Molecular Cloning

Time 2020-10-13 12:03:59

Web Name: Molecular Cloning

WebSite: http://molecularcloning.com

ID:80587

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Molecular,Cloning,

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Protocol 1: Optimizing Primer and Probe Concentrations for Use in Real-Time PCR Protocol 2: Constructing a Standard Curve Protocol 3: Quantification of DNA by Real-Time PCR Protocol 4: Quantification of RNA by Real-Time RT-PCR Protocol 5: Analysis and Normalization of Real-Time PCR Experimental Data Panel: Quantification of DNA and RNA by Real-Time Polymerase Chain Reaction631Panel: Real-Time PCR Chemistries632Panel: Instruments for Real-Time PCR639Panel: Extracting Data from a Real-Time PCR Experiment: Data Analysis and Normalization Methods641Panel: Designing Primers and Probes and Optimizing Conditions for Real-Time PCR643Panel: Constructing a Standard Curve648Panel: Performing Real-Time PCR650Panel: Performing Real-Time RT-PCR650Panel: MIQE Guidelines654Panel: Real-Time PCR Protocols654Panel: Multiplex PCR680Panel: SNP Genotyping681Chapter 10: Nucleic Acid Platform Technologies683Protocol 1: Printing Microarrays Protocol 2: Round A/Round B Amplification of DNA Protocol 3: T7 Linear Amplification of DNA (TLAD) for Nucleosomal and Other DNA 500 bp Protocol 4: Amplification of RNA Protocol 5: Direct Cyanine-dUTP Labeling of RNA Protocol 6: Indirect Aminoallyl-dUTP Labeling of RNA Protocol 7: Cyanine-dCTP Labeling of DNA Using Klenow Protocol 8: Indirect Labeling of DNA Protocol 9: Blocking Polylysines on Homemade Microarrays Protocol 10: Hybridization to Homemade Microarrays Panel: Nucleic Acid Platform Technologies683Panel: Microarray Applications685Panel: Performing Microarray Experiments688Chapter 11: DNA Sequencing735Protocol 1: Preparing Plasmid Subclones for Capillary Sequencing Protocol 2: Preparing PCR Products for Capillary Sequencing Protocol 3: Cycle-Sequencing Reactions Protocol 4: Whole Genome: Manual Library Preparation Protocol 5: Whole Genome: Automated, Nonindexed Library Preparation Protocol 6: Whole Genome: Automated, Indexed Library Preparation Protocol 7: Preparation of a 3-kb Mate-Pair Library for Illumina Sequencing Protocol 8: Preparation of an 8-kb Mate-Pair Library for Illumina Sequencing Protocol 9: RNA-Seq: RNA Conversion to cDNA and Amplification Protocol 10: Solution-Phase Exome Capture Protocol 11: Automated Size Selection Protocol 12: Library Quantification Using SYBR Green-qPCR Protocol 13: Library Quantification Using PicoGreen Fluorometry Protocol 14: Library Quantification: Fluorometric Quantitation of Double-Stranded or Single-Stranded DNA Samples Using the Qubit System Protocol 15: Preparation of Small-Fragment Libraries for 454 Sequencing Protocol 16: sstDNA Library Capture and emPCR Protocol 17: Roche/454 Sequencer: Executing a Sequencing Run Protocol 18: Validation Protocol 19: Quality Assessment of Sequence Data Protocol 20: Data Analysis Panel: DNA Sequencing735Panel: History of Sanger/Dideoxy DNA Sequencing736Panel: Next-Generation Sequencing742Panel: Overview of Next-Generation Sequencing Instruments752Panel: Sanger Sequencing versus Next-Generation Sequencing: When to Do What?760Panel: Introduction to Protocols761Panel: Additional Protocol: Automated Library Preparation789Panel: Additional Protocol: AMPure Bead Calibration821Panel: Additional Protocol: RNAClean XP Bead Cleanup (before RNA-Seq)830Panel: Additional Protocol: AMPure XP Bead Cleanup840Panel: