Basic methods in molecular biology:
Saved in:
| Main Authors: | , , |
|---|---|
| Format: | Book |
| Language: | English |
| Published: |
Norwalk, Conn.
Appleton & Lange
1994
|
| Edition: | 2. ed. |
| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Item Description: | NST: Molecular biology |
| Physical Description: | XIV, 777 S. Ill. |
| ISBN: | 0838506429 |
Staff View
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| 100 | 1 | |a Davis, Leonard G. |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Basic methods in molecular biology |c Leonard G. Davis ; W. Michael Kuehl ; James F. Battey |
| 250 | |a 2. ed. | ||
| 264 | 1 | |a Norwalk, Conn. |b Appleton & Lange |c 1994 | |
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Record in the Search Index
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| adam_text | IMAGE 1
CONTENTS
ACKNOWLEDGMENTS XIII
INTRODUCTION 1
THE MOLECULAR GENETIC REVOLUTION 1
USING THIS MANUAL 3
SAFETY CONSIDERATIONS 6
EQUIPMENT USED IN THE MOLECULAR BIOLOGY LABORATORY 9
1. GENERAL METHODS 15
1-1 SS-PHENOL/CHLOROFORM EXTRACTION AND ETHANOL PRECIPITATION 16
1-2 UV AND FLUORESCENCE SPECTROSCOPY FOR DETERMINING DNA/RNA
CONCENTRATION 22
1-3 PHOTOGRAPHING GELS OR AUTORADIOGRAMS 25
1-4 QUANTITATION OF ISOTOPES 27
1-5 AUTORADIOGRAPHY 31
1-6 SEPHADEX G50, SEPHACRYL 300HR, AND SEPHAROSE CL-4B CHROMATOGRAPHY 36
1-7 PLASTIC BAG SEALING 40
1-8 PREPARATION OF DIALYSIS MEMBRANES 43
2. BACTERIAL STRAINS AND CLONING VECTORS 45
INTRODUCTION 46
2-1 MICROBIOLOGICAL TECHNIQUES 47
V
IMAGE 2
VI CONTENTS
2-2 SOME E COLI STRAINS USEFUL IN CLONING METHODS 54
C600 C600 HFL HB101 LE392 Y1090 JM103 JM109 DH5-A DH5-AF DM1 SURE
XLL-BLUE MRF 2-3 CLONING VECTORS 59
XGTLO
XZAPII PBR322 PUC PLASMID VECTORS DESIGNED FOR IN VITRO TRANSCRIPTION:
PGEM AND
PBLUESCRIPT II CHARON 28 CHARON 4A EMBL 3 AND 4 M13
3. ENZYMES THAT MODIFY DNA AND RNA 85
3-1 RESTRICTION ENDONUCLEASES AND METHYLASES 86
3-2 OTHER ENZYMES FOR MODIFYING DNA OR RNA 96
4. IN VITRO AMPLIFICATION OF DNA USING THE POLYMERASE CHAIN REACTION
(PCR) AND THE THERMOSTABLE TAQ DNA POLYMERASE 113
INTRODUCTION 114
4-1 PCR AMPLIFICATION OF A FRAGMENT FROM PURIFIED EUKARYOTIC GENOMIC DNA
TEMPLATE: THE BASIC PROTOCOL 125
4-2 PCR AMPLIFICATION OF INSERTS FROM BACTERIOPHAGE AND PLASMID CLONES
131
4-3 PCR AMPLIFICATION FROM CDNA TEMPLATES 135
4-4 PCR AMPLIFICATION TO INCREASE THE AMOUNT OF A POPULATION OF DNA
FRAGMENTS 140
5. DNA RESTRICTION FRAGMENT ANALYSIS AND PREPARATION 147
INTRODUCTION 148
5-1 AGAROSE GEL ELECTROPHORESIS ON LARGER GELS 153
5-2 AGAROSE MINIGELS 159
IMAGE 3
CONTENTS V LL
