- Volume 1
- Chapter 1: Isolation and Quantification of DNA1
- Protocol 1: Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreps
- Protocol 2: Preparation of Plasmid DNA by Alkaline Lysis with SDS: Maxipreps
- Protocol 3: Isolating DNA from Gram-Negative Bacteria (e.g., E. coli)
- Protocol 4: Precipitation of DNA with Ethanol
- Protocol 5: Precipitation of DNA with Isopropanol
- Protocol 6: Concentrating and Desalting Nucleic Acids with Microconcentrators
- Protocol 7: Concentrating Nucleic Acids by Extraction with Butanol
- Protocol 8: Preparation of Single-Stranded Bacteriophage M13 DNA by Precipitation with Polyethylene Glycol
- Protocol 9: Plating Bacteriophage M13
- Protocol 10: Growing Bacteriophage M13 in Liquid Culture
- Protocol 11: Preparation of Double-Stranded (Replicative Form) Bacteriophage M13 DNA
- Protocol 12: Isolation of High-Molecular-Weight DNA Using Organic Solvents to Purify DNA
- Protocol 13: Isolation of High-Molecular-Weight DNA from Mammalian Cells Using Proteinase K and Phenol
- Protocol 14: A Single-Step Method for the Simultaneous Preparation of DNA, RNA, and Protein from Cells and Tissues
- Protocol 15: Preparation of Genomic DNA from Mouse Tails and Other Small Samples
- Protocol 16: Rapid Isolation of Yeast DNA
- Protocol 17: Using Ethidium Bromide to Estimate the Amount of DNA in Bands after Electrophoresis through Minigels
- Protocol 18: Estimating the Concentration of DNA by Fluorometry Using Hoechst 33258
- Protocol 19: Quantifying DNA in Solution with PicoGreen
- Panel: Isolation and Quantification of DNA1
- Panel: DNA Isolation2
- Panel: Commercial Kits for Purification of DNA3
- Panel: Quantifying DNA5
- Panel: Alternative Protocol: Isolation of DNA from Mouse Tails without Extraction by Organic Solvents61
- Panel: Alternative Protocol: One-Tube Isolation of DNA from Mouse Tails62
- Panel: DNA Extraction from Formaldehyde-Fixed Tissue Embedded in Paraffin Blocks72
- Panel: Polyethylene Glycol73
- Panel: -Complementation74
- Panel: X-Gal76
- Panel: Minimizing Damage to Large DNA Molecules77
- Panel: Spectrophotometry78
- Chapter 2: Analysis of DNA81
- Protocol 1: Agarose Gel Electrophoresis
- Protocol 2: Detection of DNA in Agarose Gels by Staining
- Protocol 3: Polyacrylamide Gel Electrophoresis
- Protocol 4: Detection of DNA in Polyacrylamide Gels by Staining
- Protocol 5: Detection of DNA in Polyacrylamide Gels by Autoradiography
- Protocol 6: Alkaline Agarose Gel Electrophoresis
- Protocol 7: Imaging: Autoradiography and Phosphorimaging
- Protocol 8: Recovery of DNA from Agarose Gels Using Glass Beads
- Protocol 9: Recovery of DNA from Low-Melting-Temperature Agarose Gels: Organic Extraction
- Protocol 10: Isolation of DNA Fragments from Polyacrylamide Gels by the Crush and Soak Method
- Protocol 11: Southern Blotting
- Protocol 12: Southern Blotting: Simultaneous Transfer of DNA from an Agarose Gel to Two Membranes
- Protocol 13: Southern Hybridization of Radiolabeled Probes to Nucleic Acids Immobilized on Membranes
- Panel: Analysis of DNA81
- Panel: Agarose Gel Electrophoresis82
- Panel: Analysis of DNA Fragments86
- Panel: Recovering DNA from Gels87
- Panel: Southern Blotting88
- Panel: Additional Protocol: Autoradiography of Alkaline Agarose Gels117
- Panel: Additional Protocol: Stripping Probes from Membranes150
- Panel: Formamide and Its Uses in Molecular Cloning153
- Panel: Rapid Hybridization Buffers155
- Chapter 3: Cloning and Transformation with Plasmid Vectors157
- Protocol 1: The Hanahan Method for Preparation and Transformation of Competent E. coli: High-Efficiency Transformation
- Protocol 2: The Inoue Method for Preparation and Transformation of Competent E. coli: Ultracompetent Cells
- Protocol 3: Easy Transformation of E. coli: Nanoparticle-Mediated Transformation
- Protocol 4: Transformation of E. coli by Electroporation
- Protocol 5: Cloning in Plasmid Vectors: Directional Cloning
- Protocol 6: Cloning in Plasmid Vectors: Blunt-End Cloning
- Protocol 7: Dephosphorylation of Plasmid DNA
- Protocol 8: Attaching Phosphorylated Adaptors/Linkers to Blunt-Ended DNAs
- Protocol 9: Cloning PCR Products: Addition of Restriction Sites to the Termini of Amplified DNA
- Protocol 10: Cloning PCR Products: Blunt-End Cloning
- Protocol 11: Cloning PCR Products: Making T Vectors
- Protocol 12: Cloning PCR Products: TA Cloning
- Protocol 13: Cloning PCR Products: TOPO TA Cloning
- Protocol 14: Screening Bacterial Colonies Using X-Gal and IPTG: -Complementation
