Introduction to molecular cloning techniques:
Gespeichert in:
| Hauptverfasser: | , |
|---|---|
| Format: | Buch |
| Sprache: | English French |
| Veröffentlicht: |
New York u.a.
VCH
1993
|
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | XVI, 298 S. Ill., graph. Darst. |
| ISBN: | 1560816139 3527896139 |
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| 240 | 1 | 0 | |a Techniques de clonage moléculaire |
| 245 | 1 | 0 | |a Introduction to molecular cloning techniques |c by Gérard Lucotte and François Baneyx |
| 264 | 1 | |a New York u.a. |b VCH |c 1993 | |
| 300 | |a XVI, 298 S. |b Ill., graph. Darst. | ||
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| 650 | 7 | |a Cloning |2 cabt | |
| 650 | 7 | |a Genetica molecular e de microorganismos |2 larpcal | |
| 650 | 4 | |a Cloning, Molecular |v Laboratory Manuals | |
| 650 | 4 | |a Molecular cloning |v Laboratory manuals | |
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Datensatz im Suchindex
| _version_ | 1804122609799921664 |
|---|---|
| adam_text | Introduction to
Molecular Cloning
Techniques
by
Girard Lucotte and Frangois Baneyx
VCH
Contents
PART I The Host 1
CHAPTER 1 Escherichia coli: a Host for Genetic
Engineering 3
1 1 Structure, Growth Conditions, Genetic Markers, and Nomenclature
1 2 Resistance to Antibiotics 5
1 3 Bacterial Growth 5
1 4 Host Restriction and Modification 7
1 5 Host Recombination 7
Experiments 7
1 1 Growth Media 7
1 2 Spreading 9
1 3 Isolation of a Single Colony 9
1 4 Use of Antibiotics 10
1 5 Storage of Bacterial Strains 11
Problems 11
1 1 Strain Phenotype 11
1 2 Characteristics of the Exponential Phase 12
vii
viii INTRODUCTION TO MOLECULAR CLONING TECHNIQUES
PART II Restriction Enzymes, Methylation, Electrophoresis
Techniques 13
CHAPTER 2 Restriction Enzymes 15
2 1 Host Restriction and Modification 15
2 2 Isolation of Type II Restriction Endonucleases 17
2 3 Properties of the Recognition Site 18
Experiments 19
2 1 Purity 19
2 2 Storage 19
2 3 Unit Definition 19
2 4 Measuring Restriction Enzyme Activity 19
2 5 Reaction Conditions 20
Problems 21
2 1 Enzymes and Extensions 21
2 2 Enzymes with Multiple Recognition Sites 21
2 3 Rotational Symmetry of Restriction Enzymes 21
2 4 Isoschizomery 22
2 5 Theoretical Frequency of Restriction Cuts 22
2 6 Theoretical Symmetrical Sites 22
2 7 Theoretical vs Actual Recognition Sequences 22
2 8 Enzymatic Units Necessary to Digest a Given Amount of DNA
CHAPTER 3 Methylation 25
3 1 Prokaryotic Methylases 25
3 2 Methylation by Eukaryotes 26
Experiments 26
3 1 Example: EcoRl Methylase 26
3 2 Unit Definition 27
3 3 Reaction Conditions 27
Problems 27
3 1 Methylation Sites 27
3 2 Selecting Restriction Enzymes to Study Adenine Methylation 27
3 3 Effect of the dam Methylase on Restriction Enzymes 27
3 4 Protecting Restriction Sites by Methylation 27
3 5 Methylation and Cleavage of Eukaryotic DNA 27
CHAPTER 4 Electrophoresis and Blotting Techniques 29
4 1 DNA Electrophoresis 29
4 2 Protein Electrophoresis 32
4 3 Blotting Techniques 33
CONTENTS f ix
Experiments 36
4 1 Electrophoresis Cells 36
4 2 Agarose 38
4 3 Electrophoresis Buffers 38
4 4 Casting DNA Gels and Loading the Samples 40
4 5 Visualization of DNA Bands 41
4 6 Migration Anomalies 41
Problems 42
4 1 DNA Molecular Weight Markers for Agarose Gels 42
4 2 DNA Molecular Weight Markers for Polyacrylamide Gels 42
4 3 Criteria for Complete Digestion of Eukaryotic DNA 43
PART III Cloning Vectors 45
CHAPTER 5 Bacteriophage X 47
5 1 Genes Involved in the Alternative Life Cycles of X 47
5 2 Gene Map 49
5 3 Plaque Formation 50