Additional Protocol: Agarose Gel Size Selection842Panel: Biotin888Panel: Magnetic Beads890Panel: Fragmenting of DNA892Chapter 12: Analysis of DNA Methylation in Mammalian Cells893Protocol 1: DNA Bisulfite Sequencing for Single-Nucleotide-Resolution DNA Methylation Detection Protocol 2: Methylation-Specific PCR for Gene-Specific DNA Methylation Detection Protocol 3: Methyl-Cytosine-Based Immunoprecipitation for DNA Methylation Analysis Protocol 4: High-Throughput Deep Sequencing for Mapping Mammalian DNA Methylation Protocol 5: Roche 454 Clonal Sequencing of Bisulfite-Converted DNA Libraries Protocol 6: Illumina Sequencing of Bisulfite-Converted DNA Libraries Panel: Analysis of DNA Methylation in Mammalian Cells893Panel: DNA Methylation Affects and Reveals Biological Phenomena894Panel: Experimental Approaches for Analysis of DNA Methylation895Panel: Advantages and Limitations of Different Approaches for Analyzing DNA Methylation898Panel: Future Perspectives899Panel: Public Domain Software for Identifying CpG Islands in Promoter and Coding Regions of Mammalian Genes937Panel: Designing Primers for the Amplification of Bisulfite-Converted Product to Perform Bisulfite Sequencing and MS-PCR939Panel: Postsequence Processing of High-Throughput Bisulfite Deep-Sequencing Data940Chapter 13: Preparation of Labeled DNA, RNA, and Oligonucleotide Probes943Protocol 1: Random Priming: Labeling of Purified DNA Fragments by Extension of Random Oligonucleotides Protocol 2: Random Priming: Labeling of DNA by Extension of Random Oligonucleotides in the Presence of Melted Agarose Protocol 3: Labeling of DNA Probes by Nick Translation Protocol 4: Labeling of DNA Probes by Polymerase Chain Reaction Protocol 5: Synthesis of Single-Stranded RNA Probes by In Vitro Transcription Protocol 6: Synthesis of cDNA Probes from mRNA Using Random Oligonucleotide Primers Protocol 7: Radiolabeling of Subtracted cDNA Probes by Random Oligonucleotide Extension Protocol 8: Labeling 3 Termini of Double-Stranded DNA Using the Klenow Fragment of E. coli DNA Polymerase I Protocol 9: Dephosphorylation of DNA Fragments with Alkaline Phosphatase Protocol 10: Phosphorylation of DNA Molecules with Protruding 5-Hydroxyl Termini Protocol 11: Phosphorylation of DNA Molecules with Dephosphorylated Blunt Ends or Recessed 5 Termini Protocol 12: Phosphorylating the 5 Termini of Oligonucleotides Using T4 Polynucleotide Kinase Protocol 13: Labeling the 3 Termini of Oligonucleotides Using Terminal Deoxynucleotidyl Transferase Protocol 14: Labeling of Synthetic Oligonucleotides Using the Klenow Fragment of E. coli DNA Polymerase I Protocol 15: Purification of Labeled Oligonucleotides by Precipitation with Ethanol Protocol 16: Purification of Labeled Oligonucleotides by Size-Exclusion Chromatography Protocol 17: Purification of Labeled Oligonucleotides by Chromatography on a Sep-Pak C18 Column Protocol 18: Hybridization of Oligonucleotide Probes in Aqueous Solutions: Washing in Buffers Containing Quaternary Ammonium Salts Panel: Preparation of Labeled DNA, RNA, and Oligonucleotide Probes943Panel: Radioactive versus Nonradioactive Labeling of Nucleic Acids944Panel: Types of Nonradioactive Detection Systems948Panel: Designing Oligonucleotides for Use as Probes953Panel: Additional Protocol: Asymmetric Probes982Panel: Additional Protocol: Using PCR to Add Promoters for Bacteriophage-Encoded RNA Polymerases to Fragments of DNA991Panel: Alternative Protocol: Synthesizing Nonradiolabeled Probes Using TdT1023Panel: