Total DNA (deoxyribonucleic acid) extraction method and kit for synchronously removing humic acid and mycoprotein

A technology of bacterial protein and extraction method, which is applied in the research field of metagenomics in soil microorganisms and general microbiology, and achieves the effect of low cost

Inactive Publication Date: 2014-03-12
INST OF MICROBIOLOGY JIANGXI ACADEMY OF SCI
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  • Application Information

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Problems solved by technology

As a main part of the ecosystem, soil microbial flora analysis is particularly important. Although the total DNA of microorganisms in the general environment is relatively easy to extract, the extraction of total DNA of soil microorganisms is affected by various soil components, especially The impact of humic acid is more difficult to carry out. Fortunately, there are many corresponding treatment measures now, and there are various soil microbial total DNA extraction methods or kits, but they always have one or another deficiency, such as convenience and simplicity. lack of sex, economy, etc.

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  • Total DNA (deoxyribonucleic acid) extraction method and kit for synchronously removing humic acid and mycoprotein
  • Total DNA (deoxyribonucleic acid) extraction method and kit for synchronously removing humic acid and mycoprotein

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Embodiment Construction

[0031] The present invention will be described in detail below with the best embodiment 。

[0032] The soil for the test was collected from the surface soil with oncomelania gathering points and no oncomelania points on grasslands in different areas of Poyang Lake District, Jiangxi Province (soil at the contact surface of oncomelania and 5cm below) 1, 2, 3, 4, 5, 8, 9, Soil samples No. 10 and No. 11.

[0033] The method for extracting total DNA is as follows:

[0034] 1) Take 0.4g soil sample and put it in a 2ml centrifuge tube, add 1ml humic acid complex solution [100 mmol / L sodium pyrophosphate (Na 4 P 2 o 7 ), 20 mmol / L EDTA -2Na and 150 mmol / L NaCl, pH9-10], shake and mix for 5 minutes to redissolve the humate in the soil, centrifuge at 12000r / min for 5 minutes, discard the supernatant to wash away the humic acid , then add 1ml of PBS buffer [140mmol / L NaCl, 2.7mmol / L KCl, 7.3mmol / L Na 2 HPO 4 and 1.5mmol / L KH 2 PO 4 , pH 7.0--7.4], stir and mix with a to...

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Abstract

The invention provides a total DNA (deoxyribonucleic acid) extraction method and a kit for synchronously removing humic acid and mycoprotein and relates to the field of research of metagenomics in soil microbiology and general microbiology. The method comprises the following steps: (1) suspending samples by utilizing a humic acid combination solution, so as to obtain sediments; (2) adding a buffer solution and proteinase K, and carrying out centrifuging to obtain supernate; (3) adding a coagulant to carry out coagulating sedimentation on the residual humic acid and mycoprotein, and carrying out centrifuging to obtain the supernate; (4) adding isopropanol to precipitate DNA; (5) adding 70% ethanol to wash the sediments, and blowing off the residual ethanol; (6) adding a DNA dissolving solution (TE) to dissolve the sediments, so as to obtain a sample, namely the total DNA. The kit comprises the following components: the humic acid combination solution, a PBS butter liquid, an extraction butter liquid, the proteinase K, a 20% SDS (sodium dodecyl sulfonate) water solution, the coagulant, a DNA washing agent, the 70% ethanol and the DNA dissolving solution. Due to the adoption of the method and the kit, the one-time extraction quantity of the total DNA is enough to carry out PCR (polymerase chain reaction) amplification for dozens of times.

Description

technical field [0001] The invention relates to the research field of soil microorganisms and metagenomics in general microbiology, specifically a method for simply and quickly extracting the total DNA of soil microorganisms (including common microorganisms). Background technique [0002] In order to reveal the differences and characteristics of the microbial flora structure, species and distribution in different ecosystems, 16SrRNA gene sequence analysis has been widely used. This requires that the total DNA of microorganisms in the qualified sample can be obtained first. As a main part of the ecosystem, soil microbial flora analysis is particularly important. Although the total DNA of microorganisms in the general environment is relatively easy to extract, the extraction of total DNA of soil microorganisms is affected by various soil components, especially The impact of humic acid is more difficult to carry out. Fortunately, there are many corresponding treatment measures...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 涂祖新
Owner INST OF MICROBIOLOGY JIANGXI ACADEMY OF SCI
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