Positional cloning by exon trapping and cDNA selection:
Saved in:
| Format: | Book |
|---|---|
| Language: | German |
| Published: |
New York [u.a.]
Wiley
1999
|
| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | X, 76 S. graph. Darst. |
| ISBN: | 0471297976 |
Staff View
MARC
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| 245 | 1 | 0 | |a Positional cloning by exon trapping and cDNA selection |c ed. by Bernhard Korn ... |
| 264 | 1 | |a New York [u.a.] |b Wiley |c 1999 | |
| 300 | |a X, 76 S. |b graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 650 | 4 | |a Exons (Genetics) | |
| 650 | 4 | |a Chromosome Mapping |x methods | |
| 650 | 4 | |a Cloning, Molecular |x methods | |
| 650 | 4 | |a DNA, Complementary | |
| 650 | 4 | |a Exons | |
| 650 | 4 | |a Exons (Gentetics) | |
| 650 | 4 | |a Gene mapping | |
| 650 | 4 | |a Molecular cloning | |
| 650 | 4 | |a Sequence Analysis, DNA |x methods | |
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Record in the Search Index
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| adam_text | Contents
Preface ix
Chapter 1 Exon Amplification 1
1.1 Introduction 1
1.1.1 The Eukaryotic Nuclear Splicing Mechanism .... 1
1.1.2 Exon Trapping Systems 3
1.1.2.1 Vectors Allowing the Trapping of Internal Exons . 3
1.1.2.2 Vectors Allowing the Trapping of 3 Terminal
Exons 4
1.1.2.3 Choice of the Exon Trap Vector 4
1.1.3 Description of the pSPL 1 Vector and Its Modified
Version, pSPL3 5
1.2 Overview of the Exon Amplification Method .... 6
1.2.1 Summary of the Procedure (Figure 1.2) 6
1.2.2 List of Components Necessary for the Exon
Amplification Experiment 7
1.3 Exon Amplification Protocol 8
1.3.1 Preparation of the Linearized pSPL3 Vector for
Cloning 8
1.3.1.1 Linearization 8
1.3.1.2 Dephosphorylation 9
1.3.1.3 Verification of the Linearized Phosphatased
Vector (L/P pSPL3) 9
1.3.2 Subcloning Genomic DNA into the
pSPL3 Vector 10
1.3.2.1 Choice and Preparation of the Genomic
Target DNA 10
1.3.2.2 Digestion of the Target Genomic DNA to
Be Inserted into pSPL3 11
1.3.2.3 Ligation into the pSPL3 Vector 11
1.3.3 Transformation of the Sublibrary 12
1.3.3.1 Preparation of Electrocompetent Cells 12
1.3.3.2 Electroporation 12
1.3.4 Analysis of the Sublibrary in the pSPL3 Vector .. 13
1.3.5 Preparation of the Pooled Sublibrary DNA for
Transfection 14
1.3.6 Transfection of the Sublibrary into Eukaryotic
COS 7 Cells 15
1.3.6.1 Propagation of COS 7 Cells 15
1.3.6.2 Transfection of COS 7 Cells by Electroporation . . 15
1.3.7 RNA Preparation 17
Contents
1.3.8 Reverse Transcription: First Strand
cDNA Synthesis 19
1.3.9 Exon Amplification 19
1.3.9.1 Primary PCR Amplification 20
1.3.9.2 Treatment of the First PCR and
Digestion with BstXI 21
1.3.9.3 Secondary PCR Amplification 21
1.3.9.4 Tertiary PCR Amplification 22
1.3.9.5 Cloning the PCR Products 23
1.3.9.5.1 ChromaspinlOO Column DNA Purification
of the PCR Product 23
1.3.9.5.2 Cloning in the pAMPl Vector 24
1.4 Analysis of the Exon Library 25
1.4.1 Evaluation of the Library by PCR Colony Testing 25
1.4.2 Identifying the artifactual Clones by Southern
Blotting 26
1.4.3 Mapping Back Candidate Exons 27
1.4.3.1 Protocol for Radiolabelling Exons 27
1.4.4 Sequencing the Exons 28
1.4.4.1 Analysis of the Sequence Data and
Identifying Genes 28
1.5 Permanent Storage of the Exon Library 30
1.6 Analysis of the Exon Library by a Strategy of
Sampling Without Replacement 31
1.6.1 Preparation of Library Filters for Screening
Purposes 31
1.6.2 Hybridizations 32
Chapter 2 cDNA Selection 33
2.1 Introduction 33
2.1.1 Conventional cDNA Screening 33
2.1.1.1 Screening of Large Genomic Regions 33
2.1.1.2 The Complexity of the Source 33
2.1.2 Hybridization in Solution 34
2.1.3 Biotinylation 35
2.1.4 Source of Genomic DNA 36
2.1.5 Source of cDNA 36
2.1.6 Blocking, Hybridization, and Elution 37
2.1.7 Analysis of cDNA Sublibraries 38
2.1.8 Conclusion and Outlook 38
2.2 Genomic Templates 39
2.2.1 Cosmid, PI, and PAC DNA Isolation 39
2.2.2 YAC DNA Isolation 40
2.3 Competition DNA 41
2.3.1 Repetitive Sequences 41
2.3.2 Vector DNA 41
2.3.3 Host DNA 42
2.3.3.1 Preparation of Yeast Genomic DNA 42
Contents
2.3.3.2 Preparation of E. coli Genomic DNA 42
2.3.3.3 Fragmentation of Competition DNA 43
2.4 cDNA Source 44
2.4.1 Biotinylation 44
2.4.2 Competition of Genomic Template DNA 45
2.4.3 First Round of cDNA Selection 45
2.4.3.1 Hybridization in Liquid (1) 45
2.4.3.2 Immobilization of Genomic Templates, Wash,
and Elution 46
