Protocol for pulsed-field gel electrophoresis via contour-clamped homogeneous electric field gels. In CHEF gels, the electric field is generated from multiple electrodes, arranged in a square of hexagonal contour around the horizontal gel and clamped to predetermined potentials. Using a combination of low field strengths, low concentrations of aragose, long switching intervals, and extended periods of electrophoresis, DNAs up to 5000 kb can be resolved. - [Read Pulsed-field Gel Electrophoresis via Contour-clamped Homogeneous Electric Field Gels Protocol]
Protocol for purification of synthetic oligonucleotides by polyacrylamide gel electrophoresis. As a rule of thumb, oligonucleotides >25 nucleotides should be purified by polyacrylamide gel electrophoresis, as should oligonucleotides of any length that yield anomalous results. After electrophoresis, the oligonucleotide is eluted from the gel and concentrated by reversed-phase chromatography on Sep-Pak C18 columns. - [Read Purification of Synthetic Oligonucleotides by Polyacrylamide Gel Electrophoresis Protocol]
Protocol for quantification of DNA methylation in electrofluidics chips. Describe Bio-COBRA, a modified protocol for Combined Bisulfite Restriction Analysis (COBRA), that incorporates an electrophoresis step in microfluidics chips. Microfluidics technology involves the handling of small amounts of liquid in miniaturized systems. - [Read Quantification of DNA Methylation in Electrofluidics Chips Protocol]
In this protocol sequences cloned in standard bacteriophage or plasmid vectors are amplified in PCRs containing primers targeted to flanking vector sequences. The amplified fragments can be analyzed by gel electrophoresis, DNA sequencing, and/or restriction mapping. Many colonies or plaques can be assayed simultaneously. - [Read Rapid Characterization of DNAs Cloned in Prokaryotic Vectors Protocol]
DNA fragments separated by electrophoresis through gels cast with low-melting-temperature agarose are recovered by melting the agarose and extracting the resulting solution with phenol:chloroform. The protocol works best for DNA fragments ranging in size from 0.5 kb to 5 kb. - [Read Recovery of DNA from Low-melting-temperature Agarose Gels: Organic Extraction Protocol]
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]
Restriction landmark genomic scanning (RLGS) is a method to detect large numbers of restriction landmarks in a single experiment. It is based on the concept that restriction enzyme sites can serve as landmarks throughout a genome. RLGS uses direct end-labeling of the genomic DNA digested with a rare-cutting restriction enzyme and high-resolution two-dimensional electrophoresis. - [Read Restriction Landmark Genomic Scanning Protocol]
Protocol for retrieval of DNA fragments from pulsed-field gels following DNA concentration. DNA contained in a slice of low-melting-temperature agarose is first concentrated by electrophoresis into a high-percentage agarose gel, and then isolated by treatment with agarase. The resulting DNA preparation is purified by microdialysis. - [Read Retrieval of DNA Fragments from Pulsed-field Gels following DNA Concentration Protocol]
SDS POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE). Ed Rybicki and Maud Purves. Dept Microbiology, University of Cape Town. PROTOCOL includes: Assembling Gel Apparatus, Resolving Gels, Stacking Gels, Electrophoresis buffer, Sample Preparation for Sds-page and Staining of Gels. - [Read SDS POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE)]
SDS-PAGE: gel electrophoresis of proteins. Very simple protocol for SDS-PAGE. Koshland Lab, Carnegie Institution of Washington, Howard Hughes Medical Institute. - [Read SDS-PAGE]
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]
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]
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]
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]
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]
Method is used to assess (roughly) the integrity of total RNA samples by visualization of discreet 18S and 28S ribosomal RNAs. Total RNA is separated by electrophoresis through a 1% agarose gel containing 1.3 ìM ethidium bromide. Binding of the ethidium bromide to the RNA allows visualization of the separated RNA molecules when the gel is exposed to ultraviolet (UV) light. - [Read Visualization of RNA Preparations on 1% Agarose Gels Protocol]
This protocol was developed for the BIORAD protein gel and transfer apparatus. The buffers can be used with any electrophoresis/transfer system. - [Read Western Blot Protocol]
Protocol is a simple, reliable method for the preparation of yeast protein extracts for analysis by polyacrylamide gel electrophoresis (PAGE) and Western blotting. - [Read Yeast Protein Extracts Protocol]
A Single Stranded Plasmid DNA Isolation Protocol describing the production and isolation of single-stranded DNA (ssDNA) using bacteriophagemid-containing bacteria and helper phage. Infection of the host cells with helper phage allows for packaging of ssDNA into bacteriophage. The ssDNA can then be isolated from phage particles.
Histone H1 Kinase Activity Assay Protocol. This protocol describes assaying kinase activity of a putative kinase using Histone H1 as the substrate. Histone H1 is the canonical kinase substrate in this type of assay. Phosphorylation of Histone H1 is assessed by SDS-Polyacrylamide gel electrophoresis followed by autoradiography.