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Method for efficiently extracting DNA (Deoxyribonucleic Acid) of microbe in soil

A soil microorganism and soil technology, applied in DNA preparation, recombinant DNA technology, etc., can solve problems such as affecting the effect of microbial diversity analysis experiments, and achieve the effect of reducing impurities

Inactive Publication Date: 2018-05-04
CHINESE RES ACAD OF ENVIRONMENTAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In particular, DNA in soil samples in mountainous areas contains a large amount of humus, lipids and sugars. If the quality of extracted DNA is not high, such as humic acid or polyphenols, it will affect the effect of subsequent microbial diversity analysis experiments.

Method used

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  • Method for efficiently extracting DNA (Deoxyribonucleic Acid) of microbe in soil
  • Method for efficiently extracting DNA (Deoxyribonucleic Acid) of microbe in soil
  • Method for efficiently extracting DNA (Deoxyribonucleic Acid) of microbe in soil

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] The extraction process of the microbial DNA of the desert soil provided by this implementation is as follows:

[0082] (1) Weigh 0.3-0.4g sample into Lysing Matrix E Tube. Add 978μl of SPB, add 122μl of MT Buffer;

[0083] (2) Close the lid of the Lysing Matrix E Tube tightly, place the centrifuge tube on a vortex mixer, shake and homogenize the material in the tube, and set the time to 40-50s. Centrifuge at 8000r / min for 15min.

[0084] (3) Aspirate the supernatant with a large pipette tip and transfer it to a 2ml microcentrifuge tube, add an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), mix by inverting, centrifuge at 15°C, 10,000rpm 10min;

[0085] (4) Transfer the supernatant to a clean centrifuge tube, add an equal volume of chloroform-isoamyl alcohol (24:1), mix by inversion, and centrifuge at 15°C, 10,000rpm for 10min;

[0086] (5) Add 250 μl of PPS, invert and mix more than 10 times, and let stand at room temperature for 10 minutes;

[0087] ...

Embodiment 2

[0098] The only difference from Example 1 is that the raw material was replaced with semi-desert soil to obtain sample microbial genomic DNA.

Embodiment 3

[0100] The difference from Example 1 is only that the raw material is replaced with farmland soil to obtain the sample microbial genomic DNA.

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Abstract

The invention relates to a method for efficiently extracting DNA (Deoxyribonucleic Acid) of a microbe in soil. The method can be used for effectively removing the interference, to the extraction of the DNA, of humus and lipid and saccharide substances by applying a method for combining organic solvent abstraction with a genomic DNA extraction kit for the microbe in the soil, which is provided by the MP (MP Biomedicals), can adapt to various soil types, can be used for effectively enriching the DNA, and is used for removing the pollution by the humus, and purity and concentration detection experimental results of the DNA show that the DNA obtained by the extraction method provided by the invention not only can meet a requirement of a downstream experiment, but also can be used for effectively decreasing an influence by the humus.

Description

technical field [0001] The invention relates to a method for extracting soil microbial genome DNA, in particular to a method for efficiently extracting soil microbial DNA. Background technique [0002] Extracting microbial DNA from soil is the first step in molecular biology research. High-quality genomic DNA is the basis for research such as detecting bacteria that cannot be cultivated by existing methods, tracking the behavior of certain target strains or recombinant genes in the natural environment, genetic diversity of microorganisms in soil and its changes with the environment ( Wang Jiaxin, 2010). So far, people have proposed many methods for extracting DNA, commonly used methods include freeze-thaw method, freeze-thaw boiling method, glass bead homogenization method, liquid nitrogen grinding method, ultrasonic disruption method, etc. (Cai Liuti, 2011). [0003] The presence of humic acid substances in the samples that often appear in the extraction may inhibit the a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003C12N15/1017
Inventor 沈奇关潇全占军臧春鑫李果赵志平
Owner CHINESE RES ACAD OF ENVIRONMENTAL SCI
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