Colony PCR 8. Ochman H., et al. The inverse PCR method involves a series of restriction digests and ligation, resulting in a looped fragment that can be primed for PCR from a single section of known sequence. Nested PCR 5. It is one of the most important biotechnological tools developed. One-step vs. Two-step RT-qPCR. PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA complementary to the offered template strand. Reverse transcriptase PCR 12. This technique was developed by Kary Mullis who was awarded the Nobel Prize in 1993 for t… Inverse PCR - Isolate partially purified chromosomal DNA using a modification of the Magic MiniPreps DNA Purification System from Promega (see atta ched) as follows. Principle and applications of . Inverse PCR is especially useful for the determination of insert locations. Band stab PCR 9. In both cases, RNA is first reverse-transcribed into cDNA, which is then used as the template for PCR amplification. . The template for the reverse primers is a restriction fragment that has been selfligated Inverse PCR functions to clone sequences flanking a known sequence. The target DNA is cleaved with a restriction endonuclease which does not cut the known sequence but cuts the unknown sequence on either side. 1). The PCR involves the primer mediated enzymatic amplification of DNA. Principles and procedure • Most PCR methods typically amplify DNA fragments of up to ~10 kilo base pairs (kb) (some techniques up to 40 kb) • A basic PCR set up requires several ... • Inverse PCR: is commonly used to identify the flanking sequences around genomic inserts. It Genetics. Inverse polymerase chain reaction (Inverse PCR) is a variant of the polymerase chain reaction that is used to amplify DNA with only one known sequence. RT-qPCR can be performed in a one-step or a two-step assay (Figure 1, Table 1).One-step assays combine reverse transcription and PCR in a single tube and buffer, using a reverse transcriptase along with a DNA polymerase. The DNA is cut with a restriction enzyme that cuts upstream and downstream of the known region but not within it. The linear piece of DNA is circularized and then amplified with primers that anneal in the known region. Inverse PCR Inverse PCR (Ochman et al., 1988) uses standard PCR (polymerase chain reaction)- primers oriented in the reverse direction of the usual orientation. Reverse Transcribed PCR. Inverse PCR enables amplification of a region of unknown sequence using primers oriented in the reverse direction. Quantitative PCR is also called real-time PCR. DNA polymerase then elongate its 3 end by adding more nucleotides to generate an extende… The enzyme involved in the synthesis of new DNA strands by binding with a single DNA strand. However, inverse PCR is still used extensively for rapid allelotyping and to determine the locations at which retroviruses, transgenes, and transposons are integrated into genomes. What is Inverse PCR, and how it works. Under low DNA concentrations, self-ligation is induced to give a circular DNA product. Inverse PCR- Inverse PCR is a method used to . The higher concentration primer continues to primer synthesis, but only of its strand. Genetic applications of an inverse Polymerase Chain Reaction . - Spin down 500 μl of an overnight culture in a 1.5 ml microfuge tube. Inverse PCR 2. [1] To identify the sites where they have entered, the known, "internal" viral or transposon sequences can be used to design primers that will amplify a small portion of the flanking, "external" genomic DNA. 11. DNA synthesis occurs outside the known DNA region. Asymmetric PCR 15. Inverse PCR allows unknown sequences to be amplified by PCR provided that they are located near a known sequence. Degenerate PCR 13. Tip: “Stitching” Fragments Together using Oligos When you need intervening sequence between two PCR products, one method is to “stitch” together several oligos. Principle of RT-PCR Reverse transcription and PCR amplification can be performed as a two-step process in a single tube or with two separate reactions. PCR is carried out as usual, with primers complementary to sections of the known internal sequence.*. The linear piece of DNA is circularized and then amplified with primers that anneal in the known region. Inverse PCR is helpful for investigating the promoter sequence of a gene; oncogenic chromosomal rearrangements such as gene fusion, translocation, and transposition; and viral gene integration. As asymmetric PCR proceeds, the lower concentration primer is quantitatively incorporated into double-stranded DNA. Real-Time qRT-PCR Introduction Real-Time qRT-PCR (Real-Time Quantitative Reverse Transcription PCR) is a major development of PCR technology that enables reliable detection and measurement of products generated during each cycle of PCR process. Assembly PCR 16. Quantitative PCR. This article describes the principle, procedure, protocol, application and limitation of reverse PCR. Polymerase chain reaction itself is the process used to amplify DNA samples, via a temperature-mediated DNA polymerase.The products can be used for sequencing or analysis, and this process is a key part of many genetics research laboratories, along with uses in DNA fingerprinting for forensics and other human genetic cases. This page was last edited on 23 September 2019, at 07:05. Random Amplified Polymorphic DNA (RAPD) Introduction Random Amplified Polymorphic DNA (RAPD) markers