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PCR Cloning Protocols: From Molecular Cloning to Genetic Engineering, 2nd edition |
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Book Info
Rutgers Univ., New Brunswick, NJ. Covers performing and optimizing PCR, cloning PCR products, mutagenesis and recombination, cloning unknown neighboring DNA, and library construction and screening. Features reproducible methods for isolating genes from all biological samples. Hardcover, comb-bound not yet available. --This text refers to the Hardcover edition.
Book Description
This completely revised second edition provides proven protocols for PCR cloning and mutagenesis. It builds on its best-selling predecessor to allow researchers of all levels to increase their success rate in their experiments. The editor has divided the book into five parts, beginning with performing and optimizing PCR to Cloning PCR products, through to mutagenesis and recombination. The final two parts cover the cloning of unknown neighboring DNA and DNA library construction and screening. Each part includes an overview that summarizes the current methods available and their underlying strategies, advantages, and disadvantages for the particular research. The techniques bring to both new and established researchers the power to apply PCR-based methodology to the cloning and modification of DNA, either through innovative protocols or by fostering individual creativity to modify and customize the protocols to best fit their own needs.
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Preface Contributors 1 Polymerase Chain Reaction: Basic Principles and Routine Practice 3 2 Computer Programs for PCR Primer Design and Analysis 19 3 Single-Step PCR Optimization Using Touchdown and Stepdown PCR Programming 31 4 XL PCR Amplification of Long Targets from Genomic DNA 37 5 Coupled One-Step Reverse Transcription and Polymerase Chain Reaction Procedure for Cloning Large cDNA Fragments 53 6 Long Distance Reverse-Transcription PCR 59 7 Increasing PCR Sensitivity for Amplification from Paraffin-Embedded Tissues 67 8 GC-Rich Template Amplification by Inverse PCR: DNA Polymerase and Solvent Effects 75 9 PCR Procedure for the Isolation of Trinucleotide Repeats 81 10 Methylation-Specific PCR 91 11 Direct Cloning of Full-Length Cell Differentially Expressed Genes by Multiple Rounds of Subtractive Hybridization Based on Long-Distance PCR and Magnetic Beads 99 12 Cloning PCR Products: An Overview 111 13 Using T4 DNA Polymerase to Generate Clonable PCR Products 121 14 Enzyme-Free Cloning of PCR Products and Fusion Protein Expression 125 15 Directional Restriction Site-Free Insertion of PCR Products into Vectors 133 16 Autosticky PCR: Directional Cloning of PCR Products with Preformed 5' Overhangs 141 17 A Rapid and Simple Procedure for Direct Cloning of PCR Products into Baculoviruses 153 18 PCR Approaches to DNA Mutagenesis and Recombination: An Overview 167 19 In-Frame Cloning of Synthetic Genes Using PCR Inserts 175 20 Megaprimer PCR 189 21 PCR-Mediated Recombination: A General Method Applied to Construct Chimeric Infectious Molecular Clones 197 22 PCR Method for Generating Multiple Mutations at Adjacent Sites 207 23 A Fast Polymerase Chain Reaction-Mediated Strategy for Introducing Repeat Expansions into CAG-Repeat Containing Genes 217 24 PCR Screening in Signature-Tagged Mutagenesis of Essential Genes 225 25 Staggered Extension Process (StEP) In Vitro Recombination 235 26 Random Mutagenesis by Whole-Plasmid PCR Amplification 241 27 PCR-Based Strategies to Clone Unknown DNA Regions from Known Foreign Integrants: An Overview 249 28 Long Distance Vectorette PCR (LDV PCR) 275 29 Nonspecific, Nested Suppression PCR Method for Isolation of Unknown Flanking DNA ("Cold-Start Method") 285 30 Inverse PCR: cDNA Cloning 293 31 Inverse PCR: Genomic DNA Cloning 301 32 Gene Cloning and Expression Profiling by Rapid Amplification of Gene Inserts with Universal Vector Primers 309 33 The Isolation of DNA Sequences Flanking Tn5 Transposon Insertions by Inverse PCR 315 34 Rapid Amplification of Genomic DNA Sequences Tagged by Insertional Mutagenesis 325 35 Isolation of Large Terminal Sequences of BAC Inserts Based on Double-Restriction-Enzyme Digestion Followed by Anchored PCR 337 36 A "Step Down" PCR-Based Technique for Walking Into and the Subsequent Direct Sequence Analysis of Flanking Genomic DNA 343 37 Use of PCR in Library Screening: An Overview 353 38 Cloning of Homologous Genes by Gene-Capture PCR 359 39 Rapid and Nonradioactive Screening of Recombinant Libraries by PCR 377 40 Rapid cDNA Cloning by PCR Screening (RC-PCR) 385 41 Generation and PCR Screening of Bacteriophage [lambda] Sublibraries Enriched for Rare Clones (the "Sublibrary Method") 391 42 PCR-Based Screening for Bacterial Artificial Chromosome Libraries 401 43 A 384-Well Microtiter-Plate-Based Template Preparation and Sequencing Method 411 44 A Microtiter-Plate-Based High Throughput PCR Product Purification Method 417 Index 423
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