Molecular cloning: a laboratory manual Volume 1
Gespeichert in:
| Hauptverfasser: | , |
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| 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, 630, I-46 Seiten Illustrationen, Diagramme |
Internformat
MARC
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| 007 | t| | ||
| 008 | 120312s2012 xx a||| |||| 00||| eng d | ||
| 035 | |a (OCoLC)785848853 | ||
| 035 | |a (DE-599)BVBBV039952689 | ||
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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 1 |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, 630, 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 | |
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| 943 | 1 | |a oai:aleph.bib-bvb.de:BVB01-024810588 | |
Datensatz im Suchindex
| _version_ | 1820497046694526976 |
|---|---|
| adam_text |
Contents
List of Tables
xxv
Preface
xxxi
Acknowledgments
xxxiii
VOLUME
1
PART
1:
ESSENTIALS
CHAPTER
1
Isolation and Quantification of
DNA
INTRODUCTION
DNA
isolation
2
Commercial Kits for Purification of
DNA 3
Quantifying
DNA 5
PROTOCOLS
1
Preparation of Plasmid
DNA
by Alkaline Lysis with
SDS: Minipreps
11
2
Preparation of Plasmid
DNA
by Alkaline Lysis with
SDS: Maxipreps
15
3
Isolating
DNA
from Gram-Negative Bacteria (e.g.,
E. coli)
19
4
Precipitation of
DNA
with
Ethanol
21
5
Precipitation of
DNA
with Isopropanol
26
6
Concentrating and Desalting Nucleic Acids with Microconcentrators
28
7
Concentrating Nucleic Acids by Extraction with
Butanol
30
8
Preparation of Single-Stranded Bacteriophage M13
DNA
by Precipitation with
Polyethylene
Glycol
31
9
Plating Bacteriophage M13
34
10
Growing Bacteriophage M13 in Liquid Culture
38
11
Preparation of Double-Stranded
(Replicative
Form) Bacteriophage M13
DNA 41
12
Isolation of High-Molecular-Weight
DNA
Using Organic Solvents to Purify
DNA 44
13
Isolation of High-Molecular-Weight
DNA
from Mammalian Cells
Using Proteinase
К
and Phenol
47
vi
/ Contents
14
A
Single-Step Method for the Simultaneous Preparation of
DNA, RNA,
and Protein
from Cells and Tissues
54
15
Preparation of Genomic
DNA
from Mouse Tails and Other Small Samples
58
•
Alternative Protocol: Isolation of
DNA
from Mouse Tails without
Extraction by Organic Solvents
61
•
Alternative Protocol: One-Tube Isolation of
DNA
from Mouse Tails
62
16
Rapid Isolation of Yeast
DNA 64
17
Using Ethidium Bromide to Estimate the Amount of
DNA in
Bands after
Electrophoresis through
Minigels
66
18
Estimating the Concentration of
DNA
by Fluorometry Using
Hoechst 33258 68
19
Quantifying
DNA in
Solution with PicoGreen
71
INFORMATION PANELS
DNA
Extraction from Formaldehyde-Fixed Tissue Embedded in Paraffin Blocks
72
Polyethylene
Glycol
73
α
-Complementation
74
X-Gal
76
Minimizing Damage to Large
DNA
Molecules
77
Spectrophotometry
78
CHAPTER
2
Analysis of
DNA 81
INTRODUCTION
Agarose Gel
Electrophoresis
82
Analysis of
DNA
Fragments
86
Recovering
DNA
from Gels
87
Southern Blotting
88
PROTOCOLS
1
Agarose Gel
Electrophoresis
94
2
Detection of
DNA
in
Agarose
Gels by Staining
99
3
Polyacrylamide Gel Electrophoresis
104
4
Detection of
DNA
in Polyacrylamide Gels by Staining
110
5
Detection of
DNA in
Polyacrylamide Gels by Autoradiography
112
6
Alkaline
Agarose Gel
Electrophoresis
114
•
Additional Protocol: Autoradiography of Alkaline
Agarose
Gels
117
7
Imaging: Autoradiography and Phosphorimaging
119
8
Recovery of
DNA
from
Agarose
Gels Using Glass Beads
125
9
Recovery of
DNA
from Low-Melting-Temperature
Agarose
Gels: Organic Extraction
127
10
Isolation of
DNA
Fragments from Polyacrylamide Gels by the Crush
and Soak Method
130
11
Southern Blotting
133
12
Southern Blotting: Simultaneous Transfer of
DNA
from an
Agarose
Gel to Two Membranes
141
Contents
I
vii
13
Southern
Hybridization of
Radiolabeled
Probes to Nucleic Acids
Immobilized on Membranes
144
•
Additional Protocol: Stripping Probes from Membranes
150
INFORMATION PANELS
Formamide and Its Uses in Molecular Cloning
153
Rapid Hybridization Buffers
155
CHAPTER
3
Cloning and Transformation with
Plasmici
Vectors
157
INTRODUCTION
Plasmid Vectors
158
Transformation
159
PROTOCOLS
1
The Hanahan Method for Preparation and Transformation of Competent f.
