Agarose gel electrophoresis is a commonly used method for isolation, identification and purification of DNA fragments. When DNA molecules are swimming in agarose gel, they have charge effect and molecular sieve effect. DNA molecules are negatively charged in pH solution above isoelectric point and move to the positive electrode in the electric field. Due to the repetitive structure of the glycophosphate backbone, the same amount of double stranded DNA has almost the same net charge, so they can move to the positive direction at the same speed.
Different concentrations of agarose gel can separate DNA fragments from 200bp to 50kb. In the agarose solution, low concentration of ethidum bromide (EB) was added into the agarose solution. The DNA band of 10NG could be detected under UV light. In the electric field, the negatively charged DNA migrated to the anode in pH8.0.
Agarose gel has the following characteristics:
(1) the molecular size of DNA in the gel matrix is inversely proportional to the value of the base logarithm, and the larger the molecule, the slower the migration.
(2) agarose concentration in a specific size of linear DNA molecule is different from agarose gel in different concentrations. The logarithm of electrophoretic mobility (U) of DNA is linearly related to gel concentration (T).
(3) When the voltage is low, the migration rate of linear DNA fragments is proportional to the applied voltage. However, with the increase of electric field intensity, the mobility of DNA fragments with different molecular weights will increase in different ranges. With the increase of voltage, the effective separation scope of agarose gel will be reduced. In order to maximize the resolution of DNA fragments larger than 2KB, the applied voltage should not exceed 5V / cm.
(4) electrophoretic behavior of electrophoresis temperature DNA in agarose gel electrophoresis is not affected by the temperature of electrophoresis. The relative migration rate of DNA fragments of different sizes does not change significantly between 4 and 30 degrees, but gel with low concentration of 0.5% or low melting point gel is relatively weak, preferably at 4 degrees.
(5) dye embedded fluorescent dye ethidium bromide was used to detect DNA in agarose gel. The dye was embedded in the stacked base pairs and elongated and notched circular DNA, making it more rigid and reducing the linear mobility by 15%.
(6) The composition of ionic strength electrophoresis buffer and its ionic strength affect the mobility of DNA electrophoresis. In the absence of ions, such as misuse of distilled water to prepare gels, the conductivity is minimal and DNA hardly moves. In high ionic strength buffer (such as 10 x electrophoresis buffer), conductance is very high and obvious heat production.
Agarose gel electrophoresis is also commonly used in isolation and identification of nucleic acids, such as DNA identification, DNA restriction endonuclease map production, etc. Because of its convenient operation, simple equipment, small sample size and high resolution, this method has become one of the common experimental methods in genetic engineering research. Agarose gel electrophoresis kit contains 10 agarose preforms, high voltage fast electrophoresis solution 15mL, DNA loading buffer6x 200 L, scissors and user manuals, with 8 hole 6*6, 6 hole 6*6, 13 hole 6*12, 18 hole 6*12 and other specifications.
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