The ion fits into a 0.34 nm (3.4 Å) opening created by this unwinding that is stabilized by hydrophobic interactions between ethidium and the nucleotide bases (see picture). The ethidium cation unwinds DNA by about 26°. In their presence, the DNA dynamically opens a space between its base pairs by unwinding. Ethidium cations have high affinity for binding DNA. Illumination ~300 nm gives the strongest fluorescence, however, many researchers illuminate with 360 nm to protect their sample from damage (360 nm gives considerably weaker fluorescence). The closer the UV illumination is to 260 nm, the more likely you are to damage the DNA. This absorbed energy is re-emitted as orange/yellow fluorescence at 590nm. This compound has UV absorbance maxima at 300 and 360 nm, and can also absorb energy from nucleotides excited at 260nm. The most common method of direct staining is the incubation of the gel in a weak solution of ethidium bromide (EtBr). Agarose, because it is safe, effective, and easy to handle, is the matrix of choice for gels for basic DNA analysis.Īfter electrophoresis is completed, the DNA must be stained in order to visualize the separated restriction fragments. The actual size of the pores in the gel influences the speed of migration, and thus this can be adjusted by varying the concentration of the matrix in the gel. The distance traveled during a defined length of time is inversely proportional to the size of the fragment. Smaller objects slip through the pores of the gel easily, while larger ones become trapped and move through more slowly. The larger the molecule, the more interaction it has with the gel matrix and the slower it moves. The rate at which a particular DNA fragment migrates through a gel is determined by its size. Since DNA is negatively charged, it migrates towards the anode. Any negatively charged molecule migrates toward the "anode" (+) end of the field, while positively charged molecules migrate toward the "cathode" (-) end. That is, a negatively charged molecule will "feel" a force pushing it against the positive-to-negative electric field lines. In an electric field E, a particle of charge q experiences a force (F) of F = qE. The solid phase has two roles: (1) it acts as a "sieve" to separate molecules according to a specific physical or chemical property and (2) it acts as a "trap" to keep separated molecules from diffusing at the conclusion of the electrophoretic treatment. Like PAGE, nearly all of the common forms of electrophoresis conduct separation in a semisolid gel matrix in which the gel consists of an aqueous phase (buffer) and a solid phase composed of a natural or artificial polymer (agarose, polyacrylamide, starch, etc.).
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