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This volume details technologies on recombinant DNA and nucleic acid manipulation that underpin much of the biological sciences and instructions on how to conduct them successfully. Chapters guide the reader through the basics of oligonucleotide synthesis and DNA sequencing; recombinant DNA plasmid work; cell-free experiments and the latest developments in CRISPR approaches to genome modification. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and methods, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.
Authoritative and cutting-edge, Gene Modification and Nucleic Acid Technologies aims to be the comprehensive guide for life scientists moving into the field of recombinant DNA and nucleic acid manipulation.
List of contents
Classical Recombinant DNA Cloning.- A Sequence and Ligation-Independent Cloning (SLIC) Procedure for the Insertion of Genes into a Plasmid Vector.- Molecular cloning using in vivo DNA assembly.- Assembling multiple fragments: The Gibson Assembly.- TA cloning approaches to cloning DNA with damaged ends DNA.- PCR-based Assembly of Gene Sequences by Thermodynamically Balanced Inside-Out (TBIO) Gene Synthesis.- Random Mutagenesis by PCR.- In vitro Site Directed Mutagenesis.- Xenopus transgenesis using the pGateway system.- CRISPR/Cas9 gene disruption studies in F0 Xenopus tadpoles: understanding development and disease in the frog.- A CRISPR/Cas based method for precise DNA integration in Xenopus laevis oocytes followed by intracytoplasmic sperm injection (ICSI) fertilisation.- A Lambda-Exonuclease SELEX Method for Generating Aptamers to Bacterial Targets. -Generation of functional-RNA arrays by in vitrotranscription and in situ RNA capture for the detection of RNA-RNA interactions.- Chemical synthesis of oligonucelotide sequences: Phosphoramidite chemistry.- Low Throughput Direct Cycle Sequencing of Polymerase Chain Reaction (PCR) Products.- Next generation Sequencing for mixed samples.- Ethics, Legality and Safety for Geneticists.
