hybridization Protocols

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

Protocol for multiple-target DNA in situ hybridization with enzyme-based cytochemical detection systems. Includes: Cell preparations; Cell processing; Probe preparation; Multiple-target in situ hybridization (ISH); ISH with separate probe and target denaturation [for probes with repetitive (e.g., Alu) elements]; Post-hybridization washes; Enzyme-based cytochemical detection; etc.. - [Read Multiple-Target DNA In Situ Hybridization with Enzyme-Based Cytochemical Detection Systems Protocol]

Category: Nucleic Acid Modification Protocols: DIG Biotin Labeled Probes Protocols

Protocol for nonradioactive in situ hybridization to polytene chromosomes with a DIG-labeled DNA probe. Includes: Labeling the hybridization probe; Preparation and denaturation of polytene chromosomes from Drosophila, Chironomus, or other species; Hybridization and detection. - [Read Nonradioactive In Situ Hybridization to Polytene Chromosomes With a DIG-Labeled DNA Probe Protocol]

Category: Fungi Protocols: Neurospora Protocols

Northern blot protocol for the detection of RNA in Neurospora. Includes: Extract RNA from tissue powder; Electropheresis and transfer RNA; Preparing the membrane for probing and preparation of a riboprob.; Hybridization. - [Read Northern blot Protocol for the Detection of RNA in Neurospora]

Category: Nucleic Acid Detection Protocols: Nucleic Acid Blotting Protocols

Protocol for northern hybridization. Protocol describes how to carry out northern hybridization at high stringency in phosphate-SDS-buffers.  Although a wide variety of formats are available, hybridization is usually performed in heat-sealable bags, roller bottles, or plastic boxes, as described here. - [Read Northern Hybridization Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

Discusses the effects of various components of the hybridization solution on the rate of renaturation and thermal stability of DNA hybrids free in solution. Includes: The main parameters that influence hybridization; Additional hybridization variables; Competition in situ hybridization; Oligonucleotide hybridization; Standard in situ hybridization conditions. - [Read Nucleic Acid Hybridization General Aspects]

Category: Real Time PCR: Real Time PCR Information

Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. Livak et al., 1995. - [Read Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for d]

Category: Drosophila Protocols: Drosophila Genetics Protocols

Optimized protocols for fluorescent in situ hybridization in Drosophila tissues. Includes: RNA Probe Preparation; Initial Embryo Fixation; Post-Fixation, Hybridization and post-Hybridization Washes; Development of FISH Signal; Mounting and Viewing of Samples; Double FISH; FISH on Dissected Tissues; RNA-Protein Double-labeling. - [Read Optimized Protocols for Fluorescent in situ Hybridization in Drosophila Tissues]

Category: Cytogenetics Protocols

An ideal method of tissue preparation ensures both good specimen morphology and that the target molecules are in the optimum state for probe access and hybridization. DNA:DNA in situ hybridization is usually carried out on chromosome spread preparations where chromosome and nuclei are released from cells and spread on a glass microscope slide. This method yields well separated and enlarged chromosomes with good morphology which can be analyzed in transmitted light or fluorescence microscopes. - [Read Preparation of Chromosome Spreads]

Category: Mouse Protocols: Mouse Genetics Protocols

Method is used chiefly to genotype transgenic and knockout mice. Each 6-10-mm snippet of mouse tail yields 50-100 µg of DNA that can be used in dot or slot blotting to detect a transgene of interest, in Southern hybridization to detect DNA fragments that are <20 kb in size, and as a template in PCRs. - [Read Preparation of Genomic DNA from Mouse Tails and Other Small Samples Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: Genomic DNA Isolation Protocols

Simple protocol is used to extract DNA from small numbers of cultured cells and from fragments of soft or bony tissues.  The method is used chiefly to genotype transgenic and knockout mice.  Each 6-10-mm snippet of mouse tail yields 50-100 µg of DNA that can be used in dot or slot blotting to detect a transgene of interest, in Southern hybridization to detect DNA fragments that are <20 kb in size, and as a template in PCRs. - [Read Preparation of Genomic DNA from Mouse Tails and Other Small Samples Protocol]

Category: Cell Organelle Protocols: Nucleus Protocols

A simplified and efficient protocol for nonradioactive in situ hybridization to polytene chromosomes with a DIG-labeled DNA probe. Includes: Labeling the hybridization probe; Preparation and denaturation of polytene chromosomes from Drosophila, Chironomus, or other species; Hybridization and detection; - [Read Protocol for Nonradioactive In Situ Hybridization to Polytene Chromosomes With DIG-labeled DNA Prob]

Category: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

The protocol given makes the method of in situ hybridization easier, faster, more reliable, and available to anyone who can operate a microscope. Includes: Labeling the hybridization probe; Preparation and denaturation of polytene chromosomes from Drosophila, Chironomus, or other species; Hybridization and detection. - [Read Protocol for Nonradioactive In Situ Hybridization to Polytene Chromosomes with a DIG-labeled DNA]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

RNA amplification by in vitro transcription protocol. Includes protocols on cDNA labeling and hybridization on cDNA microarrays. - [Read Protocol for RNA Amplification by In Vitro Transcription]