5-3 POLYACRYLAMIDE GEL ELECTROPHORESIS OF DNA RESTRICTION FRAGMENTS 162
5-4 OVERVIEW OF DNA FRAGMENT PREPARATION AND PURIFICATION METHOD 166
5-5 RECOVERY OF DNA FRAGMENTS: ELECTROELUTION 170
5-6 RECOVERY OF DNA FRAGMENTS: LOW-MELTING POINT GEL DISSOLUTION AND
FRAGMENT PURIFICATION 173
5-7 PURIFICATION OF DNA FRAGMENTS USING GLASS FINES POWDER 176
5-8 DNA FRAGMENT PURIFICATION BY SPERMINE PRECIPITATION 179
5-9 USE OF LOW-MELTING-POINT AGAROSE FOR NICK TRANSLATIONS AND LIGATIONS
181
5-10 DNA BLOT (SOUTHERN) 183
6. IN VITRO LABELING OF PROBES AND FILTER HYBRIDIZATION 189
6-1 OVERVIEW OF METHODS FOR LABELING OLIGO- AND POLYNUCLEOTIDES 190
6-2 NICK-TRANSLATED PROBES 196
6-3 LABELING DNA FRAGMENTS BY RANDOM PRIMING 200 6-4 TRANSCRIPTION OF
LABELED RNA PROBES FOR BLOT HYBRIDIZATION 204
6-5 LABELING 3 END OF DNA BY T4 POLYMERASE (EXONUCLEASE)/FILL-IN
REACTION 208
6-6 LABELING 5 ENDS OF DNA (OR RNA) WITH Y- 32 P-ATP USING T4
POLYNUCLEOTIDE KINASE 211
6-7 LABELING 3 END OF DNA WITH TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE
216
6-8 CONCEPTS OF NUCLEIC ACID HYBRIDIZATION 219
6-9 HYBRIDIZATION OF DNA POLYNUCLEOTIDE PROBES TO IMMOBILIZED NUCLEIC
ACIDS 223
6-10 HYBRIDIZING OLIGONUCLEOTIDE PROBES TO IMMOBILIZED NUCLEIC ACIDS 228
6-11 HYBRIDIZING RNA PROBES TO IMMOBILIZED NUCLEIC ACIDS 232
6-12 REMOVAL OF PROBES SO THAT FILTERS CAN BE REUSED 235
7. PLASMID DNA PREPARATION FROM E COLI HOSTS 237
INTRODUCTION 238
7-1 TRANSFORMATION OF E COLI WITH PLASMID DNA AND SELECTION OF PLASMIDS
WITH INSERTS BY A-COMPLEMENTATION 239
IMAGE 4
V L LL CONTENTS
7-2 PLASMID DNA MINIPREP 245
7-3 LARGE SCALE ALKALINE LYSIS METHOD: PLASMID PURIFICATION 249
7-4 PLASMID DNA PREPARATION: TRITON-LYSOZYME METHOD 254
8. PREPARATION OF DNA FROM LAMBDA BACTERIOPHAGE CLONES 261
INTRODUCTION 262
8-1 GROWTH OF LYTIC BACTERIOPHAGE AND SMALL-SCALE PREPARATION OF DNA 263
8-2 LARGE-SCALE PREPARATION AND PURIFICATION OF DNA FROM LAMBDA
BACTERIOPHAGE CLONES 267
8-3 PREPARATIVE GROWTH OF LYTIC BACTERIOPHAGE USING PLATE LYSATES 272
9. SUBCLONING FRAGMENTS INTO PLASMID VECTORS AND PLASMID CONSTRUCTION
277
INTRODUCTION 278
9-1 PLASMID CONSTRUCTION FROM PURIFIED VECTOR AND INSERT FRAGMENTS 287
9-2 SHOTGUN SUBCLONING FROM RESTRICTION ENZYME DIGESTS 291
9-3 BLUNTING 5 AND 3 PROTRUDING ENDS WITH T4 DNA POLYMERASE 293
9-4 MODIFICATION OF BLUNT TERMINI USING SYNTHETIC LINKERS AND ADAPTORS
296
9-5 SELECTION OF RECOMBINANTS BY COLONY
HYBRIDIZATION 302
10. PREPARATION OF GENOMIC DNA 305
INTRODUCTION 306
10-1 RAPID PREPARATION OF GENOMIC DNA FROM TISSUE OR CULTURED CELLS 307
10-2 PREPARATION OF GENOMIC DNA FROM TISSUE CULTURE CELLS 310