- Panel: Cloning and Transformation with Plasmid Vectors157
- Panel: Plasmid Vectors158
- Panel: Transformation159
- Panel: Alternative Protocol: One-Step Preparation of Competent E. coli: Transformation and Storage of Bacterial Cells in the Same Solution175
- Panel: Caring for E. coli213
- Panel: The History of Transformation of Bacteria by DNA217
- Panel: A Guide to Cloning the Products of Polymerase Chain Reactions218
- Panel: BioBricks and the Ordered Assembly of DNA Fragments225
- Panel: TOPO Tools: Creating Linear Expression Constructs with Functional Elements227
- Panel: Antibiotics229
- Panel: Adaptors232
- Panel: Linkers234
- Panel: Ligation and Ligases235
- Panel: Condensing and Crowding Reagents240
- Panel: The Discovery of Restriction Enzymes241
- Panel: Restriction Enzymes242
- Panel: Chloramphenicol245
- Panel: The ccdB Gene247
- Panel: Bacteriophage 248
- Panel: Bacteriophage M13249
- Panel: Plasmids251
- Panel: Cosmids258
- Chapter 4: Gateway Recombinational Cloning261
- Protocol 1: Propagating Gateway Vectors
- Protocol 2: Generating an ORF Entry Clone and Destination Clone
- Protocol 3: Using Multisite LR Cloning to Generate a Destination Clone
- Panel: Gateway Recombinational Cloning261
- Panel: Basic Principles and Applications of Gateway Cloning262
- Panel: Disadvantages of Gateway Cloning and Alternative Cloning Systems264
- Panel: Generating Gateway-Compatible Vectors280
- Chapter 5: Working with Bacterial Artificial Chromosomes and Other High-Capacity Vectors281
- Protocol 1: Small-Scale Isolation of BAC DNA and Verification by PCR
- Protocol 2: Large-Scale Preparation and Linearization of BAC DNA
- Protocol 3: Examination of BAC DNA Quality and Quantity by Pulsed-Field Gel Electrophoresis
- Protocol 4: Two-Step BAC Engineering: Preparation of Shuttle Vector DNA
- Protocol 5: Preparation of the A Homology Arm (A-Box) and B Homology Arm (B-Box)
- Protocol 6: Cloning of the A and B Homology Arms into the Shuttle Vector
- Protocol 7: Preparation and Verification of the Recombinant Shuttle Vector
- Protocol 8: Electroporation of Competent BAC Host Cells with the Recombinant Shuttle Vector
- Protocol 9: Verification of Cointegrates and Selection of Resolved BAC Clones
- Protocol 10: One-Step BAC Modification: Preparation of Plasmids
- Protocol 11: Preparation of the A Homology Arm (A-Box)
- Protocol 12: Cloning of the A Homology Arm into Reporter-Shuttle Vector
- Protocol 13: Transformation of the BAC Host with the RecA Vector
- Protocol 14: Transfer of the Reporter Vector into BAC/RecA Cells and Selection of Cointegrates
- Protocol 15: Growth of S. cerevisiae and Preparation of DNA
- Protocol 16: Small-Scale Preparations of Yeast DNA
- Panel: Working with Bacterial Artificial Chromosomes and Other High-Capacity Vectors281
- Panel: Development of High-Capacity Vectors: Advantages and Disadvantages282
- Panel: Working with Bacterial Artificial Chromosomes286
- Panel: CREloxP338
- Panel: Using EGFP as a Reporter342
- Panel: Primer Design for Homology Arms, Cointegration, and Resolution343
- Panel: Yeast Media344
- Chapter 6: Extraction, Purification, and Analysis of RNA from Eukaryotic Cells345
- Protocol 1: Purification of Total RNA from Mammalian Cells and Tissues
- Protocol 2: Isolation of Total RNA from Zebrafish Embryos and Adults
- Protocol 3: Total RNA Isolation from Drosophila melanogaster
- Protocol 4: Total RNA Extraction from Caenorhabditis elegans
- Protocol 5: Total RNA Extraction from Saccharomyces cerevisiae Using Hot Acid Phenol
- Protocol 6: Quantifying and Storing RNA
- Protocol 7: Precipitation of RNA with Ethanol
- Protocol 8: Removing DNA Contamination from RNA Samples by Treatment with RNase-Free DNase I
- Protocol 9: Isolation of Poly(A) Messenger RNA Using Magnetic Oligo(dT) Beads
- Protocol 10: Separation of RNA according to Size: Electrophoresis of RNA through Agarose Gels Containing Formaldehyde
- Protocol 11: Separation of RNA according to Size: Electrophoresis of RNA through Denaturing Urea Polyacrylamide Gels
- Protocol 12: Transfer and Fixation of Denatured RNA in Agarose Gels to Membranes
- Protocol 13: Transfer and Fixation of Denatured RNA in Polyacrylamide Gels to Membranes by Electrophoretic Transfer
- Protocol 14: Northern Hybridization
- Protocol 15: Dot and Slot Hybridization of Purified RNA
- Protocol 16: Mapping RNA with Nuclease S1
- Protocol 17: Ribonuclease Protection: Mapping RNA with Ribonuclease and Radiolabeled RNA Probes
