Current Protocols in Molecular Biology

cover
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Contents
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CHAPTER 1 --
Escherichia coli, Plasmids, and Bacteriophages......Page 26
E coli
......Page 0

Media Preparation and Bacteriological Tools......Page 29
Growth in Liquid Media......Page 36
Growth on Solid Media......Page 38
Selected Topics from Classical Bacterial Genetics
......Page 44
VECTORS DERIVED FROM PLASMIDS --
Introduction to Plasmid Biology......Page 58
Minipreps of Plasmid DNA......Page 75
PREPARATION OF CRUDE LYSATE BY ALKALINE LYSIS......Page 85
PREPARATION OF CRUDE LYSATE BY THE BOILING METHOD......Page 88
PREPARATION OF CRUDE LYSATE BY TRITON LYSIS......Page 89
PURIFICATION OF PLASMID DNA BY CsCl/ETHIDIUM BROMIDE
EQUILIBRIUM CENTRIFUGATION......Page 90
PLASMID DNA PURIFICATION BY PEG PRECIPITATION......Page 93
PLASMID DNA PURIFICATION BY ANION-EXCHANGE OR SIZE EXCLUSION
CHROMATOGRAPHY......Page 94
TRANSFORMATION USING CALCIUM CHLORIDE......Page 101
ONE-STEP PREPARATION AND TRANSFORMATION OF COMPETENT CELLS......Page 103
HIGH-EFFICIENCY TRANSFORMATION BY ELECTROPORATION......Page 104
DIRECT ELECTROPORETIC TRANSFER OF PLASMID DNA FROM YEAST INTO E. COLI......Page 106
Introduction to Lambda Phages......Page 111
Lambda as a Cloning Vector......Page 115
THE COSMID, A USEFUL LAMBDA DERIVED PLASMID VECTOR......Page 123
ISOLATING A SINGLE PLAQUE BY TITERING SERIAL DILUTIONS......Page 126
PHAGE TRANSFECTION AND IN VITRO PACKAGING......Page 128
MAKING A STOCK OF PHAGE BY PLATE LYSIS......Page 130
STORING PHAGE LYSATES......Page 131
PREPARING DNA BY STEP- AND EQUILIBRIUM-GRADIENT CENTRIFUGATION......Page 133
PREPARING DNA USING DEAE-CELLULOSE
COLUMN CHROMATOGRAPHY......Page 136
PREPARING DNA FROM SMALL-SCALE LIQUID LYSATES......Page 139
Introduction to Vectors Derived from
Filamentous Phages......Page 143
ISOLATING SINGLE M13-DERIVED VECTORS......Page 148
PREPARING SINGLE-STRANDED PHAGE DNA F
ROM M13-DERIVED VECTORS......Page 149
PREPARING DOUBLE-STRANDED REPLICATIVE-FORM DNA......Page 150
PREPARING SINGLE-STRANDED DNA FROM PLASMIDSUSING HELPER PHAGE......Page 151
DETERMINING SIZE OF INSERTS IN SINGLE-STRANDED VECTORS......Page 152
DETERMINING INSERT ORIENTATION......Page 153
Recombineering: Genetic Engineering in
Bacteria Using Homologous Recombination......Page 156
INTRODUCTION......Page 177
PHENOL EXTRACTION AND ETHANOL PRECIPITATION OF DNA......Page 180
PREPARATION OF BUFFERED PHENOL AND PHENOL / CHLOROFORM /
ISOAMYL ALCOHOL......Page 182
CONCENTRATION OF DNA USING BUTANOL......Page 183
REMOVAL OF RESIDUAL PHENOL, CHLOROFORM, OR BUTANOL BY
ETHER EXTRACTION......Page 184
