Method for extracting total DNA (deoxyribonucleic acid) of fungal hyphae

Abstract

The invention belongs to the field of molecular biology study of fungi, and in particular relates to a method for extracting total DNA (deoxyribonucleic acid) of fungal hyphae, which has the advantages of avoiding liquid nitrogen grinding and relatively thoroughly breaking cell walls to extract the total DNA of the fungal hyphae. The method comprises the following steps: (1) collecting the fungal hyphae; (2) putting into a DNA extracting solution, and mashing; (3) repeatedly freeze-thawing for 2 to 3 times in a low temperature environment of -80 DEG C and a water bath environment of 65 DEG C; (4) adding protease K and lysozyme for treating; (5) removing the enzymes and purifying the DNA. The treatment method is simple, and the required agents are common; a repeated freeze-thawing and mashing mode is adopted, so that a sample is prevented from liquid nitrogen grinding, and then the use in places where liquid nitrogen can not be obtained easily is facilitated; a mortar is not used, so that cross contamination is prevented; toxic reagents such as phenol, beta-mercaptoethanol and the like are not used; the whole extracting process is mild, the quality of the extracted total DNA of the fungal hyphae is high, and subsequent molecular biology studies such as PCR (polymerase chain reaction) amplification and sequencing of an ITS (internal transcribed spacer) sequence and the like can be performed.

Description

technical field [0001] The invention belongs to the field of molecular biology research of fungi, and in particular relates to a method for extracting total DNA of fungal hyphae. Background technique [0002] In the study of molecular biology of fungi, the extraction of total fungal DNA is crucial. At present, the commonly used extraction methods mainly include CTAB method, SDS extraction method, urea extraction method and enzymatic hydrolysis method. The extraction steps are generally similar, and the key lies in how to effectively break the cell wall, some of which are mechanically broken, and some are digested and removed by enzymes and other substances. Because the fungal cell wall is rich in polysaccharides and other substances, even after liquid nitrogen freeze-drying and grinding, it is difficult to be broken by ordinary extracts, and direct digestion with enzymes often does not get a good wall-removing effect. In fact, the wall-breaking method currently used by mos...

AI Technical Summary

Problems solved by technology

Because the fungal cell wall is rich in polysaccharides and other substances, even after liquid nitrogen freeze-drying and grinding, it is difficult to be broken by ordinary extracts, and direct digestion with enzymes often does not get a good wall-removing effect

In fact, the wall-breaking method currently used by most laboratories is liquid nitrogen freeze-drying and grinding, but the use of liquid nitrogen grinding is easy to frostbite the skin, requires strong grinding, time-consuming and labor-intensive, and is prone to cross-contamination due to the limitation of the number of mortars. Fungal hyphae are also easily lost during the grinding process, and the efficiency is not high, especially some units and institutions are severely limited due to the difficulty in obtaining liquid nitrogen and dry ice

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