- Protocol 18: Analysis of RNA by Primer Extension
- Panel: Extraction, Purification, and Analysis of RNA from Eukaryotic Cells345
- Panel: Introduction to the Isolation of Total RNA Using Monophasic Lysis Reagents348
- Panel: Alternative Protocol: Preparing RNA from Smaller Samples354
- Panel: Introduction to Hybridization of RNA by Northern Transfer381
- Panel: Alternative Protocol: Capillary Transfer by Downward Flow406
- Panel: Introduction to Mapping RNA420
- Panel: How to Win the Battle with RNase450
- Panel: Inhibitors of RNases451
- Panel: Diethylpyrocarbonate452
- Panel: Nuclease S1454
- Chapter 7: Polymerase Chain Reaction455
- Protocol 1: The Basic Polymerase Chain Reaction
- Protocol 2: Hot Start PCR
- Protocol 3: Touchdown PCR
- Protocol 4: PCR Amplification of GC-Rich Templates
- Protocol 5: Long and Accurate PCR (LA PCR)
- Protocol 6: Inverse PCR
- Protocol 7: Nested PCR
- Protocol 8: Amplification of cDNA Generated by Reverse Transcription of mRNA: Two-Step RT-PCR
- Protocol 9: Rapid Amplification of Sequences from the 5 Ends of mRNAs: 5-RACE
- Protocol 10: Rapid Amplification of Sequences from the 3 Ends of mRNAs: 3-RACE
- Protocol 11: Screening Colonies by PCR
- Panel: Polymerase Chain Reaction455
- Panel: The Basic Polymerase Chain Reaction456
- Panel: Design of Oligonucleotide Primers for Basic PCR462
- Panel: Detecting, Analyzing, and Quantifying mRNAs464
- Panel: Contamination in PCR466
- Panel: Taq DNA Polymerase533
- Panel: PCR in Theory537
- Panel: Ribonuclease H538
- Panel: The dut and ung Genes of E. coli539
- Panel: Terminal Transferase540
- Chapter 8: Bioinformatics541
- Protocol 1: Visualizing Genomic Annotations with the UCSC Genome Browser
- Protocol 2: Sequence Alignment and Homology Search with BLAST and ClustalW
- Protocol 3: Designing PCR Primers Using Primer3Plus
- Protocol 4: Expression Profiling by Microarray and RNA-seq
- Protocol 5: Mapping Billions of Short Reads to a Reference Genome
- Protocol 6: Identifying Regions Enriched in a ChIP-seq Data Set (Peak Finding)
- Protocol 7: Discovering cis-Regulatory Motifs
- Panel: Bioinformatics541
- Panel: Introduction to Sequence Alignment and Homology Search554
- Panel: Introduction to Expression Profiling by Microarray and RNA-seq571
- Panel: Introduction to Mapping Billions of Short Reads to a Reference Genome588
- Panel: Introduction to Peak-Finding Algorithms598
- Panel: Introduction to Motif Finding612
- Panel: Data Formats625
- Panel: Algorithms, Portals, and Methods628
- Chapter 1: Isolation and Quantification of DNA1
- Volume 2
- Chapter 9: Quantification of DNA and RNA by Real-Time Polymerase Chain Reaction631
- Protocol 1: Optimizing Primer and Probe Concentrations for Use in Real-Time PCR
- Protocol 2: Constructing a Standard Curve
- Protocol 3: Quantification of DNA by Real-Time PCR
- Protocol 4: Quantification of RNA by Real-Time RT-PCR
- Protocol 5: Analysis and Normalization of Real-Time PCR Experimental Data
- Panel: Quantification of DNA and RNA by Real-Time Polymerase Chain Reaction631
- Panel: Real-Time PCR Chemistries632
- Panel: Instruments for Real-Time PCR639
- Panel: Extracting Data from a Real-Time PCR Experiment: Data Analysis and Normalization Methods641
- Panel: Designing Primers and Probes and Optimizing Conditions for Real-Time PCR643
- Panel: Constructing a Standard Curve648
- Panel: Performing Real-Time PCR650
- Panel: Performing Real-Time RT-PCR650
- Panel: MIQE Guidelines654
- Panel: Real-Time PCR Protocols654
- Panel: Multiplex PCR680
- Panel: SNP Genotyping681
- Chapter 10: Nucleic Acid Platform Technologies683
- Protocol 1: Printing Microarrays
- Protocol 2: Round A/Round B Amplification of DNA
- Protocol 3: T7 Linear Amplification of DNA (TLAD) for Nucleosomal and Other DNA < 500 bp
- Protocol 4: Amplification of RNA
- Protocol 5: Direct Cyanine-dUTP Labeling of RNA
- Protocol 6: Indirect Aminoallyl-dUTP Labeling of RNA
- Protocol 7: Cyanine-dCTP Labeling of DNA Using Klenow
- Protocol 8: Indirect Labeling of DNA
- Protocol 9: Blocking Polylysines on Homemade Microarrays
- Protocol 10: Hybridization to Homemade Microarrays
- Panel: Nucleic Acid Platform Technologies683
- Panel: Microarray Applications685
- Panel: Performing Microarray Experiments688
- Chapter 11: DNA Sequencing735
- Protocol 1: Preparing Plasmid Subclones for Capillary Sequencing
- Protocol 2: Preparing PCR Products for Capillary Sequencing
- Protocol 3: Cycle-Sequencing Reactions