Additional Protocol: Tailing Reaction1024Panel: Additional Protocol: Modifications for Synthesizing Nonradiolabeled Probes1026Panel: Preparation of Stock Solutions of dNTPs1043Panel: E. coli DNA Polymerase I and the Klenow Fragment1044Panel: In Vitro Transcription Systems1050Panel: Alkaline Phosphatase1053Panel: Melting Temperatures1055Chapter 14: Methods for In Vitro Mutagenesis1059Protocol 1: Random Mutagenesis Using Error-Prone DNA Polymerases Protocol 2: Creating Insertions or Deletions Using Overlap Extension PCR Mutagenesis Protocol 3: In Vitro Mutagenesis Using Double-Stranded DNA Templates: Selection of Mutants with DpnI Protocol 4: Altered -Lactamase Selection Approach for Site-Directed Mutagenesis Protocol 5: Oligonucleotide-Directed Mutagenesis by Elimination of a Unique Restriction Site (USE Mutagenesis) Protocol 6: Saturation Mutagenesis by Codon Cassette Insertion Protocol 7: Random Scanning Mutagenesis Protocol 8: Multisite-Directed Mutagenesis Protocol 9: Megaprimer PCR-Based Mutagenesis Panel: Methods for In Vitro Mutagenesis1059Panel: Mutagenesis Approaches1064Panel: Research Goals1065Panel: Commercial Kits1065Panel: Domain Mutagenesis1127Panel: High-Throughput Site-Directed Mutagenesis of Plasmid DNA1128Panel: N6-Methyladenine, Dam Methylase, and Methylation-Sensitive Restriction Enzymes1129Chapter 15: Introducing Genes into Cultured Mammalian Cells1131Protocol 1: DNA Transfection Mediated by Cationic Lipid Reagents Protocol 2: Calcium-Phosphate-Mediated Transfection of Eukaryotic Cells with Plasmid DNAs Protocol 3: Calcium-Phosphate-Mediated Transfection of Cells with High-Molecular-Weight Genomic DNA Protocol 4: Transfection Mediated by DEAE-Dextran: High-Efficiency Method Protocol 5: DNA Transfection by Electroporation Protocol 6: Analysis of Cell Viability by the alamarBlue Assay Protocol 7: Analysis of Cell Viability by the Lactate Dehydrogenase Assay Protocol 8: Analysis of Cell Viability by the MTT Assay Panel: Introducing Genes into Cultured Mammalian Cells1131Panel: Transient Versus Stable Transfection1133Panel: Transfection Methods1133Panel: Transfection Controls1133Panel: Optimization and Special Considerations1136Panel: Assessing Cell Viability in Transfected Cell Lines1137Panel: Alternative Protocol: Transfection Using DOTMA and DOGS1145Panel: Additional Protocol: Histochemical Staining of Cell Monolayers for -Galactosidase1148Panel: Alternative Protocol: High-Efficiency Calcium-Phosphate-Mediated Transfection of Eukaryotic Cells with Plasmid DNAs1156Panel: Alternative Protocol: Calcium-Phosphate-Mediated Transfection of Adherent Cells1163Panel: Alternative Protocol: Calcium-Phosphate-Mediated Transfection of Cells Growing in Suspension1165Panel: Alternative Protocol: Transfection Mediated by DEAE-Dextran: Increased Cell Viability1170Panel: Optical Transfection1186Panel: Cotransformation1188Panel: Selective Agents for Stable Transformation1190Panel: Lipofection1194Panel: Linearizing Plasmids before Transfection1197Panel: Transfection of Mammalian Cells with Calcium PhosphateDNA Coprecipitates1198Panel: Chloroquine Diphosphate1200Panel: DEAE-Dextran Transfection1201Panel: Electroporation1203Chapter 16: Introducing Genes into Mammalian Cells: Viral Vectors1209Protocol 1: Construction of Recombinant Adenovirus Genomes by Direct Cloning Protocol 2: Release of the Cloned Recombinant Adenovirus Genome for Rescue and Expansion Protocol 3: Purification of the Recombinant Adenovirus by Cesium Chloride Gradient