10-3 PREPARATION OF HIGH-MOLECULAR-WEIGHT GENOMIC DNA FROM TISSUE 314
11. PREPARATION AND ANALYSIS OF RNA FROM EUKARYOTIC CELLS 319
INTRODUCTION 320
11-1 GUANIDINE ISOTHIOCYANATE/CESIUM CHLORIDE ULTRACENTRIFUGATION FOR
PREPARATION OF TOTAL RNA 322
IMAGE 5
CONTENTS IX
11-2 NONIONIC DETERGENT FOR ISOLATION OF CYTOPLASMIC (AND NUCLEAR) RNA
329
11-3 ACIDIC GUANIDINE ISOTHIOCYANATE/PHENOL/CHLOROFORM EXTRACTION FOR
ISOLATION OF RNA 335
11-4 ISOLATION OF MEMBRANE-ASSOCIATED AND NON-MEMBRANE-ASSOCIATED MRNAS
339
11-5 SELECTION OF POLY-A + RNA ON OLIGO-DT CELLULOSE 344 11-6
FORMALDEHYDE AGAROSE GEL ELECTROPHORESIS AND RNA (NORTHERN) BLOTS 350
11-7 SI NUCLEASE PROTECTION ASSAY 355
11-8 RNASE PROTECTION ASSAY FOR ANALYZING MRNA WITH CRNA PROBES 369
11-9 PRIMER EXTENSION OF SYNTHETIC OLIGONUCLEOTIDES TO MAP 5 TERMINI OF
MRNAS 378
11-10 NUCLEAR RUN-ON FOR DETERMINING RATE OF
TRANSCRIPTION 384
12. GENOMIC CLONING 397
INTRODUCTION 398
12-1 PREPARATION OF EUKARYOTIC GENOMIC DNA INSERT FOR LIGATION TO
BAMHI-DIGESTED BACTERIOPHAGE VECTORS 404
12-2 PREPARATION OF EMBL 3 VECTOR FOR CLONING INSERT WITH
BAMHI-COMPATIBLE ENDS 410
12-3 PREPARATION OF GENOMIC DNA INSERT FOR LIGATION INTO PARTIALLY
FILLED-IN XHO I-DIGESTED BACTERIOPHAGE VECTORS 414
12-4 LIGATION OF GENOMIC DNA INSERT INTO BACTERIOPHAGE CLONING VECTOR
ARMS, AND IN VITRO PACKAGING 418
12-5 TITERING AND PLATING OF THE PACKAGED (OR AMPLIFIED) LIBRARY 422
12-6 SCREENING A PLATED LIBRARY USING DNA FRAGMENT PROBES ( 100 BASE
PAIR LENGTH) LABELED BY NICK TRANSLATION OR RANDOM PRIMING 425
12-7 USE OF OLIGONUCLEOTIDE PROBES IN SCREENING GENOMIC AND CDNA
LIBRARIES: DESIGN, STRATEGY, HYBRIDIZATION, AND STRINGENCY
CONSIDERATIONS 431 12-8 AMPLIFICATION OF BACTERIOPHAGE LIBRARIES 441
12-9 PREPARATION OF DEPHOSPHORYLATED LINEAR COSMID VECTOR 443
12-10 LIGATION AND PACKAGING OF COSMID LIBRARY 447
IMAGE 6
CONTENTS
12-11 PLATING A COSMID LIBRARY AT HIGH DENSITY FOR SCREENING 451
12-12 AMPLIFICATION OF COSMID LIBRARIES 456
13. PREPARATION AND ANALYSIS OF CDNA LIBRARIES 459
INTRODUCTION 460
13-1 GENERATION OF CDNA INSERT FROM EUKARYOTIC MRNA 463
13-2 LIGATION AND PACKAGING OF CDNA INSERT INTO A,GTLO OR A-ZAPII PHAGE
AND INFECTION OF BACTERIAL HOST 476
13-3 IDENTIFYING, ISOLATING, AND CHARACTERIZING SPECIFIC RECOMBINANT
CDNA CLONES AND THE CORRESPONDING CDNA INSERTS FROM THE CDNA LIBRARY 482
13-4 SCREENING XZAPLL OR XGTLL CDNA EXPRESSION LIBRARIES WITH AN
ANTIBODY 486
13-5 PCR WITH MIXED OLIGONUCLEOTIDE PRIMERS TO OBTAIN INITIAL CDNA
CLONES 492
13-6 ANCHORED PCR TO ISOLATE THE 5 AND 3 ENDS OF CDNAS 497
14. PREPARATION OF SUBTRACTIVE CDNA 509
INTRODUCTION 510