- Protocol 4: Whole Genome: Manual Library Preparation
- Protocol 5: Whole Genome: Automated, Nonindexed Library Preparation
- Protocol 6: Whole Genome: Automated, Indexed Library Preparation
- Protocol 7: Preparation of a 3-kb Mate-Pair Library for Illumina Sequencing
- Protocol 8: Preparation of an 8-kb Mate-Pair Library for Illumina Sequencing
- Protocol 9: RNA-Seq: RNA Conversion to cDNA and Amplification
- Protocol 10: Solution-Phase Exome Capture
- Protocol 11: Automated Size Selection
- Protocol 12: Library Quantification Using SYBR Green-qPCR
- Protocol 13: Library Quantification Using PicoGreen Fluorometry
- Protocol 14: Library Quantification: Fluorometric Quantitation of Double-Stranded or Single-Stranded DNA Samples Using the Qubit System
- Protocol 15: Preparation of Small-Fragment Libraries for 454 Sequencing
- Protocol 16: sstDNA Library Capture and emPCR
- Protocol 17: Roche/454 Sequencer: Executing a Sequencing Run
- Protocol 18: Validation
- Protocol 19: Quality Assessment of Sequence Data
- Protocol 20: Data Analysis
- Panel: DNA Sequencing735
- Panel: History of Sanger/Dideoxy DNA Sequencing736
- Panel: Next-Generation Sequencing742
- Panel: Overview of Next-Generation Sequencing Instruments752
- Panel: Sanger Sequencing versus Next-Generation Sequencing: When to Do What?760
- Panel: Introduction to Protocols761
- Panel: Additional Protocol: Automated Library Preparation789
- Panel: Additional Protocol: AMPure Bead Calibration821
- Panel: Additional Protocol: RNAClean XP Bead Cleanup (before RNA-Seq)830
- Panel: Additional Protocol: AMPure XP Bead Cleanup840
- Panel: Additional Protocol: Agarose Gel Size Selection842
- Panel: Biotin888
- Panel: Magnetic Beads890
- Panel: Fragmenting of DNA892
- Chapter 12: Analysis of DNA Methylation in Mammalian Cells893
- Protocol 1: DNA Bisulfite Sequencing for Single-Nucleotide-Resolution DNA Methylation Detection
- Protocol 2: Methylation-Specific PCR for Gene-Specific DNA Methylation Detection
- Protocol 3: Methyl-Cytosine-Based Immunoprecipitation for DNA Methylation Analysis
- Protocol 4: High-Throughput Deep Sequencing for Mapping Mammalian DNA Methylation
- Protocol 5: Roche 454 Clonal Sequencing of Bisulfite-Converted DNA Libraries
- Protocol 6: Illumina Sequencing of Bisulfite-Converted DNA Libraries
- Panel: Analysis of DNA Methylation in Mammalian Cells893
- Panel: DNA Methylation Affects and Reveals Biological Phenomena894
- Panel: Experimental Approaches for Analysis of DNA Methylation895
- Panel: Advantages and Limitations of Different Approaches for Analyzing DNA Methylation898
- Panel: Future Perspectives899
- Panel: Public Domain Software for Identifying CpG Islands in Promoter and Coding Regions of Mammalian Genes937
- Panel: Designing Primers for the Amplification of Bisulfite-Converted Product to Perform Bisulfite Sequencing and MS-PCR939
- Panel: Postsequence Processing of High-Throughput Bisulfite Deep-Sequencing Data940
- Chapter 13: Preparation of Labeled DNA, RNA, and Oligonucleotide Probes943
- Protocol 1: Random Priming: Labeling of Purified DNA Fragments by Extension of Random Oligonucleotides
- Protocol 2: Random Priming: Labeling of DNA by Extension of Random Oligonucleotides in the Presence of Melted Agarose
- Protocol 3: Labeling of DNA Probes by Nick Translation
- Protocol 4: Labeling of DNA Probes by Polymerase Chain Reaction
- Protocol 5: Synthesis of Single-Stranded RNA Probes by In Vitro Transcription
- Protocol 6: Synthesis of cDNA Probes from mRNA Using Random Oligonucleotide Primers
- Protocol 7: Radiolabeling of Subtracted cDNA Probes by Random Oligonucleotide Extension
- Protocol 8: Labeling 3 Termini of Double-Stranded DNA Using the Klenow Fragment of E. coli DNA Polymerase I
- Protocol 9: Dephosphorylation of DNA Fragments with Alkaline Phosphatase
- Protocol 10: Phosphorylation of DNA Molecules with Protruding 5-Hydroxyl Termini
- Protocol 11: Phosphorylation of DNA Molecules with Dephosphorylated Blunt Ends or Recessed 5 Termini
- Protocol 12: Phosphorylating the 5 Termini of Oligonucleotides Using T4 Polynucleotide Kinase
- Protocol 13: Labeling the 3 Termini of Oligonucleotides Using Terminal Deoxynucleotidyl Transferase
- Protocol 14: Labeling of Synthetic Oligonucleotides Using the Klenow Fragment of E. coli DNA Polymerase I
- Protocol 15: Purification of Labeled Oligonucleotides by Precipitation with Ethanol
- Protocol 16: Purification of Labeled Oligonucleotides by Size-Exclusion Chromatography
