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Method for obtaining high-quality fermented grain microorganism genome DNA (deoxyribonucleic acid) by pretreatment

A pretreatment and microbial technology, applied in the field of molecular biology, can solve the problems of affecting the amplification effect, unable to guarantee the stability of the extraction abundance and effect, unfavorable analysis, etc., and achieve the effect of simple and fast method, less impurities and easy operation

Inactive Publication Date: 2012-12-12
LUZHOU PINCHUANG TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the kits are aimed at the good extraction effect of a certain type of bacteria, and the kit cannot guarantee the stability of the extraction abundance and effect in the fermented grains with multiple strains
Although the impact of impurities on the experimental operation can be reduced by diluting the template concentration in PCR amplification, it will also affect the amplification effect, which is not conducive to the subsequent analysis (microbial identification, diversity analysis, etc.)

Method used

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  • Method for obtaining high-quality fermented grain microorganism genome DNA (deoxyribonucleic acid) by pretreatment
  • Method for obtaining high-quality fermented grain microorganism genome DNA (deoxyribonucleic acid) by pretreatment
  • Method for obtaining high-quality fermented grain microorganism genome DNA (deoxyribonucleic acid) by pretreatment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Using the method of the present invention to extract the genomic DNA of microorganisms in fermented grains

[0034] Fill the wine grains into the new cellar. After the fermentation is complete, take the upper, middle and lower layers of the wine grains out of the cellar as samples.

[0035] The inventive method:

[0036] Weigh 0.5g of fermented grains samples into 50ml centrifuge tubes, add 3mL phosphate buffer solution (PBS) (137mMNaCl, 2.7mM KCl, 10mM NaCl) to the centrifuge tubes 2 HPO 4 , 2mM KH 2 PO 4 , pH8.0) and 0.3g glass beads (diameter 2mm), vortex for 5min, centrifuge at 200×g for 5min, absorb the supernatant, repeat the above operation three times, combine the supernatant, centrifuge at 12000rpm at 4°C for 5min, and take the precipitate as Genomic DNA extraction material.

[0037] Extract genomic DNA using proteinase K and SDS high-salt combination method: add 1mL DNA extraction solution (100mmol / L Tris-HCl pH8.0, 100mmol / L EDTA, 100mmol / LNa ...

Embodiment 2

[0045] Example 2 Using the method of the present invention to extract the genomic DNA of microorganisms in fermented grains

[0046] The fermented grains were filled into 50, 100, 200, and 300-year-old Luzhou-flavor liquor brewing cellars for fermentation, and the fermented grains were taken out as samples after the fermentation was completed.

[0047] Weigh 0.5g samples of fermented grains out of the cellar into 50ml centrifuge tubes, add 3mL phosphate buffer solution (PBS) (137mM NaCl, 2.7mM KCl, 10mM NaCl) to the centrifuge tubes 2 HPO 4 , 2mM KH 2 PO 4 , pH8.0) and 0.3g glass beads (2mm), vortex for 5min, centrifuge at 200×g for 5min, absorb the supernatant, repeat the above operation three times, combine the supernatant, centrifuge at 12000rpm for 5min at 4°C, and take the precipitate.

[0048] Genomic DNA was extracted using the combined method of proteinase k and CTAB: add 1mL CTAB extract solution (2%CTAB, 5mol / LNaCL, 1mol / LTris-hcl (pH=8), 0.5mol / LEDTA) and 20μL me...

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Abstract

The invention relates to a method for obtaining a high-quality fermented grain microorganism genome DNA (deoxyribonucleic acid) by pretreatment and belongs to the technical field of molecular biology. The technical scheme of the method for obtaining the high-quality fermented grain microorganism genome DNA by pretreatment comprises the following steps of: pretreatment of a fermented grain sample, and extraction of a microorganism genome DNA. The microorganism genome DNA in fermented grains extracted by using the method has high purity and few impurities, and can be used for classification of fermented grain microorganisms, diversity research and the like.

Description

technical field [0001] The invention relates to a method for obtaining high-quality fermented grains microbial genome DNA through pretreatment, and belongs to the technical field of molecular biology. Background technique [0002] The production process of Luzhou-flavor liquor is inseparable from the evolution of microbial flora. The fermented grains are the growth substrate of microorganisms in the liquor brewing process. The main raw materials include sorghum, glutinous rice, wheat, corn bran, etc. According to different processes and traditions, the raw material composition Different, it contains a lot of alcohol, acid, ester and other liquor flavor substances and other starch, polysaccharide, pigment and other substances. Microorganisms interact with the micro-ecological environment of the cellar and carry out complex material and energy metabolism in the cellar, accumulating a variety of metabolites. These metabolites are the important material basis for the formation o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 李德林许正宏敖宗华史劲松王松涛陆震鸣林锋
Owner LUZHOU PINCHUANG TECH CO LTD
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