Experiments 51
5 1 Infection and Plaque Formation 51
5 2 Plaque Excision 51
5 3 Preparation ofLysate Stock Solutions 51
Problems 52
5 1 Stages in the Lytic Cycle and Encapsidation 52
5 2 Phage Adsorption and Entry 52
5 3 Increasing Plaque Size 52
CHAPTER 6 Bacteriophage Cloning Vectors 53
6 1 Removal of Restriction Sites 53
6 2 Phage Vectors Derived from X 54
6 3 Categories of X Vectors 54
Experiments 61
6 1 Transfection and Bacterial Lysis 61
6 2 Bacteriophage Purification 63
6 3 Prophage Induction 64
6 4 Isolation of Phage X DNA 64
6 5 Preparation of Phage Arms 65
Problems 67
6 1 Cloning Capacity at Different Sites 67
6 2 Blue/Clear Plaque Selection 67
6 3 Advantages ofBamHl Insertion Vectors 67
x INTRODUCTION TO MOLECULAR CLONING TECHNIQUES
CHAPTER 7 Plasmids 69
7 1 General Properties 69
7 2 Naturally Occurring Plasmids 71
7 3 Characteristics of the Ideal Plasmid 72
7 4 Plasmid pBR322 73
7 5 Other Useful Plasmids 75
Experiments 79
7 1 Linearization of pBR322 with Pstl 79
Problems 80
7 1 Characteristics of Plasmids as Cloning Vectors 80
7 2 Advantages of Small Plasmids 80
7 3 Directed Cloning 80
7 4 The Multiple Cloning Site of pUC 18 81
CHAPTER 8 Purification of Plasmid DNA 83
8 1 Bacterial Growth and Plasmid Amplification 83
8 2 Cell Lysis 84
8 3 Plasmid DNA Purification 84
Experiments 86
8 1 Isolation of Plasmid DNA from Medium-Scale Cultures ( 200 ml)
by Ultracentrifugation on Cesium Chloride Gradients in the
Presence of Ethidium Bromide 86
8 2 Use of A50 Columns 86
8 3 Isolation of Plasmid DNA from Small-Volume Cultures by Alkaline
Lysis 87
Problems 88
8 1 Relevance of Plasmid and Chromosomal DNA Separation 88
8 2 Plasmic Conformation 88
8 3 Differential Sedimentation of Chromosomal and Plasmid DNA on
a CsCl Gradient in the Presence of Ethidium Bromide 88
8 4 Relative Position of DNA Bands Following Ultracentrifugation on
CsCl Gradient in a Fixed-Angle or Swinging-Bucket Rotor 88
8 5 Plasmid DNA Purity 88
CHAPTER 9 Transforming E coli with Plasmid DNA 89
Experiments 91
9 1 Typical Transformation Protocol 91
9 2 Storage of Competent Cells 91
Problems 92
9 1 General Transformation Scheme in Gram-Positive Organisms
9 2 Frequency of Transformation 92
CONTENTS
9 3 Calculating the Frequency of Transformation 92
9 4 Calculating the Fraction of Competent Cells 92
CHAPTER 10 In Vitro DNA Encapsidation 93
Experiments 96
Problems 96
10 1 Verifying the Genotype of Encapsidation Strains 96
10 2 Efficiency of DNA Encapsidation and Amount of Encapsidated
DNA 97
10 3 Encapsidation Selectivity vs DNA Size 97
PART IV DNA and RNA Preparation 99
CHAPTER 11 Extraction and Characterization of DNA from
Tissues 101
Experiments 102
11 1 Enzyme Preparation 102
11 2 DNA Extraction 103
11 3 Spectrophotometric Assay of DNA 103
11 4 Chemical Assay of DNA Content 103
11 5 Concentrating DNA Samples 104
11 6 Ethanol Precipitation of DNA 105
11 7 Storage of DNA Samples 105
Problems 105
11 1 Absorbance Spectrum of a DNA Sample 105
11 2 RNA Contamination 105
11 3 Total Yield of DNA Extracted from Blood 105
CHAPTER 12 Extraction of DNA from Gels 107
12 1 DNA Extraction from Polyacrylamide Gels 107
12 2 DNA Extraction from Agarose Gels 108
Experiments 109
12 1 ElutionofDNA from Acrylamide Gels 109
12 2 Electroelution from Agarose Gels Using DEAE Paper 109
12 3 Electroelution Using Dialysis Tubing 109
12 4 Elution from Low-Melting-Point Agarose 110
xii INTRODUCTION TO MOLECULAR CLONING TECHNIQUES
CHAPTER 13 Isolation and Characterization of Messenger
RNA 111
13 1 Isolation of mRNA by Affinity Chromatography 112
