Extraction method of total DNA of microorganisms from soil sample

A soil sample and extraction method technology, which is applied in the field of extraction of microbial total DNA in oil and gas field soil samples, can solve the problems of general extraction effect and low efficiency of microbial cell wall fragmentation, and achieve cost saving and optimistic application prospects

Inactive Publication Date: 2015-05-27
CHINA PETROLEUM & CHEM CORP +1
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Problems solved by technology

[0004] CN101230342A discloses a method for extracting total DNA from soil samples to improve DNA quality. In this method, the cells are first broken by enzymatic and chemical methods, and then freeze-thawed. This method is not efficient f...

Method used

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  • Extraction method of total DNA of microorganisms from soil sample

Examples

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Embodiment 1

[0024] The soil samples described in this example are sand samples near oil and gas fields. The method for extracting the total DNA of microorganisms in the soil sample of this embodiment is as follows.

[0025] (1) Weigh 0.5g of soil into a 2ml centrifuge tube, add 1ml of DNA extraction buffer (0.1M Tris-HCl[pH8.0], 0.1M EDTA[pH8.0], 0.1M Sodium Phosphate[pH8.0] ], 1.5M NaCl); mix well;

[0026] (2) Quick-freeze the centrifuge tube that has been added with soil samples and extracts in liquid nitrogen for 1 min, then bathe in water at 65°C until it melts, and repeat the freeze-thaw treatment three times; then add 25 μl proteinase K solution (10 mg / ml), Mix well; 37 ℃ water bath for 30 minutes, shake once every 5-10 minutes;

[0027] (3) Add 200 μL of 20% sodium dodecylsulfonate (SDS) solution and shake gently. Water bath at 65°C for 30 minutes, during which time shake once every 5-10 minutes. Centrifuge at 10,000×g for 10 minutes at room temperature, and transfer the superna...

Embodiment 2

[0032] The soil samples described in this example are fertile soil samples near oil and gas fields. The extraction method of total microbial DNA in the soil sample in this example is basically the same as that in Example 1, but 1 wt% cetyltrimethylammonium bromide (CTAB) is also added to the DNA extraction buffer.

Embodiment 3

[0034] The soil samples described in this example are clay samples near oil and gas fields. The extraction method of total microbial DNA in the soil sample of this embodiment is the same as the extraction method in Example 1.

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Abstract

The invention relates to an extraction method of total DNA of microorganisms from a soil sample. The extraction method particularly includes following steps: (1) adding a DNA extraction buffer solution to the soil sample and performing repetitive freeze-thawing in liquid nitrogen and hot water bath; (2) adding a protease K, performing a water bath treatment process at 37 DEG C for 30 min; (3) adding a sodium dodecyl solution, performing a water bath treatment process at 65 DEG C for 30 min; (4) performing centrifugation to remove precipitations and collect a supernate; (5) performing extraction with a mixed solution containing phenol, chloroform and isoamylol and performing centrifugation to collect a supernate; (6) performing extraction with a mixed solution containing chloroform and isoamylol and performing centrifugation to collect a supernate; (7) adding isopropanol to the supernate in the step (6) to performing precipitation and collecting a precipitation after centrifugation; (8) adding ethanol for washing the precipitation and drying the precipitation at the room temperature; (9) adding a TE buffer solution for dissolving the precipitation to obtain a DNA crude extraction; and (10) purifying the DNA crude extraction through an adsorption column, packaging the purified DNA crude extraction and storing the purified DNA crude extraction at -20 DEG C. The extraction method is especially effective when being used in samples from deep soil and dry sand which are less in biomass and in a clayed soil sample. The DNA extraction can be directly used in subsequent molecular biological detection.

Description

technical field [0001] The invention relates to a method for extracting total DNA of microorganisms in soil samples, more specifically, the invention relates to a method for extracting total DNA of microorganisms in soil samples of oil and gas fields. Background technique [0002] The traditional oil and gas microbial exploration technology uses microbial counting, enrichment culture, separation, and physiological and biochemical analysis of microbial metabolites to study the abnormality of oil and gas microorganisms in soil, thereby predicting the existence of oil and gas reservoirs. However, in recent years, many researchers at home and abroad have gradually realized that the conventional microbial isolation and culture technology has great limitations in the determination of microbial counts. 1%, many "viable but non-culturable" microorganisms could not be isolated and identified. Therefore, the traditional culture method may make the life characteristics and ecological ...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
Inventor 许科伟赵克斌任春黄欣汤玉平杨帆蒋涛
Owner CHINA PETROLEUM & CHEM CORP
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