Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose, one of the two main components of agar. The proteins may be separated by charge and/or size, and the DNA and RNA fragments by length. Biomolecules are separated by applying an electric field to move the charged molecules through an agarose matrix, and the biomolecules are separated by size in the agarose gel matrix.
Agarose is a polysaccharide, generally extracted from certain red seaweed. It is a linear polymer made up of the repeating unit of agarobiose, which is a disaccharide made up of D-galactose and 3,6-anhydro-L-galactopyranose. Agarose is one of the two principal components of agar, and is purified from agar by removing agar's other component, agaropectin.
Gel electrophoresis is a method for separation and analysis of macromolecules and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge.
The northern blot, or RNA blot, is a technique used in molecular biology research to study gene expression by detection of RNA in a sample.
Polyacrylamide gel electrophoresis (PAGE) is a technique widely used in biochemistry, forensic chemistry, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility. Electrophoretic mobility is a function of the length, conformation and charge of the molecule. Polyacrylamide gel electrophoresis is a powerful tool used to analyze RNA samples. When polyacrylamide gel is denatured after electrophoresis, it provides information on the sample composition of the RNA species.
Nucleic acid electrophoresis is an analytical technique used to separate DNA or RNA fragments by size and reactivity. Nucleic acid molecules which are to be analyzed are set upon a viscous medium, the gel, where an electric field induces the nucleic acids to migrate toward the anode. The separation of these fragments is accomplished by exploiting the mobilities with which different sized molecules are able to pass through the gel. Longer molecules migrate more slowly because they experience more resistance within the gel. Because the size of the molecule affects its mobility, smaller fragments end up nearer to the anode than longer ones in a given period. After some time, the voltage is removed and the fragmentation gradient is analyzed. For larger separations between similar sized fragments, either the voltage or run time can be increased. Extended runs across a low voltage gel yield the most accurate resolution. Voltage is, however, not the sole factor in determining electrophoresis of nucleic acids.
Ethidium bromide is an intercalating agent commonly used as a fluorescent tag in molecular biology laboratories for techniques such as agarose gel electrophoresis. It is commonly abbreviated as EtBr, which is also an abbreviation for bromoethane. To avoid confusion, some laboratories have used the abbreviation EthBr for this salt. When exposed to ultraviolet light, it will fluoresce with an orange colour, intensifying almost 20-fold after binding to DNA. Under the name homidium, it has been commonly used since the 1950s in veterinary medicine to treat trypanosomiasis in cattle. The high incidence of antimicrobial resistance makes this treatment impractical in some areas, where the related isometamidium chloride is used instead. Although ethidium bromide is said to be mutagenic on some circumstances of exposure, evidence shows that it is relatively safe and its alleged high toxicity is largely a myth.
Staining is a technique used to enhance contrast in samples, generally at the microscopic level. Stains and dyes are frequently used in histology and in the medical fields of histopathology, hematology, and cytopathology that focus on the study and diagnoses of disease at a microscopic level. Stains may be used to define biological tissues, cell populations, or organelles within individual cells.
Bromophenol blue is used as a pH indicator, an electrophoretic color marker, and a dye. It can be prepared by slowly adding excess bromine to a hot solution of phenolsulfonphthalein in glacial acetic acid.
Nile blue is a stain used in biology and histology. It may be used with live or fixed cells, and imparts a blue colour to cell nuclei.
In molecular biology, gel extraction or gel isolation is a technique used to isolate a desired fragment of intact DNA from an agarose gel following agarose gel electrophoresis. After extraction, fragments of interest can be mixed, precipitated, and enzymatically ligated together in several simple steps. This process, usually performed on plasmids, is the basis for rudimentary genetic engineering.
Orange G also called C.I. 16230, Acid Orange 10, or orange gelb is a synthetic azo dye used in histology in many staining formulations. It usually comes as a disodium salt. It has the appearance of orange crystals or powder.
