Molecular cloning: a laboratory manual Volume 2
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, 631-1333, I-46 Seiten Illustrationen, Diagramme |
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MARC
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| 035 | |a (DE-599)BVBBV039952692 | ||
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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 2 |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, 631-1333, 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:BVB01-024810591 | |
Datensatz im Suchindex
| _version_ | 1820497046470131712 |
|---|---|
| adam_text |
VOLUME
2
PART
2:
ANALYSIS AND MANIPULATION OF
DNA
AND
RNA
CHAPTER
9
Quantification of
DNA
and
RNA
by Real-Time Polymerase Chain Reaction
631
INTRODUCTION
Real-Time PCR Chemistries
632
Instruments for Real-Time PCR
639
Extracting Data from a Real-Time PCR Experiment: Data Analysis
and Normalization Methods
641
Designing Primers and Probes and Optimizing Conditions for Real-Time PCR
643
Constructing a Standard Curve
648
Performing
Real-Time PCR
650
Performing Real-Time RT-PCR
650
MIQE Guidelines
654
Real-Time PCR Protocols
654
χϋ
/ Contents
PROTOCOLS
1
Optimizing Primer and Probe Concentrations for Use in Real-Time PCR
658
2
Constructing a Standard Curve
663
3
Quantification of
DNA
by Real-Time PCR
667
4
Quantification of
RNA
by Real-Time RT-PCR
670
5
Analysis and Normalization of
Real-Time PCR
Experimental Data
674
INFORMATION PANELS
Multiplex PCR
680
SNP Genotyping
681
CHAPTER
10
Nucleic Acid Platform Technologies
683
Oliver Rando
INTRODUCTION
Microarray Applications
685
Performing Microarray Experiments
688
PROTOCOLS
1
Printing Microarrays
694
2
Round A/Round
В
Amplification of
DNA 698
3
T7 Linear Amplification of
DNA
(TLAD) for Nudeosomal
and Other
DNA < 500
bp
702
4
Amplification of
RNA
709
5
Direct Cyanine-dUTP Labeling of
RNA
715
6
Indirect Aminoallyl-dUTP Labeling of
RNA
718
7
Cyanine-dCTP Labeling of
DNA
Using Klenow
721
8
Indirect Labeling of
DNA 724
9
Blocking Polylysines on Homemade Microarrays
726
10
Hybridization to Homemade Microarrays
729
CHAPTER
11
DNA
Sequencing
735
Elaine
Mardis
and W. Richard McCombie
INTRODUCTION
History of
Sanger/Dideoxy DNA
Sequencing
736
Next-Generation Sequencing
742
Overview of Next-Generation Sequencing Instruments
752
Sänger
Sequencing versus Next-Generation Sequencing: When to Do What?
760
Introduction to Protocols
761
Contents
j
XÜi
SECTION I. LIBRARY PREPARATIONS FOR VARIOUS PLATFORMS
Capillary Sequencing
1
Preparing
Plasmici
Subclones
for Capillary Sequencing
764
2
Preparing PCR Products for Capillary Sequencing
770
3
Cycling Sequencing Reactions
773
Illumina
4
Whole Genome: Manual Library Preparation
776
5
Whole Genome: Automated, Nonindexed Library Preparation
783
•
Additional Protocol: Automated Library Preparation
789
6
Whole Genome: Automated, Indexed Library Preparation
792
7
Preparation of a 3-kb Mate-Pair Library for
Illumina
Sequencing
799
8
Preparation of an 8-kb Mate-Pair Library for
Illumina
Sequencing
808
•
Additional Protocol: AMPure Bead Calibration
821
9
