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Method for extracting mold spore dna by mechanical crushing method

A technology of mold spores and mechanical fragmentation, which is applied in the field of DNA extraction from mold spores by mechanical fragmentation, and achieves the effects of good DNA quality, reliable research basis, and high extraction efficiency.

Active Publication Date: 2020-07-28
CHINA AGRI UNIV
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Problems solved by technology

The traditional method requires a large number of samples to extract DNA, so that the existing method of extracting DNA from mold spores cannot meet this sequencing condition

Method used

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  • Method for extracting mold spore dna by mechanical crushing method
  • Method for extracting mold spore dna by mechanical crushing method
  • Method for extracting mold spore dna by mechanical crushing method

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Experimental program
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Effect test

Embodiment

[0052] 1. Experimental materials

[0053] The mold spores used in the following experiments are mainly Aspergillus niger spores. The Aspergillus niger strains were purchased from Shanghai Fuxiang Biological Company and stored in 0.9% sterile sodium chloride solution containing 15% glycerol. The freeze-dried strains were used as generation 0 , mainly the 3rd and 4th generations in the experiment. Cultivated according to conventional methods in the art.

[0054] 2. The mold spore mechanical crushing method used in the following experiments: take the mold spore liquid, add glass beads (hereinafter referred to as beads), carry out mechanical crushing (grinding) processing, and then carry out DNA extraction.

[0055] 3. The DNA extraction method used in the following experiments:

[0056] (1) Add 20 μL Proteinase K solution to the tube and mix well;

[0057] (2) Add 220 μL buffer GB, shake for 15 sec, place at 70°C for 10 min, the solution should become clear, and briefly centrifu...

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Abstract

The invention relates to a method for extracting DNA of mold spores with a mechanical breaking method. The method comprises the following steps: a mold spore liquid is taken, glass beads are added to the mold spore liquid for mechanical breaking treatment, and then DNA extraction is performed. Mold spore walls are broken with the mechanical method, DNA extraction and purification are performed through combination with a column purification method after breaking, then an extraction effect is detected with real-time fluorescence quantitative PCR and gel electrophoresis, and a technological system for precisely extracting DNA of the mold spores is established on the basis. The method is high in DNA extraction efficiency, convenient to operate, easy to control, non-toxic and harmless. The quantity of the mold spores is accurate to 10<2> for DNA extraction for the first time, study for the mold spores can be turned to precise study of single cell or a small quantity of cells from group study of a large quantity of cells, the extracted DNA is good in quality, follow-up sequencing can be met, and reliable study basis and powerful guarantee are provided for follow-up molecular biology studies.

Description

technical field [0001] The invention relates to a method for extracting mold spore DNA by a mechanical crushing method. Background technique [0002] Although there are many methods for extracting DNA, and many of them have matured day by day, for fungi with complex cell wall components, these common DNA extraction methods have limitations. The polysaccharides, pigments, nucleases and other substances rich in fungi make DNA extraction And isolation is more difficult, especially the DNA extraction of mold spores that contain a harder spore wall is more limited. [0003] On the other hand, due to the development of sequencing technology, our traditional large-sample cell sequencing method has been changed, and the analysis of a cell population has been transformed into the analysis of a single cell, which can provide us with more valuable research. For example, in different positions of the mold culture dish, the genetic composition of the mold in different positions may be d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12R1/685
CPCC12N15/1003C12Q2563/149
Inventor 罗云波黄昆仑许文涛田兰芳贺晓云郭明璋
Owner CHINA AGRI UNIV
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