- 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
Expression of Cloned Genes in P. pastoris Using the Methanol-Inducible Promoter AOX1
(Protocol summary only for purposes of this preview site)Pichia pastoris is a methylotrophic yeast capable of metabolizing methanol as its sole carbon source (Cregg et al. 1985). Growth in methanol-containing medium results in dramatic induction of genes in the alcohol oxidation pathway including alcohol oxidase (AOX), formaldehyde dehydrogenase (FLD), and dihydroxyacetone synthase (DHAS) (Cregg 2007a). These proteins may comprise up to 30 of the biomass. Investigators have exploited these methanol-dependent genes to generate tightly regulated expression vectors (Daly and Hearn 2005). Most Pichia vectors use the strong and tightly regulated AOX1 promoter to drive heterologous protein expression. The pPICZ vector is shown in Figure 1. After cloning, the heterologous gene will be flanked by the AOX1 promoter and AOX1 transcription termination sequences. These sequences are homologous to the corresponding AOX1 regions in the Pichia genome and facilitate integration of the heterologous gene into the genome via homologous recombination (Cregg et al. 1985, 1989).
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