- Protocol 17: Purification of Labeled Oligonucleotides by Chromatography on a Sep-Pak C18 Column
- Protocol 18: Hybridization of Oligonucleotide Probes in Aqueous Solutions: Washing in Buffers Containing Quaternary Ammonium Salts
- Panel: Preparation of Labeled DNA, RNA, and Oligonucleotide Probes943
- Panel: Radioactive versus Nonradioactive Labeling of Nucleic Acids944
- Panel: Types of Nonradioactive Detection Systems948
- Panel: Designing Oligonucleotides for Use as Probes953
- Panel: Additional Protocol: Asymmetric Probes982
- Panel: Additional Protocol: Using PCR to Add Promoters for Bacteriophage-Encoded RNA Polymerases to Fragments of DNA991
- Panel: Alternative Protocol: Synthesizing Nonradiolabeled Probes Using TdT1023
- Panel: Additional Protocol: Tailing Reaction1024
- Panel: Additional Protocol: Modifications for Synthesizing Nonradiolabeled Probes1026
- Panel: Preparation of Stock Solutions of dNTPs1043
- Panel: E. coli DNA Polymerase I and the Klenow Fragment1044
- Panel: In Vitro Transcription Systems1050
- Panel: Alkaline Phosphatase1053
- Panel: Melting Temperatures1055
- Chapter 14: Methods for In Vitro Mutagenesis1059
- Protocol 1: Random Mutagenesis Using Error-Prone DNA Polymerases
- Protocol 2: Creating Insertions or Deletions Using Overlap Extension PCR Mutagenesis
- Protocol 3: In Vitro Mutagenesis Using Double-Stranded DNA Templates: Selection of Mutants with DpnI
- Protocol 4: Altered -Lactamase Selection Approach for Site-Directed Mutagenesis
- Protocol 5: Oligonucleotide-Directed Mutagenesis by Elimination of a Unique Restriction Site (USE Mutagenesis)
- Protocol 6: Saturation Mutagenesis by Codon Cassette Insertion
- Protocol 7: Random Scanning Mutagenesis
- Protocol 8: Multisite-Directed Mutagenesis
- Protocol 9: Megaprimer PCR-Based Mutagenesis
- Panel: Methods for In Vitro Mutagenesis1059
- Panel: Mutagenesis Approaches1064
- Panel: Research Goals1065
- Panel: Commercial Kits1065
- Panel: Domain Mutagenesis1127
- Panel: High-Throughput Site-Directed Mutagenesis of Plasmid DNA1128
- Panel: N6-Methyladenine, Dam Methylase, and Methylation-Sensitive Restriction Enzymes1129
- Chapter 15: Introducing Genes into Cultured Mammalian Cells1131
- Protocol 1: DNA Transfection Mediated by Cationic Lipid Reagents
- Protocol 2: Calcium-Phosphate-Mediated Transfection of Eukaryotic Cells with Plasmid DNAs
- Protocol 3: Calcium-Phosphate-Mediated Transfection of Cells with High-Molecular-Weight Genomic DNA
- Protocol 4: Transfection Mediated by DEAE-Dextran: High-Efficiency Method
- Protocol 5: DNA Transfection by Electroporation
- Protocol 6: Analysis of Cell Viability by the alamarBlue Assay
- Protocol 7: Analysis of Cell Viability by the Lactate Dehydrogenase Assay
- Protocol 8: Analysis of Cell Viability by the MTT Assay
- Panel: Introducing Genes into Cultured Mammalian Cells1131
- Panel: Transient Versus Stable Transfection1133
- Panel: Transfection Methods1133
- Panel: Transfection Controls1133
- Panel: Optimization and Special Considerations1136
- Panel: Assessing Cell Viability in Transfected Cell Lines1137
- Panel: Alternative Protocol: Transfection Using DOTMA and DOGS1145
- Panel: Additional Protocol: Histochemical Staining of Cell Monolayers for -Galactosidase1148
- Panel: Alternative Protocol: High-Efficiency Calcium-Phosphate-Mediated Transfection of Eukaryotic Cells with Plasmid DNAs1156
- Panel: Alternative Protocol: Calcium-Phosphate-Mediated Transfection of Adherent Cells1163
- Panel: Alternative Protocol: Calcium-Phosphate-Mediated Transfection of Cells Growing in Suspension1165
- Panel: Alternative Protocol: Transfection Mediated by DEAE-Dextran: Increased Cell Viability1170
- Panel: Optical Transfection1186
- Panel: Cotransformation1188
- Panel: Selective Agents for Stable Transformation1190
- Panel: Lipofection1194
- Panel: Linearizing Plasmids before Transfection1197
- Panel: Transfection of Mammalian Cells with Calcium PhosphateDNA Coprecipitates1198
- Panel: Chloroquine Diphosphate1200
- Panel: DEAE-Dextran Transfection1201
- Panel: Electroporation1203
- Chapter 16: Introducing Genes into Mammalian Cells: Viral Vectors1209
- Protocol 1: Construction of Recombinant Adenovirus Genomes by Direct Cloning
- Protocol 2: Release of the Cloned Recombinant Adenovirus Genome for Rescue and Expansion
- Protocol 3: Purification of the Recombinant Adenovirus by Cesium Chloride Gradient Centrifugation
