Total DNA (Deoxyribonucleic Acid) extraction method of bottom mud containing high content of humus at river mouth

A humus and sediment technology, applied in the field of environmental microbiology and molecular biology, can solve the problem of low quality of total DNA in estuary sediment with high humus content, and achieve the effect of easy molecular biology experiment, quality improvement and wide application range

Inactive Publication Date: 2013-08-28
INST OF BIOLOGICAL RESOURCES JIANGXI ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The purpose of the present invention is to provide a method for extracting high-quality DNA from high-humus estuary bottom mud. The method is e

Method used

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  • Total DNA (Deoxyribonucleic Acid) extraction method of bottom mud containing high content of humus at river mouth
  • Total DNA (Deoxyribonucleic Acid) extraction method of bottom mud containing high content of humus at river mouth

Examples

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Effect test

Embodiment 1

[0020] The method of the present invention is used to extract the total DNA of Changjiang Estuary sediment. The sediment of Changjiang Estuary comes from the west side of 200m upstream of the confluence of Changjiang River and Le'an River. The bottom sediment of 0-3 cm in the upper layer is taken as a sample. The sediment sample is gray-black and has a high content of humus. The following are the specific implementation steps:

[0021] 1. Pretreatment of sediment samples: Weigh 10g of estuary sediment into a 50ml centrifuge tube, add 30ml of normal saline, vortex for 5min, let stand for 2min, and centrifuge at 2500r / min for 3min at low speed at 4°C, then 10000r Centrifuge at high speed / min for 8 min, discard the supernatant, and transfer the upper 1 / 2 of the pellet to another new centrifuge tube; repeat the previous step 2 times, transfer the 1.25g sediment to a 10ml centrifuge tube; wash with normal saline The sediment was centrifuged at 10,000 r / min for 8 min at 4°C, and wa...

Embodiment 2

[0029] The method of the present invention is used to extract the total DNA of the Ganjiang estuary sediment. The estuary sediment of the Ganjiang River is from the north of the 150m upstream of the confluence of the Ganjiang River and the Xiu River. The upper layer of 0-3cm sediment is taken as a sample. The sediment sample is yellow clay and contains humus, especially with high pigment content.

[0030]The specific implementation steps are the same as in Example 1.

[0031] like figure 1 As shown, the DNA electrophoresis pattern in lane 2 has a single and clear band without tailing, indicating that the DNA is of high purity and has no breaks; such as figure 2 As shown, the electrophoresis map of the PCR product in lane 2 can clearly see the target fragment of about 1380bp, indicating that the method of the present invention is used to extract the total DNA quality of the Ganjiang estuary sediment, which can be used for subsequent molecular biology research.

Embodiment 3

[0033] The soil total DNA is extracted by the method of the present invention. The soil was from Meiling, Nanchang City, Jiangxi Province. The topsoil on the mountain with a lot of fallen leaves and decay was taken as a sample. The soil sample is gray-black and contains a lot of humus.

[0034] The specific implementation steps are the same as in Example 1.

[0035] like figure 1 As shown in Figure 3, the DNA electrophoresis pattern in lane 3 has a single and clear band, and no tailing phenomenon, indicating that the DNA is of high purity and no breakage; such as figure 2 As shown in the electrophoresis map of the PCR product in lane 3, the target fragment of about 1380bp can be clearly seen, indicating that the total DNA extracted from Meiling soil by the method of the present invention is of high quality and can be used for subsequent molecular biology research.

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Abstract

The invention discloses a total DNA (Deoxyribonucleic Acid) extraction method of bottom mud containing high content of humus at the river mouth. The method comprises the following steps: washing bottom mud by stroke-physiological saline solution to remove part of impurities such humus, and meanwhile, concentrating microorganisms; then, multigelating in liquid nitrogen and a water bath at 65 DEG C by means of CTAB (Cetyltrimethyl Ammonium Bromide), proteinase K and lysozyme for cell disruption, and then adding SDS (Sodium Dodecyl Sulfonate) and insulating for 2 hours in the water bath at 65 DEG C to combine protein; adding isovolumetric phenol/chloroform/isoamylol (25:24:1) to remove protein; adding isovolumetric isopropanol to set DNA, washing precipitate with 70% ethanol, and dissolving DNA by TE (pH8.0) after airing; adding 3 times of absolute ethyl alcohol in volume to re-set DNA, washing the precipitate with 70% ethanol, dissolving the precipitated DNA by TE after airing, and storing at minus 20 DEG C for later use. The method provided by the invention is simple to operate and cheap in price. The total DNA of bottom mud extracted by the method provided by the invention has the advantages of high purity and integral biodiversity, and can be used for molecular biology study and microbial diversity analysis.

Description

technical field [0001] The invention belongs to the technical field of environmental microbiology and molecular biology, and specifically describes a high-efficiency method for extracting total DNA from estuarine sediments with high humus content. Background technique [0002] Estuarine sediments, like land surface soils, are rich in microorganisms, but most of these microorganisms cannot be cultivated in the laboratory, so research work cannot be carried out. Research offers hope. In recent years, there have been many reports on the extraction method of soil microbial DNA, but there are very few related reports on the effective extraction method of microbial DNA from ditch river sediments. The large amount of humus contained in the soil is the main reason for the difficulty in extracting high-quality DNA of soil microorganisms. However, the impurities such as humus contained in the sediments of Gouhe rivers are more complex, so the extraction of high-quality DNA of microor...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 王东升黄江丽丁建南丁一尊田晓娟张志红黄黄
Owner INST OF BIOLOGICAL RESOURCES JIANGXI ACAD OF SCI
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