13 2 Analysis of RNA Samples by Sedimentation on Sucrose Gradients
13 3 Assay of mRNA Content by Hybridization to Poly(U) 113
Experiments 115
13 1 Isolation of Total Cellular RNA 115
13 2 Spectrophotometric Assay of RNA 115
13 3 Affinity Chromatography on Oligo(dT) Columns 115
13 4 Sedimentation of RNA Solutions on Sucrose Gradients 116
13 5 Assay ofthePoly(A) Content 116
Problems 116
13 1 RNA Integrity 116
13 2 Calculating the Concentration of Total Extracted RNA 117
13 3 Poly(A) Yield 117
13 4 Poly(A) Contamination by Ribosomal RNA 117
CHAPTER 14 RNA Electrophoresis 119
Experiments 120
14 1 Sample Preparation 120
14 2 Methylmercuric Hydroxide Gels 120
14 3 Formaldehyde Gels 120
14 4 UseofGlyoxal 120
14 5 Preparative Electrophoresis Using Low-Melting-Point Agarose in
the Presence of Methylmercuric Hydroxide 121
14 6 Formamide Polyacrylamide Gels 121
14 7 Urea Polyacrylamide Gels 121
CHAPTER 15 In Vitro Protein Synthesis 123
15 1 Different Types of In Vitro Translation Systems 123
15 2 Optimization of the Reaction Conditions 124
15 3 Analysis of the Translation Products 125
Experiments 127
Problems 129
15 1 Effect of the Potassium Concentration on the Efficiency of
Translation 129
15 2 Advantages of [3SS]-Methionine 129
15 3 Assaying Different Incorporations 130
15 4 Assaying the Amount of Immunoprecipitated Protein 132
15 5 Characterization by Electrophoresis 132
CONTENTS i xiii
CHAPTER 16 Enriching for Specific mRNA Molecules
16 1 Tissue Selection 133
16 2 Preparation of Polysomes and Extraction of Polysomal RNA 133
16 3 Affinity Chromatography of Oligo(dT) Columns 135
Experiments 135
16 1 Polysome Isolation 135
16 2 Extraction of Polysomal RNA 137
Problems 137
16 1 Polysome Integrity 137
16 2 Artifacts in the Sucrose Gradient 137
16 3 Purification of Albumin mRNA by Second Affinity
Chromatography of the Poly(A)+ Fraction 137
CHAPTER 17 Complementary DNA Synthesis 139
17 1 Synthesis of cDNA from Poly(A)+mRNA 139
17 2 Synthesis of Double-Stranded DNA 141
17 3 SI Nuclease Digestion 143
17 4 Selection of cDNA According to Size 144
17 5 Isolation of Full-Length cDNA 144
Experiments 145
17 1 Estimating the Required Amount of Reverse Transcriptase 145
17 2 Synthesis of the First cDNA Strand 146
17 3 Synthesis of the Second cDNA Strand 147
17 4 SI Nuclease Digestion 148
Problems 149
17 1 Yield of Single-Stranded cDNA 149
17 2 Yield of Double-Stranded cDNA 150
17 3 Verifying the cDNA Digestion by SI Nuclease 150
17 4 Heterogeneous Nature of Double-Stranded cDNA Prepared from
Total Liver Poly(A)+ mRNA 150
17 5 Incomplete Double-Stranded cDNA 150
PARTV Cloning Techniques 151
CHAPTER 18 Ligation 153
18 1 Ligases 153
18 2 DNA Molecules Involved in Ligations 154
18 3 In Vivo vs In Vitro Ligation 154
18 4 Ligation Parameters 156
18 5 Increasing the Cloning Efficiency by the Simultaneous Use of Two
Restriction Enzymes 158
xiv INTRODUCTION TO MOLECULAR CLONING TECHNIQUES
18 6 Treatment of Linearized DNA with Alkaline Phosphatase 159
18 7 Blunt-End Ligations 159
Experiments 160
18 1 Typical Ligation Protocol 160
18 2 Blunt-End Ligation 160
18 3 Vector Dephosphorylation 160
Problems 160
18 1 Schematic Representation of Ligation Reactions 160
18 2 Efficiency of Ligation and Nature of Overhangs 160
18 3 Restriction Enzymes and Ligation 161
18 4 Determination of End Concentrations 161
18 5 Calculating^ 161
18 6 Influence of the DNA Concentration on the j/i Ratio 161
CHAPTER 19 Cloning by Addition of Complementary
Homopolymeric Sequences 163
Experiments 166
19 1 Synthesis of Poly(dC) Homopolymeric Tracts at the 3 End of the
Double-Stranded cDNA Molecules 166
