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Total DNA extraction method for researching microbial community structure in vinegar residue substrate

A technology of microbial community and extraction method, which is applied in the field of total DNA extraction to study the structure of microbial community in vinegar grain soilless culture substrate, can solve the problems of time-consuming and laborious, and increase the complexity of purification treatment methods, and achieve the effect of shortening the DNA extraction time

Active Publication Date: 2013-04-17
JIANGSU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These purification treatments add complexity and are time-consuming and labor-intensive

Method used

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  • Total DNA extraction method for researching microbial community structure in vinegar residue substrate
  • Total DNA extraction method for researching microbial community structure in vinegar residue substrate
  • Total DNA extraction method for researching microbial community structure in vinegar residue substrate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 、 Extraction of total DNA from vinegar grain matrix

[0036] (1) Weigh 0.5 g of distilled vinegar matrix into a 10 ml sterile centrifuge tube, add 2.5 ml of extraction buffer and 12.5 μl of proteinase K (20 mg / ml) into the centrifuge tube. The formula of DNA extraction buffer is: 10 mM Tris-Hcl (pH 8.0), 100 mM Sodium EDTA (pH 8.0), 100 mM Sodium phosphate (pH 8.0), 1.5 M Nacl, 1% CTAB, 0.5% β-mercaptoethanol , 0.5% PVP.

[0037] (2) Shake the above centrifuge tube at 37°C, 225 rpm for 30 min on a shaker.

[0038] (3) Add 250 μl of SDS with a mass fraction of 20% to the centrifuge tube taken out of the shaker, vortex for 10 s with a vortexer, and then place the centrifuge tube in a water bath at 65°C for 1 hour, during which time every 15-20 min Shake once. After the water bath, the centrifuge tube was centrifuged at 10000 g for 10 min at 4 °C, and the supernatant was taken into a new centrifuge tube.

[0039] (4) Add 1.25 ml of extraction buffer and 125 ...

Embodiment 2

[0047] Electrophoresis detection and PCR amplification of embodiment 2, DNA

[0048] The total DNA obtained in Example 1 was electrophoresed on an agarose gel with a mass fraction of 1%, the molecular weight marker (Marker) used was λ DNA / Hind III, the electrophoresis buffer was 1×TAE, and the voltage used was 10 V / cm, after electrophoresis for 30 min, the agarose gel was photographed in the coagulation imaging system to obtain the electrophoresis map of DNA (such as figure 1 shown). The DNA band is single without tailing.

[0049] The 16S rDNA of bacteria and the 18S rDNA of fungi were used for PCR amplification and product detection. The specific steps are as follows:

[0050] (1) Select the commonly used primer pair for bacterial 16S rDNA amplification F341: 5'-CCT ACG GGA GGC AGC AG -3' (SEQ ID No.1), 907R: 5'-CCG TCA ATT CCT TTG AGT TT-3' (SEQ ID No.2), as an amplification primer. 50 μl PCR reaction system: 10 × PCR Buffer (including mg 2+ ) solution 5 μl, dNTP 10...

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Abstract

The invention relates to a total DNA (Deoxyribose Nucleic Acid) extraction method for researching a microbial community structure in a vinegar residue substrate. The total DNA extraction method comprises the following steps of: firstly, adding DNA extraction buffer solution comprising 0.5 percent of beta-mercaptoethanol and 0.5 percent of polyvinylpyrrolidone (PVP) into the vinegar residue substrate; adding protease K; placing the mixture on a shaking table with a temperature of 37 DEG C to shake for 30 minutes; after enabling microorganisms in the substrate to be sufficiently dissolved in the DNA extraction buffer solution, cracking microbial cells in the vinegar residue substrate by sodium dodecyl sulfate (SDS) with mass fraction of 20 percent; carrying out extraction by chloroform and isoamyl alcohol according to a ratio of 24:1 so as to remove proteins; after removing humic acid and phenolic substances by PVP with mass fraction of 2 percent, carrying out sedimentation by precooled isopropanol; and finally, obtaining high-quality DNA which can be directly used for PCR (Polymerase Chain Reaction) amplification.

Description

technical field [0001] The invention belongs to the field of agricultural microorganisms and biotechnology, and in particular relates to a method for extracting total DNA for studying the microbial community structure in a soilless cultivation matrix of vinegar grains. Background technique [0002] Microorganisms are a very important component of soilless culture substrates, so the study of microorganisms in soilless cultivation substrates is an important direction in the field of soilless cultivation substrates, and it is of great significance to promote the development of soilless cultivation substrates. Commonly used methods for microbial community research include: culture isolation method, phospholipid fatty acid method, and BIOLOG plate method, etc., but the above methods have their own defects. The culture and separation method is only suitable for the research of cultivable microorganisms. Since more than 99% of the microorganisms in the environment are non-culturabl...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68C12Q1/04
Inventor 李萍萍林英赵青松杜道林王纪章
Owner JIANGSU UNIV
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