Current protocols Molecular Biology

Introduction  .pdf......Page 1
Unit 1.1 Media Preparation and Bacteriological Tools.pdf......Page 4
Unit 1.2 Growth in Liquid Media.pdf......Page 11
Unit 1.3 Growth on Solid Media   .pdf......Page 13
Unit 1.4 Selected Topics from Classical Bacterial Genetics .pdf......Page 19
Unit 1.5 Introduction to Plasmid Biology.pdf......Page 33
Unit 1.6 Minipreps of Plasmid DNA.pdf......Page 50
Unit 1.7 Large-Scale Preparation of Plasmid DNA.pdf......Page 60
Unit 1.8 Introduction of Plasmid DNA into Cells  .pdf......Page 76
Unit 1.9 Introduction to Lambda Phages.pdf......Page 86
Unit 1.10 Lambda as a Cloning Vector .pdf......Page 90
Unit 1.11 Plating Lambda Phage to Generate Plaques.pdf......Page 101
Unit 1.12 Growing Lambda-Derived Vectors.pdf......Page 105
Unit 1.13 Preparing Lambda DNA from Phage Lysates .pdf......Page 108
Unit 1.14 Introduction to Vectors Derived from Filamentous Phages.pdf......Page 118
Unit 1.15 Preparing and Using M13-Derived Vectors  .pdf......Page 123
Introduction .pdf......Page 131
Unit 2.1A Purification and Concentration of DNA from Aqueous Solutions .pdf......Page 134
Unit 2.1B Purification of DNA by Anion-Exchange Chromatography  .pdf......Page 144
Unit 2.2 Preparation of Genomic DNA from Mammalian Tissue.pdf......Page 152
Unit 2.3 Preparation of Genomic DNA from Plant Tissue.pdf......Page 155
Unit 2.4 Preparation of Genomic DNA from Bacteria.pdf......Page 162
Unit 2.5A Agarose Gel Electrophoresis.pdf......Page 167
Unit 2.5B Pulsed-Field Gel Electrophoresis.pdf......Page 176
Unit 2.6 Isolation and Purification of Large DNA Restriction Fragments from Agarose Gels.pdf......Page 185
Unit 2.7 Separation of Small DNA Fragments by Conventional Gel Electrophoresis.pdf......Page 197
Unit 2.8 Capillary Electrophoresis of DNA.pdf......Page 205
Unit 2.9A Southern Blotting.pdf......Page 222
Unit 2.9B Dot and Slot Blotting of DNA.pdf......Page 237
Unit 2.10 Hybridization Analysis of DNA Blots.pdf......Page 243
Unit 2.11.pdf......Page 259
Unit 2.12.pdf......Page 284
Introduction.pdf......Page 291
Unit 3.1 Digestion of DNA with Restriction Endonucleases.pdf......Page 293
Unit 3.2 Mapping by Multiple Endonuclease Digestions.pdf......Page 314
Unit 3.3 Mapping by Partial Endonuclease Digestions.pdf......Page 319
Unit 3.4 Reagents and Radioisotopes Used to Manipulate Nucleic Acids.pdf......Page 321
Unit 3.5 DNA-Dependent DNA Polymerases .pdf......Page 332
Unit 3.6 Template-Independent DNA Polymerases.pdf......Page 347
Unit 3.7 RNA-Dependent DNA Polymerases.pdf......Page 349
Unit 3.8 DNA-Dependent RNA Polymerases.pdf......Page 352
Unit 3.9 DNA-Independent RNA Polymerases.pdf......Page 356
Unit 3.10 Phosphatases and Kinases .pdf......Page 358
Unit 3.11 Exonucleases.pdf......Page 363
Unit 3.12 Endonucleases.pdf......Page 367
Unit 3.13 Ribonucleases.pdf......Page 373
Unit 3.14 DNA Ligases.pdf......Page 376
Unit 3.15 RNA Ligases.pdf......Page 380
Unit 3.16 Subcloning of DNA Fragments   .pdf......Page 382
Unit 3.17 Constructing Recombinant DNA Molecules by the Polymerase Chain Reaction.pdf......Page 393
Unit 3.18 Labeling and Colorimetric Detection of Nonisotopic Probes.pdf......Page 403
Unit 3.19 Chemiluminescent Detection of Nonisotopic Probes.pdf......Page 412
Introduction .pdf......Page 420
Unit 4.1 Preparation of Cytoplasmic RNA from Tissue Culture Cells.pdf......Page 422
Unit 4.2 Guanidine Methods for Total RNA Preparation .pdf......Page 427
Unit 4.3 Phenol-SDS Method for Plant RNA Preparation.pdf......Page 436
Unit 4.4 Preparation of Bacterial RNA.pdf......Page 440
Unit 4.5 Preparation of Poly(A)+ RNA .pdf......Page 447
Unit 4.6 S1 Analysis of Messenger RNA Using Single-Stranded DNA Probes.pdf......Page 450
