Extraction method of total genome DNA from microbes

An extraction method and genome technology, applied in the fields of environmental science and biotechnology science, can solve the problems of low DNA purity and long operation time, and achieve the effect of accurate abundance and diversity, simple operation and short time-consuming

Inactive Publication Date: 2012-04-11
EAST CHINA NORMAL UNIVERSITY
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  • Abstract
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Problems solved by technology

[0003] The invention solves the problems of long operation time and low purity of the extracted DNA in the extraction of total microbial genome DNA from the bottom sludge of cultured pon

Method used

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  • Extraction method of total genome DNA from microbes
  • Extraction method of total genome DNA from microbes
  • Extraction method of total genome DNA from microbes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0043] Example: Microbial DNA Extraction from Shrimp Culture Pond Bottom Mud in Caojing Breeding Base, Jinshan District, Shanghai

[0044] 1. Pre-treatment of sediment samples

[0045] 1.1 Take 0.2 g of sediment sample and add 2 mL of sterilized TENP washing solution (50 mmol / L Tris, 20 mmol / L EDTA, 100 mmol / L NaCl, 1% w / v polyvinylpyrrolidone, pH = 10) Vortex to mix, bathe in 65°C water for 3 minutes, centrifuge at 8000rpm for 5 minutes, and take the precipitate. Repeat step 1 2-3 times until the supernatant becomes more transparent.

[0046] 2. Lysis of microbial cell walls in sediment samples

[0047] 2.1 Add about 6 glass beads with a diameter of 1 mm to the precipitate obtained in step 1.1, and 1 mL of lysate (1M Tris-HCl, 0.5M EDTA, 0.5M Na 3 PO4, 1.5M NaCl, 1% CTAB) and 300 μL of 50 mg / mL lysozyme solution, vortexed for 5-10 min, and placed in a water bath at 37 °C for more than 1 h.

[0048] 3. Acquisition of crude DNA from sediment samples

[0049]3.1 Add 60 ...

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Abstract

The invention discloses an extraction method of total genome DNA from microbes, comprising the following steps of: adding a lysis buffer and lysozyme to a sample to be extracted to enable bacterial cells to fully rupture and release DNA; and extracting the obtained crude extract DNA by phenol/chloroform/isoamyl alcohol extracting solutions to obtain purified total genome DNA of microbes. The extraction method disclosed by the invention has the beneficial effects of big DNA release amount, simple experiment conditions, low cost and the like.

Description

technical field [0001] The invention belongs to the fields of environmental science and biotechnology science, and in particular relates to a simple method for extracting total genome DNA of microorganisms. The method obtains the genome of microorganisms in a sample by means of biotechnology means. Background technique [0002] Due to the complexity of its components, it is difficult to obtain DNA in large quantities and with high purity when extracting DNA from environmental samples. Compared with the extraction of microbial DNA in environmental water samples, it is more difficult to extract microbial DNA from the sediment of aquaculture ponds. On the one hand, the microbial species in the bottom mud of aquaculture ponds is more abundant, which increases the requirement for lysing cell walls during the DNA extraction process; It affects the purity of the extracted DNA, which in turn affects subsequent experimental operations such as PCR. Therefore, fully cleaving the micr...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 王亭芳常忠义高红亮赵云龙金风杰
Owner EAST CHINA NORMAL UNIVERSITY
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