- Protocol 4: Characterization of the Purified Recombinant Adenovirus for Viral Genome Structure by Restriction Enzyme Digestions
- Protocol 5: Measuring the Infectious Titer of Recombinant Adenovirus Using TCID50 End-Point Dilution and qPCR
- Protocol 6: Detection Assay for Replication-Competent Adenovirus by Concentration Passage and Real-Time qPCR
- Protocol 7: Production of rAAVs by Transient Transfection
- Protocol 8: Purification of rAAVs by Cesium Chloride Gradient Sedimentation
- Protocol 9: Purification of rAAVs by Iodixanol Gradient Centrifugation
- Protocol 10: Purification of rAAV2s by Heparin Column Affinity Chromatography
- Protocol 11: Enrichment of Fully Packaged Virions in Column-Purified rAAV Preparations by Iodixanol Gradient Centrifugation Followed by Anion-Exchange Column Chromatography
- Protocol 12: Titration of rAAV Genome Copy Number Using Real-Time qPCR
- Protocol 13: Sensitive Determination of Infectious Titer of rAAVs Using TCID50 End-Point Dilution and qPCR
- Protocol 14: Analysis of rAAV Sample Morphology Using Negative Staining and High-Resolution Electron Microscopy
- Protocol 15: Analysis of rAAV Purity Using Silver-Stained SDS-PAGE
- Protocol 16: Production of High-Titer Retrovirus and Lentivirus Vectors
- Protocol 17: Titration of Lentivirus Vectors
- Protocol 18: Monitoring Lentivirus Vector Stocks for Replication-Competent Viruses
- Panel: Introducing Genes into Mammalian Cells: Viral Vectors1209
- Panel: Factors to Consider When Choosing a Viral Vector1211
- Panel: The Major Types of Viruses Currently Used as Vectors1212
- Panel: In Vivo Expression1221
- Panel: Adenovirus Vectors1221
- Panel: Adeno-Associated Virus Vectors1224
- Panel: Retrovirus and Lentivirus Vectors1227
- Panel: Additional Protocol: Preparation of a DNA Standard for qPCR1262
- Panel: Adenovirus Vectors1322
- Panel: AAV Vectors1323
- Panel: Lentivirus Vectors1324
- Panel: Basic Elements in Viral Vectors1326
- Panel: Assays Done in Transduced Cells1328
- Panel: Transgene Expression Cassettes1330
- Chapter 9: Quantification of DNA and RNA by Real-Time Polymerase Chain Reaction631
- Volume 3
- Chapter 17: Analysis of Gene Regulation Using Reporter Systems1335
- Protocol 1: Assay for -Galactosidase in Extracts of Mammalian Cells
- Protocol 2: Single Luciferase Reporter Assay
- Protocol 3: Dual Luciferase Reporter Assay
- Protocol 4: Using ELISA to Measure GFP Production
- Protocol 5: Generation of Cell Lines with Tetracycline-Regulated Gene Expression
- Panel: Analysis of Gene Regulation Using Reporter Systems1335
- Panel: Introduction to Reporter Systems1336
- Panel: Reporter Genes Used in the Analysis of Regulatory Elements1336
- Panel: Assaying for -Galactosidase in Extracts of Mammalian Cells1338
- Panel: Assaying for Luciferase in Extracts of Mammalian Cells1339
- Panel: Tetracycline-Responsive Expression Systems1341
- Panel: Additional Protocol: Chemiluminescent Assay for -Galactosidase Activity1350
- Panel: Additional Protocol: Selecting Stable Clones via Limiting Dilution of Suspension Cells1378
- Panel: Fluorescent Proteins1381
- Panel: Epitope Tagging1394
- Panel: -Galactosidase1401
- Panel: Luciferase1406
- Panel: Tetracycline1409
- Chapter 18: RNA Interference and Small RNA Analysis1415
- Protocol 1: Preparation of siRNA Duplexes
- Protocol 2: RNAi in Mammalian Cells by siRNA Duplex Transfection
- Protocol 3: RNAi in Drosophila S2 Cells by siRNA Duplex Transfection
- Protocol 4: Preparation of dsRNAs by In Vitro Transcription
- Protocol 5: RNAi in Drosophila S2 Cells by dsRNA Soaking
- Protocol 6: RNAi in Drosophila S2 Cells by dsRNA Transfection
- Protocol 7: Analysis of Small RNAs by Northern Hybridization
- Protocol 8: Analysis of Small RNAs by Quantitative Reverse Transcription PCR
- Protocol 9: Construction of Small RNA Libraries for High-Throughput Sequencing
- Protocol 10: Preparation of Antisense Oligonucleotides to Inhibit miRNA Function
- Protocol 11: Inhibiting miRNA Function by Antisense Oligonucleotides in Cultured Mammalian Cells
- Protocol 12: Inhibiting miRNA Function by Antisense Oligonucleotides in Drosophila S2 Cells
- Panel: RNA Interference and Small RNA Analysis1415
- Panel: Reverse Genetics by RNAi1419
- Panel: Analysis of Small RNAs1425
- Panel: Genome-Wide RNA Interference: Functional Genomics in the Postgenomics Era1472
- Panel: StarFire Probes1478
- Chapter 19: Expressing Cloned Genes for Protein Production, Purification, and Analysis1481
