Method for extracting total DNA of microorganism in liquor Daqu

A technology of microorganisms and Daqu, applied in DNA preparation, recombinant DNA technology, sugar derivatives, etc., can solve the problems of certain difficulties in extraction methods, large types of microorganisms, and difficulty, and achieve the effect of simple separation and identification.

Active Publication Date: 2010-03-31
贵州国台酒业集团股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the complex components contained in Daqu samples and the huge variety of microorganisms, it is difficult to extract high-quality microbial total DNA from them.
With the development of microbial ecology,

Method used

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  • Method for extracting total DNA of microorganism in liquor Daqu

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Take 5g Daqu + 25mL phosphate buffer (pH8.0) + 0.1%PVPP, + 1mL soil temperature (80 or 60) shaker for 30min, ultrasonic for 6-7min, let stand for 2-5min, and precipitate at low speed (1000rpm) centrifuge for 5min , take out the supernatant, mix, centrifuge at high speed (9000rpm) for 8min, remove the supernatant, then add phosphate buffer (PVPP), 25mL, break up and centrifuge at high speed (9000rpm) for 8min, remove the supernatant.

[0033] Add 15-25mL extract solution (the components of the extract solution are: 100mM Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride) pH8.0, 100mM sodium EDTA (disodium ethylenediaminetetraacetate) pH8.0, 100mM Sodium Phosphate pH 8.0, 1.5M NaCl and 1% CTAB (Cetyltrimethylammonium Bromide)), shake well. Add 50ul of 10mg / mL protease K, 37°C, 200rpm, shake horizontally for 30min, shake up and down once in the middle; add 1ml of 50mg / mL lysozyme, add 10ml of 20% SDS after shaking (addition is 4% of the total volume) ), incubated in...

Embodiment 2

[0035] Take 3g Daqu + 25mL phosphate buffer (pH8.0) + 0.05% PVPP, + 1mL Tuwen-60 shaker for 30min, ultrasonic for 6-7min, let stand for 2-5min, centrifuge at low speed (1000rpm) for 5min, remove the supernatant , mix, centrifuge at high speed (9000rpm) for 8min, remove the supernatant, then add phosphate buffer (PVPP), 25mL, break up and centrifuge at high speed (9000rpm) for 8min, remove the supernatant.

[0036] Add 15ml of extract solution (the components of the extract solution are: 100mM Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride) pH8.0, 100mM sodium EDTA (disodium ethylenediaminetetraacetic acid) pH8.0, 100mM phosphoric acid Sodium pH8.0, 1.5M NaCl and 1% CTAB (cetyltrimethylammonium bromide)) plus 10mg / mL proteinase K 100ul, 37°C, 200rpm, shake horizontally for 30min, shake up and down once in the middle; 1ml of 50mg / mL lysozyme, after shaking, add 3ml of 10% SDS, incubate in a water bath at 50°C for 2 hours, slowly invert the centrifuge tube every 15-20min...

Embodiment 3

[0038] Take 8g Daqu + 25mL phosphate buffer solution (pH8.0) + 0.2% PVPP, + 1mL Tuwen-80 shaker for 30min, ultrasonic for 6-7min, let stand for 2-5min, precipitate at low speed (1000rpm) centrifuge for 5min, take out the supernatant , mix, centrifuge at high speed (9000rpm) for 8min, remove the supernatant, then add phosphate buffer (PVPP), 25mL, break up and centrifuge at high speed (9000rpm) for 8min, remove the supernatant.

[0039]Weigh 5g Daqu and place it in a 50ml centrifuge tube, add 15ml of extract (the extract composition is: 100mM Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride) pH8.0, 100mM sodium EDTA (ethylene di Disodium amine tetraacetate) pH8.0, 100mM sodium phosphate pH8.0, 1.5M NaCl and 1% CTAB (hexadecyltrimethylammonium bromide)) plus 10mg / mL proteinase K 100ul, 37°C, 200rpm , shake horizontally for 30 minutes, shake up and down once in the middle; add 1ml of 50mg / mL lysozyme, add 3ml of 10% SDS after shaking, incubate in a water bath at 50°C for 2...

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Abstract

The invention relates to a method for extracting total DNA of microorganism in liquor Daqu, which is characterized in that, the liquor Daqu is pre-treated before extracting DNA, and the pre-treatment comprises the following steps: adding pre-treatment liquid into Daqu: the phosphoric acid buffer solution contains 0.05-0.2w/v% PVPP (polyvinylpolypyrrolidone) and 3-5w/v% tween-80 or tween-60, pH is8.0, wherein the weight volume ratio between Daqu and pre-treatment liquid is 3-8:25; shaking; executing ultrasound treatment; centrifuging at low speed after standing; centrifuging at a low speed after standing; removing supernatant, and adding the phosphoric acid buffer solution containing 0.05-0.2w/v% PVPP (polyvinylpolypyrrolidone) into the deposition; centrifuging at a high speed after scatter, removing supernatant for obtaining sample. The method provided by the invention is simple.

Description

technical field [0001] The invention relates to a method for extracting microbial total DNA, in particular to a method for extracting microbial total DNA from liquor Daqu. Background technique [0002] Chinese liquor Daqu is a saccharification and fermentation agent for the production of liquor. Although the predecessors have done a lot of research on the analysis of Daqu microorganisms, the detection and classification methods of bacteria based on morphological and physiological indicators are relatively complicated, and the results obtained The conclusion inevitably deviates from the actual situation. Using the traditional laboratory isolation and culture method, due to the complexity of the environment and the limitation of culture conditions, it is believed that the microorganisms that can be cultured by the existing technology only account for about 1% of the total number of microorganisms, and the traditional laboratory isolation and culture method cannot be timely It...

Claims

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Application Information

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IPC IPC(8): C07H21/02C12N15/10
Inventor 梁慧珍李长文魏纪平张春辉
Owner 贵州国台酒业集团股份有限公司
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