RNA-Seq:
RNA
Conversion to cDNA and Amplification
824
•
Additional Protocol: RNACIean XP Bead Cleanup (before RNA-Seq)
830
10
Solution-Phase
Exorne
Capture
831
•
Additional Protocol: AMPure XP Bead Cleanup
840
•
Additional Protocol:
Agarose Gel
Size Selection
842
11
Automated Size Selection
844
12
Library Quantification Using SYBR Green-qPCR
848
13
Library Quantification Using PicoGreen Fluorometry
852
14
Library Quantification: Fluorometric Quantitation of Double-Stranded or
Single-Stranded
DNA
Samples Using the Qubit System
857
Pyrosequencing
15
Preparation of Small-Fragment Libraries for
454
Sequencing
859
16
sstDNA Library Capture and emPCR
866
17
Roche/454 Sequencer: Executing a Sequencing Run
874
SECTION II. POSTSEQUENCING ANALYSES
18
Validation
883
19
Quality Assessment of Sequence Data
885
20
Data Analysis
887
INFORMATION PANELS
Biotin
888
Magnetic Beads
890
Fragmenting of
DNA 892
CHAPTER
12
Analysis of
DNA
Methylation in Mammalian Cells
893
Paul M. Lizardi, Qin Yan, and
Narendra Wajapeyee
INTRODUCTION
DNA
Methylation Affects and Reveals Biological Phenomena
894
Experimental Approaches for Analysis of
DNA
Methylation
895
XIV
/ Contents
Advantages and Limitations of Different Approaches for Analyzing
DNA Methylation 898
Future Perspectives
^9
PROTOCOLS
1 DNA
Bisulfite Sequencing for
Single-Nucleotide-Resolution DNA
Methylation
Detection
900
2
Methylation-Specific PCR for Gene-Specific
DNA
Methylation Detection
907
3
Methyl-Cytosine-Based Immunoprecipitation for
DNA
Methylation Analysis
911
4
High-Throughput Deep Sequencing for Mapping Mammalian
DNA
Methylation
915
5
Roche
454
Clonal Sequencing of
Bisulfite-Converted DNA
Libraries
927
6
Illumina
Sequencing of Bisulfite-Converted
DNA
Libraries
932
INFORMATION PANELS
Public Domain Software for Identifying CpG Islands in Promoter and Coding Regions
of Mammalian Genes
937
Designing Primers for the Amplification of Bisulfite-Converted Product to Perform
Bisulfite Sequencing and MS-PCR
939
Postsequence Processing of High-Throughput Bisulfite Deep-Sequencing Data
940
CHAPTER
13
Preparation of Labeled
DNA, RNA,
and Oligonucleotide Probes
943
INTRODUCTION
Radioactive versus Nonradioactive Labeling of Nucleic Acids
944
Types of Nonradioactive Detection Systems
948
Designing Oligonucleotides for Use as Probes
953
PROTOCOLS
1
Random Priming: Labeling of Purified
DNA
Fragments by Extension of Random
Oligonucleotides %5
2
Random Priming: Labeling of
DNA
by Extension of Random Oligonucleotides in the
Presence of Melted
Agarose
971
3
Labeling of
DNA
Probes by Nick Translation
974
4
Labeling of
DNA
Probes by Polymerase Chain Reaction
978
•
Additional Protocol: Asymmetric Probes
982
5
Synthesis of Single-Stranded
RNA
Probes by In Vitro Transcription
984
•
Additional Protocol: Using PCR to Add Promoters for Bacteriophage-Encoded
RNA Polymerases
to Fragments of
DNA 991
6
Synthesis of cDNA Probes from mRNA Using Random Oligonucleotide Primers
994
7
Radiolabeling of Subtracted cDNA Probes by Random Oligonucleotide Extension
997
8
Labeling
3'
Termini of Double-Stranded
DNA
Using the Klenow Fragment of
f.