14-1 FIRST-STRAND CDNA SYNTHESIS AND HYBRIDIZATION TO MRNA 515
14-2 SEPARATION OF SINGLE- AND DOUBLE-STRANDED POLYNUCLEOTIDES BY
HYDROXYAPATITE CHROMATOGRAPHY 523
14-3 POSITIVE SELECTION AND GENERATION OF DOUBLE-STRANDED CDNA 530
14-4 PCR AMPLIFICATION OF SUBTRACTIVE CDNA 534
14-5 USE OF AMPLIFIED SUBTRACTIVE CDNA AS AN INSERT FOR A SUBTRACTIVE
CDNA LIBRARY OR AS A HYBRIDIZATION PROBE 541
15. CHAIN TERMINATION SEQUENCING 549
INTRODUCTION 550
15- 1A PREPARATION OF INSERT FOR M13 OR PLASMID SUBCLONING USING
SUCCESSIVE SAZ31 EXONUCLEASE DIGESTION 560
X
IMAGE 7
CONTENTS XI
15-IB UNIDIRECTIONAL NESTED DELETIONS USING EXONUCLEASE III FOR
OVERLAPPING CHAIN TERMINATION SEQUENCING IN A PLASMID OR M13 SUBCLONE
568
15-2 PREPARATION OF M 13 MP 18 OR MP 19 RF DNA 574
15-3 SUBCLONING FRAGMENTS FOR SEQUENCE ANALYSIS INTO M 13 VECTORS:
PREPARATION OF VECTOR AND INSERT FRAGMENTS FOR LIGATION 577
15-4 TRANSFORMATION OF M13 MP VECTORS AND RECOMBINANTS INTO E COLI HOST
581
15-5 SCREENING M13 SUBCLONES WITH A RADIOLABELED PROBE TO SELECT INSERTS
FOR SEQUENCING 584
15-6 PREPARATION OF SINGLE-STRANDED M13 DNA 587
15-7 PREPARATION OF 6% AND 8% POLYACRYLAMIDE, 8 M UREA DENATURING GELS
FOR DNA SEQUENCE ANALYSIS 590 15-8 CHAIN TERMINATION SEQUENCING OF
SINGLE- AND DOUBLE-STRANDED TEMPLATES USING MODIFIED T7
DNA POLYMERASE 594
15-9 THERMAL CYCLER DNA SEQUENCING 604
16. TRANSFECTION OF MAMMALIAN CELLS 611
INTRODUCTION 612
16-1 CALCIUM PHOSPHATE TRANSFECTION OF NONADHERENT AND ADHERENT CELLS
624
16-2 DEAE DEXTRAN-MEDIATED TRANSFECTION OF NONADHERENT AND ADHERENT
MAMMALIAN CELLS 630
16-3 LIPOSOME-MEDIATED TRANSFECTION OF NONADHERENT AND ADHERENT CELLS
633
16-4 ELECTROPORATION 636
16-5 SELECTION OF TRANSFECTED MAMMALIAN CELLS BY RESISTANCE TO THE
AMINOGLYCOSIDE G418 641
16-6 CHLORAMPHENICOL ACETYLTRANSFERASE (CAT) ASSAY 647 16-7 LUCIFERASE
REPORTER ASSAY 653
17. BASIC METHODS OF PROTEIN ANALYSIS 659
INTRODUCTION 660
17-1 ELECTROPHORESIS OF PROTEINS ON SODIUM DODECYL SULFATE
POLYACRYLAMIDE GELS 661
17-2 BIOSYNTHETIC LABELING AND IMMUNOPRECIPITATION OF PROTEINS IN TISSUE
CULTURE CELLS AND CELL-FREE SYSTEMS 669
17-3 WESTERN BLOTTING OF PROTEINS 680
IMAGE 8
XU CONTENTS
18. IN SITU HYBRIDIZATION 691
18-1 IN SITU HYBRIDIZATION WITH CRNA PROBES 692
18-2 IN SITU HYBRIDIZATION WITH OLIGONUCLEOTIDE PROBES 705
19. TRANSGENIC MOUSE PREPARATION 711
20. DETECTION AND IN VITRO GENERATION OF SPECIFIC MUTATIONS IN GENES AND
CDNAS 723
INTRODUCTION 724
20-1 SINGLE-STRAND CONFORMATION POLYMORPHISM (SSCP) TO DETECT MUTATIONS
726
20-2 THREE-OLIGONUCLEOTIDE PCR TO GENERATE CHIMERIC (OR SPECIFICALLY
DELETED) DNA MOLECULES 733
20-3 SITE-DIRECTED MUTAGENESIS 738