Unit 4.7 Ribonuclease Protection Assay.pdf......Page 463
Unit 4.8 Primer Extension.pdf......Page 471
Unit 4.9 Analysis of RNA by Northern and Slot Blot Hybridization.pdf......Page 476
Unit 4.10 Identification of Newly Transcribed RNA.pdf......Page 495
Introduction.pdf......Page 506
Unit 5.1 Genomic DNA Libraries.pdf......Page 509
Unit 5.2 cDNA Libraries.pdf......Page 512
Unit 5.3 Size Fractionation Using Sucrose Gradients.pdf......Page 513
Unit 5.4 Size Fractionation Using Agarose Gels.pdf......Page 521
Unit 5.5 Conversion of mRNA into Double-Stranded cDNA.pdf......Page 525
Unit 5.6 Ligation of Linkers or Adapters to Double-Stranded cDNA.pdf......Page 539
Unit 5.7 Production of a Genomic DNA Library   .pdf......Page 549
Unit 5.8A Production of a Complete cDNA Library.pdf......Page 553
Unit 5.9 Construction of Bacterial Artificial Chromosome (BAC-PAC) Libraries .pdf......Page 561
Unit 5.10 Amplification of a Bacteriophage Library.pdf......Page 594
Unit 5.11 Amplification of Cosmid and Plasmid Libraries .pdf......Page 597
Introduction .pdf......Page 599
Unit 6.1 Plating and Transferring Bacteriophage Libraries.pdf......Page 602
Unit 6.2 Plating and Transferring Cosmid and Plasmid Libraries.pdf......Page 606
Unit 6.3 Using DNA Fragments as Probes.pdf......Page 610
Unit 6.4 Using Synthetic Oligonucleotides as Probes.pdf......Page 616
Unit 6.5 Purification of Bacteriophage Clones.pdf......Page 626
Unit 6.6 Purification of Cosmid and Plasmid Clones .pdf......Page 628
Unit 6.7 Immunoscreening of Fusion Proteins Produced in Lambda Plaques.pdf......Page 629
Unit 6.8 Immunoscreening after Hybrid Selection and Translation.pdf......Page 635
Unit 6.9 Overview of Strategies for Screening YAC Libraries and Analyzing YAC Clones.pdf......Page 640
Unit 6.10 Analysis of Isolated YAC Clones.pdf......Page 647
Unit 6.11 Use of Monoclonal Antibodies for Expression Cloning.pdf......Page 666
Unit 6.12 Recombination-Based Assay (RBA) for Screening Bacteriophage Lambda Libraries.pdf......Page 682
Introduction .pdf......Page 694
Unit 7.1 DNA Sequencing Strategies .pdf......Page 709
Unit 7.2 Constructing Nested Deletions for Use in DNA Sequencing.pdf......Page 716
Unit 7.3 Preparation of Templates for DNA Sequencing.pdf......Page 736
Unit 7.4A DNA Sequencing by the Dideoxy Method.pdf......Page 746
Unit 7.4B Dideoxy DNA Sequencing with Chemiluminescent Detection.pdf......Page 785
Unit 7.5 DNA Sequencing by the Chemical Method   .pdf......Page 799
Unit 7.6 Denaturing Gel Electrophoresis for Sequencing.pdf......Page 810
Unit 7.7 Computer Manipulation of DNA and Protein Sequences.pdf......Page 823
Introduction .pdf......Page 846
Unit 8.1 Oligonucleotide-Directed Mutagenesis without Phenotypic Selection.pdf......Page 848
Unit 8.2A Mutagenesis with Degenerate Oligonucleotides- Creating Numerous Mutations in a Small DNA Sequence.pdf......Page 854
Unit 8.2B Gene Synthesis- Assembly of Target Sequences Using Mutually Priming Long Oligonucleotides.pdf......Page 861
Unit 8.3 Random Mutagenesis by PCR  .pdf......Page 867
Unit 8.4 Linker-Scanning Mutagenesis of DNA.pdf......Page 876
Unit 8.5 Directed Mutagenesis Using the Polymerase Chain Reaction.pdf......Page 883
Introduction.pdf......Page 893
Unit 9.1 Calcium Phosphate Transfection  .pdf......Page 898
Unit 9.2 Transfection Using DEAE-Dextran .pdf......Page 909
Unit 9.3 Transfection by Electroporation.pdf......Page 919
Unit 9.4 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents .pdf......Page 925
Unit 9.5 Selection of Transfected Mammalian Cells.pdf......Page 942
Unit 9.6 Overview of Genetic Reporter Systems.pdf......Page 961
