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In Vitro Mutagenesis Protocols 3rd Edition by Miguel Alcalde, Jeff Braman ISBN 1607616521 9781607616528

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In Vitro Mutagenesis Protocols Third Edition Miguel Alcalde (Auth.) Digital Instant Download

Author(s): Miguel Alcalde (auth.), Jeff Braman (eds.)
ISBN(s): 9781607616528, 1607616521
Edition: 3
File Details: PDF, 11.06 MB
Year: 2010
Language: english
SKU: EB-2183294 Category: Tags: ,

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ISBN 10: 1607616521
ISBN 13: 9781607616528
Author: Miguel Alcalde, Jeff Braman

In the post-genomic era, in vitro mutagenesis has emerged as a critically important tool for establishing the functions of components of the proteome. The third edition of In Vitro Mutagenesis Protocols represents a practical toolbox containing protocols vital to advancing our understanding of the connection between nucleotide sequence and sequence function. Fully updated from the previous editions, this volume contains a variety of specialty tools successfully employed to unravel the intricacies of protein-protein interaction, protein structure-function, protein regulation of biological processes, and protein activity, as well as a novel section on mutagenesis methods for unique microbes as a guide to the generalization of mutagenesis strategies for a host of microbial systems. Written in the highly successful Methods in Molecular Biology™ series format, chapters include brief introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and expert tips on troubleshooting and avoiding known pitfalls.

Authoritative and up-to-date, In Vitro Mutagenesis Protocols, Third Edition offers today’s researchers a valuable compendium of reliable and powerful techniques with which to illuminate the proteome and its rich web of biological implications.

In Vitro Mutagenesis Protocols 3rd Table of contents:

Chapter 1: Introduction to In Vitro Mutagenesis

  • 1.1 Basic Concepts of Mutagenesis

  • 1.2 Types of Mutagenesis: Site-Directed, Random, and Saturation Mutagenesis

  • 1.3 Applications of In Vitro Mutagenesis in Functional Genomics

  • 1.4 Overview of Common Methods and Technologies

  • 1.5 Experimental Design and Optimization

Chapter 2: Preparation of DNA Templates for Mutagenesis

  • 2.1 Isolation and Purification of Plasmid DNA

  • 2.2 Linearization of Plasmids

  • 2.3 PCR Amplification of DNA Templates

  • 2.4 Design of Mutagenic Oligonucleotides

  • 2.5 Preparation of dsDNA for Mutagenesis

  • 2.6 Troubleshooting Common Problems

Chapter 3: Oligonucleotide-Based Site-Directed Mutagenesis

  • 3.1 Introduction to Site-Directed Mutagenesis (SDM)

  • 3.2 Principles of Oligonucleotide-Directed Mutagenesis

  • 3.3 Use of PCR for Site-Directed Mutagenesis

  • 3.4 QuikChange Mutagenesis

  • 3.5 Overlap Extension Mutagenesis

  • 3.6 Mutagenesis Using Long-Range PCR

  • 3.7 Tips and Troubleshooting

Chapter 4: Error-Prone PCR and Random Mutagenesis

  • 4.1 Overview of Error-Prone PCR

  • 4.2 Chemical and Enzyme-Based Mutagenesis Strategies

  • 4.3 Protocols for Error-Prone PCR

  • 4.4 Screening for Random Mutants

  • 4.5 Applications of Error-Prone PCR in Directed Evolution

  • 4.6 Troubleshooting Random Mutagenesis

Chapter 5: In Vitro Mutagenesis Using the Gibson Assembly Method

  • 5.1 Overview of Gibson Assembly for Mutagenesis

  • 5.2 Protocol for Gibson Assembly Mutagenesis

  • 5.3 Advantages and Applications of Gibson Assembly

  • 5.4 Troubleshooting Gibson Assembly Mutagenesis

  • 5.5 Case Studies and Examples

Chapter 6: CRISPR-Cas9 and Genome Editing for Mutagenesis

  • 6.1 Overview of CRISPR-Cas9 Technology

  • 6.2 Design and Construction of CRISPR Guides

  • 6.3 In Vitro Mutagenesis with CRISPR-Cas9

  • 6.4 Off-Target Effects and How to Minimize Them

  • 6.5 Applications of CRISPR in Mutagenesis

  • 6.6 Validation and Screening of Mutants

Chapter 7: Mutagenesis in Eukaryotic Systems

  • 7.1 Overview of Mutagenesis in Eukaryotic Cells

  • 7.2 Transfection Techniques for Mutagenesis

  • 7.3 CRISPR and RNAi Applications in Eukaryotic Cells

  • 7.4 Site-Directed Mutagenesis in Yeast and Mammalian Cells

  • 7.5 Selection and Screening of Mutants in Eukaryotic Cells

  • 7.6 Troubleshooting Eukaryotic Mutagenesis

Chapter 8: Screening and Characterization of Mutants

  • 8.1 Screening Strategies for Mutagenesis (Colony PCR, Restriction Digests)

  • 8.2 DNA Sequencing for Mutant Identification

  • 8.3 Functional Assays for Mutant Analysis

  • 8.4 Phenotypic Screening: Case Studies

  • 8.5 Troubleshooting and Optimizing Screening Methods

Chapter 9: High-Throughput Mutagenesis and Screening

  • 9.1 Introduction to High-Throughput Technologies

  • 9.2 Automation in Mutagenesis Protocols

  • 9.3 Robotic Screening Systems

  • 9.4 Next-Generation Sequencing (NGS) for Mutant Characterization

  • 9.5 Data Analysis and Bioinformatics in High-Throughput Screening

  • 9.6 Case Studies in High-Throughput Mutagenesis

Chapter 10: Applications of In Vitro Mutagenesis

  • 10.1 Directed Evolution of Proteins

  • 10.2 Gene Function Analysis and Gene Knockdown

  • 10.3 Mutagenesis in Drug Discovery and Development

  • 10.4 Industrial Applications: Enzyme Engineering

  • 10.5 Clinical Applications in Gene Therapy

Chapter 11: Troubleshooting and Optimization of Mutagenesis Protocols

  • 11.1 Common Problems in In Vitro Mutagenesis

  • 11.2 Strategies for Optimizing PCR-Based Mutagenesis

  • 11.3 Addressing Low Efficiency or Failure of Mutagenesis

  • 11.4 Protocol Modifications for Difficult Templates

  • 11.5 Troubleshooting Guide: Step-by-Step Solutions

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Tags: Miguel Alcalde, Jeff Braman, Mutagenesis Protocols