coli:
High-Efficiency Transformation
162
2
The Inoue Method for Preparation and Transformation of Competent
E. coli:
"Ultracompetent" Cells
168
3
Easy Transformation of f.
coli: Nanoparticle-Mediated
Transformation
173
•
Alternative Protocol: One-Step Preparation of Competent
£
coli:
Transformation
and Storage of Bacterial Cells in the Same Solution
175
4
Transformation of
E. coli
by Electroporation
177
5
Cloning in Plasmid Vectors: Directional Cloning
183
6
Cloning in Plasmid Vectors: Blunt-End Cloning
186
7
Dephosphorylation of Plasmid
DNA 189
8
Attaching Phosphorylated Adaptors/Linkers to Blunt-Ended DNAs
192
9
Cloning PCR Products: Addition of Restriction Sites to the Termini of Amplified
DNA 194
10
Cloning PCR Products: Blunt-End Cloning
197
11
Cloning PCR Products: Making
Τ
Vectors
200
12
Cloning PCR Products:
TA
Cloning
203
13
Cloning PCR Products:
TOPO TA
Cloning
206
14
Screening Bacterial Colonies Using
Х
-Cal
and IPTG:
α
-Complementation
210
INFORMATION PANELS
Caring for
E. coli
213
The History of Transformation of Bacteria by
DNA 217
A Guide to Cloning the Products of Polymerase Chain Reactions
218
BioBricks and the Ordered Assembly of
DNA
Fragments
225
TOPO
Tools: Creating Linear Expression Constructs with Functional Elements
227
Antibiotics
229
Adaptors
232
Linkers
234
Ligation and
Ligases
235
viii
/ Contents
Condensing and Crowding Reagents
240
The Discovery of Restriction Enzymes
241
Restriction Enzymes
242
Chloramphenicol
245
The ccdB Gene
247
Bacteriophage
λ
248
Bacteriophage M13
249
Plasmids
251
Cosmids
258
CHAPTER
4
Gateway Recombinational Cloning
261
John S. Reece-Hoyes and
Albertba
¡.M. Walhout
INTRODUCTION
Basic Principles and Applications of Gateway Cloning
262
Disadvantages of Gateway Cloning and Alternative Cloning Systems
264
PROTOCOLS
1
Propagating Gateway Vectors
267
2
Generating an
ORF
Entry Clone and Destination Clone
270
3
Using Multisite LR Cloning to Generate a Destination Clone
277
INFORMATION PANEL
Generating Gateway-Compatible Vectors
2Ö0
CHAPTER
5
Working with Bacterial Artificial Chromosomes and Other High-Capacity Vectors
281
Nathaniel Heintz and Shiaoching Cong
INTRODUCTION
Development of High-Capacity Vectors: Advantages and Disadvantages
282
Working with Bacterial Artificial Chromosomes
286
PROTOCOLS
1
Small-Scale Isolation of
ВАС
DNA
and Verification by PCR
294
2
Large-Scale Preparation and Linearization of
ВАС
DNA 297
3
Examination of
ВАС
DNA
Quality and Quantity by Pulsed-Field Gel
Electrophoresis
301
4
Two-Step
ВАС
Engineering: Preparation of Shuttle Vector
DNA 303
5
Preparation of the A Homology Arm
(А
-Box)
and
В
Homology Arm (B-Box)
306
6
Cloning of the A and
В
Homology Arms into the Shuttle Vector
309
7
Preparation and Verification of the
Recombinant
Shuttle Vector
312
Contents
j
ІХ
8 Electroporation
of Competent
ВАС
Host Cells with the
Recombinant
Shuttle Vector
315
9
Verification of Cointegrates and Selection of Resolved
ВАС
Clones
317
10
One-Step
ВАС
Modification: Preparation of Plasmids
321
11
Preparation of the A Homology Arm (A-Box)
324
12
Cloning of the A Homology Arm into Reporter-Shuttle Vector
326
13
Transformation of the
ВАС
Host with the RecA Vector
329
14
Transfer of the Reporter Vector into BAC/RecA Cells and Selection
of Cointegrates
331
15
Growth
oí
S. cerevisiae
and Preparation of
DNA 334
16
Small-Scale Preparations of Yeast
DNA 336
INFORMATION PANELS
CRE-/OXP
338
Using ECFP as a Reporter
342
Primer Design for Homology Arms, Cointegration, and Resolution
343
Yeast Media
344
CHAPTER
6
Extraction, Purification, and Analysis of
RNA
from Eukaryotic Cells
345
INTRODUCTION
PROTOCOLS
Introduction to the Isolation of Total
RNA
Using Monophasic Lysis Reagents
348
1
Purification of Total
RNA
from Mammalian Cells and Tissues
351
•
Alternative Protocol: Preparing
RNA
from Smaller Samples
354
2
Isolation of Total
RNA
from Zebrafish Embryos and Adults
355
3
Total
RNA
Isolation from
Drosophila melanogaster
357
4
Total
RNA
Extraction from Caenorhabditis
