Method for extracting microbial total DNA of cotton dreg fermentation sample

A technology of cottonseed meal fermentation and extraction method, which is applied in the field of biological genetic engineering and can solve problems such as difficulty in extracting total microbial DNA

Inactive Publication Date: 2011-06-22
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Cotton meal and its fermentation products are special samples, which contain a large amount of proteins, polysaccharides and phenols, making it extremely difficult to extract the total DNA of microorganisms, especially the phenolic substances contained in cotton meal It has a serious impact on the subsequent molecular manipulation of the extracted DNA. However, there is no research and method for the extraction of total microbial DNA from such samples at home and abroad.

Method used

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  • Method for extracting microbial total DNA of cotton dreg fermentation sample
  • Method for extracting microbial total DNA of cotton dreg fermentation sample
  • Method for extracting microbial total DNA of cotton dreg fermentation sample

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1: (extraction method 1 of cottonseed meal fermentation sample microbial total DNA)

[0032] (1) Weigh 0.5 g of cottonseed meal fermentation sample into a sterilized 10mL eppendoff tube, add 3mL of cottonseed meal decolorization buffer, mix on a vortex mixer for 2min, bathe in 37°C water for 10min, vortex again for 2min, 9000 Centrifuge at × g for 10 min, suck off the supernatant with a pipette; repeat the above steps 3 times until the color of the upper liquid turns from brown to light yellow, discard the upper liquid, and keep the precipitate;

[0033] The described cottonseed meal decolorization buffer solution is prepared with deionized water, and its composition is: 50mmol L -1 Tris-Cl, 50mmol·L -1 EDTA, 300mmol·L -1 NaCl, 0.01g·mL -1 PVP (polyvinylpyrrolidone), 0.01g mL -1 Na 2 CO 3 , and adjust the pH to 9.5.

[0034] (2) Add 1mL of cottonseed meal fermentation sample microbial DNA extraction buffer to the precipitate in step (1), and then add...

Embodiment 2

[0043] Embodiment 2: (extraction method 2 of cottonseed meal fermentation sample microbial total DNA)

[0044] (1) Weigh 1 g of cottonseed meal fermentation sample into a sterilized 10 mL eppendoff tube, add 4 mL -1 Cottonseed meal decolorization buffer, mixed on a vortex mixer for 3 minutes, 40°C water bath for 10 minutes, vortex mixed for 2 minutes, centrifuged at 8000×g for 15 minutes, and sucked off the supernatant with a pipette gun; repeat the above steps twice until the upper layer liquid Until the color turns from brown to light yellow, discard the upper liquid after centrifugation and keep the precipitate;

[0045] The described cottonseed meal decolorization buffer solution is prepared with deionized water, and its composition is: 75mmol L -1 Tris-Cl, 35mmol·L -1 EDTA, 200mmol·L -1 NaCl, 0.02g·mL -1 PVP (polyvinylpyrrolidone), 0.02g mL -1 Na 2 CO 3 , and adjust the pH to 10.

[0046] (2) Add 2 mL of cottonseed meal fermentation sample microbial DNA extrac...

Embodiment 3

[0055] Embodiment 3: (extraction method 3 of cottonseed meal fermentation sample microbial total DNA)

[0056] (1) Weigh 2g of cottonseed meal fermentation sample into a sterilized 10mL eppendoff tube, add 5mL of cottonseed meal decolorization buffer, mix on a vortex mixer for 3min, place in a water bath at 45°C for 15min, and then mix on a vortex mixer 2min, centrifuge at 6000×g for 15min, absorb the upper layer liquid with a pipette gun, repeat the above steps 5 times, the color of the supernatant liquid becomes lighter, discard the upper layer liquid after centrifugation, and keep the precipitate;

[0057] Cottonseed meal decolorization buffer is prepared with deionized water, the composition is: 100mmol L -1 Tris-Cl, 20mmol·L -1 EDTA, 100mmol·L -1 NaCl, 0.03g·mL -1 PVP (polyvinylpyrrolidone), 0.03g mL -1 Na 2 CO 3 , and adjust the pH to 10.5.

[0058] (2) Add 2 mL of cottonseed meal fermentation sample microbial DNA extraction buffer into the eppendoff tube, and...

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Abstract

The invention discloses a method for extracting microbial total DNA of a cotton dreg fermentation sample, which belongs to the technical field of bio-genetic engineering. The method aims at the adverse effects caused by protein, polysaccharide and phenol substances contained in a cotton dreg and a fermentation product in the process of extracting the microbial total DNA and the subsequent molecular operations, and is a method which is used for extracting the microbial total DNA at high quality and consists of key techniques of phenol removal through decolorization, protein and polysaccharide removal through repeated extraction, moderate wall breaking of microbial cells and the like. The method has the characteristics of strong pertinence, simplicity, practicability, lower extraction cost and the like, and the obtained DNA can be directly applied to PCR amplification and enzyme cutting reaction, thus the method establishes a foundation for researching microbial fermentation cotton dregdetoxification by using a molecular ecological method. The method can be applied to units or fields relating to cotton dreg comprehensive development technical study.

Description

technical field [0001] The invention relates to the technical field of biological genetic engineering, in particular to a method for extracting total DNA of microorganisms in a cotton meal fermentation sample. Background technique [0002] Cotton meal is a by-product of extracting cottonseed oil. It generally contains 33% to 42% crude protein. It is a plant protein resource second only to soybean meal in output. However, because cotton meal contains free gossypol, which is toxic to animal organisms, its utilization value and usage are severely limited. The method of microbial fermentation can not only detoxify cotton meal, but also improve the protein content and nutritional value of cotton meal, so it has become an important technology at present. Among them, the screening of fermentation strains and the optimization of fermentation conditions are two very important aspects in the detoxification technology of microbial fermentation cotton meal. In order to eliminate the i...

Claims

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Application Information

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IPC IPC(8): C07H21/04C12N15/10
Inventor 王新汤江武姚晓红吴逸飞
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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