Centrifugation Protocol 4: Characterization of the Purified Recombinant Adenovirus for Viral Genome Structure by Restriction Enzyme Digestions Protocol 5: Measuring the Infectious Titer of Recombinant Adenovirus Using TCID50 End-Point Dilution and qPCR Protocol 6: Detection Assay for Replication-Competent Adenovirus by Concentration Passage and Real-Time qPCR Protocol 7: Production of rAAVs by Transient Transfection Protocol 8: Purification of rAAVs by Cesium Chloride Gradient Sedimentation Protocol 9: Purification of rAAVs by Iodixanol Gradient Centrifugation Protocol 10: Purification of rAAV2s by Heparin Column Affinity Chromatography Protocol 11: Enrichment of Fully Packaged Virions in Column-Purified rAAV Preparations by Iodixanol Gradient Centrifugation Followed by Anion-Exchange Column Chromatography Protocol 12: Titration of rAAV Genome Copy Number Using Real-Time qPCR Protocol 13: Sensitive Determination of Infectious Titer of rAAVs Using TCID50 End-Point Dilution and qPCR Protocol 14: Analysis of rAAV Sample Morphology Using Negative Staining and High-Resolution Electron Microscopy Protocol 15: Analysis of rAAV Purity Using Silver-Stained SDS-PAGE Protocol 16: Production of High-Titer Retrovirus and Lentivirus Vectors Protocol 17: Titration of Lentivirus Vectors Protocol 18: Monitoring Lentivirus Vector Stocks for Replication-Competent Viruses Panel: Introducing Genes into Mammalian Cells: Viral Vectors1209Panel: Factors to Consider When Choosing a Viral Vector1211Panel: The Major Types of Viruses Currently Used as Vectors1212Panel: In Vivo Expression1221Panel: Adenovirus Vectors1221Panel: Adeno-Associated Virus Vectors1224Panel: Retrovirus and Lentivirus Vectors1227Panel: Additional Protocol: Preparation of a DNA Standard for qPCR1262Panel: Adenovirus Vectors1322Panel: AAV Vectors1323Panel: Lentivirus Vectors1324Panel: Basic Elements in Viral Vectors1326Panel: Assays Done in Transduced Cells1328Panel: Transgene Expression Cassettes1330Protocol 1: Assay for -Galactosidase in Extracts of Mammalian Cells Protocol 2: Single Luciferase Reporter Assay Protocol 3: Dual Luciferase Reporter Assay Protocol 4: Using ELISA to Measure GFP Production Protocol 5: Generation of Cell Lines with Tetracycline-Regulated Gene Expression Panel: Analysis of Gene Regulation Using Reporter Systems1335Panel: Introduction to Reporter Systems1336Panel: Reporter Genes Used in the Analysis of Regulatory Elements1336Panel: Assaying for -Galactosidase in Extracts of Mammalian Cells1338Panel: Assaying for Luciferase in Extracts of Mammalian Cells1339Panel: Tetracycline-Responsive Expression Systems1341Panel: Additional Protocol: Chemiluminescent Assay for -Galactosidase Activity1350Panel: Additional Protocol: Selecting Stable Clones via Limiting Dilution of Suspension Cells1378Panel: Fluorescent Proteins1381Panel: Epitope Tagging1394Panel: -Galactosidase1401Panel: Luciferase1406Panel: Tetracycline1409Chapter 18: RNA Interference and Small RNA Analysis1415Protocol 1: Preparation of siRNA Duplexes Protocol 2: RNAi in Mammalian Cells by siRNA Duplex Transfection Protocol 3: RNAi in Drosophila S2 Cells by siRNA Duplex Transfection Protocol 4: Preparation of dsRNAs by In Vitro Transcription Protocol 5: RNAi in Drosophila S2 Cells by dsRNA Soaking Protocol 6: RNAi in Drosophila S2 Cells by dsRNA Transfection Protocol 7: Analysis of Small RNAs by Northern Hybridization Protocol 8: Analysis of Small RNAs by Quantitative Reverse Transcription PCR Protocol 9: Construction of Small