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Bibliographic Details
Main Authors:	Green, Michael R. 1954- (Author), Sambrook, Joseph 1939-2019 (Author)
Format:	Book
Language:	English
Published:	Cold Spring Harbor, New York Cold Spring Harbor Laboratory Press [2012]
Edition:	Fourth edition
Subjects:	cloning
Links:	http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=024810600&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA
Physical Description:	xxxiii, 1335 - 1890, I-46 Seiten Illustrationen, Diagramme

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adam_text	VOLUME 3 PART 4: GENE EXPRESSION CHAPTER 17 Analysis of Gene Regulation Using Reporter Systems 1335 Pradeep D. Uchil, Arvindhan Nagarajan, and Priti Kumar INTRODUCTION Introduction to Reporter Systems 1336 Reporter Genes Used in the Analysis of Regulatory Elements 1336 Assaying for ß-Galactosidase in Extracts of Mammalian Cells 1338 Assaying for Luciferase in Extracts of Mammalian Cells 1339 Tetracycline-Responsive Expression Systems 1341 PROTOCOLS 1 Assay for ß-Galactosidase in Extracts of Mammalian Cells 1346 • Additional Protocol: Chemiluminescent Assay for ß-Galactosidase Activity 1350 2 Single Luciferase Reporter Assay 1354 3 Dual Luciferase Reporter Assay 1359 4 Using ELISA to Measure GFP Production 1366 5 Generation of Cell Lines with Tetracycline-Regulated Gene Expression 1370 • Additional Protocol: Selecting Stable Clones via Limiting Dilution of Suspension Cells 1378 INFORMATION PANELS Fluorescent Proteins 1381 Epitope Tagging 1394 ß-Galactosidase 1401 Luciferase 1406 Tetracycline Contents I xix CHAPTER 18 RNA Interference and Small RNA Analysis 1415 Chengjian Li and Phillip D. Zamore INTRODUCTION Reverse Genetics by RNAi 1419 Analysis of Small RNAs 1425 PROTOCOLS 1 Preparation of siRNA Duplexes 1431 2 RNAi in Mammalian Cells by siRNA Duplex Transfection 1434 3 RNAi in Drosophila S2 Cells by siRNA Duplex Transfection 1437 4 Preparation of dsRNAs by In Vitro Transcription 1440 5 RNAi in Drosophila S2 Cells by dsRNA Soaking 1445 6 RNAi in Drosophila S2 Cells by dsRNA Transfection 1447 7 Analysis of Small RNAs by Northern Hybridization 1448 8 Analysis of Small RNAs by Quantitative Reverse Transcription PCR 1453 9 Construction of Small RNA Libraries for High-Throughput Sequencing 1456 10 Preparation of Antisense Oligonucleotides to Inhibit miRNA Function 1466 11 Inhibiting miRNA Function by Antisense Oligonucleotides in Cultured Mammalian Cells 1468 12 Inhibiting miRNA Function by Antisense Oligonucleotides in Drosophila S2 Cells 1470 INFORMATION PANEL Genome-Wide RNA Interference: Functional Genomics in the Postgenomics Era 1472 StarFire Probes 1478 CHAPTER 19 Expressing Cloned Genes for Protein Production, Purification, and Analysis 1481 Clara L. Kielkopf, William Bauer, and lna L. Urbatsch INTRODUCTION Choosing an Expression System 1483 Choosing an Appropriate Expression Vector 1488 Fusion Proteins 1499 Optimization of Expression of Foreign Proteins 1503 PROTOCOLS 1 Expression of Cloned Genes in E. coli Using IPTG-lnducible Promoters 1508 • Additional Protocol: Small-Scale Test for Soluble Target Protein Expression 1514 • Alternative Protocol: Expression of Cloned Genes in f. coli Using the Arabinose BAD Promoter 1520 • Alternative Protocol: Subcellular Localization of Signal Peptide Fusion Proteins 1522 XX / Contents 2 Expression of Cloned Genes Using the Baculovirus Expression System 1527 • Additional Protocol: Plaque Assay to Determine the Titer of the Baculovirus Stock 1535 • Alternative Protocol: Production of