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Method for extracting high molecular genome from bacterium

A high-molecular-weight, genomic technology, applied in the field of molecular biology of microorganisms, to achieve good repeatability and simple operation

Inactive Publication Date: 2007-04-25
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In order to solve the deficiencies in the existing bacterial extraction methods, the present invention provides a method for extracting high-molecular-weight genomes from bacteria

Method used

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  • Method for extracting high molecular genome from bacterium

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Experimental program
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Embodiment 1

[0022] Collect fresh cells with a wet weight of about 1 g, suspend them with TE, and centrifuge at 10,000 rpm for 2 min;

[0023] Remove the supernatant, and add 480ulTE to the pellet to suspend the bacteria;

[0024] Add 0.05ml lysozyme to the suspension, invert and mix well, and incubate at 37°C for 1h. (suitable for Gram-positive bacteria, Gram-negative bacteria directly enter step 4);

[0025] Add 40ul of 20% SDS and 5ul of proteinase K, mix by inverting, and incubate at 37°C for 1h;

[0026] Add 200ul of CTAB / NaCl solution, mix by inverting gently, and incubate at 65°C for 10min;

[0027] Add an equal volume of chloroform:isoamyl alcohol (24:1) and gently invert to mix, and centrifuge at 12000rpm for 5min;

[0028] Transfer the supernatant to a new centrifuge tube carefully with a pipette tip cut into a wide mouth;

[0029] Add an equal volume of isopropanol and gently invert to mix, use a sterilized toothpick to pick out the flocs and transfer them to a new centrifug...

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Abstract

The invention discloses a method of extracting high molecular genome from the bacteria. The method contains the steps of: breaking cell wall using lysozyme +protease +SDS or protease +SDS, extracting in TE buffer (removing proteins and polysaccharides), and preciping, washing, drying and dissolving. The invention begins with the factors influencing extracted DNA fragment length and obtains high molecular DNA fragment longer than fragments extracted by conventional method. The genomic DNA extracted in the method has more than 40 kb with OD260 / OD280 between 1.8 and 2.0 detecting by absorbance. The method has the advantages of good repeatability and simple operation and can meet the need of the above molecular operation.

Description

technical field [0001] The invention relates to a method for extracting bacterial genomes, through which the genomes of macromolecules and large fragments can be extracted to meet the needs of many molecular biology analyzes and belong to the field of molecular biology of microorganisms. Background technique [0002] Due to the advantages of fast growth, simple structure, easy cultivation, wide variety, and wide distribution of bacteria, the elucidation of many important life activities is carried out with bacteria as the research object, and many production practices use bacteria as carriers. It has been realized that bacteria, as a kind of extremely important and extensive microbial resources, have shown irreplaceable functions and values ​​in medicine, chemical industry, metallurgy, environmental protection, resources and other aspects. At present, the research on bacteria is going deeper and deeper to the molecular level. Such as molecular cloning, RFLP analysis, librar...

Claims

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Application Information

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IPC IPC(8): C12N15/31
Inventor 柳建设夏乐先邱冠周高健闫颖曾嘉
Owner CENT SOUTH UNIV
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