DNA PURIFICATION USING SILICA MEMBRANE SPIN COLUMNS......Page 185
PURIFICATION AND CONCENTRATION OF RNA AND DILUTE SOLUTIONS OF DNA......Page 186
REMOVAL OF LOW-MOLECULAR-WEIGHT OLIGONUCLEOTIDES AND
TRIPHOSPHATES BY ETHANOL PRECIPITATION......Page 187
Purification of DNA by Anion-Exchange Chromatography......Page 190
Preparation of Genomic DNA from Mammalian Tissue......Page 198
PREPARATION OF PLANT DNA USING CSCL CENTRIFUGATION......Page 201
PREPARATION OF PLANT DNA USING CTAB......Page 203
MINIPREP OF BACTERIAL GENOMIC DNA......Page 208
REMOVAL OF POLYSACCHARIDES FROM
EXISTING GENOMIC DNA PREPS......Page 209
LARGE-SCALE CsCl PREP OF BACTERIAL GENOMIC DNA......Page 210
RESOLUTION OF DNA FRAGMENTS ON STANDARD AGAROSE GELS......Page 213
MINIGELS AND MIDIGELS......Page 215
PHOTOGRAPHY OF DNA IN AGAROSE GELS......Page 216
FIELD-INVERSION ELECTROPHORESIS......Page 222
CHEF ELECTROPHORESIS......Page 223
PREPARATION OF HIGH-MOLECULAR-WEIGHT DNA SAMPLES AND SIZE MARKERS......Page 224
ELECTROELUTION FROM AGAROSE GELS......Page 231
ELECTROPHORESIS ONTO NA-45 PAPER......Page 234
ISOLATION OF DNA FRAGMENTS USING LOW GELLING / MELTING TEMPERATURE AGAROSE GELS......Page 235
RECOVERY OF DNA FROM LOW GELLING / MELTING TEMPERATURE AGAROSE GELS USING BETA-AGARASE DIGESTION......Page 236
RECOVERY OF DNA FROM AGAROSE USING SILICA MEMBRANE SPIN
COLUMNS......Page 237
REMOVAL OF OLIGONUCLEOTIDE FRAGMENTS USING A SEPHACRYL S-300 COLUMN......Page 238
RAPID ESTIMATION OF DNA CONCENTRATION BY ETHIDIUM BROMIDE DOT QUANTITATION......Page 239
NONDENATURING POLYACRYLAMIDE GEL ELECTROPHORESIS......Page 243
ELECTROELUTION OF SMALL DNA FRAGMENTS FROM
POLYACRYLAMIDE GELS......Page 247
SIEVING AGAROSE GEL ELECTROPHORESIS......Page 248
Capillary Electrophoresis of DNA......Page 251
INSTRUMENTATION......Page 252
SEPARATION THEORY......Page 253
STRATEGIC PLANNING......Page 254
SEPARATION OF OLIGONUCLEOTIDES......Page 255
QUANTITATIVE PCR ANALYSIS......Page 257
GENOTYPING......Page 259
Southern Blotting......Page 268
SOUTHERN BLOTTING ONTO A NYLON OR NITROCELLULOSE MEMBRANE WITH HIGH-SALT BUFFER......Page 269
CALIBRATION OF A UV TRANSILLUMINATOR......Page 273
SOUTHERN BLOTTING BY DOWNWARD CAPILLARY TRANSFER......Page 274
ELECTROBLOTTING FROM A POLYACRYLAMIDE GELTO A NYLON MEMBRANE......Page 276
Dot and Slot Blotting of DNA......Page 282
DOT AND SLOT BLOTTING OF DNA ONTO UNCHARGED NYLON AND NITROCELLULOSE MEMBRANES USING A MANIFOLD......Page 284
MANUAL PREPARATION OF A DNA DOT BLOT......Page 286
Hybridization Analysis of DNA Blots......Page 289
HYBRIDIZATION ANALYSIS OF A DNA BLOT WITH A RADIOLABELED DNA PROBE......Page 290
HYBRIDIZATION ANALYSIS OF A DNA BLOT WITH A RADIOLABELED RNA PROBE......Page 292