RNA Libraries for High-Throughput Sequencing Protocol 10: Preparation of Antisense Oligonucleotides to Inhibit miRNA Function Protocol 11: Inhibiting miRNA Function by Antisense Oligonucleotides in Cultured Mammalian Cells Protocol 12: Inhibiting miRNA Function by Antisense Oligonucleotides in Drosophila S2 Cells Panel: RNA Interference and Small RNA Analysis1415Panel: Reverse Genetics by RNAi1419Panel: Analysis of Small RNAs1425Panel: Genome-Wide RNA Interference: Functional Genomics in the Postgenomics Era1472Panel: StarFire Probes1478Chapter 19: Expressing Cloned Genes for Protein Production, Purification, and Analysis1481Protocol 1: Expression of Cloned Genes in E. coli Using IPTG-Inducible Promoters Protocol 2: Expression of Cloned Genes Using the Baculovirus Expression System Protocol 3: Expression of Cloned Genes in P. pastoris Using the Methanol-Inducible Promoter AOX1 Protocol 4: Preparation of Cell Extract for Purification of Soluble Proteins Expressed in E. coli Protocol 5: Purification of Polyhistidine-Tagged Proteins by Immobilized Metal Affinity Chromatography Protocol 6: Purification of Fusion Proteins by Affinity Chromatography on Glutathione Resin Protocol 7: Solubilization of Expressed Proteins from Inclusion Bodies Protocol 8: SDS-PAGE of Proteins Protocol 9: Analysis of Proteins by Immunoblotting Protocol 10: Methods for Measuring the Concentrations of Proteins Panel: Expressing Cloned Genes for Protein Production, Purification, and Analysis1481Panel: Choosing an Expression System1483Panel: Choosing an Appropriate Expression Vector1488Panel: Fusion Proteins1499Panel: Optimization of Expression of Foreign Proteins1503Panel: Additional Protocol: Small-Scale Test for Soluble Target Protein Expression1514Panel: Alternative Protocol: Expression of Cloned Genes in E. coli Using the Arabinose BAD Promoter1520Panel: Alternative Protocol: Subcellular Localization of Signal Peptide Fusion Proteins1522Panel: Additional Protocol: Plaque Assay to Determine the Titer of the Baculovirus Stock1535Panel: Alternative Protocol: Production of Bacmid DNA for Transfection into Insect Cells1538Panel: Additional Protocol: Cryostorage of Yeast Cultures1553Panel: Additional Protocol: Lysis of Yeast Cells Using Glass Beads1564Panel: Alternative Protocol: Preparation of E. coil Cell Extract Using Gentle, Heat-Induced Enzymatic Lysis1566Panel: Alternative Protocol: Preparation of E. coli Cell Extract Using FreezeThaw with Enzymatic Lysis by Lysozyme1568Panel: Additional Protocol: Regenerating and Cleaning the Ni2NTA Resin1579Panel: Alternative Protocol: Fast Performance Liquid Chromatography Purification of Histidine-Tagged Proteins1581Panel: Alternative Protocol: Variations of Staining SDSPolyacrylamide Gels with Coomassie Brilliant Blue1609Panel: Alternative Protocol: Staining SDSPolyacrylamide Gels with Silver Salts1611Panel: Considerations for Membrane Protein Purification1632Panel: Historical Footnote: Coomassie Brilliant Blue1636Chapter 20: Cross-Linking Technologies for Analysis of Chromatin Structure and Function1637Protocol 1: Formaldehyde Cross-Linking Protocol 2: Preparation of Cross-Linked Chromatin for ChIP Protocol 3: ChIP Protocol 4: ChIPQuantitative PCR (ChIP-qPCR) Protocol 5: ChIP-chip Protocol 6: ChIP-seq Protocol 7: Generation of 3C Libraries from Cross-Linked Cells Protocol 8: Generation of ChIP-loop Libraries Protocol 9: Generation of Control Ligation Product Libraries Protocol 10: PCR Detection of 3C Ligation Products Present