- Protocol 1: Expression of Cloned Genes in E. coli Using IPTG-Inducible Promoters
- Protocol 2: Expression of Cloned Genes Using the Baculovirus Expression System
- Protocol 3: Expression of Cloned Genes in P. pastoris Using the Methanol-Inducible Promoter AOX1
- Protocol 4: Preparation of Cell Extract for Purification of Soluble Proteins Expressed in E. coli
- Protocol 5: Purification of Polyhistidine-Tagged Proteins by Immobilized Metal Affinity Chromatography
- Protocol 6: Purification of Fusion Proteins by Affinity Chromatography on Glutathione Resin
- Protocol 7: Solubilization of Expressed Proteins from Inclusion Bodies
- Protocol 8: SDS-PAGE of Proteins
- Protocol 9: Analysis of Proteins by Immunoblotting
- Protocol 10: Methods for Measuring the Concentrations of Proteins
- Panel: Expressing Cloned Genes for Protein Production, Purification, and Analysis1481
- Panel: Choosing an Expression System1483
- Panel: Choosing an Appropriate Expression Vector1488
- Panel: Fusion Proteins1499
- Panel: Optimization of Expression of Foreign Proteins1503
- Panel: Additional Protocol: Small-Scale Test for Soluble Target Protein Expression1514
- Panel: Alternative Protocol: Expression of Cloned Genes in E. coli Using the Arabinose BAD Promoter1520
- Panel: Alternative Protocol: Subcellular Localization of Signal Peptide Fusion Proteins1522
- Panel: Additional Protocol: Plaque Assay to Determine the Titer of the Baculovirus Stock1535
- Panel: Alternative Protocol: Production of Bacmid DNA for Transfection into Insect Cells1538
- Panel: Additional Protocol: Cryostorage of Yeast Cultures1553
- Panel: Additional Protocol: Lysis of Yeast Cells Using Glass Beads1564
- Panel: Alternative Protocol: Preparation of E. coil Cell Extract Using Gentle, Heat-Induced Enzymatic Lysis1566
- Panel: Alternative Protocol: Preparation of E. coli Cell Extract Using FreezeThaw with Enzymatic Lysis by Lysozyme1568
- Panel: Additional Protocol: Regenerating and Cleaning the Ni2NTA Resin1579
- Panel: Alternative Protocol: Fast Performance Liquid Chromatography Purification of Histidine-Tagged Proteins1581
- Panel: Alternative Protocol: Variations of Staining SDSPolyacrylamide Gels with Coomassie Brilliant Blue1609
- Panel: Alternative Protocol: Staining SDSPolyacrylamide Gels with Silver Salts1611
- Panel: Considerations for Membrane Protein Purification1632
- Panel: Historical Footnote: Coomassie Brilliant Blue1636
- Chapter 20: Cross-Linking Technologies for Analysis of Chromatin Structure and Function1637
- Protocol 1: Formaldehyde Cross-Linking
- Protocol 2: Preparation of Cross-Linked Chromatin for ChIP
- Protocol 3: ChIP
- Protocol 4: ChIPQuantitative PCR (ChIP-qPCR)
- Protocol 5: ChIP-chip
- Protocol 6: ChIP-seq
- Protocol 7: Generation of 3C Libraries from Cross-Linked Cells
- Protocol 8: Generation of ChIP-loop Libraries
- Protocol 9: Generation of Control Ligation Product Libraries
- Protocol 10: PCR Detection of 3C Ligation Products Present in 3C, ChIP-loop, and Control Libraries: Library Titration and Interaction Frequency Analysis
- Protocol 11: 4C Analysis of 3C, ChIP-loop, and Control Libraries
- Protocol 12: 5C Analysis of 3C, ChIP-loop, and Control Libraries
- Panel: Cross-Linking Technologies for Analysis of Chromatin Structure and Function1637
- Panel: Formaldehyde Cross-Linking to Interrogate Genomic Interactions1638
- Panel: ChIP Analysis of ProteinDNA Interactions1638
- Panel: 3C-Based Chromatin Interaction Analyses1641
- Panel: Formaldehyde1701
- Panel: What Is Captured by 3C-Based Assays?1702
- Chapter 21: Mapping of In Vivo RNA-Binding Sites by UV-Cross-Linking Immunoprecipitation (CLIP)1703
- Protocol 1: Optimization of Immunoprecipitation Stringency for CLIP
- Protocol 2: UV Cross-Linking of Live Cells and Lysate Preparation
- Protocol 3: RNase Titration, Immunoprecipitation, and SDS-PAGE
- Protocol 4: 3-Linker Ligation and Size Selection by SDS-PAGE
- Protocol 5: Isolation of the RNA Tags, 5-Linker Ligation, and Reverse Transcription PCR Amplification
- Protocol 6: Sequencing of RNA CLIP Tags
- Protocol 7: Gel Purification and Storage of RNA Linkers
- Panel: Mapping of In Vivo RNA-Binding Sites by UV-Cross-Linking Immunoprecipitation (CLIP)1703
- Panel: The Cross-Linking Immunoprecipitation Method1706
- Panel: High-Throughput Sequencing (HITS) CLIP1708
- Panel: Validation of CLIP Results1708
- Panel: CLIP Method Variations1709
- Panel: General Considerations in Planning CLIP Experiments1710
