Basic Cloning Procedures

This manual introduces the reader to bacic methods used in the isolation, cloning and analysis of genetic material. The protocols include RT-PCR amplification, gene cloning, hybridization analysis and sequencing of nucleic acids, PCR-based site-specific mutagenesis, analysis of protein DNA-specific interaction, cell-free protein synthesis and product electrophoretic and immunological analysis. Each protocol includes short background information, a detailed description of the necessary materials, step-by-step procedures, a troubleshooting guide and useful practical hints. Front Matter....Pages i-vii cDNA Synthesis and Cloning....Pages 1-21 DNA Sequencing....Pages 22-42 RNA In Vitro Synthesis by Phage T7 DNA-Dependent RNA Polymerase....Pages 43-52 PCR-Based Site-Specific Mutagenesis....Pages 53-67 Analysis of Specific Protein-DNA Interactions....Pages 68-103 Gel Electrophoresis, Transfer and Hybridization of Nucleic Acids....Pages 104-134 Immunological Methods for Analysis of Recombinant Proteins....Pages 135-144 Protein Synthesis in Cell-Free Systems....Pages 145-163 Back Matter....Pages 165-169 Introduces the basic methods in the isolation, cloning and analysis of genetic material. The protocols include RT-PCR amplification, gene cloning, hybridization analysis and sequencing of nucleic acids, PCR-based site-specific mutagenesis, analysis of protein DNA-specific interaction.