Unit 9.7A Isotopic Assays for Reporter Gene Activity.pdf......Page 973
Unit 9.7B Nonisotopic Assays for Reporter Gene Activity.pdf......Page 984
Unit 9.7C Use of the A. Victoria Green Fluorescent Protein to Study Protein Dynamics in Vivo.pdf......Page 994
Unit 9.8 Direct Analysis of RNA after Transfection .pdf......Page 1001
Unit 9.9 Overview of the Retrovirus Transduction System.pdf......Page 1003
Unit 9.10 Preparation of a Specific Retrovirus Producer Cell Line.pdf......Page 1019
Unit 9.11 Transient Transfection Methods for Preparation of High-Titer Retroviral Supernatants.pdf......Page 1032
Unit 9.12 Large-Scale Preparation and Concentration of Retrovirus Stocks.pdf......Page 1050
Unit 9.13 Detection of Helper Virus in Retrovirus Stocks .pdf......Page 1056
Unit 9.14 Retrovirus Infection of Cells In Vitro and In Vivo  .pdf......Page 1063
Unit 9.15 Human Somatic Cell Gene Targeting.pdf......Page 1069
Introduction.pdf......Page 1089
Unit 10.1A Spectrophotometric and Colorimetric Determination of Protein Concentration .pdf......Page 1109
Unit 10.1B Quantitative Amino Acid Analysis.pdf......Page 1119
Unit 10.2A One-Dimensional SDS Gel Electrophoresis of Proteins  .pdf......Page 1122
Unit 10.2B One-Dimensional Electrophoresis Using Nondenaturing Conditions.pdf......Page 1157
Unit 10.3 Two-Dimensional Gel Electrophoresis Using the ISO-DALT System.pdf......Page 1168
Unit 10.4 Two-Dimensional Gel Electrophoresis.pdf......Page 1180
Unit 10.5 Overview of Digital Electrophoresis Analysis .pdf......Page 1203
Unit 10.6 Staining Proteins in Gels.pdf......Page 1228
Unit 10.7 Detection of Proteins on Blot Transfer Membranes.pdf......Page 1253
Unit 10.8 Immunoblotting and Immunodetection.pdf......Page 1259
Unit 10.9 Gel-Filtration Chromatography.pdf......Page 1283
Unit 10.10 Ion-Exchange Chromatography.pdf......Page 1315
Unit 10.11A Immunoaffinity Chromatography .pdf......Page 1345
Unit 10.11B Metal-Chelate Affinity Chromatography.pdf......Page 1354
Unit 10.12 HPLC of Peptides and Proteins- Preparation and System Set-Up.pdf......Page 1369
Unit 10.13 HPLC of Peptides and Proteins- Standard Operating Conditions.pdf......Page 1378
Unit 10.14 Reversed-Phase Isolation of Peptides .pdf......Page 1410
Unit 10.15 Purification of Recombinant Proteins and Study of Protein Interaction by Epitope Tagging.pdf......Page 1427
Unit 10.16 Immunoprecipitation.pdf......Page 1436
Unit 10.17 Synthesizing Proteins In Vitro by Transcription and Translation of Cloned Genes  .pdf......Page 1465
Unit 10.18 Metabolic Labeling with Amino Acids.pdf......Page 1470
Unit 10.19 Isolation of Proteins for Microsequence Analysis.pdf......Page 1480
Unit 10.20 Capillary Electrophoresis of Proteins and Peptides.pdf......Page 1492
Unit 10.21 Overview of Peptide and Protein Analysis by Mass Spectrometry.pdf......Page 1505
Unit 10.22 Protein Identification and Characterization by Mass Spectrometry.pdf......Page 1532
Unit 10.23 Difference Gel Electrophoresis (DIGE) Using CyDye DIGE Fluor Minimal Dyes.pdf......Page 1556
Introduction.pdf......Page 1564
Unit 11.1 Conjugation of Enzymes to Antibodies.pdf......Page 1567
Unit 11.2 Enzyme-Linked Immunosorbent Assays (ELISA).pdf......Page 1574
Unit 11.3 Isotype Determination of Antibodies.pdf......Page 1596
Unit 11.4 Immunization of Mice .pdf......Page 1602
Unit 11.5 Preparation of Myeloma Cells .pdf......Page 1607
Unit 11.6 Preparation of Mouse Feeder Cells for Fusion and Cloning.pdf......Page 1610
Unit 11.7 Fusion of Myeloma Cells with Immune Spleen Cells .pdf......Page 1613
Unit 11.8 Cloning of Hybridoma Cell Lines by Limiting Dilution.pdf......Page 1617
Unit 11.9 Freezing and Recovery of Hybridoma Cell Lines.pdf......Page 1619