elegans
359
5
Total
RNA
Extraction from Saccharomyces cerevisiae Using Hot
Acid Phenol
362
6
Quantifying and Storing
RNA
365
7
Precipitation of
RNA
with
Ethanol
372
8
Removing
DNA
Contamination from
RNA
Samples by Treatment with
RNase-Free DNase I
375
9
Isolation of Poly(A)+ Messenger
RNA
Using Magnetic Oligo(dT) Beads
377
Introduction to Hybridization of
RNA
by Northern Transfer
381
10
Separation of
RNA
according to Size: Electrophoresis of
RNA
through
Agarose
Gels
Containing Formaldehyde
388
11
Separation of
RNA
according to Size: Electrophoresis of
RNA
through Denaturing Urea
Polyacrylamide Gels
393
12
Transfer and Fixation of Denatured
RNA
in
Agarose
Gels to Membranes
401
•
Alternative Protocol: Capillary Transfer by Downward Flow
406
Χ
/ Contents
13 Transfer
and Fixation of Denatured
RNA
in Polyacrylamide Gels to Membranes by
Electrophoretic Transfer
408
14
Northern Hybridization
410
15
Dot and Slot Hybridization of Purified
RNA
415
Introduction to Mapping
RNA
420
16
Mapping
RNA
with Nuclease S1
430
17
Ribonuclease Protection: Mapping
RNA
with Ribonuclease and
Radiolabeled
RNA
Probes
437
18
Analysis of
RNA
by Primer Extension
445
INFORMATION PANELS
How to Win the Battle with RNase
450
Inhibitors of RNases
451
Diethylpyrocarbonate
452
Nuclease S1
454
CHAPTER
7
Polymerase Chain Reaction
455
INTRODUCTION
The Basic Polymerase Chain Reaction
456
Design of Oligonucleotide Primers for Basic PCR
462
Detecting, Analyzing, and Quantifying mRNAs
464
Contamination in PCR
466
PROTOCOLS
1
The Basic Polymerase Chain Reaction
470
2
Hot Start PCR
477
3
Touchdown PCR
481
4
PCR Amplification of GC-Rich Templates
484
5
Long and Accurate PCR (LA PCR)
490
6
Inverse PCR
494
7
Nested PCR
499
8
Amplification of cDNA Generated by Reverse Transcription of mRNA: Two-Step
RT-PCR
503
9
Rapid Amplification of Sequences from the
5'
Ends of mRNAs: 5'-RACE
515
10
Rapid Amplification of Sequences from the
3'
Ends of mRNAs:
З'
-RACE
523
11
Screening Colonies by PCR
531
INFORMATION PANELS
Taq
DNA
Polymerase
533
PCR in Theory
537
Ribonuclease
Η
538
The
dut
and
ung
Genes of
E. coli
539
Terminal
Transferase
540
Contents
I
xi
CHAPTER
8
Bioinformatics
541
jui-Hung Hung and Zhiping Weng
INTRODUCTION
PROTOCOLS
1
Visualizing Genomic Annotations with the UCSC Genome Browser
544
Introduction to Sequence Alignment and Homology Search
554
2
Sequence Alignment and Homology Search with BLAST and ClustalW
557
3
Designing PCR Primers Using Primer3Plus
564
Introduction to Expression Profiling by Microarray and RNA-seq
571
4
Expression Profiling by Microarray and RNA-seq
577
Introduction to Mapping Billions of Short Reads to a Reference Genome
588
5
Mapping Billions of Short Reads to a Reference Genome
591
Introduction to Peak-Finding Algorithms
598
6
Identifying Regions Enriched in a ChlP-seq Data Set (Peak Finding)
604
Introduction to Motif Finding
612
7
Discovering as-Regulatory Motifs
617
INFORMATION PANELS
Data Formats
625
Algorithms, Portals, and Methods
628 |
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| language | English |
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| spelling | Green, Michael R. 1954- Verfasser (DE-588)1024045439 aut Molecular cloning a laboratory manual Volume 1 Michael R. Green, Joseph Sambrook Fourth edition Cold Spring Harbor, New York Cold Spring Harbor Laboratory Press [2012] xxxiii, 630, 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 1 Digitalisierung UB Regensburg application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=024810588&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 1 Michael R. Green, Joseph Sambrook |
| title_fullStr | Molecular cloning a laboratory manual Volume 1 Michael R. Green, Joseph Sambrook |
| title_full_unstemmed | Molecular cloning a laboratory manual Volume 1 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=024810588&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| volume_link | (DE-604)BV039952684 |
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