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Method for extracting soil microbial DNA

A soil microorganism and extraction method technology, applied in the field of soil microorganism DNA extraction, can solve the problems of complex and numerous chemical reagent operations, and achieve the effect of major application prospects.

Inactive Publication Date: 2007-06-13
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to overcome the shortcomings of the existing soil DNA extraction involving numerous chemical reagents and complicated operations, and to provide a soil DNA method that can remove a large amount of humus and protein, and the obtained soil DNA can be directly used for molecular operations such as PCR. Extraction Method

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  • Method for extracting soil microbial DNA

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Embodiment 1: the extraction of soil DNA

[0025] step:

[0026] (1) Soil microbial cell lysis:

[0027] 1) Place 5g of soil in an Erlenmeyer flask containing 10ml of soil extraction buffer (100mM Tris-HCl, 100mM EDTA, 1.5M NaCl, pH8.0), add 2mg proteinase K, 0.1mgPVPP and 5g small glass to the extract Beads, shake at 55°C for 30min (180rpm);

[0028] 2) Add 1ml of 100g / LSDS and continue shaking for 1min;

[0029] 3) After the sample was transferred to a 65° C. warm bath for 1 hour, it was centrifuged at 6000 rpm for 10 minutes, and the supernatant was taken to obtain a cell lysate.

[0030] (2) Obtaining of crude DNA solution:

[0031] 1) Add 1 / 2 volume of PEG20000 (300g / L PEG20000 mixed with an equal volume of 1.6M NaCl) to the cell lysate, and precipitate at room temperature for 2 hours;

[0032] 2) Centrifuge at room temperature for 20 min at 10,000 rpm to collect the precipitate;

[0033] 3) Dissolve the precipitate with 0.5ml of TE to obtain a crude DNA solu...

Embodiment 2

[0040] Embodiment 2: comparative experiment

[0041]In the control method, PVPP was not added to the DNA extraction buffer, PEG20000 was not used for the initial DNA precipitation, but isopropanol was used instead, and other operating steps remained unchanged.

[0042] The purity of DNA extracted by the two extraction methods was compared. The OD260 / OD230 and OD260 / OD280 values ​​of the DNA solution obtained by the control method and the method of the present invention were measured respectively with a spectrophotometer, and the results are shown in Table 1. As can be seen from Table 1, the OD260 / OD230 and OD260 / 280 of the soil DNA extracted by the control method are only 0.87 and 1.17, which are far lower than the standard 2.0 and 1.6, indicating that the content of humus and protein in the DNA solution is very high. Molecular manipulation cannot be carried out without further purification treatment; and the DNA solution extracted by the method of the present invention, OD26...

Embodiment 3

[0046] Embodiment 3: the extraction of the microbial DNA in other types of soil

[0047] The method described in Example 1 was used to extract DNA from soils from Sichuan (taken from Mount Emei), Hangzhou, Zhejiang, and Tonglu, Zhejiang. DNA was extracted from soil from three different places, and the OD260 / OD230 and OD260 / OD280 values ​​of the obtained DNA solutions were measured with a spectrophotometer, and the results are shown in Table 2. The results in table 2 show that with the method described in the summary of the invention, the OD260 / OD230 of the soil DNA in different regions extracted is all close to 2.0, indicating that the humic substance content in the DNA solution is very low; OD260 / 280 is close to 1.6, indicating that the DNA solution The protein content in it is very low. Therefore, the method of the present invention is also suitable for extracting other types of soil microbial DNA.

[0048] Table 2: OD260 / OD230, OD230,

[0049] OD260 / OD...

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Abstract

This invention relates to an extracting method of soil microorganism DNA. The stated method includes steps as following: cell disruption of soil microorganism, obtaining crude DNA solution, obtaining soil microorganism DNA; the stated method of this invention is using PVPP, protease K etc for valid combination. After organic solvent such as PEG20000, isopropyl alcohol and so on are precipitated, use spectrophotometer to determine value of OD260 / OD230, OD260 / OD280 close to standard value. It can be directly used for molecule operation, possessing great application perspective.

Description

(1) Technical field [0001] The invention relates to a method for extracting soil microbial DNA. (2) Background technology [0002] The number and types of microorganisms in the soil are very large. It is estimated that 1g of soil contains 10 10 above microorganisms. For a long time, the study of the diversity of soil microbial communities has been carried out through traditional pure culture methods, and only 0.001% to 10% of them can grow on nutrient plates. On the other hand, the microorganisms that are isolated and cultured are usually not what the isolaters expected Certain dominant species in natural habitats. Obviously, traditional pure culture techniques have major limitations, and the results cannot accurately reflect the true composition of the microbial community in the system. In recent years, the rapid development of molecular biology has enabled researchers to analyze and study complex microbial ecosystems at the genomic DNA level instead of relying on the is...

Claims

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Application Information

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IPC IPC(8): C07H21/00C07H1/08
Inventor 钱海丰程秋霞王智烨徐茜许皓
Owner ZHEJIANG UNIV OF TECH
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