Unit 11.10 Production of Monoclonal Antibody Supernatant and Ascites Fluid.pdf......Page 1622
Unit 11.11 Purification of Monoclonal Antibodies.pdf......Page 1630
Unit 11.12 Production of Polyclonal Antisera.pdf......Page 1635
Unit 11.13 In Vitro Antibody Production.pdf......Page 1644
Unit 11.14 Purification of Immunoglobulin G Fraction from Antiserum, Ascites Fluid, or Hybridoma Supernatant .pdf......Page 1661
Unit 11.15 Introduction to Peptide Synthesis .pdf......Page 1666
Unit 11.16 Synthetic Peptides for Production of Antibodies that Recognize Intact Proteins.pdf......Page 1675
Unit 11.17 Determination of the Specific Antibody Titer.pdf......Page 1694
Unit 11.18 Identification of Polyol-Responsive Monoclonal Antibodies for Use in Immunoaffinity Chromatography .pdf......Page 1707
Introduction .pdf......Page 1716
Unit 12.1 Preparation of Nuclear and Cytoplasmic Extracts from Mammalian Cells.pdf......Page 1720
Unit 12.2 Mobility Shift DNA-Binding Assay Using Gel Electrophoresis.pdf......Page 1729
Unit 12.3 Methylation and Uracil Interference Assays for Analysis of Protein-DNA Interactions.pdf......Page 1740
Unit 12.4 DNase I Footprint Analysis of Protein-DNA Binding .pdf......Page 1747
Unit 12.5 UV Crosslinking of Proteins to Nucleic Acids.pdf......Page 1763
Unit 12.6 Purification of DNA-Binding Proteins Using Biotin-Streptavidin Affinity Systems.pdf......Page 1771
Unit 12.7 Detection, Purification, and Characterization of cDNA Clones Encoding DNA-Binding Proteins .pdf......Page 1780
Unit 12.8 Rapid Separation of Protein-Bound DNA from Free DNA Using Nitrocellulose Filters.pdf......Page 1790
Unit 12.9 Analysis of DNA-Protein Interactions Using Proteins Synthesized In Vitro from Cloned Genes.pdf......Page 1799
Unit 12.10 Purification of Sequence-Specific DNA-Binding Proteins by Affinity Chromatography.pdf......Page 1803
Unit 12.11 Determination of Protein-DNA Sequence Specificity by PCR-Assisted Binding-Site Selection.pdf......Page 1821
Unit 12.12 Yeast One-Hybrid Screening for DNA-Protein Interactions .pdf......Page 1832
Introduction.pdf......Page 1854
Unit 13.1 Preparation of Yeast Media.pdf......Page 1857
Unit 13.2 Growth and Manipulation of Yeast.pdf......Page 1864
Unit 13.3 Genome-Wide Transposon Mutagenesis in Yeast .pdf......Page 1876
Unit 13.4 Yeast Cloning Vectors and Genes .pdf......Page 1891
Unit 13.6 Yeast Vectors and Assays for Expression of Cloned Genes.pdf......Page 1901
Unit 13.7 Introduction of DNA into Yeast Cells.pdf......Page 1907
Unit 13.8 Cloning Yeast Genes by Complementation.pdf......Page 1917
Unit 13.9 Manipulation of Plasmids from Yeast Cells.pdf......Page 1921
Unit 13.10 Manipulation of Cloned Yeast DNA .pdf......Page 1927
Unit 13.11 Preparation of Yeast DNA.pdf......Page 1941
Unit 13.12 Preparation of Yeast RNA.pdf......Page 1945
Unit 13.13 Preparation of Protein Extracts from Yeast.pdf......Page 1950
Unit 13.14 Overview of Schizosaccharomyces pombe.pdf......Page 1959
Unit 13.15 S. pombe Strain Maintenance and Media.pdf......Page 1962
Unit 13.16 Growth and Manipulation of S. pombe .pdf......Page 1967
Unit 13.17 Introduction of DNA into S. pombe Cells.pdf......Page 1984
Introduction  .pdf......Page 1992
Unit 14.1 Fixation, Embedding, and Sectioning of Tissues, Embryos, and Single Cells.pdf......Page 1994
Unit 14.2 Cryosectioning.pdf......Page 2002
Unit 14.3 In Situ Hybridization to Cellular RNA.pdf......Page 2010
Unit 14.4 Detection of Hybridized Probe  .pdf......Page 2026
Unit 14.5 Counterstaining and Mounting of Autoradiographed In Situ Hybridization Slides.pdf......Page 2029
Unit 14.6 Immunohistochemistry.pdf......Page 2034
Unit 14.7 In situ Hybridization and Detection Using Nonisotopic Probes.pdf......Page 2047
