RNA生物化学手册2015wiley

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RNA生物化学手册2015wiley

书名Handbook of RNA Biochemistry
作者Roland K. Hartmann,
Albrecht Bindereif, Astrid Sch¨on,
and Eric Westhof
页数1371
Print ISBN: 978-3-527-32776-8
ePDF ISBN: 978-3-527-65055-2
ePub ISBN: 978-3-527-65054-5
mobi ISBN: 978-3-527-65053-8
oBook ISBN: 978-3-527-64706-4
出版社 wiley
Contents
Preface XXXV
List of Contributors XXXVII
Part I RNA Synthesis and Detection 1
1 Enzymatic RNA Synthesis Using Bacteriophage T7 RNA Polymerase 3
Markus G¨oßringer, Dominik Helmecke, Karen K¨ohler, Astrid Sch¨on,
Leif A. Kirsebom, Albrecht Bindereif, and Roland K. Hartmann
1.1 Introduction 3
1.2 Description of Method – T7 Transcription In vitro 4
1.2.1 Templates 4
1.2.1.1 Strategy (i): Insertion into a Plasmid 4
1.2.1.2 Strategy (ii): Direct Use of Templates Generated by PCR 5
1.2.1.3 Strategy (iii): Annealing of a T7 Promoter DNA Oligonucleotide
to a Single-Stranded Template 5
1.2.2 Special Demands on the RNA Product 5
1.2.2.1 Homogeneous 5 and 3 Ends, Small RNAs, Functional Groups
at the 5 End 5
1.2.2.2 Modified Substrates 6
1.3 Transcription Protocols 8
1.3.1 Transcription with Unmodified Nucleotides 9
1.3.2 Transcription with 2-Fluoro-Modified Nucleotides 16
1.3.3 T7 Transcripts with 5-Cap Structures 17
1.3.4 Purification 18
1.4 Troubleshooting 20
1.4.1 Low or No Product Yield 20
1.5 Rapid Preparation of T7 RNA Polymerase 21
1.5.1 Required Material 21
1.5.1.1 Medium 21
1.5.1.2 Buffers and Solutions 21
1.5.1.3 Electrophoresis and Chromatography 22
1.5.2 Procedure 22
1.5.2.1 Cell Growth, Induction, and Test for Expression of T7 RNAP 22