Agarose gel electrophoresis and its influencing factors Agarose or polyacrylamide gel electrophoresis is the standard method for separation, identification and purification of DNA fragments. This technique is simple and quick to operate, and can distinguish DNA fragments that cannot be separated by other methods (such as density gradient centrifugation). When stained with a low concentration of fluorescent intercalating dye Ethidiumbromide (EB), at least 1-10 ng of DNA bands can be detected under ultraviolet light, so that the location of DNA fragments in the gel can be determined. In addition, specific DNA bands can also be recovered from the gel after electrophoresis for use in future cloning operations. Agarose and polyacrylamide can be made into various shapes, sizes and porosities. Separation of DNA fragments in agarose gel has a wide range of sizes, and different concentrations of agarose gel can separate DNA fragments from 200 bp to nearly 50 kb in length. Agarose is usually electrophoresed in a horizontal device under an electric field of constant intensity and direction. Polyacrylamide is better for separating small DNA (5-500 bp), and its resolution is extremely high, even DNA fragments with a difference of 1 bp can be separated. Polyacrylamide gel electrophoresis is fast and can hold a relatively large amount of DNA, but it is more difficult to prepare and handle than agarose gel. The polyacrylamide gel is electrophoresed using a vertical device. At present, the general laboratory mostly uses agarose horizontal flat-plate gel electrophoresis device for DNA electrophoresis. Agarose is mainly used as a solid support matrix in DNA preparation electrophoresis, and its density depends on the concentration of agarose. In an electric field, negatively charged DNA migrates to the anode at a neutral pH, and its migration rate is determined by the following factors: 1. The molecular size of DNA: linear double-stranded DNA molecules in agarose gel at a certain concentration The migration rate is inversely proportional to the logarithm of the molecular weight of DNA. The larger the molecule, the greater the resistance and the harder it is to creep through the pores of the gel, so the migration is slower. 2. Agarose concentration A linear DNA molecule of a given size, its migration speed is different in different concentrations of agarose gel. The logarithm of the DNA electrophoretic mobility is linearly related to the gel concentration. The choice of gel concentration depends on the size of the DNA molecule. The gel concentration required to separate DNA fragments smaller than 0.5 kb is 1.2 ~ 1.5%, the gel concentration required to separate DNA molecules larger than 10 kb is 0.3-0.7%, and the gel concentration required between DNA fragments between the two is 0.8 ~ 1.0%. 3. Conformation of DNA molecules When a DNA molecule is in a different conformation, the distance it moves in the electric field is not only related to molecular weight, but also to its own conformation. Linear, open-loop and supercoiled DNA of the same molecular weight move in agarose gels at different speeds. Supercoiled DNA moves the fastest and linear double-stranded DNA moves the slowest. For example, when identifying the purity of the plasmid by electrophoresis, it is found that there are several DNA bands on the gel. It is difficult to determine whether it is caused by a different conformation of the plasmid DNA or because it contains other DNA. The DNA bands can be recovered one by one from the agarose gel. Dicer is hydrolyzed separately and then electrophoresed. If the same DNA pattern appears on the gel, it is the same DNA. 4. When the power supply voltage is low, the migration rate of linear DNA fragments is proportional to the applied voltage. However, as the electric field strength increases, the mobility of DNA fragments of different molecular weights will increase by different amplitudes. The larger the fragment, the greater the increase in mobility due to the increase in field strength, so the voltage increases and the agarose coagulation The effective separation range of glue will be reduced. To maximize the resolution of DNA fragments larger than 2 kb, the applied voltage must not exceed 5 v / cm. 5. The presence of intercalating dye The fluorescent dye ethidium bromide is used to detect DNA in agarose gel. The dye will intercalate between the stacked base pairs and elongate the linear and gapped circular DNA to make it more rigid Strong, will also reduce the linear DNA migration rate by 15%. 6. Ionic strength affects the composition of the electrophoresis buffer and its ionic strength affects the electrophoretic mobility of DNA. In the absence of ions (such as the use of distilled water to prepare a gel), the conductivity is minimal, and the DNA hardly moves. In a buffer with high ionic strength (such as the addition of 10 × electrophoresis buffer by mistake), the conductivity is very high and obvious Heat, when severe, can cause the gel to melt or the DNA to denature. For natural double-stranded DNA, several commonly used electrophoresis buffers are TAE [containing EDTA (pH8.0) and Tris-acetic acid], TBE (Tris-boric acid and EDTA), TPE (Tris-phosphate and EDTA), generally formulated Into a concentrated mother liquor and store at room temperature.
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