Category: Fungi Protocols: Neurospora Protocols

Protocol guide for the N. crassa yeast artificial chromosome library. Includes: Chromosome Walking; Hybridization screening of the YAC library; YAC restriction mapping and contig building; Preparation of chromosomal DNA plugs of YAC clones; Partial restriction enzyme digestion of YAC DNA plugs; Using CHEF gel analysis to resolve YAC clones; Southern Hybridization; Isolation of terminal restriction fragments from cloned DNA inserts in YAC clones; etc. - [Read Protocol Guide for the N. crassa Yeast Artificial Chromosome Library]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

In vitro transcription reactions employing T3, T7 or SP6 phage-encoded RNA polymerases are widely used to synthesize RNA from recombinant vectors containing appropriate promoters. Production of large amounts of specific RNA is valuable in the preparation of hybridization probes and in vitro translation studies; in the synthesis of ribozymes, rRNA, SRP, antisense RNA and substrates for RNA splicing; and in RNA-protein interaction studies. - [Read Protocol: Purification of In Vitro Synthesized mRNA with Microcon or Centricon Centrifugal Filters]

Category: Nucleic Acid Modification Protocols: Nucleic Acid Radiolabeling Protocols

In this procedure, synthesis of cDNA is carried out in the presence of saturating concentrations of all four dNTPs and trace amounts of a single radiolabeled dNTP.  After subtraction hybridization, the enriched single-stranded cDNA is radiolabeled to high specific activity in a second synthetic reaction by extension of random oligonucleotide primers using the Klenow fragment of E. coli DNA polymerase. - [Read Radiolabeling of Subtracted cDNA Probes by Random Oligonucleotide Extension Protocol]

Category: Nucleic Acid Modification Protocols: Random Primer Labeling Protocols

Protocol for random primer labeling of poly A and RNA. Includes: Random primer reaction; cDNA purification; Hybridization; Washing; Stripping of membranes. - [Read Random Primer Labeling of PolyA and RNA Protocol]

Category: Yeast Protocols: Yeast Genetics Protocols: Yeast Replication Protocols

Protocol for replication timing by comparative hybridization. - [Read Replication Timing by Comparative Hybridization Protocol]

Category: Nucleic Acid Electrophoresis Protocols: Pulsed Field Gel Electrophoresis Protocols

Protocol for restriction endonuclease digestion of DNA in agarose plugs. Genomic DNA isolated from mammalian, yeast, or bacterial cells can be digested with restriction endonucleases by incubating agarose plugs containing the DNA in the presence of the desired enzyme.  After digestion, the DNA can be fractionated by pulsed-field gel electrophoresis and either isolated from the gel or analyzed by Southern Hybridization. - [Read Restriction Endonuclease Digestion of DNA in Agarose Plugs Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

This protocol describes a method for reverse transcriptase (RT) in situ PCR. In situ PCR differs from PCR in situ hybridization in the inclusion of a reporter molecule in the amplification step. The two steps of RT in situ PCR that differ from in situ PCR are overnight digestion in RNase-free DNase that is performed after protease digestion, and an RT step, prior to in situ PCR. - [Read Reverse Transcriptase In Situ PCR Protocol]

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

Protocol describes a method for reverse transcriptase (RT) in situ PCR.  In situ PCR differs from PCR in situ hybridization in the inclusion of a reporter molecule in the amplification step.  The two steps of RT in situ PCR that differ from in situ PCR are overnight digestion in Rnase-free Dnase that is performed after protease digestion, and an RT step, prior to in situ PCR. - [Read Reverse Transcriptase In Situ PCR Protocol]

Category: C. elegans Protocols

Protocol for RNA in situ hybridization of dissected gonads with digoxygenin-labeled probes. Includes: Preparation of single-stranded probes from cloned cDNA; Dissections and Fixation; Proteinase K digestion; Hybridization; Making a Mount. - [Read RNA In Situ Hybridization of Dissected Gonads with Digoxygenin-Labeled Probes Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Tissue Sections Protocols

Protocol for RNA whole mount in situ hybridization. Includes: Embryo preparation; Prehybridization and Hybridization; Post-hybridization washes, blocking, and antibody incubation; Post-antibody washes; Color development. - [Read RNA Whole Mount In Situ Hybridization Protocol]

Category: Cytogenetics Protocols: RNA In Situ Hybridization Protocols

RNA-RNA in situ hybridization using DIG-labeled probes: the effect of high molecular weight polyvinyl alcohol on the alkaline phosphatase indoxyl-nitroblue tetrazolium reaction. RNA-RNA in situ hybridization protocol using alkaline phosphatase-conjugated digoxigenin-(DIG-) labeled probes is presented. The addition of polyvinyl alcohol (PVA) of high molecular weight (40 – 100 kD) to the BCIP-NBT detection system enhances the alkaline phosphatase reaction and prevents... - [Read RNA-RNA In Situ Hybridization Using DIG-Labeled Probes Protocol]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

Run-on transcription monitors the regulation of transcription in isolated nuclei. Protocol includes: Preparation of Nuclei, Run-On Transcription Reaction, Alkaline Hydrolysis of Run-Off Transcripts, Preparation of Filters and Hybridization. Solutions included: DNase Solution, Hybridization Mix, Denhardt Solution (100X) etc. - [Read Run- On Transcription Protocol]