hybridization Protocols

Category: DNA Protocols: Cosmid Library Protocols

Unamplified cosmid libraries are plated at high density onto 150-mm nitrocellulose or nylon filters and screened by hybridization - [Read Screening an Unamplified Cosmid Library by Hybridization: Plating the Library onto Filters Protocol]

Category: Bacterial Protocols

Bacterial colonies growing on agar plates are transferred en masse to nitrocellulose filters. The spatial arrangement of colonies on the plates is preserved on the filters. After transfer, the filters are processed for hybridization to an appropriate radiolabeled probe while the original (master) plate is incubated for a few hours to allow the bacterial colonies to regrow in their original positions. - [Read Screening Bacterial Colonies by Hybridization: Intermediate Numbers Protocol]

Category: Bacterial Protocols

This procedure is used to plate, replicate, and subsequently screen large numbers of bacterial colonies (up to 2 x 104 colonies per 150-mm plate or 104 colonies per 90-mm plate). - [Read Screening Bacterial Colonies by Hybridization: Large Numbers Protocol]

Category: Bacterial Protocols

Protocol used to screen a small number of bacterial colonies (<200) that are dispersed over several agar plates and are to be screened by hybridization to the same radiolabeled probe. The colonies are gridded onto a master plate and onto a nitrocellulose or nylon filter laid on the surface of a second agar plate. - [Read Screening Bacterial Colonies by Hybridization: Small Numbers Protocol]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Plaques formed by M13 bacteriophages or bacterial colonies transformed by plasmids carrying specific mutations can be detected by hybridization, using a radiolabeled oligonucleotide that forms a perfect duplex with the mutant sequence. Hybridization is carried out under conditions of low stringency that allow the radiolabeled oligonucleotide to anneal to both mutant and wild-type DNAs. - [Read Screening Recombinant Clones for Site-directed Mutagenesis by Hybridization to Radiolabeled Oligos]

Category: Nucleic Acid Electrophoresis Protocols: RNA Electrophoresis Protocols

Separation of RNAs according to size is the first stage in northern blotting and hybridization.  The method described in this protocol uses glyoxal to denature the RNA, ethidium bromide to stain it, and agarose gel electrophoresis to separate the resulting glyoxal-RNA-ethidium adducts. - [Read Separation of RNA According to Size: Electrophoresis of Glyoxylated RNA through Agarose Gels]

Category: Nucleic Acid Electrophoresis Protocols: RNA Electrophoresis Protocols

Separation of RNAs according to size is the first stage in northern blotting and hybridization.  The method described in this protocol uses formaldehyde to denature the RNA, ethidium bromide to stain it, and electrophoresis through agarose gels containing 2.2 M formamide to separate the resulting formaldehyde-RNA-ethidium adducts. - [Read Separation of RNA According to Size: Electrophoresis of RNA through Agarose Gels Containing Formalde]

Category: Cytogenetics Protocols: Fluorescent In Situ Hybridization FISH Protocol

Protocol for SKY/FISH of previously G-banded slides. Includes: G-Band Slide Pretreatment; G-Band Slide Denaturation and Hybridization. - [Read SKY/FISH of Previously G-Banded Slides Protocol]

Category: Cytogenetics Protocols: Spectral Karyotyping SKY Protocols

Protocol for SKY/FISH of previously G-Banded slides. Includes: G-Band Slide Pretreatment; G-Band Slide Denaturation and Hybridization. - [Read SKY/FISH of Previously G-Banded Slides Protocol]

Category: Nucleic Acid Detection Protocols: Nucleic Acid Blotting Protocols

Protocol for southern hybridization of radiolabeled probes to nucleic acids immobilized on membranes. Protocol describes how to carry out Southern hybridizations at high stringency in phosphate-SDS buffers.  Although a wide variety of formats are available, most Southern hybridizations are carried out in heat-sealable bags, roller bottles, or plastic boxes. - [Read Southern Hybridization of Radiolabeled Probes to Nucleic Acids Immobilized on Membranes Protocol]

Category: Microarray Protocols: Microarray Hybridization Protocols

Protocol for spotted microarray hybridization and scan. Includes: Setup Hybridization; Wash Slides; Scan Slides. - [Read Spotted Microarray Hybridization and Scan Protocol]

Category: PCR Protocols: cDNA Synthesis Protocols

Protocol describes the preparation of subtracted cDNA probes by hybridization to an mRNA driver, followed by purification of the single-stranded radiolabeled cDNA by hydroxyapatite chromatography.  Before preparing the probe, it is a good idea to have filters (which contain the cDNA library to be screened) ready to hybridize. - [Read Synthesis of Radiolabeled, Subtracted cDNA Probes Using Oligo(dT) as a Primer Protocol]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Technique yields a heterogeneous population of short radiolabeled molecules 200-300 nucleotides in length. These probes are synthesized, as in Synthesis of Single-stranded DNA Probes of Defined Length from Bacteriophage M13 Templates, by extension of an oligonucleotide primer on a single-stranded DNA template. The radiolabeled products of the reaction are then separated from the template by electrophoresis through a denaturing gel from which they are eluted directly into hybridization buffer. - [Read Synthesis of Single-stranded DNA Probes of Heterogeneous Length from Bacteriophage M13 Templates]

