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    Home > Biochemistry News > Biotechnology News > Agarose gel electrophoresis detects RNA quality.

    Agarose gel electrophoresis detects RNA quality.

    • Last Update: 2020-10-26
    • Source: Internet
    • Author: User
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    related topics .One. Principle
    the tendency of charged matter to move in an electric field is called electrophoresis. Among them,
    gel electrophoresis
    because of its simple operation, fast, sensitive and other advantages, making it the separation, identification and purification of
    nucleic acid
    the preferred standard method. Similar to
    protein
    , nucleic acid molecules are also dissophed molecules of both sexes. At pH3.5, the amino group on the base is dissopheded, while only the first phosphoric acid in the three phosphoric acid groups is dissopheded, the entire molecule is positively charged and swims towards the negative pole in the electric field; The charge density of nucleic acid molecules of different sizes and compositions is about the same, and the migration rate of each nucleic acid molecule is very small and difficult to separate when the freestyle is moving. Therefore, the use of gel medium adapted to the concentration as an electrophoretic support, play the function of molecular sieve, so that different molecular size and composition of different nucleic acid molecules swimming rate appears a large difference, to achieve the purpose of separation.
    1) Four major factors affecting swimming:
    (1) The primary factor affecting swimming is the physical properties of the electrophoresis sample: how much charge,
    molecule
    size, particle shape and spatial structure. Generally speaking, the higher the density of charge charge of particles, the faster the swimming rate, the larger the physical shape of the particles, the greater the friction with the support, the smaller the swimming rate. That is, the swimming rate is inversely inversely related to the molecular size and media viscosity of the particles, and is directly related to the charge charged by the particles.
    (2) Support media:
    . DNA
    gel electrophoresis often uses two supporting materials:
    agar
    and polyacrylamide gel. The molecular screen hole size of the gel is adjusted by the change of concentration of these two media, and the nucleic acid pieces of different molecular weights are separated. Agarose has a large aperture that separates nucleic acid pieces with a length of 100bp to 60kb, and polyacrylamide gels have a small aperture to separate nucleic acids from small pieces (5-50bp).
    (3) electric field strength: the voltage drop of the unit support length between the poles of the electrophoresis field is the electric field strength or voltage gradient. The greater the intensity of the electric field, the faster the swimming rate of the charged particles, but the effective separation range of the gel decreases with the increase of voltage. At low voltages, the swimming rate of linear DNA molecules is directly related to the voltage. The electric field strength of the general gel electrophoresis does not exceed 5V/cm.
    (4) buffer ion strength: buffer is a conductor in the electrophoresis field, its type, pH, ion concentration directly affects the efficiency of electrophoresis. Tris. Cl buffer system, because Cl-swimming speed is much faster than sample molecules, easy to cause the phenomenon of band inequality, so commonly used TAE, TBE, TPE three buffer systems. The pH of the buffer directly affects the degree of DNA dissocation and charge density, and the farther away the pH of the buffer is from the isoelectric point of the nucleic acid sample, the more charge the sample carries, the faster the swimming speed. Nucleic acid electrophoresis buffer, often using alkaline or neutral conditions, so that nucleic acid molecules with negative charge, swimming towards the positive pole. The ion strength of the buffer is inversely inversely related to the velocity of the sample swimming, and the most appropriate ion strength of electrophoresis is generally between 0.02-0.2.
    2)
    , a color-coded marker is often used to indicate the migration process of the sample during electrophoresis. There are two kinds of indicators commonly used in nucleic acid electrophoresis: bromophenol blue-purple; The molecular weight of bromophenol orchids is 670Da, in different concentrations of gels, the rate of migration is basically the same, its molecular sieve effect is small, similar to free electrophoresis, so it is widely used as an indicator. The molecular weight of xylene is 554.6Da, carries less charge than bromophenol orchids, the migration rate in the gel is slower than bromophenol orchids, is often used in polyacrylamide gel electrophoresis, there are also bromphenol orchid and xylene blue mixed applications. The indicator is generally added to the sample buffer, in order to allow the sample to sink into the glue hole, but also add the appropriate amount of sucrose, polysucrose 400 or glycerol to increase the proportion.
    3) staining agent
    nucleic acid after dyeing to show the band type, the most commonly used is the bromine ethyl ingot dyeing method. Ethyl bromide (EB) is a fluorescent dye that can be embedded between the paired bases of a double-stranded nucleic acid and emits red fluorescence when stimulated by ultraviolet light. The energy that excites fluorescence comes from two aspects, one is that nucleic acid absorbs ultraviolet light with a wavelength of 260nm and transmits energy to ethyl bromide ingots, and the other is the EB itself, which is combined in DNA molecules, mainly absorbs ultraviolet energy at wavelengths of 300nm and 360nm, and comes from these two sources of energy, which eventually excites EB to emit visible
    spectral
    fluorescent orange regions with a wavelength of 590nm. The EB in the EB-DNA complex emits 10 times more fluorescence than the EB itself in the free gel, so the electrophoresis band type of nucleic acid can be clearly observed without washing the background. Typically, EB with a final concentration of 0.5 μg/ml is added to the gel to observe the migration of nucleic acids at any time during electrophoresis, which is used in general
    nucleic acid
    .
    EB light easy to decompose, it should be stored in brown bottles at 4 degrees C conditions. Single-stranded DNA, RNA molecules often exist in their own pairing of double-stranded regions, can also be embedded in EB molecules, but the amount of embedding is small, therefore, low fluorescence, its minimum detection is 0.1 sg.two. Materials and Methods
    1 Materials
    RNA Samples
    2 Instruments, Appliances
    Electrophoresis, Electrophoresis Tank, Gel Sample Combs, Microwave Ovens,
    Liquidators
    , etc.
    3
    Reagents
    (1) 1×TAE Electrophoretic Buffer
    (2) Ethyl Bromide (EB)
    .
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