An electrophoretic mobility shift assay (EMSA) or mobility shift electrophoresis, also referred as a gel shift assay, gel mobility shift assay, band shift assay, or gel retardation assay, is a common affinity electrophoresis technique used to study protein–DNA or protein–RNA interactions. This procedure can determine if a protein or mixture of proteins is capable of binding to a given DNA or RNA sequence, and can sometimes indicate if more than one protein molecule is involved in the binding complex. Gel shift assays are often performed in vitro concurrently with DNase footprinting, primer extension, and promoter-probe experiments when studying transcription initiation, DNA gang replication, DNA repair or RNA processing and maturation, as well as pre-mRNA splicing. Although precursors can be found in earlier literature, most current assays are based on methods described by Garner and Revzin and Fried and Crothers.
Acridine orange is an organic compound that serves as a nucleic acid-selective fluorescent dye with cationic properties useful for cell cycle determination. Acridine orange is cell-permeable, which allows the dye to interact with DNA by intercalation, or RNA via electrostatic attractions. When bound to DNA, acridine orange is very similar spectrally to an organic compound known as fluorescein. Acridine orange and fluorescein have a maximum excitation at 502nm and 525 nm (green). When acridine orange associates with RNA, the fluorescent dye experiences a maximum excitation shift from 525 nm (green) to 460 nm (blue). The shift in maximum excitation also produces a maximum emission of 650 nm (red). Acridine orange is able to withstand low pH environments, allowing the fluorescent dye to penetrate acidic organelles such as lysosomes and phagolysosomes that are membrane-bound organelles essential for acid hydrolysis or for producing products of phagocytosis of apoptotic cells. Acridine orange is used in epifluorescence microscopy and flow cytometry. The ability to penetrate the cell membranes of acidic organelles and cationic properties of acridine orange allows the dye to differentiate between various types of cells. The shift in maximum excitation and emission wavelengths provides a foundation to predict the wavelength at which the cells will stain.
SYBR Green I (SG) is an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. The SYBR family of dyes is produced by Molecular Probes Inc., now owned by Thermo Fisher Scientific. SYBR Green I binds to DNA. The resulting DNA-dye-complex best absorbs 497 nanometer blue light and emits green light. The stain preferentially binds to double-stranded DNA, but will stain single-stranded (ss) DNA with lower performance. SYBR Green can also stain RNA with a lower performance than ssDNA.
A gel doc, also known as a gel documentation system, gel image system or gel imager, refers to equipment widely used in molecular biology laboratories for the imaging and documentation of nucleic acid and protein suspended within polyacrylamide or agarose gels. These gels are typically stained with ethidium bromide or other nucleic acid stains such as GelGreen. Generally, a gel doc includes an ultraviolet (UV) light transilluminator, a hood or a darkroom to shield external light sources and protect the user from UV exposure, and a CCD camera for image capturing. With advancement in CMOS camera sensors like Sony's Pregius and Exmor series, low light capable cameras made of these sensors are also being incorporated in gel documentation systems
GelRed is an intercalating nucleic acid stain used in molecular genetics for agarose gel DNA electrophoresis. GelRed structurally consists of two ethidium subunits that are bridged by a linear oxygenated spacer.
SYBR Safe is a cyanine dye used as a nucleic acid stain in molecular biology. SYBR Safe is one of a number of SYBR dyes made by the Life Technologies Corporation. SYBR Safe binds to DNA. The resulting DNA-dye-complex absorbs blue light and emits green light.
Stains-all is a carbocyanine dye, which stains anionic proteins, nucleic acids, anionic polysaccharides and other anionic molecules.
SYBR Gold is an asymmetrical cyanine dye. It can be used as a stain for double-stranded DNA, single-stranded DNA, and RNA. SYBR Gold is the most sensitive fluorescent stain of the SYBR family of dyes for the detection of nucleic acids. The SYBR family of dyes is produced by Molecular Probes Inc., now owned by Thermo Fisher Scientific