APPENDIX I: PREPARATION OF STOCK SOLUTIONS 745
APPENDIX II: GENERALLY USEFUL INFORMATION 752
APPENDIX III: SUPPLIERS OF REAGENTS AND EQUIPMENT 758
INDEX 763
|
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| indexdate | 2024-07-09T17:41:12Z |
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| language | English |
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| spelling | Davis, Leonard G. Verfasser aut Basic methods in molecular biology Leonard G. Davis ; W. Michael Kuehl ; James F. Battey 2. ed. Norwalk, Conn. Appleton & Lange 1994 XIV, 777 S. Ill. txt rdacontent n rdamedia nc rdacarrier NST: Molecular biology Biologie moléculaire - Méthodologie Biología molecular Molecular Biology methods Molecular biology Methodology Molekularbiologie (DE-588)4039983-7 gnd rswk-swf Methode (DE-588)4038971-6 gnd rswk-swf Molekularbiologie (DE-588)4039983-7 s Methode (DE-588)4038971-6 s DE-604 Kuehl, W. M. Verfasser aut Battey, James F. Verfasser aut SWB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=006489846&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Davis, Leonard G. Kuehl, W. M. Battey, James F. Basic methods in molecular biology Biologie moléculaire - Méthodologie Biología molecular Molecular Biology methods Molecular biology Methodology Molekularbiologie (DE-588)4039983-7 gnd Methode (DE-588)4038971-6 gnd |
| subject_GND | (DE-588)4039983-7 (DE-588)4038971-6 |
| title | Basic methods in molecular biology |
| title_auth | Basic methods in molecular biology |
| title_exact_search | Basic methods in molecular biology |
| title_full | Basic methods in molecular biology Leonard G. Davis ; W. Michael Kuehl ; James F. Battey |
| title_fullStr | Basic methods in molecular biology Leonard G. Davis ; W. Michael Kuehl ; James F. Battey |
| title_full_unstemmed | Basic methods in molecular biology Leonard G. Davis ; W. Michael Kuehl ; James F. Battey |
| title_short | Basic methods in molecular biology |
| title_sort | basic methods in molecular biology |
| topic | Biologie moléculaire - Méthodologie Biología molecular Molecular Biology methods Molecular biology Methodology Molekularbiologie (DE-588)4039983-7 gnd Methode (DE-588)4038971-6 gnd |
| topic_facet | Biologie moléculaire - Méthodologie Biología molecular Molecular Biology methods Molecular biology Methodology Molekularbiologie Methode |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=006489846&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT davisleonardg basicmethodsinmolecularbiology AT kuehlwm basicmethodsinmolecularbiology AT batteyjamesf basicmethodsinmolecularbiology |