19 2 Preparing pBR322 for the Cloning Step 167
19 3 Hybridization and Formation of the Recombinant Plasmid
Problems 167
19 1 Number of dNTP Residues Synthesized per 3 End 167
19 2 Cloning at the Hindlll Site of pBR322 Using dA/dT Tracts 168
19 3 Number of 3 Ends Available for Generating Homopolymeric
Tracts 168
CHAPTER 20 Cloning by Addition of Synthetic Sequences
20 1 Generating Blunt Ends from Staggered Ends 169
20 2 Synthetic Linkers 170
20 3 Adaptors 173
Experiments 173
20 1 Phosphorylation of Synthetic Linkers 173
20 2 Ligation of a Phosphorylated Linker to a Blunt-Ended cDNA
Molecule 173
Problems 174
20 1 Regenerating a Restriction Site with Polymerase 174
20 2 Linker Purity 176
20 3 Linker Sites 176
20 4 Hexameric Linkers 176
20 5 Linker Manipulations 176
CONTENTS , xv
20 6 Insertion of a Hpall Fragment in a Vector Linearized with EcoRI
20 7 Adaptor Sites 176
20 8 Use of a Single-Stranded Adaptor 176
20 9 Simultaneous Use of Two Adaptors 177
20 10 Adaptor-Mediated cDNA Cioning 177
PART VI Libraries 179
CHAPTER 21 Genomic Libraries 181
21 1 Example: Construction of a Human Genomic Library 182
21 2 Steps Involved in the Construction of a Genomic Library 182
21 3 EMBL Vectors 184
Experiments 186
21 1 DNA Fractionation on a Sodium Chloride Gradient 186
21 2 Preparation of the Phage Arms and Removal of the Central
Fragment 186
21 3 Ligation of Passenger DNA to the Phage Arms 187
Problems 187
21 1 Ideal Genomic Library 187
21 2 Number of EcdSl Recombinants to Obtain a Given Sequence
21 3 Number of 20-kbp Fragments to Obtain a Complete Library
21 4 Probability of Obtaining a Complete Genomic Library 188
21 5 Complete Genomic Libraries in Different Organisms 188
21 6 Lower Theoretical Limit for the Construction of a Genomic
Library 188
21 7 Advantages of EMBL Vectors 188
CHAPTER 22 cDNA Libraries 189
22 1 Requirements for a Complete cDNA Library 189
22 2 Eliminating the S1 Nuclease Step 191
22 3 Cloning cDNA into XgtlO, Xgtll, and XZAPII 194
Experiments 196
22 1 Synthesis of a Full-Length Complementary Strand 196
22 2 Using XgtlO and Xgtll 197
Problems 198
22 1 Importance of cDNA Libraries 198
22 2 Obtaining Full-Length cDNA Molecules 198
22 3 pUC Cloning Vectors 198
22 4 Length of the Complementary cDNA Strand 198
22 5 XgtlO vs Xgtll 198
xvi INTRODUCTION TO MOLECULAR CLONING TECHNIQUES
CHAPTER 23 Cosmid Libraries 199
Experiments 203
23 1 Digestion of Genomic DNA with Sau3A 203
23 2 Isolation of Restriction Fragments about 45 kDA in size 204
23 3 Cosmid Preparation and Ligation 204
Problems 204
23 1 Theoretical Cloning Capacity of Cosmids 204
23 2 Features of SauiA 204
23 3 Complete Cosmid Genomic Library 205
APPENDICES 207
1 Commonly Used Bacterial Strains 207
2 Some of the Known Restriction Enzymes 209
3 Examples of Isoschizomers 219
4 Methylation Chart 223
5 Detailed Genetic and Physical Map of XDNA 227
6 Simplified Restriction Map of XDNA 229
7 Map of the First 21 Vectors of the Charon Series 231
8 Complete Nucleotide Sequence of pBR322 235
9 Detailed Restriction Map of pBR322 239
10 Structure of the pUC Plasmid Series 241
11 ProteinaseK 243
12 Extraction of Lymphocyte DNA 245
13 RNaseA 247
14 DNasel 249
15 Preparation of Rabbit Reticulocyte Extract 251
16 SI Nuclease 253
17 Reverse Transcriptase 255
18 T4DNALigase 257
19 E coli Alkaline Phosphatase 259
20 Deoxynucleotidyl Terminal Transferase 261
21 The Polymerase Chain Reaction 263
22 Example of Adaptor Molecules of Different Sizes 265
23 Ribonuclease H 267
24 Characteristics of a Few Commonly Used Cosmids 269
ANSWERS 271
REFERENCES 287
