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Extraction method of total DNA of metagenome of fish enteric microorganisms and kit

A technology of gut microbes and metagenomics, which is applied in the field of extracting total DNA from fish gut microbes metagenomic library construction, can solve problems such as unsatisfactory, and achieve the effect of improving integrity and simplifying processing

Active Publication Date: 2017-05-31
QINGDAO AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, conventional DNA extraction methods cannot meet the requirements for the construction of fish intestinal microbial metagenomic libraries

Method used

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  • Extraction method of total DNA of metagenome of fish enteric microorganisms and kit
  • Extraction method of total DNA of metagenome of fish enteric microorganisms and kit
  • Extraction method of total DNA of metagenome of fish enteric microorganisms and kit

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Embodiment Construction

[0033] The present invention provides a method and a kit for extracting the total DNA of the metagenome of fish intestinal microorganisms. In order to make the purpose, technical scheme and effect of the present invention clearer and clearer, the present invention will be further described in detail below. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

[0034] The invention provides a kit for the method of extracting the total DNA of the metagenome of fish intestinal microorganisms, which comprises:

[0035] Solution A: 100mM / L Tris-HCl, 1mM / L EDTA, pH 8.0;

[0036] Solution B: 100mM / L Tris-HCl, 20mM / L EDTA, 100mM / L NaCl, 0.1g / L PVP, 100mM / L guanidine isothiocyanate, 0.1% (v / v) Triton-X100, 0.1mM PMSF, 1% (v / v) β-mercaptoethanol, pH 8.5;

[0037] Solution C: 100mM / L Tris-HCl, 1mM / L EDTA, 0.9% NaCl, 5mM / L DTT, pH 8.0;

[0038] Solution D: 50mg / ml proteinase K, 20mg / ml l...

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Abstract

The invention provides an extraction method of total DNA of metagenome of fish enteric microorganisms and a kit. The extraction method comprises the following steps: (1) fish sample sampling and fixing treatment of microorganisms on the surface of the fish; (2) acquisition of enteric microorganisms and collection of thalli; (3) embedding and cracking of cells; (4) extraction of DNA; and (5) DNA preservation. The kit comprises the following main components: a solution A which is a mixed solution of Tris and EDTA; a solution B which is a mixed solution of Tris, EDTA, NaCL, PVP, guanidinium isothiocyanate, Triton-X100, PMSF, and beta-mercaptoethanol; a solution C which is a mixed solution of Tris, EDTA, NaCl and DTT; a solution D which is a mixed solution of proteinaseK, a lysozyme, Tris, NaCl and SDS; a solution E which is a mixed solution of Tris saturated phenol and chloroform; F which is isopropanol; and G which is an ethanol aqueous solution. The DNA fragment obtained by the invention is high in purity, and the content of DNA of a host and the microorganisms on the surface of the fish is small.

Description

technical field [0001] The invention relates to the field of DAN extraction, in particular to an extraction method and a kit for constructing a total DNA of a fish intestinal microbial metagenomic library. Background technique [0002] At present, traditional microbial research methods can only cultivate 0.1% to 1% of environmental microorganisms, which will not be able to analyze and utilize the composition and function of microbial communities as a whole. Metagenomic library technology can avoid the restriction on the pure culture of microorganisms, obtain DNA molecules directly from the environment, and provide an important means for human beings to further utilize microbial resources through sequencing or library screening. The extraction and purification of DNA is the first and most critical step in the construction of a metagenomic library. [0003] Fish intestinal microbes have very important research value, and can be used to study fish health and nutrient metabolis...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 朱丹程凡升
Owner QINGDAO AGRI UNIV
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