LEADER 04644nam 2200433 450 001 9910830656003321 005 20231005165859.0 010 $a3-527-64706-6 024 7 $a10.1002/9783527647064 035 $a(CKB)3580000000001182 035 $a(NjHacI)993580000000001182 035 $a(EXLCZ)993580000000001182 100 $a20231005d2014 uy 0 101 0 $aeng 135 $aur||||||||||| 181 $ctxt$2rdacontent 182 $cc$2rdamedia 183 $acr$2rdacarrier 200 00$aHandbook of RNA biochemistry /$fedited by Roland K. Hartmann 205 $aSecond edition. 210 1$aWeinheim :$cJohn Wiley & Sons, Inc.,$d2014. 215 $a1 online resource (xlii, 1314 pages) 311 $a3-527-32764-9 327 $aCover; Title Page; Copyright; Contents; Preface; List of Contributors; Part I RNA Synthesis and Detection; Chapter 1 Enzymatic RNA Synthesis Using Bacteriophage T7 RNA Polymerase; 1.1 Introduction; 1.2 Description of Method -- T7 Transcription In vitro; 1.2.1 Templates; 1.2.1.1 Strategy (i): Insertion into a Plasmid; 1.2.1.2 Strategy (ii): Direct Use of Templates Generated by PCR; 1.2.1.3 Strategy (iii): Annealing of a T7 Promoter DNA Oligonucleotide to a Single-Stranded Template; 1.2.2 Special Demands on the RNA Product 1.2.2.1 Homogeneous 5' and 3' Ends, Small RNAs, Functional Groups at the 5' End1.2.2.2 Modified Substrates; 1.3 Transcription Protocols; 1.3.1 Transcription with Unmodified Nucleotides; 1.3.2 Transcription with 2' -Fluoro-Modified Nucleotides; 1.3.3 T7 Transcripts with 5' -- Cap Structures; 1.3.4 Purification; 1.4 Troubleshooting; 1.4.1 Low or No Product Yield; 1.5 Rapid Preparation of T7 RNA Polymerase; 1.5.1 Required Material; 1.5.1.1 Medium; 1.5.1.2 Buffers and Solutions; 1.5.1.3 Electrophoresis and Chromatography; 1.5.2 Procedure 1.5.2.1 Cell Growth, Induction, and Test for Expression of T7 RNAP1.5.2.2 Purification of T7 RNAP; 1.5.3 Notes and Troubleshooting; References; Chapter 2 Production of RNAs with Homogeneous 5' -- and 3' -Ends; 2.1 Introduction; 2.2 Description of Approach; 2.2.1 Cis-Cleaving Autocatalytic Ribozyme Cassettes; 2.2.1.1 The 5' -Cassette; 2.2.1.2 The 3' -Cassette; 2.2.1.3 Purification of Released RNA Product and Conversion of End Groups; 2.2.2 Trans-Cleaving Ribozymes for the Generation of Homogeneous 3' Ends; 2.2.3 Further Strategies toward Homogeneous Ends 2.3 Critical Experimental Steps, Changeable Parameters, Troubleshooting2.3.1 Construction of Cis-Cleaving 5'- and 3'-Cassettes; 2.4 PCR Protocols; 2.5 Potential Problems; References; Chapter 3 RNA Ligation; 3.1 General Introduction; 3.1.1 T4 Polynucleotide Ligases; 3.1.2 Reaction Mechanism; 3.1.3 Advantages of T4 DNA Ligase for RNA Ligation; 3.1.4 Chapter Structure; 3.2 RNA Ligation Using T4 DNA Ligase (T4 Dnl); 3.2.1 Overview of the RNA Ligation Method Using the T4 DNA Ligase (T4 Dnl); 3.2.2 Large-Scale Transcription and Purification of RNAs 3.2.3 Generating Homogeneous Acceptor 3' -Ends for Ligation3.2.4 Site-Directed Cleavage with RNase H; 3.2.5 Dephosphorylation and Phosphorylation of RNAs; 3.2.6 RNA Ligation; 3.2.7 Troubleshooting; 3.3 Simultaneous Splint Ligation of Five RNA Fragments to Generate RNAs for FRET Experiments; 3.3.1 Introduction; 3.3.2 Construct Design; 3.3.3 Troubleshooting; 3.3.3.1 Low Overall Ligation Efficiency; 3.3.3.2 Undesired Ligation By-products; 3.3.3.3 RNA Degradation; 3.4 T4 RNA Ligase(s); 3.4.1 Introduction; 3.4.2 Mechanism and Substrate Specificity; 3.4.2.1 Early Studies. 330 $aThe second edition of a highly acclaimed handbook and ready reference. Unmatched in its breadth and quality, around 100 specialists from all over the world share their up-to-date expertise and experiences, including hundreds of protocols, complete with explanations, and hitherto unpublished troubleshooting hints. They cover all modern techniques for the handling, analysis and modification of RNAs and their complexes with proteins. Throughout, they bear the practising bench scientist in mind, providing quick and reliable access to a plethora of solutions for practical questions of RNA research. 517 $aHandbook of RNA Biochemistry: Second, Completely Revised and Enlarged Edition 517 $aHandbook of RNA Biochemistry 606 $aBiochemistry$vHandbooks, manuals, etc 606 $aBiochemistry$yOutlines, syllabi, etc 615 0$aBiochemistry 615 0$aBiochemistry 676 $a574.192 702 $aHartmann$b Roland K. 801 0$bNjHacI 801 1$bNjHacl 906 $aBOOK 912 $a9910830656003321 996 $aHandbook of RNA biochemistry$91420155 997 $aUNINA