Unit 14.8 In situ Polymerase Chain Reaction and Hybridization to Detect Low-Abundance Nucleic Acid Targets.pdf......Page 2061
Unit 14.9 Whole-Mount In Situ Hybridization and Detection of RNAs in Vertebrate Embryos and Isolated Organs.pdf......Page 2085
Unit 14.10 Principles and Application of Fluorescence Microscopy   .pdf......Page 2109
Unit 14.11 Basic Confocal Microscopy  .pdf......Page 2120
Unit 14.12 Measurement of In Situ Hybridization.pdf......Page 2132
Unit 14.13 Morphological, Biochemical, and Flow Cytometric Assays of Apoptosis.pdf......Page 2141
Unit 14.14 Whole-Mount Histochemical Detection of -Galactosidase Activity.pdf......Page 2162
Unit 14.15 Overview of Image Analysis, Image Importing, and Image Processing using Freeware   .pdf......Page 2170
Introduction.pdf......Page 2179
Unit 15.1 Enzymatic Amplification of DNA by PCR- Standard Procedures and Optimization.pdf......Page 2182
Unit 15.2 Direct DNA Sequencing of PCR Products.pdf......Page 2196
Unit 15.3 Ligation-Mediated PCR for Genomic Sequencing and Footprinting.pdf......Page 2209
Unit 15.4 Molecular Cloning of PCR Products.pdf......Page 2235
Unit 15.5 Enzymatic Amplification of RNA by PCR (RT-PCR).pdf......Page 2246
Unit 15.6 cDNA Amplification Using One-Sided (Anchored) PCR.pdf......Page 2252
Unit 15.7 Quantitation of Rare DNAs by PCR.pdf......Page 2262
Unit 15.8 High-Throughput Real-Time Quantitative Reverse Transcription PCR.pdf......Page 2270
Introduction.pdf......Page 2291
Unit 16.1 Overview of Protein Expression in E. coli .pdf......Page 2295
Unit 16.2 Expression Using the T7 RNA Polymerase-Promoter System.pdf......Page 2298
Unit 16.3 Expression Using Vectors with Phage  Regulatory Sequences.pdf......Page 2309
Unit 16.4A Introduction to Expression by Fusion Protein Vectors.pdf......Page 2320
Unit 16.4B Enzymatic and Chemical Cleavage of Fusion Proteins.pdf......Page 2324
Unit 16.5 Expression and Purification of lacZ and trpE Fusion Proteins.pdf......Page 2337
Unit 16.6 Expression and Purification of Maltose-Binding Protein Fusions.pdf......Page 2343
Unit 16.7 Expression and Purification of Glutathione-S-Transferase Fusion Proteins .pdf......Page 2357
Unit 16.8 Expression and Purification of Thioredoxin Fusion Proteins .pdf......Page 2364
Unit 16.9 Overview of the Baculovirus Expression System.pdf......Page 2378
Unit 16.10 Maintenance of Insect Cell Cultures and Generation of Recombinant Baculoviruses.pdf......Page 2388
Unit 16.11 Expression and Purification of Recombinant Proteins Using the Baculovirus System.pdf......Page 2407
Unit 16.12 Transient Expression of Proteins Using COS Cells .pdf......Page 2421
Unit 16.13 Expression and Purification of Epitope-Tagged Multisubunit Protein Complexes from Mammalian Cells.pdf......Page 2428
Unit 16.14 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System.pdf......Page 2445
Unit 16.15 Overview of the Vaccinia Virus Expression System .pdf......Page 2454
Unit 16.16 Preparation of Cell Cultures and Vaccinia Virus Stocks  .pdf......Page 2459
Unit 16.17 Generation of Recombinant Vaccinia Viruses.pdf......Page 2472
Unit 16.18 Characterization of Recombinant Vaccinia Viruses and Their Products.pdf......Page 2491
Unit 16.19 Gene Expression Using the Vaccinia Virus-T7 RNA Polymerase Hybrid System.pdf......Page 2502
Unit 16.20 Expression of Proteins Using Semliki Forest Virus Vectors  .pdf......Page 2513
Unit 16.21 Overview of the HIV-1 Lentiviral Vector System.pdf......Page 2529
Unit 16.22 Generation of HIV-1-Based Lentiviral Vector Particles.pdf......Page 2544
Unit 16.23 Amplification Using CHO Cell Expression Vectors.pdf......Page 2559
Unit 16.24 Helper-Dependent Adenoviral Vectors.pdf......Page 2572