are DNA fragments from PCR amplification of random segments of genomic DNA with single primer of arbitrary nucleotide sequence.. How It Works. Inverse PCR Molecular Cloning, also known as Maniatis, has served as the foundation of technical expertise in labs worldwide for 30 years. cDNA synthesis (aka reverse transcription or RT): cDNA is a … Learn how and when to remove this template message, "Genetic applications of an inverse polymerase chain reaction", Reverse transcription polymerase chain reaction, Overlap extension polymerase chain reaction, Multiplex ligation-dependent probe amplification, co-amplification at lower denaturation temperature-PCR, https://en.wikipedia.org/w/index.php?title=Inverse_polymerase_chain_reaction&oldid=917307749, Articles lacking in-text citations from April 2012, Creative Commons Attribution-ShareAlike License, A target region with an internal section of known sequence and unknown flanking regions is identified, Genomic DNA is digested into fragments of a few. This procedure of inverse PCR (IPCR) has many applications in molecular genetics, for example, the amplification and identification of sequences flanking transposable elements. These days, sequencing would in most cases be the method of choice to characterize an unknown segment of DNA. One limitation of conventional PCR is that it requires primers complementary to both termini of the target DNA, but this method allows PCR to be carried out even if only one sequence is available from which primers may be designed. The inverse PCR method involves a series of restriction digests and ligation, resulting in a looped fragment that can be primed for PCR from a single section of known sequence. DNA polymerase is the key enzyme that is present behind the whole process. Protocol 3: Isolating DNA from Gram-Negative Bacteria (e.g., 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 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, 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 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, Protocol 1: The Hanahan Method for Preparation and Transformation of Competent, Protocol 2: The Inoue Method for Preparation and Transformation of Competent, 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, Protocol 2: Generating an ORF Entry Clone and Destination Clone, Protocol 3: Using Multisite LR Cloning to Generate a Destination Clone, 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 16: Small-Scale Preparations of Yeast DNA, Protocol 1: Purification of Total RNA from Mammalian Cells and Tissues, Protocol 2: Isolation of Total RNA from Zebrafish Embryos and Adults, Protocol 7: Precipitation of RNA with Ethanol, Protocol 8: Removing DNA Contamination from RNA Samples by Treatment with RNase-Free DNase I, 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 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, Protocol 1: The Basic Polymerase Chain Reaction, Protocol 4: PCR Amplification of GC-Rich Templates, Protocol 5: Long and Accurate PCR (LA 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 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 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, 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 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 9: Blocking Polylysines on Homemade Microarrays, Protocol 10: Hybridization to Homemade Microarrays, Protocol 1: Preparing Plasmid Subclones for Capillary Sequencing, Protocol 2: Preparing PCR Products for Capillary Sequencing, 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 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 19: Quality Assessment of Sequence Data, 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, 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, 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, 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 C, Protocol 18: Hybridization of Oligonucleotide Probes in Aqueous Solutions: Washing in Buffers Containing Quaternary Ammonium Salts, 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 8: Multisite-Directed Mutagenesis, Protocol 9: Megaprimer PCR-Based Mutagenesis, 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, 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 TCID, 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 TCID, 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, 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, Protocol 1: Preparation of siRNA Duplexes, Protocol 2: RNAi in Mammalian Cells by siRNA Duplex Transfection, Protocol 4: Preparation of dsRNAs by In Vitro Transcription, 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, Protocol 1: Expression of Cloned Genes in, Protocol 2: Expression of Cloned Genes Using the Baculovirus Expression System, Protocol 3: Expression of Cloned Genes in, Protocol 4: Preparation of Cell Extract for Purification of Soluble Proteins Expressed in, 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 9: Analysis of Proteins by Immunoblotting, Protocol 10: Methods for Measuring the Concentrations of Proteins, Protocol 2: Preparation of Cross-Linked Chromatin for ChIP, Protocol 4: ChIPQuantitative PCR (ChIP-qPCR), 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, 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 7: Gel Purification and Storage of RNA Linkers, 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, Chapter 1: Isolation