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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.
Borax, also known as sodium borate, sodium borate decahydrate or sodium tetraborate decahydrate, is a hydrate salt of boric acid. Commonly available in powder or granular form, it dissolves in water to make a basic, aqueous solution. It is soluble and has many industrial and household applications as a component in a wide range of products. Applications include as a pesticide; metal soldering; glaze and enamel manufacturing; tanning of skins and hides; artificial aging of wood; as a preservative against wood fungus; analytical chemistry as a buffering agent; and pharmaceutic aid as an alkalizer.
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.
TBE may refer to:
Tris, or tris(hydroxymethyl)aminomethane, or known during medical use as tromethamine or THAM, is an organic compound with the formula (HOCH2)3CNH2. It is extensively used in biochemistry and molecular biology as a component of buffer solutions such as in TAE and TBE buffers, especially for solutions of nucleic acids. It contains a primary amine and thus undergoes the reactions associated with typical amines, e.g. condensations with aldehydes. Tris also complexes with metal ions in solution. In medicine, tromethamine is occasionally used as a drug, given in intensive care for its properties as a buffer for the treatment of severe metabolic acidosis in specific circumstances. Some medications are formulated as the "tromethamine salt" including hemabate (carboprost as trometamol salt), and "ketorolac trometamol".
A lysis buffer is a buffer solution used for the purpose of breaking open cells for use in molecular biology experiments that analyze the labile macromolecules of the cells. Most lysis buffers contain buffering salts and ionic salts to regulate the pH and osmolarity of the lysate. Sometimes detergents are added to break up membrane structures. For lysis buffers targeted at protein extraction, protease inhibitors are often included, and in difficult cases may be almost required. Lysis buffers can be used on both animal and plant tissue 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.
Lithium acetate (CH3COOLi) is a salt of lithium and acetic acid.
Phosphate-buffered saline is a buffer solution commonly used in biological research. It is a water-based salt solution containing disodium hydrogen phosphate, sodium chloride and, in some formulations, potassium chloride and potassium dihydrogen phosphate. The buffer helps to maintain a constant pH. The osmolarity and ion concentrations of the solutions match those of the human body (isotonic).
TE buffer is a commonly used buffer solution in molecular biology, especially in procedures involving DNA, cDNA or RNA. "TE" is derived from its components: Tris, a common pH buffer, and EDTA, a molecule that chelates cations like Mg2+. The purpose of TE buffer is to solubilize DNA or RNA, while protecting it from degradation.
TAE buffer is a buffer solution containing a mixture of Tris base, acetic acid and EDTA.
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.
Alkaline lysis or alkaline extraction is a method used in molecular biology to isolate plasmid DNA from bacteria.
A molecular-weight size marker, also referred to as a protein ladder, DNA ladder, or RNA ladder, is a set of standards that are used to identify the approximate size of a molecule run on a gel during electrophoresis, using the principle that molecular weight is inversely proportional to migration rate through a gel matrix. Therefore, when used in gel electrophoresis, markers effectively provide a logarithmic scale by which to estimate the size of the other fragments.
SB buffer is a buffer solution used in agarose and polyacrylamide gel electrophoresis for the separation of nucleic acids such as DNA and RNA. "SB" is a commercial trademark of Faster Better Media LLC for their sodium boric acid-based conductive medium, which is based on the publications of Brody and Kern.
LB buffer, also known as lithium borate buffer, is a buffer solution used in agarose electrophoresis, typically for the separation of nucleic acids such as DNA and RNA. It is made up of Lithium borate.
An electrophoretic color marker is used to monitor the progress of agarose gel electrophoresis and polyacrylamide gel electrophoresis (PAGE) since DNA, RNA, and most proteins are colourless. They are also referred to as tracking dyes, and are frequently present in loading dyes as well as molecular weight ladders.
TSE or Tris/Saline/EDTA, is a buffer solution containing a mixture of Tris base, Sodium chloride and EDTA.
References
- ↑ Brody, J.R.; Kern, S.E. (2004). "History and principles of conductive media for standard DNA electrophoresis" (PDF). Anal Biochem. 333 (1): 1–13. doi:10.1016/j.ab.2004.05.054. PMID 15351274.
- ↑ https://www.eeb.ucla.edu/Faculty/Barber/Web%20Protocols/Protocol7.pdf [ bare URL PDF ]