Bacmid DNA for Transfection into Insect Cells 1538 3 Expression of Cloned Genes in P. pastoris Using the Methanol-lnducible Promoter AOX1 1542 • Additional Protocol: Cryostorage of Yeast Cultures 1553 4 Preparation of Cell Extract for Purification of Soluble Proteins Expressed in E. coli 1558 • Additional Protocol: Lysis of Yeast Cells Using Glass Beads 1564 • Alternative Protocol: Preparation of E. coil Cell Extract Using Gentle, Heat-Induced Enzymatic Lysis 1566 • Alternative Protocol: Preparation of E. coli Cell Extract Using Freeze-Thaw with Enzymatic Lysis by Lysozyme 1568 5 Purification of Polyhistidine-Tagged Proteins by Immobilized Metal Affinity Chromatography 1571 • Additional Protocol: Regenerating and Cleaning the Ni^-NTA Resin 1579 • Alternative Protocol: Fast Performance Liquid Chromatography Purification of Histidine-Tagged Proteins 1581 6 Purification of Fusion Proteins by Affinity Chromatography on Glutathione Resin 1586 7 Solubilization of Expressed Proteins from Inclusion Bodies 1593 8 SDS-PAGE of Proteins 1599 • Alternative Protocol: Variations of Staining SDS-Polyacrylamide Gels with Coomassie Brilliant Blue 1609 • Alternative Protocol: Staining SDS-Polyacrylamide Gels with Silver Salts 1611 9 Analysis of Proteins by Irnmunoblotting 1616 10 Methods for Measuring the Concentrations of Proteins 1625 INFORMATION PANELS Considerations for Membrane Protein Purification 1632 Historical Footnote: Coomassie Brilliant Blue 1636 PART 5: INTERACTION ANALYSIS CHAPTER 20 Cross-Linking Technologies for Analysis of Chromatin Structure and Function 1637 Tae Hoon Kim and Job Dekker INTRODUCTION Formaldehyde Cross-Linking to Interrogate Genomic Interactions 1638 ChIP Analysis of Protein-DNA Interactions 1638 ЗС -Based Chromatin Interaction Analyses 1641 PROTOCOLS 1 Formaldehyde Cross-Linking 1649 2 Preparation of Cross-Linked Chromatin for ChIP 1652 Contents / xxi 3 СЫР 1655 4 ChlP-Quantitative PCR (ChlP-qPCR) 1659 5 ChlP-chip 1661 6 ChlP-seq 1669 7 Generation of 3C Libraries from Cross-Linked Cells 1674 8 Generation of ChlP-loop Libraries 1679 9 Generation of Control Ligation Product Libraries 1684 10 PCR Detection of ЗС Ligation Products Present in 3C, ChlP-loop, and Control Libraries: Library Titration and Interaction Frequency Analysis 1687 11 4C Analysis of ЗС, ChlP-loop, and Control Libraries 1692 12 5C Analysis of 3C, ChlP-loop, and Control Libraries 1696 INFORMATION PANELS Formaldehyde 1701 What Is Captured by ЗС -Based Assays? 1702 CHAPTER 21 Mapping of In Vivo RNA-Binding Sites by U V-Cross-Linking Immunoprecipitation (CLIP) 1703 Jennifer C. Darnell, Aldo Mele, Ka Ying Sharon Hung, and Robert B. Darnell INTRODUCTION The Cross-Linking Immunoprecipitation Method 1706 High-Throughput Sequencing (HITS) CLIP 1708 Validation of CLIP Results 1708 CLIP Method Variations 1709 General Considerations in Planning CLIP Experiments 1710 PROTOCOLS 1 Optimization of Immunoprecipitation Stringency for CLIP 1713 2 UV Cross-Linking of Live Cells and Lysáte Preparation 1720 3 RNase Titration, Immunoprecipitation, and SDS-PAGE 1724 4 З' -Linker Ligation and Size Selection by SDS-PAGE 1734 • Alternative Protocol: S'-End Labeling of Dephosphorylated RL3 Linker 1738 5 Isolation of the RNA Tags, 5'-Linker Ligation, and Reverse Transcription PCR Amplification 1741 6 Sequencing of RNA CLIP Tags 1751 7 Gel Purification and Storage of RNA Linkers 1753 INFORMATION PANELS Mechanism and Specificity of UV-Protein Cross-Linking 1756 H ITS-CLI Ρ Data Analysis 1758 xxii / Contents CHAPTER 22 Gateway-Compatible Yeast One-Hybrid