REMOVAL OF PROBES FROM HYBRIDIZED MEMBRANES......Page 294
INTRODUCTION TO CHEMICAL NUCLEIC ACID SYNTHESIS......Page 305
STRATEGIES FOR NUCLEIC ACID SYNTHESIS......Page 309
STRATEGIES FOR OLIGONUCLEOTIDE PURIFICATION......Page 318
MONITORING DNA SYNTHESIS USING THE TRITYL ASSAY......Page 320
USING THE TRITYL ASSAY FOR TROUBLESHOOTING......Page 321
DEPROTECTION OF DNA OLIGONUCLEOTIDES......Page 322
DEPROTECTION OF RNA OLIGONUCLEOTIDES......Page 323
Purification of Oligonucleotides Using Denaturing Polyacrylamide Gel
Electrophoresis......Page 330
INTRODUCTION......Page 337
DIGESTING A SINGLE DNA SAMPLE WITH A SINGLE RESTRICTION ENDONUCLEASE......Page 338
DIGESTING DNA WITH MULTIPLE RESTRICTION ENDONUCLEASES......Page 339
PARTIAL DIGESTION OF DNA WITH RESTRICTION
ENDONUCLEASES......Page 340
METHYLATION OF DNA......Page 342
Mapping by Multiple Endonuclease Digestions......Page 360
Mapping by Partial Endonuclease Digestions......Page 365
STOCK SOLUTIONS......Page 367
10× ENZYME BUFFERS......Page 368
ENZYME REACTION CONDITIONS AND APPLICATIONS......Page 369
NUCLEOSIDE TRIPHOSPHATES......Page 372
Measuring Radioactivity in DNA and RNA by Acid Precipitation......Page 373
Separating Radioactively Labeled DNA from Unincorporated dNTP Precursors by Column Chromatography......Page 374
Spin-Column Procedure for Separating Radioactively Labeled DNA from Unincorporated dNTP Precursors......Page 376
DNA-Dependent DNA Polymerases......Page 378
ESCHERICHIA COLI DNA POLYMERASE I......Page 380
Uniform Labeling of DNA by Nick Translation......Page 381
Labeling the 3′ Ends of DNA......Page 384
Labeling of DNA by Random Oligonucleotide–Primed Synthesis......Page 386
Buffer Compatibility......Page 388
NATIVE T7 DNA POLYMERASE......Page 389
MODIFIED T7 DNA POLYMERASE......Page 390
TAQ DNA POLYMERASE......Page 391
TERMINAL DEOXYNUCLEOTIDYLTRANSFERASE......Page 393
REVERSE TRANSCRIPTASE......Page 395
ESCHERICHIA COLI RNA POLYMERASE......Page 398
PHAGE RNA POLYMERASES: SP6, T7, T3......Page 399
POLY(A) POLYMERASE......Page 402
ALKALINE PHOSPHATASES: BACTERIAL ALKALINE
PHOSPHATASE AND CALF INTESTINE PHOSPHATASE......Page 404
T4 POLYNUCLEOTIDE KINASE......Page 405
Labeling 5′ Ends by the Forward Reaction......Page 406
Labeling 5′ Termini by the Exchange Reaction......Page 407
Exonuclease VII (exo VII)
......Page 409
T7 Gene 6 Exonuclease......Page 410
Exonuclease III (exo III)......Page 411
BAL 31 NUCLEASE......Page 413
S1 NUCLEASE......Page 414
MUNG BEAN NUCLEASE......Page 415
MICROCOCCAL NUCLEASE......Page 416
DEOXYRIBONUCLEASE I (DNase I)......Page 417
RNase-Free DNase I......Page 418
RIBONUCLEASE A......Page 419
RIBONUCLEASE T1......Page 420
T4 DNA LIGASE......Page 422
ESCHERICHIA COLI DNA LIGASE......Page 424
