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Method for economically and rapidly extracting microbial genome DNA in fermented grains

A microbial and genomic technology, applied in the field of winemaking, to achieve the effect of low labor intensity, saving time and cost, and low cost

Active Publication Date: 2017-02-15
KWEICHOW MOUTAI COMPANY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the existing problems in the extraction of genomic DNA from fermented grains in the prior art, in order to economically and quickly extract the genomic DNA of microorganisms in fermented grains in the research and development of liquor production, the present invention provides an economical, rapid and high-quality extraction method for genomic DNA of microorganisms in fermented grains method

Method used

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  • Method for economically and rapidly extracting microbial genome DNA in fermented grains
  • Method for economically and rapidly extracting microbial genome DNA in fermented grains
  • Method for economically and rapidly extracting microbial genome DNA in fermented grains

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Extraction of Microorganism Genome DNA from Fermented Grains

[0071] 1. After sterilizing and drying the 16-mesh quartz sand, weigh 18.5g and put it into a 50mL sterile centrifuge tube.

[0072] 2. Weigh 7.5g of fermented grains in a centrifuge tube, add 20mL of CTAB extract, oscillate with a homogenizer for 1min, and centrifuge at 10,000rpm for 10min. .

[0073] 3. Transfer the supernatant to a 50mL sterile centrifuge tube, add 0.8-1.2 times the volume of P:C:I (phenol:chloroform:isoamyl alcohol=25:24:1), shake well, and centrifuge at 11000rpm for 20min.

[0074] 4. Transfer the supernatant to a 50mL sterile centrifuge tube, add 1.5-2.5 times the volume of silica gel membrane binding solution, shake well; filter with a 15mL adsorption column, discard the filtrate until the end of the filter.

[0075] 5. Add 3mL of 70% ethanol to the adsorption column, filter and discard the filtrate.

[0076] 6. Repeat step 5 once.

[0077] 7. Centrifuge the empty tube a...

Embodiment 2

[0079] Example 2 Extraction of Microorganism Genome DNA from Fermented Grains

[0080] 1. After sterilizing and drying the 12-mesh quartz sand, weigh 15g and put it into a 50mL sterile centrifuge tube.

[0081] 2. Weigh 6g of fermented grains sample into a centrifuge tube, add 16mL of CTAB extract, oscillate with a homogenizer for 1min, and centrifuge at 10,000rpm for 10min. .

[0082] 3. Transfer the supernatant to a 50mL sterile centrifuge tube, add an equal volume of P:C:I (phenol:chloroform:isoamyl alcohol=25:24:1), shake well, and centrifuge at 11000rpm for 20min.

[0083] 4. Transfer the supernatant to a 50mL sterile centrifuge tube, add 2 times the volume of silica gel membrane binding solution, shake well; filter with a 15mL adsorption column, discard the filtrate until the end of the filter.

[0084] 5. Add 2.5mL 70% ethanol to the adsorption column, filter and discard the filtrate.

[0085] 6. Repeat step 5 once.

[0086] 7. Centrifuge the empty tube at 10000rpm ...

Embodiment 3

[0088] Example 3 Extraction of Microorganism Genome DNA from Fermented Grains

[0089] 1. After sterilizing and drying 20-mesh quartz sand, weigh 22g and put it into a 50mL sterile centrifuge tube.

[0090] 2. Weigh 9g of fermented grains sample into a centrifuge tube, add 24mL of CTAB extract, shake with a homogenizer for 1min, and centrifuge at 10,000rpm for 10min. .

[0091] 3. Transfer the supernatant to a 50mL sterile centrifuge tube, add an equal volume of P:C:I (phenol:chloroform:isoamyl alcohol=25:24:1), shake well, and centrifuge at 11000rpm for 20min.

[0092] 4. Transfer the supernatant to a 50mL sterile centrifuge tube, add 2 times the volume of silica gel membrane binding solution, shake well; filter with a 15mL adsorption column, discard the filtrate until the end of the filter.

[0093] 5. Add 3.5mL 70% ethanol to the adsorption column, filter and discard the filtrate.

[0094] 6. Repeat step 5 once.

[0095] 7. Centrifuge the empty tube at 10000rpm for 2min...

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Abstract

The invention relates to the technical field of brewing of Baijiu, and in particular to extraction of microbial genome DNA in fermented grains. The method comprises the following steps: (1) wall breaking of microbial cells in the fermented grains: weighing a fermented grain sample and quartz sand, adding lysate, and conducting shaking centrifugation so as to obtain a coarse nucleic acid extracting solution; and (2) nucleic acid purification: adding a mixture of phenol: chloroform: isoamylol (P.C.I), conducting centrifugation to remove protein, adding a silica gel membrane binding solution, conducting filtering by virtue of a nucleic acid purification column, eluting the nucleic acid purification column by virtue of 70% ethanol, adding de-ionized water or a TE solution and preserving the DNA. The method is simple and convenient to operate, low in cost consumption, low in labor intensity and time-saving, and the extracted microbial genome in the fermented grains is high in quality and is applicable to such molecular biology analysis as next generation sequencing, fluorescent quantitative PCR and the like.

Description

technical field [0001] The invention belongs to the technical field of brewing, and in particular relates to an economical and rapid method for extracting microbial genome DNA from fermented grains in liquor production enterprises. Background technique [0002] The extraction of microbial genome DNA from fermented grains is a necessary condition for the study of microbial composition, microbial function, microbial metabolism, and the relationship between microorganisms and the environment during liquor brewing. Therefore, the extraction speed and quality of genomic DNA from fermented grains play an important role in the production and research of liquor. [0003] At present, the methods for extracting microbial genome DNA from fermented grains can be divided into portable and kit methods. The portable method uses liquid nitrogen to grind and break the wall or enzymatically breaks the wall, phenol:chloroform:isoamyl alcohol (P.C.I) purification, isopropanol or ethanol precip...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 晏培胡传旺卢建军杨帆方芳王莉陈坚
Owner KWEICHOW MOUTAI COMPANY
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