Molecular cloning: a laboratory manual Volume 3
Gespeichert in:
| Hauptverfasser: | , |
|---|---|
| Format: | Buch |
| Sprache: | English |
| Veröffentlicht: |
Cold Spring Harbor, New York
Cold Spring Harbor Laboratory Press
[2012]
|
| Ausgabe: | Fourth edition |
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | xxxiii, 1335 - 1890, I-46 Seiten Illustrationen, Diagramme |
Internformat
MARC
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| 035 | |a (DE-599)BVBBV039952702 | ||
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| 100 | 1 | |a Green, Michael R. |d 1954- |e Verfasser |0 (DE-588)1024045439 |4 aut | |
| 245 | 1 | 0 | |a Molecular cloning |b a laboratory manual |n Volume 3 |c Michael R. Green, Joseph Sambrook |
| 250 | |a Fourth edition | ||
| 264 | 1 | |a Cold Spring Harbor, New York |b Cold Spring Harbor Laboratory Press |c [2012] | |
| 300 | |a xxxiii, 1335 - 1890, I-46 Seiten |b Illustrationen, Diagramme | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 650 | 7 | |a cloning |2 cabt | |
| 700 | 1 | |a Sambrook, Joseph |d 1939-2019 |e Verfasser |0 (DE-588)1158752814 |4 aut | |
| 773 | 0 | 8 | |w (DE-604)BV039952684 |g 3 |
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Datensatz im Suchindex
| _version_ | 1804148924382969856 |
|---|---|
| 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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| spelling | Green, Michael R. 1954- Verfasser (DE-588)1024045439 aut Molecular cloning a laboratory manual Volume 3 Michael R. Green, Joseph Sambrook Fourth edition Cold Spring Harbor, New York Cold Spring Harbor Laboratory Press [2012] xxxiii, 1335 - 1890, I-46 Seiten Illustrationen, Diagramme txt rdacontent n rdamedia nc rdacarrier cloning cabt Sambrook, Joseph 1939-2019 Verfasser (DE-588)1158752814 aut (DE-604)BV039952684 3 Digitalisierung UB Regensburg application/pdf 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 Inhaltsverzeichnis |
| 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 |
| url | 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 |
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