T4 RNA LIGASE......Page 426
Subcloning of DNA Fragments......Page 428
LIGATION OF DNA FRAGMENTS IN GEL SLICES......Page 430
SUBCLONING DNA FRAGMENTS......Page 439
PREPARATION OF BIOTINYLATED PROBES BY NICK TRANSLATION......Page 449
PREPARATION OF BIOTINYLATED PROBES BY RANDOM OLIGONUCLEOTIDE–PRIMED SYNTHESIS......Page 451
COLORIMETRIC DETECTION OF BIOTINYLATED PROBES......Page 452
PREPARATION AND DETECTION OF DIGOXIGENIN-LABELED DNA PROBES......Page 453
CHEMILUMINESCENT DETECTION OF BIOTINYLATED PROBES......Page 458
CHEMILUMINESCENT DETECTION OF DIGOXIGENIN-LABELED PROBES......Page 461
CALIBRATING AN ULTRAVIOLET LIGHT SOURCE......Page 462
Recombinational Cloning......Page 466
AMPLIFICATION OF TARGET GENES BY PCR......Page 467
RAPID GEL PURIFICATION OF PCR PRODUCTS......Page 470
CAPTURE OF ORFS TO MAKE MASTER CLONES FOR THE GATEWAY SYSTEM: BP REACTION......Page 471
CAPTURE OF ORFS TO MAKE MASTER CLONES FOR THE CREATOR SYSTEM: IN-FUSION CLONING......Page 473
GENERATING GATEWAY EXPRESSION CLONES......Page 475
GENERATING CREATOR EXPRESSION CLONES......Page 478
INTRODUCTION......Page 488
PREPARATION OF RNA FROM EUKARYOTIC AND PROKARYOTIC CELLS......Page 490
Preparation of Cytoplasmic RNA from Tissue Culture Cells......Page 491
REMOVAL OF CONTAMINATING DNA......Page 493
SINGLE-STEP RNA ISOLATION FROM CULTURED CELLS OR TISSUES......Page 495
CsCl PURIFICATION OF RNA FROM CULTURED CELLS......Page 497
CsCl PURIFICATION OF RNA FROM TISSUE......Page 499
Phenol/SDS Method for Plant RNA Preparation......Page 504
ISOLATION OF HIGH-QUALITY RNA FROM GRAM-NEGATIVE BACTERIA......Page 508
ISOLATION OF RNA FROM GRAM-POSITIVE BACTERIA......Page 510
RAPID ISOLATION OF RNA FROM GRAM-NEGATIVE BACTERIA......Page 511
Preparation of Poly(A)+ RNA......Page 515
S1 Analysis of Messenger RNA Using Single-Stranded DNA Probes......Page 518
S1 ANALYSIS OF mRNA USING M13 TEMPLATE......Page 519
PREPARATION OF SINGLE-STRANDED END-LABELED PROBE AND S1 ANALYSIS OF mRNA......Page 523
QUANTITATIVE S1 ANALYSIS OF mRNA USING OLIGONUCLEOTIDE PROBES......Page 524
CONTROLS FOR QUANTITATIVE S1 ANALYSIS OF mRNA......Page 527
Ribonuclease Protection Assay......Page 531
GEL PURIFICATION OF RNA PROBES......Page 533
PREPARATION OF TEMPLATE DNA......Page 534
Primer Extension......Page 539
Analysis of RNA by Northern and Slot Blot Hybridization......Page 544
NORTHERN HYBRIDIZATION OF RNA FRACTIONATED BY AGAROSE-FORMALDEHYDE GEL ELECTROPHORESIS......Page 545
NORTHERN HYBRIDIZATION OF RNA DENATURED BY GLYOXAL / DMSO TREATMENT......Page 551
NORTHERN HYBRIDIZATION OF UNFRACTIONATED RNA IMMOBILIZED BY SLOT BLOTTING......Page 552
NORTHERN HYBRIDIZATION OF SMALL RNA FRACTIONATED BY POLYACRYLAMIDE GEL ELECTROPHORESIS......Page 554