and Quantification of DNA1, Panel: Isolation and Quantification of DNA1, Panel: Commercial Kits for Purification of DNA3, 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: Minimizing Damage to Large DNA Molecules77, Panel: Additional Protocol: Autoradiography of Alkaline Agarose Gels117, Panel: Additional Protocol: Stripping Probes from Membranes150, Panel: Formamide and Its Uses in Molecular Cloning153, Chapter 3: Cloning and Transformation with Plasmid Vectors157, Panel: Cloning and Transformation with Plasmid Vectors157, Panel: Alternative Protocol: One-Step Preparation of Competent, 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: Condensing and Crowding Reagents240, Panel: The Discovery of Restriction Enzymes241, Chapter 4: Gateway Recombinational Cloning261, 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, 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: Primer Design for Homology Arms, Cointegration, and Resolution343, Chapter 6: Extraction, Purification, and Analysis of RNA from Eukaryotic Cells345, 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: How to Win the Battle with RNase450, Panel: The Basic Polymerase Chain Reaction456, Panel: Design of Oligonucleotide Primers for Basic PCR462, Panel: Detecting, Analyzing, and Quantifying mRNAs464, 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: Algorithms, Portals, and Methods628, Chapter 9: Quantification of DNA and RNA by Real-Time Polymerase Chain Reaction631, Panel: Quantification of DNA and RNA by Real-Time Polymerase Chain Reaction631, 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, Chapter 10: Nucleic Acid Platform Technologies683, Panel: Nucleic Acid Platform Technologies683, Panel: Performing Microarray Experiments688, Panel: History of Sanger/Dideoxy DNA Sequencing736, Panel: Overview of Next-Generation Sequencing Instruments752, Panel: Sanger Sequencing versus Next-Generation Sequencing: When to Do What?760, 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, Chapter 12: Analysis of DNA Methylation in Mammalian Cells893, 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: 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, 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: In Vitro Transcription Systems1050, Chapter 14: Methods for In Vitro Mutagenesis1059, Panel: Methods for In Vitro Mutagenesis1059, Panel: High-Throughput Site-Directed Mutagenesis of Plasmid DNA1128, Chapter 15: Introducing Genes into Cultured Mammalian Cells1131, Panel: Introducing Genes into Cultured Mammalian Cells1131, Panel: Transient Versus Stable Transfection1133, 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: Selective Agents for Stable Transformation1190, Panel: Linearizing Plasmids before Transfection1197, Panel: Transfection of Mammalian Cells with Calcium PhosphateDNA Coprecipitates1198, Chapter 16: Introducing Genes into Mammalian Cells: Viral Vectors1209, 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: Adeno-Associated Virus Vectors1224, Panel: Retrovirus and Lentivirus Vectors1227, Panel: Additional Protocol: Preparation of a DNA Standard for qPCR1262, Panel: Basic Elements in Viral Vectors1326, Panel: Assays Done in Transduced Cells1328, Panel: Transgene Expression Cassettes1330, Chapter 17: Analysis of Gene Regulation Using Reporter Systems1335, 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, Chapter 18: RNA Interference and Small RNA Analysis1415, Panel: RNA Interference and Small RNA Analysis1415, Panel: Genome-Wide RNA Interference: Functional Genomics in the Postgenomics Era1472, Chapter 19: Expressing Cloned Genes for Protein Production, Purification, and Analysis1481, Panel: Expressing Cloned Genes for Protein Production, Purification, and Analysis1481, Panel: Choosing an Expression System1483, Panel: Choosing an Appropriate Expression Vector1488, 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, 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, Panel: Additional Protocol: Regenerating and Cleaning the Ni, 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, 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: What Is Captured by 3C-Based Assays?1702, Chapter 21: Mapping of In Vivo RNA-Binding Sites by UV-Cross-Linking Immunoprecipitation (CLIP)1703, 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: 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, Chapter 22: Gateway-Compatible Yeast One-Hybrid and Two-Hybrid Assays1761, 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: Protocols for Yeast One-Hybrid and Two-Hybrid Systems1770, Panel: Alternative Protocol: Creating Entry Clones from DNA-Baits Generated by Annealing Primers1782, Panel: Choosing a Vector and a Yeast Strain1809, Panel: Replica-Plating and Replica-Cleaning Using Velvets1810, Panel: Phosphate Buffers (Gomori Buffers)1830, Panel: Phenol:Chloroform:Isoamyl Alcohol (25:24:1)1834, Panel: Blocking Agents Used for Nucleic Acid Hybridization1836, Panel: Blocking Agents Used for Western Blotting1836, Panel: Siliconizing Glassware, Plasticware, and Glass Wool1843, Panel: Preparation of RNase-Free Glassware1844, 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: Chemiluminescent Enzyme Assays1861, Panel: Commercial Reagents, Kits, and Luminometers1863, Panel: Immunoglobulin-Binding Proteins: Proteins A, G, and L1879, Panel: General Safety and Hazardous Material1885. 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