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Extraction method of microorganism genome DNA

An extraction method and genome technology, applied in the field of molecular biology, can solve the problems of low genome concentration, pollution, and inability to meet the third-generation sequencing, and achieve the effect of high concentration, less time-consuming, and a single band

Active Publication Date: 2015-12-09
SHANGHAI PASSION BIOTECHNOLOGY CO LTD
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, the concentration of genomes extracted by these methods is very low, and most of them are seriously polluted, which cannot meet the requirements of third-generation sequencing

Method used

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  • Extraction method of microorganism genome DNA
  • Extraction method of microorganism genome DNA
  • Extraction method of microorganism genome DNA

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Embodiment 1

[0019] The following uses the method provided by the present invention to extract genomic DNA from the following 9 sample cells:

[0020]

[0021] Experimental operation:

[0022] 1. Preparation of STES buffer and TE buffer:

[0023] STES buffer: 0.2M Tris-Hcl (pH=8.0), 0.5M Nacl, 0.01 MEDTA (pH=8.0), 0.1% SDS, prepare 100ml, and autoclave.

[0024] TE buffer: 10mMTris-Hcl and 1mM EDTA.

[0025] 2. Extraction steps:

[0026] 1) Take 2ml of the bacterial solution that reaches the exponential phase, centrifuge at the maximum speed, and collect the bacteria;

[0027] 2) Aspirate and discard the culture solution, add 1ml 1xPBS to resuspend the bacteria, centrifuge at maximum speed, and collect the bacteria;

[0028] 3) Suction the culture medium and add 200ul STES buffer to resuspend;

[0029] 4) Add 200mg glass beads to the bacterial suspension, add 80ul TE buffer to each tube;

[0030] 5) Add an equal volume of phenol / chloroform solution (wherein, phenol:chloroform=25:24 by volume), cover the ...

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Abstract

The invention belongs to the technical field of molecular biology, and discloses an extraction method of microorganism genome DNA. The extraction method comprises the following steps: (1) collecting sample thallus, adding a STES buffer solution, re-suspending, adding glass beads, and dissolving by virtue of a TE buffer solution; (2) adding an equal volume of phenol / chloroform solution, oscillating, mixing, and primarily centrifuging; (3) adding an equal volume of chloroform / isoamylol solution into an upper layer of water after the primary centrifuging, mixing, and secondarily centrifuging; (4) adding an equal volume of isopropanol or twice volume of anhydrous ethanol into an upper layer of water after the secondary centrifuging, precipitating under the room temperature, tertiarily centrifuging, and collecting precipitates; (5) washing the precipitates by utilizing ethanol, re-centrifuging to obtain DNA precipitates, dissolving the DNA precipitates by utilizing sterile water, carrying out the magnetic-ball passivation, to obtain extracted microorganism genome DNA. The genome DNA obtained by virtue of the method has advantages of high concentration, single banding and no pollution, and satisfies the three-generation sequencing requirement.

Description

Technical field [0001] The invention belongs to the technical field of molecular biology, and particularly relates to a method for extracting microbial genomes applied in molecular sequencing technology. Background technique [0002] In recent years, the genome research of pathogenic microorganisms has made rapid progress. Through genomic research, it is possible to fundamentally reveal all the genes of microorganisms. Not only can new genes be discovered, but also new gene interactions, new regulatory factors, etc. can be discovered. This research will enable humans to grasp the pathogenic mechanism and laws of pathogenic microorganisms from a higher level, so as to develop new preparations, vaccines and medicines for the diagnosis, prevention and treatment of microbial infections. [0003] With the continuous development of sequencing technology, the second-generation high-throughput sequencing technology has been widely used in various research fields, but its shortcomings have...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 孙子奎丁方美王锋刘艳艳范伟兵江畅
Owner SHANGHAI PASSION BIOTECHNOLOGY CO LTD
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