NORTHERN HYBRIDIZATION OF RNA USING CHURCH’S HYBRIDIZATION BUFFER......Page 557
NUCLEAR RUNOFF TRANSCRIPTION IN MAMMALIAN CELLS......Page 563
ISOLATION OF NUCLEI BY DOUNCE HOMOGENIZATION......Page 566
ISOLATION OF NUCLEI BY SUCROSE GRADIENT CENTRIFUGATION......Page 568
PREPARATION OF NITROCELLULOSE FILTERS FOR NUCLEAR RUNOFF TRANSCRIPTION ASSAY......Page 569
INTRODUCTION......Page 574
REPRESENTATION AND RANDOMNESS......Page 577
VECTORS FOR GENOMIC DNA LIBRARIES......Page 578
cDNA Libraries......Page 580
PREPARATION OF INSERT DNA FROM GENOMIC DNA......Page 581
SUCROSE GRADIENT PREPARATION OF SIZE-SELECTED DNA......Page 582
PARTIAL ENZYME DIGESTION......Page 584
COMPLETE ENZYME DIGESTION......Page 585
ELECTROPHORESIS ON THE BULL’S-EYE AGAROSE GEL APPARATUS......Page 589
PREPARATION OF INSERT DNA FROM MESSENGER RNA......Page 593
CONVERSION OF mRNA INTO BLUNT-ENDED DOUBLE-STRANDED cDNA......Page 594
CONVERSION OF mRNA INTO DOUBLE-STRANDED cDNA FOR DIRECTIONAL CLONING......Page 598
METHYLATION OF cDNA AND LIGATION OF LINKERS......Page 607
LIGATION OF BstXI SYNTHETIC ADAPTERS......Page 610
PREPARATION OF A CL-4B COLUMN......Page 611
Production of a Genomic DNA Library......Page 617
LIGATION AND TRANSFECTION FOR A COMPLETE LAMBDA
PHAGE LIBRARY......Page 621
LIGATION AND TRANSFORMATION FOR A COMPLETE PLASMID LIBRARY......Page 623
EVALUATION OF A cDNA LIBRARY......Page 625
PREPARATION OF TEST INSERT FOR EVALUATION
OF PHOSPHATASED PHAGE ARMS......Page 626
Construction of Bacterial Artificial Chromosome (BAC/PAC) Libraries......Page 629
PREPARATION OF BAC/PAC CLONES USING pCYPAC2, pPAC4, OR pBACe3.6 VECTOR......Page 631
PREPARATION OF BAC/PAC VECTOR FOR CLONING......Page 636
PREPARATION OF HIGH-MOLECULAR-WEIGHT DNA FROM LYMPHOCYTES IN AGAROSE BLOCKS......Page 640
PREPARATION OF HIGH-MOLECULAR-WEIGHT DNA FROM ANIMAL TISSUE CELLS IN AGAROSE BLOCKS......Page 642
PARTIAL DIGESTION AND SIZE FRACTIONATION OF GENOMIC DNA
......Page 643
MODIFIED ALKALINE LYSIS MINIPREP FOR RECOVERY OF DNA FROM BAC/PAC CLONES......Page 650
Amplification of a Bacteriophage Library......Page 662
Amplification of Cosmid and Plasmid Libraries......Page 665
INTRODUCTION......Page 667
Plating and Transferring Bacteriophage Libraries......Page 670
Plating and Transferring Cosmid and Plasmid Libraries......Page 674
HYBRIDIZATION IN FORMAMIDE......Page 678
HYBRIDIZATION IN AQUEOUS SOLUTION......Page 680
HYBRIDIZATION IN SODIUM CHLORIDE/SODIUM CITRATE (SSC)......Page 684
HYBRIDIZATION IN TETRAMETHYLAMMONIUM CHLORIDE (TMAC)......Page 686
Purification of Bacteriophage Clones......Page 694
Purification of Cosmid and Plasmid Clones......Page 696
SCREENING A Lambda
gt11 EXPRESSION LIBRARY WITH ANTIBODIES......Page 697