INDEX 293
|
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| id | DE-604.BV008224030 |
| illustrated | Illustrated |
| indexdate | 2024-07-09T17:16:36Z |
| institution | BVB |
| isbn | 1560816139 3527896139 |
| language | English French |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-005427613 |
| oclc_num | 28148192 |
| open_access_boolean | |
| owner | DE-355 DE-BY-UBR DE-703 DE-91G DE-BY-TUM DE-M49 DE-BY-TUM DE-29T DE-20 DE-11 DE-188 DE-B768 |
| owner_facet | DE-355 DE-BY-UBR DE-703 DE-91G DE-BY-TUM DE-M49 DE-BY-TUM DE-29T DE-20 DE-11 DE-188 DE-B768 |
| physical | XVI, 298 S. Ill., graph. Darst. |
| publishDate | 1993 |
| publishDateSearch | 1993 |
| publishDateSort | 1993 |
| publisher | VCH |
| record_format | marc |
| spelling | Lucotte, Gérard Verfasser aut Techniques de clonage moléculaire Introduction to molecular cloning techniques by Gérard Lucotte and François Baneyx New York u.a. VCH 1993 XVI, 298 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Cloning cabt Genetica molecular e de microorganismos larpcal Cloning, Molecular Laboratory Manuals Molecular cloning Laboratory manuals Escherichia coli (DE-588)4070959-0 gnd rswk-swf Gentechnologie (DE-588)4071722-7 gnd rswk-swf DNS-Klonierung (DE-588)4250249-4 gnd rswk-swf Genklonierung (DE-588)4123275-6 gnd rswk-swf DNS-Klonierung (DE-588)4250249-4 s DE-604 Genklonierung (DE-588)4123275-6 s Gentechnologie (DE-588)4071722-7 s Escherichia coli (DE-588)4070959-0 s Baneyx, François Verfasser aut HEBIS Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=005427613&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Lucotte, Gérard Baneyx, François Introduction to molecular cloning techniques Cloning cabt Genetica molecular e de microorganismos larpcal Cloning, Molecular Laboratory Manuals Molecular cloning Laboratory manuals Escherichia coli (DE-588)4070959-0 gnd Gentechnologie (DE-588)4071722-7 gnd DNS-Klonierung (DE-588)4250249-4 gnd Genklonierung (DE-588)4123275-6 gnd |
| subject_GND | (DE-588)4070959-0 (DE-588)4071722-7 (DE-588)4250249-4 (DE-588)4123275-6 |
| title | Introduction to molecular cloning techniques |
| title_alt | Techniques de clonage moléculaire |
| title_auth | Introduction to molecular cloning techniques |
| title_exact_search | Introduction to molecular cloning techniques |
| title_full | Introduction to molecular cloning techniques by Gérard Lucotte and François Baneyx |
| title_fullStr | Introduction to molecular cloning techniques by Gérard Lucotte and François Baneyx |
| title_full_unstemmed | Introduction to molecular cloning techniques by Gérard Lucotte and François Baneyx |
| title_short | Introduction to molecular cloning techniques |
| title_sort | introduction to molecular cloning techniques |
| topic | Cloning cabt Genetica molecular e de microorganismos larpcal Cloning, Molecular Laboratory Manuals Molecular cloning Laboratory manuals Escherichia coli (DE-588)4070959-0 gnd Gentechnologie (DE-588)4071722-7 gnd DNS-Klonierung (DE-588)4250249-4 gnd Genklonierung (DE-588)4123275-6 gnd |
| topic_facet | Cloning Genetica molecular e de microorganismos Cloning, Molecular Laboratory Manuals Molecular cloning Laboratory manuals Escherichia coli Gentechnologie DNS-Klonierung Genklonierung |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=005427613&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT lucottegerard techniquesdeclonagemoleculaire AT baneyxfrancois techniquesdeclonagemoleculaire AT lucottegerard introductiontomolecularcloningtechniques AT baneyxfrancois introductiontomolecularcloningtechniques |