coli
DNA
Polymerase I 1003
9
Dephosphorylation of
DNA
Fragments with Alkaline Phosphatase
1009
10
Phosphorylation of
DNA
Molecules with Protruding 5'Hydroxyl Termini
1012
Contents
I XV
11 Phosphorylation
of
DNA
Molecules with Dephosphorylated Blunt Ends or
Recessed
5'
Termini
1015
12
Phosphorylating the
5'
Termini of Oligonucleotides Using T4 Polynucleotide Kinase
1018
13
Labeling the
3'
Termini of Oligonucleotides Using Terminal Deoxynucleotidyl
Transferase
1021
•
Alternative Protocol: Synthesizing Nonradiolabeled Probes Using TdT
1023
•
Additional Protocol: Tailing Reaction
1024
•
Additional Protocol: Modifications for Synthesizing Nonradiolabeled Probes
1026
14
Labeling of Synthetic Oligonucleotides Using the Klenow Fragment of
E. coli
DNA
Polymerase
і
1028
15
Purification of Labeled Oligonucleotides by Precipitation with
Ethanol
1032
16
Purification of Labeled Oligonucleotides by Size-Exclusion Chromatography
1034
17
Purification of Labeled Oligonucleotides by Chromatography on a
Sep
-Рак
C^ Column
1037
18
Hybridization of Oligonucleotide Probes in Aqueous Solutions: Washing in Buffers
Containing Quaternary Ammonium Salts
1039
INFORMATION PANELS
Preparation of Stock Solutions of dNTPs
1043
E. coli
DNA
Polymerase I and the Klenow Fragment
1044
In Vitro Transcription Systems
1050
Alkaline Phosphatase
1053
Melting Temperatures
1055
CHAPTER
14
Methods for In Vitro Mutagenesis
1059
Matteo Forloni, Alex Y. Liu, and
Narendra Wajapeyee
INTRODUCTION
Mutagenesis Approaches
1064
Research Goals
1065
Commercial Kits
1065
PROTOCOLS
1
Random Mutagenesis Using Error-Prone
DNA Polymerases 1068
2
Creating Insertions or Deletions Using Overlap Extension PCR Mutagenesis
1080
3
In Vitro Mutagenesis Using Double-Stranded
DNA
Templates: Selection
of Mutants with Dpnl
1087
4
Altered
ß-Lactamase
Selection Approach for Site-Directed Mutagenesis
1095
5
Oligonucleotide-Directed Mutagenesis by Elimination of a Unique Restriction Site
(USE Mutagenesis)
1102
6
Saturation Mutagenesis by Codon Cassette Insertion
1108
7
Random Scanning Mutagenesis
1115
8
Multisite-Directed Mutagenesis
1120
9
Megaprimer PCR-Based Mutagenesis
1124
xvi / Contents
INFORMATION PANELS
1127
Domain Mutagenesis
High-Throughput Site-Directed
Mutagenesis
of
Plasmid DNA 1128
N6-Methyladenine, Dam Methylase, and Methylation-Sensitive
Restriction Enzymes
PART
3:
INTRODUCING GENES INTO CELLS
CHAPTER
15
Introducing Genes into Cultured Mammalian Cells
1131
Priti Kumar, Arvindhan Nagarajan, and Pradeep D. Uchil
INTRODUCTION
Transient Versus Stable Transfection
1133
Transfection Methods
1133
Transfection Controls
1133
Optimization and Special Considerations
1136
Assessing Cell Viability in Transfected Cell Lines
1137
PROTOCOLS
1 DNA
Transfection Mediated by Cationic
Lipid
Reagents
1139
•
Alternative Protocol: Transfection Using DOTMA and DOCS
1145
•
Additional Protocol: Histochemical Staining of Cell Monolayers for
ß-Calactosidase 1148
2
Calcium-Phosphate-Mediated Transfection of Eukaryotic Cells with Plasmid DNAs
1150
•
Alternative Protocol: High-Efficiency Calcium-Phosphate-Mediated Transfection of
Eukaryotic Cells with Plasmid DNAs
1156
3
Calcium-Phosphate-Mediated Transfection of Cells with High-Molecular-Weight
Genomic
DNA 1160
•
Alternative Protocol: Calcium-Phosphate-Mediated Transfection of Adherent Cells
1163
•
Alternative Protocol: Calcium-Phosphate-Mediated Transfection of Cells Growing
in Suspension
1165
4
Transfection Mediated by DEAE-Dextran: High-Efficiency Method
1167
•
Alternative Protocol: Transfection Mediated by DEAE-Dextran: Increased
Cell Viability H70
5 DNA
Transfection by Electroporation
1173
6
Analysis of Cell Viability by the alamarBlue Assay
1177
7
Analysis of Cell Viability by the
Lactate
Dehydrogenase Assay
1180
8
Analysis of Cell Viability by the MTT Assay
П83
INFORMATION PANELS
Optical Transfection
Cotransformation
Selective Agents for Stable Transformation
1190
Lipofection 1194
Contents
I
xvii