Introduction   .pdf......Page 2595
Unit 17.1 Special Considerations for Glycoproteins and Their Purification  .pdf......Page 2605
Unit 17.2 Special Considerations for Proteoglycans and Glycosaminoglycans and Their Purification.pdf......Page 2615
Unit 17.3 Special Considerations for Glycolipids and Their Purification.pdf......Page 2624
Unit 17.4 Metabolic Radiolabeling of Animal Cell Glycoconjugates .pdf......Page 2637
Unit 17.5 Chemical Labeling of Carbohydrates by Oxidation and Sodium Borohydride Reduction.pdf......Page 2653
Unit 17.6 Detection and Analysis of Proteins Modified by O-Linked N-Acetylglucosamine .pdf......Page 2661
Unit 17.7 Lectin Analysis of Proteins Blotted onto Filters.pdf......Page 2686
Unit 17.8 Detection of Glycophospholipid Anchors on Proteins.pdf......Page 2694
Unit 17.9 Direct Chemical Analysis of Glycoconjugates for Carbohydrates.pdf......Page 2707
Unit 17.10A Inhibition of N-Linked Glycosylation.pdf......Page 2718
Unit 17.10B Inhibition of Glycolipid Biosynthesis.pdf......Page 2727
Unit 17.11 Synthetic Glycosides as Primers of Oligosaccharide Biosynthesis and Inhibitors of Glycoprotein and Proteoglycan Assembly.pdf......Page 2732
Unit 17.12 Sialidases.pdf......Page 2738
Unit 17.13A Endoglycosidase and Glycoamidase Release of N-Linked Oligosaccharides.pdf......Page 2746
Unit 17.13B Analysis of Glycosaminoglycans with Polysaccharide Lyases .pdf......Page 2772
Unit 17.14A Preparation of Glycopeptides  .pdf......Page 2788
Unit 17.14B Detection of Individual Glycosylation Sites on Glycoproteins.pdf......Page 2797
Unit 17.15A -Elimination for Release of O-Linked Glycosaminoglycans from Proteoglycans.pdf......Page 2803
Unit 17.15B -Elimination for Release of O-GalNAc-Linked Oligosaccharides from Glycoproteins and Glycopeptides .pdf......Page 2806
Unit 17.16 Acid Hydrolysis for Release of Monosaccharides.pdf......Page 2812
Unit 17.17A Enzymatic Release of Oligosaccharides from Glycolipids.pdf......Page 2823
Unit 17.17B Endo--Galactosidases and Keratanase.pdf......Page 2828
Unit 17.18 Analysis of Monosaccharides.pdf......Page 2836
Unit 17.19A Total Compositional Analysis by High-Performance Liquid Chromatography or Gas-Liquid Chromatography .pdf......Page 2855
Unit 17.19B Composition of Labeled Monosaccharides from Glycosaminoglycans    .pdf......Page 2880
Unit 17.20 Analysis of Oligosaccharide Negative Charge by Anion-Exchange Chromatography.pdf......Page 2887
Unit 17.21A HPLC Methods for the Fractionation and Analysis of Negatively Charged Oligosaccharides and Gangliosides.pdf......Page 2894
Unit 17.21B Fractionation and Analysis of Neutral Oligosaccharides by HPLC .pdf......Page 2902
Unit 17.22A Nitrous Acid Degradation of Glycosaminoglycans.pdf......Page 2907
Unit 17.22B Analysis of Disaccharides and Tetrasaccharides Released from Glycosaminoglycans.pdf......Page 2912
Unit 17.23 Analysis of Sulfate Esters by Solvolysis or Hydrolysis.pdf......Page 2924
Introduction.pdf......Page 2932
Unit 18.1 Overview of Protein Phosphorylation.pdf......Page 2934
Unit 18.2 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation.pdf......Page 2939
Unit 18.3 Phosphoamino Acid Analysis.pdf......Page 2947
Unit 18.4 Analysis of Phosphorylation of Unlabeled Proteins.pdf......Page 2955
Unit 18.5 Detection of Phosphorylation by Enzymatic Techniques.pdf......Page 2962
Unit 18.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides.pdf......Page 2971
Unit 18.7 Assays of Protein Kinases Using Exogenous Substrates    .pdf......Page 2990
Unit 18.8 Permeabilization Strategies to Study Protein Phosphorylation.pdf......Page 3012
Unit 18.9 Phosphopeptide Mapping and Identification of Phosphorylation Sites.pdf......Page 3031