INDUCTION OF FUSION PROTEIN EXPRESSION WITH IPTG PRIOR TO SCREENING WITH ANTIBODIES......Page 700
Immunoscreening after Hybrid Selectionand Translation......Page 703
GENERATING YAC LIBRARIES......Page 708
DESIGNING A LOCUS-SPECIFIC PCR ASSAY FOR SCREENING......Page 709
ANALYZING INDIVIDUAL YAC CLONES......Page 711
CONSTRUCTION AND ANALYSIS OF A YAC-INSERT SUBLIBRARY......Page 713
PROPAGATION AND STORAGE OF YAC-CONTAINING
YEAST STRAINS......Page 715
PREPARATION OF YAC-CONTAINING DNA FROM YEAST CLONES FOR ANALYSIS BY SOUTHERN BLOTTING......Page 716
PREPARATION OF YEAST CHROMOSOMES IN AGAROSE PLUGS FOR PULSED-FIELD GEL ELECTROPHORESIS......Page 718
END-FRAGMENT ANALYSIS USING PCR AMPLIFICATION......Page 720
END-FRAGMENT ANALYSIS BY SUBCLONING INTO A BACTERIAL PLASMID VECTOR......Page 723
DESIGN AND PREPARATION OF pUC19-ES and pUC19-HS
SUBCLONING VECTOR......Page 725
PREPARATION OF HIGH-MOLECULAR-WEIGHT YAC-CONTAINING YEAST DNA IN SOLUTION......Page 727
PREPARATION AND ANALYSIS OF A YAC-INSERT SUBLIBRARY......Page 728
ISOLATION OF cDNA CLONES ENCODING CELL-SURFACE ANTIGENS......Page 734
PREPARATION OF ANTIBODY-COATED PLATES......Page 739
ISOLATION OF cDNA CLONES ENCODING INTRACELLULAR ANTIGENS......Page 740
PREPARATION OF POLYVINYLIDENE-WRAPPED PLATES......Page 743
Recombination-Based Assay (RBA) for
Screening Bacteriophage Lambda Libraries......Page 750
INTRODUCTION......Page 762
DIDEOXY (SANGER)
SEQUENCING......Page 763
CHEMICAL (MAXAM-GILBERT)SEQUENCING......Page 766
CHOOSING BETWEEN DIDEOXY AND CHEMICAL SEQUENCING METHODS......Page 767
DEVELOPMENTS IN SEQUENCING TECHNOLOGY......Page 768
DIDEOXY SEQUENCING......Page 777
CHEMICAL SEQUENCING......Page 781
USING EXONUCLEASE III TO CONSTRUCT UNIDIRECTIONAL DELETIONS......Page 784
USING BAL 31 NUCLEASE TO CONSTRUCT NESTED DELETIONS......Page 791
PREPARATION OF M13mp SEQUENCING VECTOR DNA FOR SUBCLONING OF BAL 31–DIGESTED DNA FRAGMENTS......Page 800
PREPARATION OF SINGLE-STRANDED M13 PHAGE DNA......Page 804
PREPARATION OF DNA FROM SMALL-SCALE LYSATES......Page 806
MINIPREP OF RECOMBINANT pSP64CS OR pSP65CS PLASMID DNA FOR CHEMICAL SEQUENCING......Page 808
MINIPREP OF DOUBLE-STRANDED PLASMID DNA FOR DIDEOXY SEQUENCING......Page 809
ALKALI DENATURATION OF DOUBLE-STRANDED PLASMID DNA FOR DIDEOXY SEQUENCING......Page 810
PREPARATION OF PLASMID DNA FROM AN E. COLI COLONY OR PHAGE DNA FROM A PLAQUE FOR THERMAL CYCLE SEQUENCING......Page 811
DNA Sequencing by the Dideoxy Method......Page 814
LABELING/TERMINATION SEQUENCING REACTIONS USING SEQUENASE......Page 815
USING Mn2+ IN THE LABELING/TERMINATION REACTIONS......Page 817
SEQUENCING BY THE SANGER PROCEDURE USING KLENOW FRAGMENT......Page 819