in 3C, ChIP-loop, and Control Libraries: Library Titration and Interaction Frequency Analysis Protocol 11: 4C Analysis of 3C, ChIP-loop, and Control Libraries Protocol 12: 5C Analysis of 3C, ChIP-loop, and Control Libraries Panel: Cross-Linking Technologies for Analysis of Chromatin Structure and Function1637Panel: Formaldehyde Cross-Linking to Interrogate Genomic Interactions1638Panel: ChIP Analysis of ProteinDNA Interactions1638Panel: 3C-Based Chromatin Interaction Analyses1641Panel: Formaldehyde1701Panel: What Is Captured by 3C-Based Assays?1702Chapter 21: Mapping of In Vivo RNA-Binding Sites by UV-Cross-Linking Immunoprecipitation (CLIP)1703Protocol 1: Optimization of Immunoprecipitation Stringency for CLIP Protocol 2: UV Cross-Linking of Live Cells and Lysate Preparation Protocol 3: RNase Titration, Immunoprecipitation, and SDS-PAGE Protocol 4: 3-Linker Ligation and Size Selection by SDS-PAGE Protocol 5: Isolation of the RNA Tags, 5-Linker Ligation, and Reverse Transcription PCR Amplification Protocol 6: Sequencing of RNA CLIP Tags Protocol 7: Gel Purification and Storage of RNA Linkers Panel: Mapping of In Vivo RNA-Binding Sites by UV-Cross-Linking Immunoprecipitation (CLIP)1703Panel: The Cross-Linking Immunoprecipitation Method1706Panel: High-Throughput Sequencing (HITS) CLIP1708Panel: Validation of CLIP Results1708Panel: CLIP Method Variations1709Panel: General Considerations in Planning CLIP Experiments1710Panel: Alternative Protocol: 5-End Labeling of Dephosphorylated RL3 Linker1738Panel: Mechanism and Specificity of UV-Protein Cross-Linking1756Panel: HITS-CLIP Data Analysis1758Chapter 22: Gateway-Compatible Yeast One-Hybrid and Two-Hybrid Assays1761Protocol 1: Generating Yeast One-Hybrid DNA-Bait Strains Protocol 2: Generating Yeast Two-Hybrid Bait Strains Protocol 3: Identifying Interactors from an Activation Domain Prey Library Protocol 4: High-Efficiency Yeast Transformation Protocol 5: Colony Lift Colorimetric Assay for -Galactosidase Activity Protocol 6: Yeast Colony PCR Panel: Gateway-Compatible Yeast One-Hybrid and Two-Hybrid Assays1761Panel: The Yeast Two-Hybrid (Y2H) System: Concept and Methodology1763Panel: The Yeast One-Hybrid (Y1H) System: Concept and Methodology1767Panel: Y2H and Y1H Assays: Advantages and Disadvantages1768Panel: False Positives1769Panel: Protocols for Yeast One-Hybrid and Two-Hybrid Systems1770Panel: Alternative Protocol: Creating Entry Clones from DNA-Baits Generated by Annealing Primers1782Panel: Why Integrate DNA-Baits?1808Panel: Choosing a Vector and a Yeast Strain1809Panel: Replica-Plating and Replica-Cleaning Using Velvets1810Panel: Reagents and Buffers1811Panel: Tris Buffers1828Panel: Good Buffers1829Panel: Phosphate Buffers (Gomori Buffers)1830Panel: Phenol1834Panel: Equilibration of Phenol1834Panel: Phenol:Chloroform:Isoamyl Alcohol (25:24:1)1834Panel: Deionization of Formamide1834Panel: Blocking Agents Used for Nucleic Acid Hybridization1836Panel: Blocking Agents Used for Western Blotting1836Panel: Commonly Used Techniques1843Panel: Siliconizing Glassware, Plasticware, and Glass Wool1843Panel: Preparation of RNase-Free Glassware1844Panel: Hemocytometry Counting1846Panel: Viability Staining1847Panel: Precipitation of Nucleic Acids with Trichloroacetic Acid1849Panel: Removing Ethidium Bromide from DNA1851Panel: Disposing of Ethidium Bromide1851Panel: Decontamination of Concentrated Solutions of Ethidium Bromide (Solutions Containing 0.5 mg/mL)1851Panel: Decontamination of Dilute Solutions of