Unit 18.10 Use of Protein Phosphatase Inhibitors.pdf......Page 3059
Unit 18.11 Design and Use of Analog-Sensitive Protein Kinases.pdf......Page 3072
Unit 18.12 Detection of MAP Kinase Signaling.pdf......Page 3091
Introduction.pdf......Page 3114
Unit 19.1 Internet Basics for Biologists.pdf......Page 3116
Unit 19.2 Sequence Databases- Integrated Information Retrieval and Data Submission.pdf......Page 3132
Unit 19.3 Sequence Similarity Searching Using the BLAST Family of Programs  .pdf......Page 3152
Unit 19.4 Protein Databases on the Internet.pdf......Page 3181
Unit 19.5 Basic Protein Sequence Analysis.pdf......Page 3196
Introduction.pdf......Page 3220
Unit 20.1 Interaction Trap-wo-Hybrid System to Identify Interacting Proteins.pdf......Page 3226
Unit 20.2 Affinity Purification of Proteins Binding to GST Fusion Proteins.pdf......Page 3266
Unit 20.3 Phage-Based Expression Cloning to Identify Interacting Proteins.pdf......Page 3276
Unit 20.4 Surface Plasmon Resonance for Measurements of Biological Interest .pdf......Page 3285
Unit 20.5 Detection of Protein-Protein Interactions by Coprecipitation.pdf......Page 3297
Unit 20.6 Identification of Protein Interactions by Far Western Analysis.pdf......Page 3306
Unit 20.7 Two-Hybrid Dual Bait System.pdf......Page 3316
Unit 20.8 Interaction Trap-Two-Hybrid System to Identify Loss-of-Interaction Mutant Proteins .pdf......Page 3353
Introduction.pdf......Page 3363
Unit 21.1 Micrococcal Nuclease Analysis of Chromatin Structure .pdf......Page 3365
Unit 21.2 Separation of Histone Variants and Post-Translationally Modified Isoforms by Triton.pdf......Page 3382
Unit 21.3 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific Genomic Sequences In Vivo.pdf......Page 3392
Unit 21.4 DNase I and Hydroxyl Radical Characterization of Chromatin Complexes.pdf......Page 3425
Unit 21.5 Isolation of Histones and Nucleosome Cores from Mammalian Cells.pdf......Page 3434
Unit 21.6 Assembly of Nucleosomal Templates by Salt Dialysis.pdf......Page 3446
Unit 21.7 Chromatin Assembly Using Drosophila Systems.pdf......Page 3462
Unit 21.8 Analysis of Protein Co-Occupancy by Quantitative Sequential Chromatin Immunoprecipitation .pdf......Page 3489
Unit 21.9 Defining In Vivo Targets of Nuclear Proteins by Chromatin Immunoprecipitation and Microarray Analysis.pdf......Page 3496
Introduction  .pdf......Page 3503
Unit 22.1 Overview of Nucleic Acid Arrays.pdf......Page 3504
Unit 22.2 Preparation of mRNA for Expression Monitoring.pdf......Page 3511
Unit 22.3 Profiling Human Gene Expression with cDNA Microarrays.pdf......Page 3524
Unit 22.4 Overview of mRNA Expression Profiling Using Microarrays.pdf......Page 3550
Unit 22.5 Pattern Discovery in Expression Profiling Data.pdf......Page 3558
Introduction.pdf......Page 3569
Unit 23.1 Overview of Gene Targeting by Homologous Recombination.pdf......Page 3571
Unit 23.2 Mouse Embryo Fibroblast (MEF) Feeder Cell Preparation.pdf......Page 3582
Unit 23.3 Mouse Embryonic Stem (ES) Cell Culture.pdf......Page 3589
Unit 23.4 Mouse Embryonic Stem (ES) Cell Isolation .pdf......Page 3595
Unit 23.5 Production of a Heterozygous Mutant Cell Line by Homologous Recombination.pdf......Page 3604
Unit 23.6 Production of a Homozygous Mutant Embryonic Stem Cell Line.pdf......Page 3615
Unit 23.7 Chimeric Mouse Production by Microinjection .pdf......Page 3618
Unit 23.8 Mouse Colony Management .pdf......Page 3638
Unit 23.9 Transgenic Mouse Production By Zygote Injection   .pdf......Page 3649
Unit 23.10 Transgenic Mouse Colony Management .pdf......Page 3678
Unit 23.11 Modification and Production of BAC Transgenes.pdf......Page 3686
