denatured Protocols

Category: Immunology

Protocol for detection of autoantibodies with self-assembling radiolabeled antigen tetramers. Details how to produce radiolabeled antigen-streptavidin tetramers for detection of antibodies by immunoprecipitation. Optionally, the antigen tetramers can be denatured to compare responses to folded and unfolded antigen in the same system. This technique can be applied to a large or small number of samples, and a given sample can be simultaneously assayed with multiple antigens. - [Read Detection of Autoantibodies with Self-Assembling Radiolabeled Antigen Tetramers Protocol]

Category: Nucleic Acid Detection Protocols: Nucleic Acid Blotting Protocols

Protocol for dot and slot hybridization of purified RNA. Dot blotting of RNA is best carried out using purified preparations of RNA that are denatured with glyoxal or formaldehyde immediately before loading onto a nylon membrane through a vacuum manifold. - [Read Dot and Slot Hybridization of Purified RNA Protocol]

Category: Cloning Protocols: Mutagenesis Protocols

Protocol for in vitro mutagenesis using double-stranded DNA templates. Two oligonucleotides are used to prime DNA synthesis catalyzed by a high-fidelity thermostable polymerase on a denatured plasmid template. The two oligonucleotides both contain the desired mutation and occupy the same starting and ending positions on opposite strands of the plasmid DNA. - [Read In Vitro Mutagenesis Using Double-stranded DNA Templates: Selection of Mutants with DpnI]

Category: DNA Protocols: DNA Sequencing Protocols: Dideoxy Mediated Sequencing Protocols

Protocol describes how double-stranded plasmid DNA is denatured by alkali and annealed to an appropriate oligonucleotide primer, in preparation for dideoxy-sequencing reactions catalyzed by Sequenase. - [Read Preparing Denatured Templates for Sequencing by Dideoxy-mediated Chain Termination Protocol]

Category: Genetics and Genomics: Cancer Genetics Protocols: Single Strand Conformational Polymorphism(SSCP) Protocols

Single-strand confirmation polymorphism analysis (SSCP) is a powerful and robust method for the detection of DNA sequence changes (single-base substitutions) based on shifts in electrophoretic mobility. In this protocol, the target sequence is simultaneously labeled and amplified, then heat-denatured and resolved by non-denaturing polyacrylamide gel electrophoresis. - [Read Single-Strand Conformation Polymorphism Analysis Protocol]

Category: DNA Protein Interactions Protocols: Electrophoretic Mobility Shift Assays EMSA Protocols

The target sequence is simultaneously labeled and amplified, then heat-denatured and resolved by non-denaturing polyacrylamide gel electrophoresis.  Differences in sequence alter the conformation of the DNA and hence its electrophoretic mobility and, because of the high resolution of polyacrylamide gels, most conformational changes caused by subtle changes in sequence can be detected. - [Read Single-Strand Conformation Polymorphism Analysis Protocol]

Category: Nucleic Acid Detection Protocols: Nucleic Acid Blotting Protocols

Protocol describes the first stages of Southern blotting: digestion of genomic DNA with one or more restriction enzymes, separation of the resulting fragments by electrophoresis through an agarose gel, and transfer of the denatured fragments to a membrane by downward capillary transfer. - [Read Southern Blotting: Capillary Transfer of DNA to Membranes Protocol]

Category: Nucleic Acid Electrophoresis Protocols: RNA Electrophoresis Protocols

Protocol describes the transfer of RNA from agarose gels to neutral or positively charged nylon membranes, using upward capillary flow of neutral or alkaline buffers.  RNA becomes covalently fixed to positively charged nylon membranes during transfer in alkaline buffers.  However, treatment by UV irradiation or heating is required to fix RNA to neutral membranes. - [Read Transfer and Fixation of Denatured RNA to Membranes Protocol]