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Soil sample total DNA extraction method for improving DNA quality

A soil sample and extraction method technology, applied in the field of soil microbiology, can solve the problems of limited removal capacity and large difference in DNA quality, and achieve the effects of small cost increase, easy PCR amplification process and wide application range

Inactive Publication Date: 2008-07-30
SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complexity of environmental samples, the quality of DNA obtained by different DNA extraction methods varies greatly
For soil samples rich in organic matter content, the traditional "mechanical wall breaking + enzymatic action" extraction method has limited ability to remove impurities such as humic acid contained in the soil

Method used

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  • Soil sample total DNA extraction method for improving DNA quality

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] 1) Accurately weigh 0.5g of northeast black soil soil sample, place it in a 2mL sterilized centrifuge tube, add 0.3g of quartz sand, and then add 800-1000μL of DNA extraction buffer (100mM Tris-HCl[pH8.0], 100mM EDTA [pH8.0], 100 mM sodium phosphate [pH8.0], 1.5M NaCl, 1% CTAB) and 5 μL proteinase K (20 mg / mL). Vortex and vibrate for 10 minutes to fully disperse the soil aggregate structure and fully mix with DNA extraction buffer and other components. Place in a 37°C water bath for 1 hour. During the water bath process, turn the sample over and mix several times every 15 minutes; the phosphate buffer It is 0.147g sodium dihydrogen phosphate; 5.08g disodium hydrogen phosphate heptahydrate can be dissolved in 200 ml of water.

[0022] 2) Add 200 μL of 10% SDS solution to the reaction solution after shaking, vortex to mix, and then freeze and thaw repeatedly 3 times. The freezing time at ℃ is 30 minutes, and the melting time at 65 ℃ is 20 minutes. Then put it in a 65°C ...

Embodiment 2

[0030]1) Accurately weigh 1.0g of Changbai Mountain Korean pine forest soil sample, place it in a 5mL sterilized centrifuge tube, add 0.5g of quartz sand, and then add 1500μL of DNA extraction buffer (100mM Tris-HCl[pH7.5], 100mM sodium EDTA [pH7.5], 100 mM sodium phosphate [pH7.5], 1.5M NaCl, 1% CTAB) and 15 μL proteinase K (20 mg / mL). Vortex and vibrate for 20 minutes to fully disperse the soil aggregate structure and fully mix it with DNA extraction buffer and other components. Place it in a 37°C water bath for 1.5 hours. The solution is 0.735g of sodium dihydrogen phosphate and 25.4g of disodium hydrogen phosphate heptahydrate dissolved in 1000 ml of water.

[0031] 2) Add 400 μL of 10% SDS solution to the reaction solution after shaking, vortex to mix, and then freeze and thaw repeatedly 3 times. 40min, melting time at 65°C is 10min. . Then put it in a 65°C water bath for 2 hours, and gently shake it every 15-20 minutes to fully lyse the cell wall; then centrifuge at 1...

Embodiment 3

[0039] The difference from Example 1 is:

[0040] 1) Accurately weigh 5g of soil sample, add 1g of quartz sand, and then add 5mL of DNA extraction buffer (50mM Tris-HCl[pH7.5], 80mM sodium EDTA[pH7.5], 60mM phosphate buffer (sodium phosphate) [pH7.5], 2.5M NaCl, 2% CTAB) and 50 μL proteinase K (20 mg / mL). Vortex and vibrate for 15 minutes to fully disperse the soil aggregate structure and fully mix with DNA extraction buffer and other components, place in a 30°C water bath for 2 hours, and turn the sample over and mix several times every 5 minutes during the water bath;

[0041] 2) Add 2000 μL of 10% SDS solution to the reaction solution after shaking, centrifuge at 8000×g for 30 min at room temperature, and collect the supernatant in a new sterilized centrifuge tube.

[0042] Add 600 μL of DNA extraction buffer and 40 μL of 10% SDS solution to the obtained precipitate, re-shock, dissolve the precipitate, and place it in a 65°C water bath for 50-60 min, centrifuge at 8000×g f...

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Abstract

The invention relates to soil microbiology, in particular to a method of extracting the total DNA of the soil sample, which can improve the quality of DNA. The method includes the following steps: adding DNA extracting buffer to the soil sample, and then adding protease K; freeze thawing under -65 to 65 DEG C and centrifuging; discarding the precipitate; collecting the supernatant liquid; extracting and centrifuging by the same volume of the solution of phenol, chloroform and isoamylol, reserving the water phase; extracting one more time by the solution of chloroform and isoamylol; adding sodium acetate and cold isopropanol, and then centrifuging and collecting the DNA precipitate; washing by ethanol and drying in room temperature; adding TE buffer to dissolve, and gaining the DNA coarse extract; adding DNA extracting buffer in the coarse extract; and then adding the same volume of the solution of phenol, chloroform and isoamylol; repeating the preceding steps such as precipitating, washing and dissolving; finally, gaining the DNA of better quality. The method adopted by the invention can effectively remove the impurity in the DNA, which evidently increases the purity and quality of DNA.

Description

technical field [0001] The invention relates to soil microbiology, in particular to a method for extracting total DNA from soil samples for improving DNA quality. Background technique [0002] Many studies have shown that under the current technical level, most of the microorganisms (>95%) in the natural ecological environment cannot be successfully cultivated in the laboratory. Since the medium cannot meet the real habitat conditions of most microorganisms, pure culture technology only selectively allows those microbial groups that grow faster and adapt to changing conditions to grow and reproduce preferentially. Microorganisms obtained by pure culture techniques cannot represent the true microbial community structure composition in the environment. [0003] In the past 20 years, many molecular biology and molecular ecology techniques based on nucleic acid (or 16S rRNA gene sequence diversity) have been continuously developed and gradually matured, providing strong tech...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 史荣久莫旭华徐慧杨伟超张颖
Owner SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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