Recombinant and In Vitro RNA Synthesis: Methods and Protocols(941 Methods in Molecular Biology)

The discovery of catalytic RNAs in the mid-1980s marked the beginning of a new era in RNA biology and an ever increasing appreciation of the diverse and critical roles played by this fascinating molecule. In Recombinant and In Vitro RNA Synthesis: Methods and Protocols, expert researchers in the field detail approaches from the inception of a new RNA project to the final sample ready for analysis. These experimental protocols combine established work-horse approaches with some of the latest innovative methods for RNA synthesis and purification. Written in the highly successful Methods in Molecular Biology™ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls.   Authoritative and practical, Recombinant and In Vitro RNA Synthesis: Methods and Protocols aids scientists in continuing to study the fascinating world of RNA.

Table of Contents:
Purification of RNA Expressed in vivo Inserted in a tRNA Scaffold.-Selective RNase H Cleavage of Target RNAs  from a tRNA Scaffold.- Preparation of Long Templates for RNA in vitro Transcription by Recursive PCR.- General Protocols for Preparation of Plasmid DNA Template, RNA in vitro Transcription and RNA Purification by Denaturing PAGE.- Preparation of Short RNA by in vitro Transcription.- Native RNA Purification by Gel Filtration Chromatography.- Cis-acting Ribozymes for the Production of RNA in vitro Transcripts with Defined 5’ and 3’ Ends.- Trans-acting Antigenomic HDV Ribozyme for Production of in vitro Transcripts with Homogenous 3' Ends.- Rapid Preparation of RNA Samples using DNA-affinity Chromatography and DNAzyme Methods.-Preparation of lN-GST Fusion Protein for Affinity Immobilization of RNA.- Affinity Purification of RNA using an ARiBo Tag.- Plasmid Template Design and in vitro Transcription of Short RNAs within a ‘Structure Cassette’ for Structure Probing Experiments.- In Vitro Transcription of Modified RNAs.- End-Labeling Oligonucleotides with Chemical Tags After Synthesis.- High-Purity Enzymatic Synthesis of  Site-Specifically Modified tRNA.- Se-derivatized RNAs for X-ray Crystallography.- Biosynthetic Preparation of 13C/15N-labelled rNTPs for High-resolution NMR Studies of RNAs.- Preparative Separation of Ribonucleoside Monophosphates by Ion-Pair Reverse-Phase HPLC.- Splint Ligation of RNA with T4 DNA Ligase.