Linearizing Plasmids before Transfection
1197
Transfection of Mammalian Cells with Calcium Phosphate-DNA Coprecipitates
1198
Chloroquine Diphosphate
1200
DEAE-Dextran Transfection
1201
Electroporation
1203
CHAPTER
16
Introducing Genes into Mammalian Cells: Viral Vectors
1209
Guangping
Gao
and Miguel Sena-Esteves
INTRODUCTION
Factors to Consider When Choosing a Viral Vector
1211
The Major Types of Viruses Currently Used as Vectors
1212
In Vivo Expression
1221
Adenovirus Vectors
1221
Adeno-Associated Virus Vectors
1224
Retrovirus
and Lentivirus Vectors
1227
PROTOCOLS
1
Construction of
Recombinant
Adenovirus Genomes by Direct Cloning
1233
2
Release of the Cloned
Recombinant
Adenovirus Genome for Rescue and Expansion
1238
3
Purification of the
Recombinant
Adenovirus by Cesium Chloride Gradient
Centrifugation
1244
4
Characterization of the Purified
Recombinant
Adenovirus Vector for Viral Genome
Structure by Restriction Enzyme Digestions
1249
5
Measuring the Infectious
Titer
of
Recombinant
Adenovirus Vectors Using
TCID50 End-Point Dilution and qPCR
1253
•
Additional Protocol: Preparation of
a
DNA
Standard for qPCR
1262
6
Detection Assay for Replication-Competent Adenovirus by Concentration
Passage and Real-Time qPCR
1264
7
Production of rAAVs by Transient Transfection
1274
8
Purification of rAAVs by Cesium Chloride Gradient Sedimentation
1278
9
Purification of rAAVs by lodixanol Gradient Centrifugation
1283
10
Purification of rAAV2s by Heparin Column Affinity Chromatography
1287
11
Enrichment of Fully Packaged
Virions in
Column-Purified rAAV Preparations
by lodixanol Gradient Centrifugation Followed by Anion-Exchange Column
Chromatography
1290
12 Titration
of rAAV Genome Copy Number Using Real-Time qPCR
1294
13
Sensitive Determination of Infectious
Titer
of rAAVs Using TCID50
End-Point Dilution and qPCR
1298
14
Analysis of rAAV Sample Morphology Using Negative Staining and
High-Resolution Electron Microscopy
1301
15
Analysis of rAAV Purity Using Silver-Stained SDS-PAGE
1304
16
Production of High-Titer
Retrovirus
and Lentivirus Vectors
1307
xviii / Contents
17 Titration
of
Lentivirus
Vectors
1314
18 Monitoring Lentivirus
Vector
Stocks
for Replication-Competent Viruses
1319
INFORMATION PANELS
Adenovirus
Vectors
1322
AAV Vectors
1323
Lentivirus
Vectors
1324
Basic Elements in
Viral Vectors
1326
Assays Done in Transduced Cells
1328
Transgene
Expression Cassettes
1330 |
| any_adam_object | 1 |
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| illustrated | Illustrated |
| indexdate | 2025-01-06T11:01:15Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-024810591 |
| oclc_num | 785848854 |
| open_access_boolean | |
| owner | DE-20 DE-703 DE-29T DE-91G DE-BY-TUM DE-11 DE-578 DE-19 DE-BY-UBM DE-Er8 DE-355 DE-BY-UBR DE-384 DE-M49 DE-BY-TUM DE-188 DE-N32 DE-83 |
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| physical | xxxiii, 631-1333, I-46 Seiten Illustrationen, Diagramme |
| publishDate | 2012 |
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| publisher | Cold Spring Harbor Laboratory Press |
| record_format | marc |
| spelling | Green, Michael R. 1954- Verfasser (DE-588)1024045439 aut Molecular cloning a laboratory manual Volume 2 Michael R. Green, Joseph Sambrook Fourth edition Cold Spring Harbor, New York Cold Spring Harbor Laboratory Press [2012] xxxiii, 631-1333, 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 2 Digitalisierung UB Regensburg application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=024810591&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 2 Michael R. Green, Joseph Sambrook |
| title_fullStr | Molecular cloning a laboratory manual Volume 2 Michael R. Green, Joseph Sambrook |
| title_full_unstemmed | Molecular cloning a laboratory manual Volume 2 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=024810591&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| volume_link | (DE-604)BV039952684 |
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