and Two-Hybrid Assays 1761 John S. Reece-Hoyes and Albertha J.M. Walhout INTRODUCTION The Yeast Two-Hybrid (Y2H) System: Concept and Methodology 1763 The Yeast One-Hybrid (Y1H) System: Concept and Methodology 1767 Y2H and Y1H Assays: Advantages and Disadvantages 1768 False Positives 1769 Protocols for Yeast One-Hybrid and Two-Hybrid Systems 1770 PROTOCOLS 1 Generating Yeast One-Hybrid DNA-Bait Strains 1773 • Alternative Protocol: Creating Entry Clones from DNA-Baits Generated by Annealing Primers 1782 2 Generating Yeast Two-Hybrid Bait Strains 1785 3 Identifying Interactors from an Activation Domain Prey Library 1792 4 High-Efficiency Yeast Transformation 1799 5 Colony Lift Colorimetric Assay for ß-Galactosidase Activity 1803 6 Yeast Colony PCR 1805 INFORMATION PANELS Why Integrate DNA-Baits? 1808 Choosing a Vector and a Yeast Strain 1809 Replica-Plating and Replica-Cleaning Using Velvets 1810 APPENDICES APPENDIX 1 Reagents and Buffers 1811 RECIPES 1811 BUFFERS 1828 Tris Buffers 1828 Good Buffers 1829 Phosphate Buffers (Gomori Buffers) 1830 ACIDS AND BASES 1833 ORGANIC REAGENTS 1834 Phenol 1834 Equilibration of Phenol 1834 Phenol:Chloroform:lsoamyl Alcohol (25:24:1) 1834 Deionization of Formamide 1834 Contents I xxüi ANTIBIOTICS 1835 BLOCKING AGENTS 1836 Blocking Agents Used for Nucleic Acid Hybridization 1836 Blocking Agents Used for Western Blotting 1836 BACTERIAL STORAGE MEDIA 1839 PERIODIC TABLE 1840 REAGENTS AND BUFFERS INDEX 1841 APPENDIX 2 Commonly Used Techniques 1843 PREPARATION OF GLASSWARE AND PLASTICWARE 1843 Siliconizing Glassware, Plasticware, and Glass Wool 1843 Preparation of RNase-Free Glassware 1844 PREPARATION OF DIALYSIS TUBING 1845 ESTIMATION OF CELL NUMBER 1846 Hemocytometry Counting 1846 Viability Staining 1847 MEASUREMENT OF RADIOACTIVITY IN NUCLEIC ACIDS 1849 Precipitation of Nucleic Acids with Trichloroacetic Acid 1849 DECONTAMINATION OF SOLUTIONS CONTAINING ETHIDIUM BROMIDE 1851 Removing Ethidium Bromide from DNA 1851 Disposing of Ethidium Bromide 1851 Decontamination of Concentrated Solutions of Ethidium Bromide (Solutions Containing >0.5 mg/mL) 1851 Decontamination of Dilute Solutions of Ethidium Bromide (e.g., Electrophoresis Buffer Containing 0.5 μg/mL Ethidium Bromide) 1852 Commercial Decontamination Kits 1852 APPENDIX 3 Detection Systems 1855 STAINING NUCLEIC ACIDS 1855 Ethidium Bromide 1855 Methylene Blue 1857 SYBR Dyes 1857 CHEMILUMINESCENCE 1859 Chemiluminescent Labels 1860 Chemiluminescent Enzyme Assays 1861 Commercial Reagents, Kits, and Luminometers 1863 XXIV Contents INFORMATION PANELS Horseradish Peroxidase 1865 Digoxygenin 1869 BCIP 1873 AMPPD 1876 Immunoglobulin-Binding Proteins: Proteins A, C, and L 1879 APPENDIX 4 General Safety and Hazardous Material 1885 Index 1-1
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spellingShingle	Green, Michael R. 1954- Sambrook, Joseph 1939-2019 Molecular cloning a laboratory manual cloning cabt
title	Molecular cloning a laboratory manual
title_auth	Molecular cloning a laboratory manual
title_exact_search	Molecular cloning a laboratory manual
title_full	Molecular cloning a laboratory manual Volume 3 Michael R. Green, Joseph Sambrook
title_fullStr	Molecular cloning a laboratory manual Volume 3 Michael R. Green, Joseph Sambrook
title_full_unstemmed	Molecular cloning a laboratory manual Volume 3 Michael R. Green, Joseph Sambrook
title_short	Molecular cloning
title_sort	molecular cloning a laboratory manual
title_sub	a laboratory manual
topic	cloning cabt
topic_facet	cloning
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