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General and reliable extraction method of total DNA of soil and application thereof

An extraction method and soil technology, applied in DNA preparation, recombinant DNA technology and other directions, can solve problems such as different experimental conditions and experimental purposes, and achieve the effect of reducing extraction steps, low extraction cost, universal and reliable

Inactive Publication Date: 2010-07-14
DALIAN NATIONALITIES UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although there have been many research reports on soil DNA extraction methods, none of them can be applied to all soil samples, and most of them require re-purification, and none of them can meet different experimental conditions and experiments. Purpose

Method used

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  • General and reliable extraction method of total DNA of soil and application thereof
  • General and reliable extraction method of total DNA of soil and application thereof
  • General and reliable extraction method of total DNA of soil and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Utilize the method described in the present invention to extract the soil total DNA of Dongjiagou protected land in Dalian:

[0032] (1) Soil collection and pretreatment:

[0033] The Dongjiagou Cucumber Reserve in Dalian adopts the five-point sampling method to remove the surface soil, take a 10-20cm soil sample, put it in a sterilized paper bag and bring it back to the laboratory, grind the fine soil sample with a mortar, and pass through a 200-mesh standard sieve. Wash 5 g of the soil sample with 10 mL of 0.12 mol / L sodium phosphate buffer solution with pH 8.0, centrifuge at 8000 r / min for 10 min, and wash the precipitate once more.

[0034] (2) lysed cells:

[0035] Add 5 mL of solution I (0.15 mol / L NaCl, 0.1 mol / L EDTA, 15 mg / ml lysozym, pH 8.0) to the precipitate in step (1), bathe in water at 37°C for 10 min, then add 5 mL of solution II (0.1 mol / L NaCl , 0.5mol / L Tris-HCl, 10% SDS, pH 8.0), in a water bath at 65°C for 1 hour, shaken every 15 minutes, and cent...

Embodiment 2

[0041] Utilize method described in the present invention to extract the total DNA of Tongniuling forest soil in Dalian Development Zone:

[0042] (1) Soil collection and pretreatment:

[0043] Remove the fallen leaves and humus layer from the Tongniuling forest in Dalian Development Zone, take a 10-20cm soil sample, put it in a sterilized paper bag and bring it back to the laboratory, remove the larger stones, grind the fine soil sample with a mortar, and pass the 200-mesh standard screen. Wash 5 g of the soil sample with 10 mL of 0.12 mol / L sodium phosphate buffer solution with pH 8.0, centrifuge at 8000 r / min for 10 min, and wash the precipitate once more.

[0044] (2) lysed cells:

[0045] Add 5 mL of solution I (0.15 mol / L NaCl, 0.1 mol / L EDTA, 15 mg / ml lysozym, pH 8.0) to the precipitate in step (1), bathe in water at 37°C for 10 min, then add 5 mL of solution II (0.1 mol / L NaCl , 0.5mol / L Tris-HCl, 10% SDS, pH 8.0), in a water bath at 65°C for 1 hour, shaken every 15 ...

Embodiment 3

[0051] Utilize the method described in the present invention to extract the total DNA of Dalian offshore sludge soil:

[0052] (1) Sludge collection and pretreatment:

[0053] Take a boat from Boshiwan, Dalian Development Zone, into the sea, collect about 10m of sea mud, put it into a sterile plastic bag, and bring it back to the laboratory, remove larger sand particles, and wash the sludge with 10mL of 0.12mol / L sodium phosphate buffer solution with a pH of 8.0 5 g of the sample was centrifuged at 8000 r / min for 10 min, and the precipitate was washed once more.

[0054] (2) lysed cells:

[0055] Add 5 mL of solution I (0.15 mol / L NaCl, 0.1 mol / L EDTA, 15 mg / ml lysozym, pH 8.0) to the precipitate in step (1), bathe in water at 37°C for 10 min, then add 5 mL of solution II (0.1 mol / L NaCl , 0.5mol / L Tris-HCl, 10% SDS, pH 8.0), in a water bath at 65°C for 1 hour, shaken every 15 minutes, and centrifuged for 10 minutes (6000r / min) to discard the precipitate. Add 2.7mL 5mol / L N...

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Abstract

The invention relates to a general and reliable extraction method of total DNA of soil and application thereof, belonging to the field of molecular biology. The soil contains very rich microbial resources, but if the traditional pure culture technique is utilized, only 1 percent of microbes can be cultured, while the microbes incapable of being cultured are the main body of microbial diversity of the soil, thus bringing great limitation to the development and utilization of the diverse resources of the microbes. The extraction method provided by the study uses solution I, solution II, potassium acetate and magnesium chloride to remove the impurities of humic acid and the like, can meet the needs of different types of soil samples, can obtain DNA which has high quality and large fragment and is suitable for following cloning experiment, and lays the foundation for studying the microbial diversity of the soil (sludge) sample in various environments. The extraction process does not need to be purified, thus shortening the extraction step and reducing the extraction cost.

Description

technical field [0001] The invention relates to a soil total DNA extraction method, in particular to a universal and reliable soil total DNA extraction method and its application in various soil samples. Background technique [0002] Soil contains very rich microbial resources, but due to the limitation of research methods and means, most of the microorganisms in soil are still unknown. Molecular microbial ecology studies have shown that only 1% of microorganisms can be cultured using traditional pure culture techniques, and the uncultured microorganisms are the main body of soil microbial diversity (Amann RI. et al, 1995), which gives The development and utilization of microbial diversity resources has brought great limitations. [0003] One of the technical keys to studying unculturable microorganisms in soil at the molecular level is to obtain molecularly manipulated metagenomic DNA from various soil samples. Obtaining high-purity, large fragments, and complete soil mic...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 刘秋于基成胡英畅闫建芳
Owner DALIAN NATIONALITIES UNIVERSITY
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