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Soil microbe genome DNA extracting method

A technology of soil microorganisms and extraction methods, applied in the direction of recombinant DNA technology, biochemical equipment and methods, DNA preparation, etc., can solve the problems of pollution and high purity, and achieve the effects of cost reduction, high purity and wide application range

Inactive Publication Date: 2009-02-25
SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The DNA obtained by this method will be polluted by extracellular DNA and humic acid in the soil, and its purity is not as high as that of the indirect method, but more DNA can be obtained

Method used

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  • Soil microbe genome DNA extracting method
  • Soil microbe genome DNA extracting method
  • Soil microbe genome DNA extracting method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Taking the black soil of farmland in Northeast China as an example (see Table 2), three methods were used to extract the genomic DNA of soil microorganisms:

[0024] Table 2 Physical and chemical properties of tested soil / g·kg -1

[0025]

[0026] Method one is the method provided by the invention:

[0027] 1) Preliminary lysing of microbial cells: Take 0.5g of soil and 0.5g of quartz sand and put them into a 1.5ml screw cap centrifuge tube, add 850μl of extraction buffer, and shake for 40S with a nucleic acid extractor (Q-biogene, USA, model FP220) at a speed of 40 seconds. It is 5.5m / s.

[0028] 2) Further lysis of microbial cells: 50 μl of lysozyme with a concentration of 100 mg / ml was added to the initially lyzed cell solution and mixed evenly. After incubating at 37° C. for 30 minutes, 100 μl of 20% (w / v) SDS was added, mixed evenly, and then Incubate at 65°C for 30 minutes, then centrifuge at 12,000 rpm for 5 minutes at room temperature, and collect the supe...

Embodiment 2

[0038] The genomic DNA extracted by the three methods in Example 1 was used as a template to amplify a partial fragment of the 16S rRNA gene, and the primers for PCR amplification were 27F:5'-GAGAGTTTGATCCTGGCTCAG-3'519R:5'-ATTACCGCGGCTGCTGG-3', in the preceding A GC clamp was added to the 5′ end of the primer: 5′-cgcccgccgcgcgcggcgggcggggcgggggcccggggg-3′. The PCR reaction system is: buffer solution: 100mM Tris, 100mM EDTA and 1.5M NaCl, pH8.0; MgCl 2 The final reaction concentration is 2.0 mM; the final reaction concentration of dNTP is 0.2 mM; the final reaction concentration of the front primer P1 is 0.5 μM; the final reaction concentration of the rear primer P2 is 0.5 μM; Taq enzyme (Takara Company) 1 U;

[0039] PCR reaction conditions: pre-denaturation: 94°C, 5min; denaturation: 94°C, 10s, annealing: 63°C, 30s, 10 cycles, each cycle annealing temperature decreased by 1°C. Extension: 68°C, 2min; denaturation: 94°C, 2min, annealing: 53°C, 30s, 25 cycles; extension: 68°C,...

Embodiment 3

[0042] Adopt the method of the present invention to extract four kinds of soils simultaneously: paddy field soil, upland umber soil, straw mat soil, upland black soil, the first three kinds of soil samples are collected to the Shenyang Ecological Station of the Shenyang Institute of Ecology, Chinese Academy of Sciences, and the black soil is collected to the Heilongjiang Agricultural Ecological Experiment of the Chinese Academy of Sciences stand. The extracted DNA was subjected to agarose gel electrophoresis, the gel concentration was 6%, the voltage was 50V, and the electrophoresis was performed for 2 hours, and the concentration and purity of the extracted nucleic acid were detected simultaneously (see Table 4).

[0043] Table 4. Four kinds of soil genomic DNA extraction results

[0044] sample Paddy soil Upland Brown Soil bedding soil Dry field black soil DNA yield (μg / ml) 107.5 84.2 62.5 150.1 OD 260nm / OD 280nm 1.84 1.88 1.99 1.78 ...

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Abstract

The invention relates to the technical field of molecular biology, in particular to a method for extracting soil microbial genome DNA. The method comprises the following concrete steps: a soil sample and quartz sand are mixed and put into extracting buffer solution in a centrifugal tube, and the centrifugal tube is placed in a nucleic acid extraction apparatus for vibrating for 40-60 seconds, or on a turbine mixer for vibrating for 10-20 minutes; the mixture obtained is added with 50-100mul of lysozyme, treated by warm bath at the temperature of 30-40 DEG C for 30-60min, added with 75-125mul of SDS and mixed evenly, and further treated by the warm bath at the temperature of 50-60 DEG C for 30-60min, and supernatant is obtained after centrifugation; and the supernatant is further extracted and purified to obtain the soil microbial genome DNA. The soil microbial genome DNA extracted by the method has high purity, the OD260nm / OD280nm can reach over 1.6, the output can be up to 100ug / ml, and the whole process takes about 10 hours; the method can be directly used for PCR reproduction without purification kit for further purification in the whole process; and by taking the obtained DNA as a template, and the target product can be smoothly reproduced.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for extracting soil microbial genome DNA. Background technique [0002] Soil microorganisms are the most active part of the soil ecosystem and play an important role in maintaining soil ecological functions. Soil microorganisms are very sensitive to changes in soil quality. Therefore, soil microorganisms have always been considered as one of the biological indicators most closely related to soil quality and sustainable development. The destruction of the environment and its potential risks provide a basis. At present, the microorganisms that can be cultivated in the laboratory are less than 1% of the total number of microorganisms in nature, and there are still quite a few strains that have not been recognized by humans because they cannot be cultivated artificially. The introduction of molecular biology technology has reduced the dependence on cultivation tec...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N15/31
Inventor 苏振成张惠文李新宇吴敏娜张成刚
Owner SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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