2.4.3.3 Size Selection of Eluted cDNA 47
2.4.3.3.1 Buffer Exchange of Chromaspin400 Column 48
2.4.3.4. PCR of First Round of cDNA Selection 48
2.4.3.4.1 Analyze PCR Products on Gel 49
2.4.4 Second Round of cDNA Selection 50
2.4.4.1 Hybridization in Liquid (2): Wash and
Elution of cDNA 50
2.4.4.2 PCR of the Second Round of cDNA Selection ... 51
2.4.4.2.1 Analyze PCR Products on Gel 51
2.4.4.3 Size Selection of Amplified cDNA 52
2.4.4.3.1 Analyze PCR Products on Gel 52
2.4.5 Cloning of PCR Products 53
2.4.5.1 Annealing of PCR Product with pAMP 53
2.4.5.2 Transformation 53
2.4.5.2.1 Preparation of CaCl2 Competent Cells 54
2.4.5.2.2 Transformation with CaCl2 Competent Cells .... 54
2.5 Clone Analysis 55
2.5.1 Individual Clones 55
2.5.1.1 Evaluation of cDNA Insert Size 55
2.5.1.2 Determination of Sequence 56
2.5.2 cDNA Sublibrary 56
2.6 Analysis by Hybridization 56
2.6.1 Random Oligonucleotide Labelling 56
2.6.1.1 Removal of Unincorporated Nucleotides 57
2.6.2 Competition of Radioactive Probe 57
2.6.3 Hybridization and Wash 57
Chapter 3 Maps, Materials, and Equipment Specifications 59
3.1 Map of pSPL3 59
3.2 The CloneAmp System 60
3.3 Alkaline Lysis DNA Preparation 67
3.3.1 Alkaline Lysis Solutions 67
3.3.2 Procedure 67
3.4 Materials 68
3.4.1 Oligonucleotides 68
3.4.2 Buffers and Solutions 69
References 75
Index 77
|
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| indexdate | 2024-07-09T18:28:47Z |
| institution | BVB |
| isbn | 0471297976 |
| language | German |
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| physical | X, 76 S. graph. Darst. |
| publishDate | 1999 |
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| spelling | Positional cloning by exon trapping and cDNA selection ed. by Bernhard Korn ... New York [u.a.] Wiley 1999 X, 76 S. graph. Darst. txt rdacontent n rdamedia nc rdacarrier Exons (Genetics) Chromosome Mapping methods Cloning, Molecular methods DNA, Complementary Exons Exons (Gentetics) Gene mapping Molecular cloning Sequence Analysis, DNA methods cDNS (DE-588)4212297-1 gnd rswk-swf Genklonierung (DE-588)4123275-6 gnd rswk-swf Exon (DE-588)4201879-1 gnd rswk-swf Genkartierung (DE-588)4123274-4 gnd rswk-swf Klonierung (DE-588)4192480-0 gnd rswk-swf Exon (DE-588)4201879-1 s Klonierung (DE-588)4192480-0 s cDNS (DE-588)4212297-1 s DE-604 Genklonierung (DE-588)4123275-6 s Genkartierung (DE-588)4123274-4 s Korn, Bernhard Sonstige oth HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=008489656&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Positional cloning by exon trapping and cDNA selection Exons (Genetics) Chromosome Mapping methods Cloning, Molecular methods DNA, Complementary Exons Exons (Gentetics) Gene mapping Molecular cloning Sequence Analysis, DNA methods cDNS (DE-588)4212297-1 gnd Genklonierung (DE-588)4123275-6 gnd Exon (DE-588)4201879-1 gnd Genkartierung (DE-588)4123274-4 gnd Klonierung (DE-588)4192480-0 gnd |
| subject_GND | (DE-588)4212297-1 (DE-588)4123275-6 (DE-588)4201879-1 (DE-588)4123274-4 (DE-588)4192480-0 |
| title | Positional cloning by exon trapping and cDNA selection |
| title_auth | Positional cloning by exon trapping and cDNA selection |
| title_exact_search | Positional cloning by exon trapping and cDNA selection |
| title_full | Positional cloning by exon trapping and cDNA selection ed. by Bernhard Korn ... |
| title_fullStr | Positional cloning by exon trapping and cDNA selection ed. by Bernhard Korn ... |
| title_full_unstemmed | Positional cloning by exon trapping and cDNA selection ed. by Bernhard Korn ... |
| title_short | Positional cloning by exon trapping and cDNA selection |
| title_sort | positional cloning by exon trapping and cdna selection |
| topic | Exons (Genetics) Chromosome Mapping methods Cloning, Molecular methods DNA, Complementary Exons Exons (Gentetics) Gene mapping Molecular cloning Sequence Analysis, DNA methods cDNS (DE-588)4212297-1 gnd Genklonierung (DE-588)4123275-6 gnd Exon (DE-588)4201879-1 gnd Genkartierung (DE-588)4123274-4 gnd Klonierung (DE-588)4192480-0 gnd |
| topic_facet | Exons (Genetics) Chromosome Mapping methods Cloning, Molecular methods DNA, Complementary Exons Exons (Gentetics) Gene mapping Molecular cloning Sequence Analysis, DNA methods cDNS Genklonierung Exon Genkartierung Klonierung |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=008489656&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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