- Panel: Alternative Protocol: 5-End Labeling of Dephosphorylated RL3 Linker1738
- Panel: Mechanism and Specificity of UV-Protein Cross-Linking1756
- Panel: HITS-CLIP Data Analysis1758
- Chapter 22: Gateway-Compatible Yeast One-Hybrid and Two-Hybrid Assays1761
- Protocol 1: Generating Yeast One-Hybrid DNA-Bait Strains
- Protocol 2: Generating Yeast Two-Hybrid Bait Strains
- Protocol 3: Identifying Interactors from an Activation Domain Prey Library
- Protocol 4: High-Efficiency Yeast Transformation
- Protocol 5: Colony Lift Colorimetric Assay for -Galactosidase Activity
- Protocol 6: Yeast Colony PCR
- Panel: Gateway-Compatible Yeast One-Hybrid and Two-Hybrid Assays1761
- Panel: The Yeast Two-Hybrid (Y2H) System: Concept and Methodology1763
- Panel: The Yeast One-Hybrid (Y1H) System: Concept and Methodology1767
- Panel: Y2H and Y1H Assays: Advantages and Disadvantages1768
- Panel: False Positives1769
- Panel: Protocols for Yeast One-Hybrid and Two-Hybrid Systems1770
- Panel: Alternative Protocol: Creating Entry Clones from DNA-Baits Generated by Annealing Primers1782
- Panel: Why Integrate DNA-Baits?1808
- Panel: Choosing a Vector and a Yeast Strain1809
- Panel: Replica-Plating and Replica-Cleaning Using Velvets1810
- Panel: Reagents and Buffers1811
- Panel: Tris Buffers1828
- Panel: Good Buffers1829
- Panel: Phosphate Buffers (Gomori Buffers)1830
- Panel: Phenol1834
- Panel: Equilibration of Phenol1834
- Panel: Phenol:Chloroform:Isoamyl Alcohol (25:24:1)1834
- Panel: Deionization of Formamide1834
- Panel: Blocking Agents Used for Nucleic Acid Hybridization1836
- Panel: Blocking Agents Used for Western Blotting1836
- Panel: Commonly Used Techniques1843
- Panel: Siliconizing Glassware, Plasticware, and Glass Wool1843
- Panel: Preparation of RNase-Free Glassware1844
- Panel: Hemocytometry Counting1846
- Panel: Viability Staining1847
- Panel: Precipitation of Nucleic Acids with Trichloroacetic Acid1849
- Panel: Removing Ethidium Bromide from DNA1851
- Panel: Disposing of Ethidium Bromide1851
- Panel: Decontamination of Concentrated Solutions of Ethidium Bromide (Solutions Containing >0.5 mg/mL)1851
- Panel: Decontamination of Dilute Solutions of Ethidium Bromide (e.g., Electrophoresis Buffer Containing 0.5 g/mL Ethidium Bromide)1852
- Panel: Commercial Decontamination Kits1852
- Panel: Detection Systems1855
- Panel: Ethidium Bromide1855
- Panel: Methylene Blue1857
- Panel: SYBR Dyes1857
- Panel: Chemiluminescent Labels1860
- Panel: Chemiluminescent Enzyme Assays1861
- Panel: Commercial Reagents, Kits, and Luminometers1863
- Panel: Horseradish Peroxidase1865
- Panel: Digoxygenin1869
- Panel: BCIP1873
- Panel: AMPPD1876
- Panel: Immunoglobulin-Binding Proteins: Proteins A, G, and L1879
- Panel: General Safety and Hazardous Material1885
- Chapter 17: Analysis of Gene Regulation Using Reporter Systems1335
Formaldehyde Cross-Linking
(Protocol summary only for purposes of this preview site)Formaldehyde cross-linking of DNA to associated proteins is a relatively straightforward method, but it is also the most critical step in the ChIP and 3C analyses. Although formaldehyde is a highly permeable cross-linker, its maximum cross-linking efficiencies are estimated to be at 1 for mammalian cells because reactivity is limited to amines. Therefore, a relatively large number of cells are required for 3C and ChIP-based assays. Five hundred million cross-linked diploid cells are equivalent to 1.66 fmol of the genome. Thus, only 100 amol of genomic copies is analyzed for one ChIP assay. Because the quality of cross-linked chromatin can vary, even when generated under near-identical conditions, it is preferable to generate multiple large batches. Different cell types require different cross-linking conditions and approaches. Suspension cells are easier to cross-link and harvest because they are not attached to the growth surfaces. Formaldehyde can simply be added to the medium. Cross-linked cells are then harvested by centrifugation. When cross-linking cells that are induced with growth factors and signaling molecules, a careful plan for cross-linking and harvesting cells should be formulated before the start of the experiment. For adherent cells, decide whether to cross-link before or after harvesting them from the plates. Note that adherent cells might be difficult to recover completely when cross-linking is carried out when the cells are attached to the plates,
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