Category: Histology Protocols: Tissue Sample Sectioning

Tissue sectioning and in situ hybridization as prepared by Simon Dunn PDF. Includes Reagents and Solution recipes. Weis Lab, Virginia. - [Read Tissue sectioning and in situ hybridization as prepared by Simon Dunn PDF]

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

This protocol fixes and prepares embryos for in situ hybridization to visualize transcript expression patterns. It is a modification of the method developed by Tautz and Pfeifle for whole-mount in situ analysis of embryos. Use of the standard hybridization protocol on RNAi-treated embryos results in high background staining, which makes visualization of transcript expression patterns practically impossible. The following modifications eliminate this problem and allow visualization of transcript. - [Read Transcript In Situ Hybridization of Whole-Mount Embryos for Phenotype Analysis of RNAi-Treated]

Category: Microarray Protocols: Microarray Hybridization Protocols

Protocol details the preparation of fluorescently labeled target samples and hybridization of these samples to a microarray of Agilent inkjet-deposited cDNAs.  The procedure requires a minimum of 5 mg of purified total RNA as starting material. Includes: First Strand cDNA Synthesis; Second Strand cDNA Synthesis; cDNA Cleanup and Precipitation; In vitro Transcription; Cleanup and Quantification of in vitro Transcribed RNA; Fluorescent Labeling of the Target Samples. - [Read Transcript Profiling by Microarray Analysis Protocol.]

Category: Microarray Protocols: Microarray DNA Labeling Protocols

Protocol details the preparation of fluorescently labeled target samples and hybridization of these samples to a microarray of Agilent inkjet-deposited cDNAs. The procedure requires a minimum of 5 mg of purified total RNA as starting material. - [Read Transcript Profiling by Microarray Analysis—Agilent Protocol]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

To transfer phage DNA to nylon membranes so that a phage library can be screened by hybridization. - [Read Transfer of Phage DNA from Plaques to Nylon Membrane or Nitrocellulose Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Tissue Sections Protocols

Protocol for visualizing miRNAs in adult skeletal muscle by hybridization in situ. Includes: Visualizing miRNAs in adult skeletal muscle by hybridization in situ; Probe preparation; Tissue preparation; Fixation; Deproteinization with Proteinase K; Acetylation of the tissue; Dehydration; Hybridization step; Post-Hybridization Washes (Step 1-6 should be performed on shaker); Signal Detection (Steps should be performed on a shaker); Post-Detection Washes. - [Read Visualizing miRNAs in Adult Skeletal Muscle by Hybridization In Situ Protocol]

Category: Cytogenetics Protocols: RNA In Situ Hybridization Protocols

Protocol for visualizing miRNAs in adult skeletal muscle by hybridization in situ. Includes: Visualizing miRNAs in adult skeletal muscle by hybridization in situ; Probe preparation; Tissue preparation; Fixation; Deproteinization with Proteinase K; Acetylation of the tissue; Dehydration; Hybridization step; Post-Hybridization Washes (Step 1-6 should be performed on shaker); Signal Detection (Steps should be performed on a shaker); Post-Detection Washes. - [Read Visualizing miRNAs in Adult Skeletal Muscle by Hybridization in situ Protocol]

Category: Plant Biology Protocols: Arabidopsis Genetics Protocols

Protocol for whole mount fluorescence in situ hybridization (FISH) of repetitive DNA sequences on interphase nuclei of the small cruciferous plant Arabidopsis thaliana. Includes: Seed sterilization and germination; Tissue fixation; Labeling of the probe DNA; Pretreatment; In situ hybridization; Pre-absorption of antibodies; Posthybridization washes; Immunocytochemical detection; Direct detection; Indirect detection; Staining and mounting; Fluorescence microscopy. - [Read Whole Mount Fluorescence in Situ Hybridization (FISH) of Repetitive DNA Sequences on Interphase]

Category: Drosophila Protocols: Drosophila Genetics Protocols

Protocol for whole mount in situ hybridization for the detection of mRNA in Drosophila embryos. Includes: Embryo collection and fixation; Prehybridization treatment; Hybridization; Washing and detection. - [Read Whole Mount In Situ Hybridization for the Detection of mRNA in Drosophila Embryos Protocol]

Category: Mouse Protocols: Mouse Embryology Protocols

Protocol for whole mount in situ hybridization of mouse. Incluses: Preparation of the probe; General Comments; Preparation of embryos; In Situ hybridization. - [Read Whole Mount In Situ Hybridization of Mouse Protocol]

Category: C. elegans Protocols

Protocol for whole-mount in situ hybridization for the detection of RNA in C. elegans embryos. Includes: Collection of Embryos; Permeabilization and fixation of embryos; Prehybridization; Hybridization; Post-hybridization washes; Probe Detection; Double-labeling; Interpretation. - [Read Whole-Mount In Situ Hybridization for the Detection of RNA in C. elegans Embryos Protocol]

Category: Xenopus Protocols: Xenopus Genetics Protocols

Protocols for Xenopus whole mount in situ hybridization. Includes: Protocols for Making Antisense RNA probe (dig labelled); Protocols for In Situ Hyb. - [Read Xenopus Whole Mount In Situ Hybridization Protocols]