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Method for extracting microbial total DNA from soil being low in biomass and enriched in humus

A humus and biomass technology, used in biochemical equipment and methods, microbial determination/inspection, DNA preparation, etc., can solve the problem of unsatisfactory DNA purity and integrity and inability to extract large-quality humus-rich soil microbial DNA and other problems, to achieve the effect of saving experimental steps and time, good application prospects, and high DNA yield

Inactive Publication Date: 2017-12-15
广东美格基因科技有限公司
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Problems solved by technology

[0004] The purpose of the present invention is to: overcome the problems that existing methods cannot extract large-quality humus-rich soil microbial DNA, and the extracted DNA has unsatisfactory purity and integrity, and provide a method that can efficiently extract large-quality soil DNA, and The extracted DNA has high purity and good integrity A method for extracting microbial total DNA from soil with low biomass and rich in humus

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  • Method for extracting microbial total DNA from soil being low in biomass and enriched in humus

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Experimental program
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Embodiment

[0025] The soil with low biomass and rich in humus obtained from the experimental plot in Alaska, USA was used as the sample for the experiment.

[0026] A. Preliminary extraction of total DNA

[0027] 1. Weigh 5g of soil sample into a sterile mortar and add 5g of sterile sand. Add liquid nitrogen to the mortar to cool the mortar and sterile sand.

[0028] 2. Add more liquid nitrogen to cover the sterile sand and sample, and start grinding after the liquid nitrogen has evaporated. Keep samples frozen while grinding.

[0029] 3. Repeat the above liquid nitrogen grinding step twice.

[0030] 4. Transfer the still frozen sample to a sterile 50mL centrifuge tube and immediately store it in a -80°C freezer for later use.

[0031] 5. Remove the ground sample from the freezer. Add 16.5 mL of DNA extraction buffer and 10 µL of proteinase K to each 50 mL tube. 37°C water bath for 40 minutes (invert the centrifuge tube every 10 minutes).

[0032] 6. Add 1.83mL of 20% SDS and bath...

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Abstract

The invention discloses a method for extracting microbial total DNA from soil being low in biomass and enriched in humus. The method disclosed by the invention comprises the following steps: (1) grinding a soil sample which is low in biomass and enriched in humus by liquid nitrogen so as to split microbial cells in the sample; (2) further splitting the microbial cells in water baths with different temperatures by using a DNA extraction buffer solution, protease K and 20% of SDS, carrying out degeneration on proteins, and centrifuging; (3) precipitating DNA through isopropanol in a low-temperature state so as to obtain a crude DNA product; and (4) purifying the crude DNA product obtained in the step (3) by using a QIAGEN soil DNA extraction kit, thereby obtaining the microbial total DNA, wherein the pH value of the DNA extraction buffer solution is 8.0, and the DNA extraction buffer solution comprises 0.1M of NaH2PO4, 0.1M of Na2HPO4, 0.1M of EDTA (Ethylene Diamine Tetraacetic Acid), 0.1M of Tris-HCl, 1.5M of NaCl and 1% of CTAB (Cetyltrimethyl Ammonium Bromide); and the concentration of the protease K is 10mg / mL. According to the method disclosed by the invention, high-efficiency high-mass soil DNA extraction can be performed, and the extracted DNA is high in purity and excellent in integrity and has excellent application prospects.

Description

technical field [0001] The invention belongs to the technical field of gene extraction, and more specifically relates to a method for extracting microbial total DNA from soil with low biomass and rich in humus. Background technique [0002] Soil is the most widely distributed habitat type in terrestrial ecosystems. Soil contains microbial communities composed of a large number of microbial individuals, and these microbial communities play a very important role in the biogeochemical cycle of terrestrial ecosystems. Analyzing the structure and function of soil microbial communities and their dynamic changes will help to understand the flow of material and energy in terrestrial ecosystems, and play an important role in understanding ecological processes. At present, the microorganisms that can be cultivated account for less than 1% of the total microorganisms. With the rapid development of molecular biology, genomics, and bioinformatics, researchers no longer rely on culture ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003C12Q2521/537
Inventor 邝嘉良束文圣叶脉徐卓菲李猛张传伦
Owner 广东美格基因科技有限公司
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