduction to Reporter Systems1336Reporter Genes Used in the Analysis of Regulatory Elements1336Assaying for b-Galactosidase in Extracts of Mammalian Cells1338Assaying for Luciferase in Extracts of Mammalian Cells1339Tetracycline-Responsive Expression Systems1341PROTOCOLS1Assay for b-Galactosidase in Extracts of Mammalian Cells1346† Additional Protocol: Chemiluminescent Assay for b-Galactosidase Activity13502Single Luciferase Reporter Assay13543Dual Luciferase Reporter Assay13594Using ELISA to Measure GFP Production13665Generation of Cell Lines
Molecular cloning : a laboratory manual / Michael R. Green, Joseph Sambrook. - 4th ed. p. cm. Rev. ed. of: Molecular cloning : a laboratory manual / Joseph Sambrook, David W. Russell. 2001. Includes bibliographical references and index. ISBN 978-1-936113-41-5 (cloth) - ISBN 978-1-936113-42-2 (pbk.) 1. Molecular cloning-Laboratory manuals. I.