USING TAQ DNA POLYMERASE IN THE SANGER PROCEDURE......Page 820
ONE-STEP SEQUENCING REACTIONS USING 5′-END-LABELED PRIMERS......Page 821
THERMAL CYCLE SEQUENCING REACTIONS USING α-LABELED NUCLEOTIDES......Page 822
THERMAL CYCLE SEQUENCING REACTIONS USING 5′-END-LABELED PRIMERS......Page 824
CYCLE SEQUENCING USING FLUORESCENCE DYE-LABELED PRIMER (DYE PRIMER) OR TERMINATOR (DYE TERMINATOR)......Page 825
DNA SEQUENCING USING BIOTINYLATED PRIMERS WITH CHEMILUMINESCENT DETECTION......Page 853
TWO-STEP (INDIRECT) DETECTION USING STREPTAVIDIN AND BIOTINYLATED ALKALINE PHOSPHATASE......Page 857
SEQUENCING WITH HAPTEN-LABELED PRIMERS AND DETECTION WITH ANTIBODY-ALKALINE PHOSPHATASE CONJUGATES......Page 858
STRATEGIC PLANNING......Page 867
CHEMICAL SEQUENCING USING 32P-LABELED DNA......Page 868
Tth111I DIGESTION AND END LABELING......Page 873
POURING, RUNNING, AND PROCESSING SEQUENCING GELS......Page 878
BUFFER-GRADIENT SEQUENCING GELS......Page 884
ELECTROLYTE-GRADIENT SEQUENCING GELS......Page 885
FORMAMIDE-CONTAINING SEQUENCING GELS......Page 886
SEQUENCE DATA ENTRY......Page 891
SEQUENCE DATA VERIFICATION......Page 895
RESTRICTION MAPPING......Page 897
PREDICTION OF NUCLEIC ACID STRUCTURE......Page 898
OLIGONUCLEOTIDE DESIGN STRATEGY......Page 899
IDENTIFICATION OF PROTEIN-CODING REGIONS......Page 901
GENETIC SEQUENCE DATABASES AND OTHER ELECTRONIC RESOURCES AVAILABLE TO MOLECULAR BIOLOGISTS......Page 905
CHAPTER 8 -- Mutagenesis of Cloned DNA......Page 914
Oligonucleotide-Directed Mutagenesis without Phenotypic Selection......Page 916
Mutagenesis with Degenerate Oligonucleotides: A Creating Numerous Mutations in a Small DNA Sequence......Page 922
Gene Synthesis: Assembly of Target Sequences Using Mutually Priming Long Oligonucleotides......Page 929
STRATEGIC PLANNING......Page 935
MUTAGENESIS OF A DNA SEQUENCE......Page 937
MUTAGENIZING A LIBRARY OF SEQUENCES......Page 939
Linker-Scanning Mutagenesis of DNA......Page 944
LINKER SCANNING USING NESTED DELETIONS AND COMPLEMENTARY OLIGONUCLEOTIDES......Page 945
LINKER SCANNING USING OLIGONUCLEOTIDE-DIRECTED MUTAGENESIS......Page 948
INTRODUCTION OF RESTRICTION ENDONUCLEASE SITES BY PCR......Page 951
INTRODUCTION OF POINT MUTATIONS BY PCR......Page 955
INTRODUCTION OF A POINT MUTATION BY SEQUENTIAL PCR STEPS......Page 957
CHAPTER 9 -- Introduction of DNA into Mammalian Cells......Page 961
CHAPTER 10 --
Analysis of Proteins......Page 1157
CHAPTER 11 --
Immunology......Page 1644
CHAPTER 12
-- DNA-Protein Interactions......Page 1796
CHAPTER 13 --
Yeast......Page 1934
CHAPTER 14 -- In Situ Hybridization and Immunohistochemistry......Page 2072
CHAPTER 15 -- The Polymerase Chain Reaction......Page 2272
CHAPTER 16 --
Protein Expression......Page 2384