Recombinant and in Vitro RNA Synthesis: Methods and Protocols

ISBN-13: 9781627031127 / Angielski / Twarda / 2012 / 274 str.

Graeme L. Conn

Opis

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.

Spis treści

1. Purification of RNA Expressed in vivo Inserted in a tRNA Scaffold

 

Luc Ponchon and Frédéric Dardel

 

 

 

2. Selective RNase H Cleavage of Target RNAs  from a tRNA Scaffold

 

Luc Ponchon, Geneviève Beauvais, Sylvie Nonin-Lecomte and Frédéric Dardel

 

 

 

3. Preparation of Long Templates for RNA in vitro Transcription by Recursive PCR

 

Jessica C. Bowman, Bahareh Azizi, Timothy K. Lenz, Poorna Roy, and Loren D. Williams

 

 

 

4. General Protocols for Preparation of Plasmid DNA Template, RNA in vitro Transcription and RNA Purification by Denaturing PAGE

 

Jo L. Linpinsel and Graeme L. Conn

 

 

 

5. Preparation of Short RNA by in vitro Transcription

 

Cheng Lu and Pingwei Li

 

 

 

6. Native RNA Purification by Gel Filtration Chromatography

 

Evan P. Booy, Hui Meng and Sean A. McKenna

 

 

7. Cis-acting Ribozymes for the Production of RNA in vitro Transcripts with Defined 5' and 3' Ends

 

Johanna M. Avis, Graeme L. Conn, and Scott C. Walker

 

 

 

8. Trans-acting Antigenomic HDV Ribozyme for Production of in vitro Transcripts with Homogenous 3' Ends

 

Milena Szafraniec, Leszek Blaszczyk, Jan Wrzesinski, and Jerzy Ciesiolka

 

 

 

9.  Rapid Preparation of RNA Samples using DNA-affinity Chromatography and DNAzyme Methods

 

Hae-Kap Cheong, Eunha Hwang, and Chaejoon Cheong

 

 

 

10.  Preparation of lN-GST Fusion Protein for Affinity Immobilization of RNA

 

Geneviève Di Tomasso, Philipe Lampron, James G. Omichinski and Pascale Legault

 

 

 

11. Affinity Purification of RNA using an ARiBo Tag

 

Geneviève Di Tomasso, Pierre Dagenais, Alexandre Desjardins, Alexis Rompré-Brodeur, Vanessa Delfosse and Pascale Legault

 

 

 

12. Plasmid Template Design and in vitro Transcription of Short RNAs within a ‘Structure Cassette’ for Structure Probing Experiments

 

Virginia K. Vachon and Graeme L. Conn

 

 

 

13. In Vitro Transcription of Modified RNAs

 

Stephanie L. Moon and Jeffrey Wilusz

 

 

 

14. End-Labeling Oligonucleotides with Chemical Tags After Synthesis

 

N. Ruth Zearfoss and Sean P. Ryder

 

 

 

15. High-Purity Enzymatic Synthesis of  Site-Specifically Modified tRNA

 

Ya-Ming Hou

 

 

 

16. Se-derivatized RNAs for X-ray Crystallography

 

Lina Lin and Zhen Huang

 

 

 

17. Biosynthetic Preparation of 13C/15N-labelled rNTPs for High-resolution NMR Studies of RNAs

 

Luigi Martino and Maria R Conte

 

 

 

18. Preparative Separation of Ribonucleoside Monophosphates by Ion-Pair Reverse-Phase HPLC

 

Pierre Dagenais and Pascale Legault

  

19. Splint Ligation of RNA with T4 DNA Ligase

 

Christopher J. Kershaw and Raymond T. O’Keefe