Ethidium Bromide (e.g., Electrophoresis Buffer Containing 0.5 g/mL Ethidium Bromide)1852Panel: Commercial Decontamination Kits1852Panel: Detection Systems1855Panel: Ethidium Bromide1855Panel: Methylene Blue1857Panel: SYBR Dyes1857Panel: Chemiluminescent Labels1860Panel: Chemiluminescent Enzyme Assays1861Panel: Commercial Reagents, Kits, and Luminometers1863Panel: Horseradish Peroxidase1865Panel: Digoxygenin1869Panel: BCIP1873Panel: AMPPD1876Panel: Immunoglobulin-Binding Proteins: Proteins A, G, and L1879Panel: General Safety and Hazardous Material1885Click here for free access to Chapter 10 in its entiretyMolecular Cloning: A Laboratory Manual (Fourth Edition)Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Molecular Cloning is the new gold standard the one indispensable molecular biology laboratory manual and reference source.Highlights of the new edition: Extensive new content: 12 entirely new chapters are devoted to the most exciting current research strategies, including epigenetic analysis, RNA interference, genome sequencing, and bioinformaticsExpanded scope: the nucleic-acid-based techniques selected for inclusion have promoted recent advances in gene transfer, protein expression, RNA analysis, and expression of cloned genesClassic content: 10 original core chapters have been updated to reflect developments and innovations in standard techniques and to introduce new cutting-edge protocolsEasy-to-follow format: the previous editions’ renowned attention to detail and accuracy are fully retainedEssential appendices: an up-to-date collection of reagents, vectors, media, detection systems, and commonly used techniques are includedExpanded authorship: chapters and protocols have been specifically commissioned from renowned experts at leading institutions Any basic research laboratory using molecular biology techniques will benefit from having a copy on hand of the newly published Third Edition of Molecular Cloning: A Laboratory Manual...the first two editions of this book have been staples of molecular biology with a proven reputation for accuracy and thoroughness. The Scientist In every kitchen there is at least one indispensable cookbook...Molecular Cloning: A Laboratory Manual fills the same niche in the laboratory (with) information to help both the inexperienced and the advanced user. (It) has once again established its primacy as the molecular laboratory manual and is likely to be found on lab benches...around the world. —Trends in Neurosciences Molecular Cloning: A Laboratory Manual has always been the laboratory mainstay for protocols and techniques. It has a pure-bred ancestry, and the new edition does not disappoint. (It) includes information panels at the end of each chapter that describe the principles behind the protocols.... The addition of this information extends Molecular Cloning from an essential laboratory resource into a new realm, one merging the previous prototype with a modern molecular monograph...the next generation of Molecular Cloning not only carries on the proud heritage of the first two editions but also admirably expands on that tradition to provide a truly essential laboratory manual. Trends in MicrobiologyGet alerts about special offers from Cold Spring Harbor Laboratory Press free to your Inbox

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Molecular Cloning, also known as Maniatis, has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential.

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