Agarose gel can distinguish DNA fragments with a difference of about 100bp, and its resolution is lower than that of polyacrylamide gel, but it is easy to prepare and has a wide range of separation, which is especially suitable for the separation of large segments of DNA.The range of DNA separation by ordinary agarose gel is 0.2-20kb, and DNA fragments as high as 10^7bp can be separated by using pulsed electrophoresis.
Characteristics of agarose gels
Natural agar (agar) is a polysaccharide that consists mainly of agarose (agarose, about 80%) and agar gum (agaropectin). Agarose is composed of galactose and its derivatives of neutral substances, uncharged, while agar gum is a strong acidic polysaccharide containing sulfate and carboxylic acid groups, due to the charge of these groups, under the action of the electric field can produce a strong electroosmosis phenomenon, coupled with sulfate can be affected by the role of certain proteins and electrophoretic speed and separation effect. Therefore, at present, agarose is mostly used as electrophoretic support for plate electrophoresis, and its advantages are as follows.
(1) Agarose gel electrophoresis is easy to operate, the electrophoresis speed is fast, and the samples can be electrophoresed without prior treatment.
(2) The structure of agarose gel is uniform, the water content is large (about 98%~99%), approximate free electrophoresis, the sample diffusion is more free current, and the adsorption of the sample is extremely small, so the electrophoresis pattern is clear, high resolution and good repeatability.
(3) Agarose is transparent without ultraviolet absorption, and the electrophoresis process and results can be directly detected and quantitatively determined by ultraviolet light.
(4) The zones after electrophoresis are easy to be stained, and the sample is very easy to be eluted, which is convenient for quantitative determination. The dry film can be preserved for a long time.
At present, agarose is commonly used as electrophoretic support to separate proteins and isoenzymes. The combination of agarose electrophoresis and immunochemistry has led to the development of immunoelectrophoresis technology, which is capable of identifying complex systems that cannot be identified by other methods, and due to the establishment of the ultra-micro technology, 0.1ug of protein can be detected.
Agarose gel electrophoresis is also commonly used in the separation and identification of nucleic acids, such as DNA identification, DNA restriction endonuclease mapping. Because this method is easy to operate, simple equipment, requires a small amount of sample, high resolution, has become one of the commonly used experimental methods in genetic engineering research.