Unit 23.12 Regulation of Transgene Expression Using Tetracycline.pdf......Page 3701
Unit 24.1 Overview of Agents from Combinatorial Nucleic Acid and Protein Libraries.pdf......Page 3709
Unit 24.2 Design, Synthesis, and Amplification of DNA Pools for Construction of Combinatorial Pools and Libraries.pdf......Page 3710
Unit 24.3 In Vitro Selection of RNA Aptamers to a Protein Target by Filter Immobilization   .pdf......Page 3734
Unit 24.4 Peptide Aptamers- Dominant Genetic Agents for Forward and Reverse Analysis of Cellular Processes.pdf......Page 3759
Unit 24.5 Protein Selection Using mRNA Display .pdf......Page 3784
Introduction.pdf......Page 3818
Unit 25A.1 Laser Capture Microdissection.pdf......Page 3820
Unit 25A.2 Preparation of Single Cells from Solid Tissues for Analysis by PCR.pdf......Page 3844
Unit 25B.1 Production of a Subtracted cDNA Library.pdf......Page 3859
Unit 25B.2 PCR-Based Subtractive cDNA Cloning  .pdf......Page 3867
Unit 25B.3 Differential Display of mRNA by PCR.pdf......Page 3887
Unit 25B.4 Restriction-Mediated Differential Display (RMDD).pdf......Page 3897
Unit 25B.5 AFLP-Based Transcript Profiling.pdf......Page 3914
Unit 25B.6 Serial Analysis of Gene Expression (SAGE)  .pdf......Page 3930
Unit 25B.7 Representational Difference Analysis.pdf......Page 3959
Unit 25B.8 Gene Expression Analysis of a Single or Few Cells .pdf......Page 3971
Introduction.pdf......Page 3989
Unit 26.1 Overview of RNA Interference and Related Processes.pdf......Page 3990
Unit 26.2 Gene Silencing by RNAi in Mammalian Cells  .pdf......Page 3996
Unit 26.3 RNA Interference in Caenorhabditis Elegans.pdf......Page 4010
Unit 26.4 Cloning of Small RNA Molecules.pdf......Page 4030
Unit 26.5 RNA Interference in Cultured Drosophila Cells.pdf......Page 4048
Introduction .pdf......Page 4055
Unit 27.1 Agarose Gel Separation-Isolation of RNA-Protein Complexes.pdf......Page 4056
Unit 27.2 Identification of RNA Binding Proteins by UV Cross-Linking   .pdf......Page 4061
Unit 27.3 Purification of Functional RNA-Protein Complexes using MS2-MBP.pdf......Page 4071
Introduction.pdf......Page 4078
Unit 28.1 Preparation, Culture, and Immortalization of Mouse Embryonic Fibroblasts .pdf......Page 4079
Unit 28.2 Isolation and Immortalization of Lymphocytes.pdf......Page 4087
Unit 28.3 Establishment and Culture of Human Skin Fibroblasts.pdf......Page 4092
1A Common Abbreviations.pdf......Page 4101
1B Useful Measurements and Data  .pdf......Page 4109
1C Characteristics of Amino Acids.pdf......Page 4110
1D Characteristics of Nucleic Acids.pdf......Page 4122
1E Radioactivity.pdf......Page 4133
1F Safe Use of Radioisotopes .pdf......Page 4136
1G Centrifuges and Rotors.pdf......Page 4147
1H Safe Use of Hazardous Chemicals .pdf......Page 4153
1I Commonly Used Detergents.pdf......Page 4186
1J Common Conversion Factors.pdf......Page 4189
1K Compendium of Drugs Commonly Used in Molecular Biology Research.pdf......Page 4197
2 Commonly Used Reagents and Equipment .pdf......Page 4223
3A Detection and Quantitation of Radiolabeled Proteins and DNA in Gels and Blots.pdf......Page 4231
3B Silanizing Glassware.pdf......Page 4241
3C Dialysis and Ultrafiltration.pdf......Page 4243
3D Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy  .pdf......Page 4250
3E Introduction of Restriction Enzyme Recognition Sequences by Silent Mutation .pdf......Page 4262
3F Techniques for Mammalian Cell Tissue Culture.pdf......Page 4265
3G Importing Biological Materials.pdf......Page 4279
3H Kinetic Assay Methods.pdf......Page 4282
3I Statistics for the Molecular Biologist- Group Comparisons .pdf......Page 4293
4 Selected Suppliers of Reagents and Equipment .pdf......Page 4315
5 Vectors .pdf......Page 4350