Kit and method for extracting microbial DNA

A kit and microbial technology, applied in the field of molecular biology, can solve the problems of unsuitable for high-throughput DNA sample extraction, time-consuming, complicated operation steps, etc., and achieve the effects of efficient growth inhibition, rapid extraction, and high throughput

Inactive Publication Date: 2019-07-19
成都罗宁生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The direct method uses the method of directly lysing the microbial cells in the sample. This method mainly obtains a crude DNA extract, which can be used directly or further purified according to subsequent test applications. However, this type of method requires high temperature treatment and usually requires temperature control equipment. The indirect method needs to mechanically break the sample, sample homogenate, lyse the cell wall and cell membrane with different buffer solutions, and finally obtain DNA through ethanol precipitation or adsorption and purification of hydroxyl / carboxyl materials, but this method requires more professional instruments , and the operation steps are complicated and time-consuming, so it is not suitable for the extraction of high-throughput DNA samples

Method used

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  • Kit and method for extracting microbial DNA
  • Kit and method for extracting microbial DNA
  • Kit and method for extracting microbial DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Kits for extracting microbial DNA include:

[0029] Lysis buffer: 0.002% of 5-chloro-2-methyl-4-isothiazolin-3-one, 0.001% of 2-methyl-4-isothiazolin-3-one, 0.01mM RNaseA, 0.5% of Polyvinylpyrrolidone, 150 mM 2-(N-morpholino)ethanesulfonic acid, 0.5% bromophenol red, 0.25% xylene cyanol FF. When configuring, dissolve all reagents in distilled water according to the proportion, and mix well.

[0030] Equilibrium solution: 0.1% of 3-(2-aminoethylamino)propyltrimethoxysilane, 0.25% of 1-(4-sulfonic acid phenyl)-4-(4-sulfonic acid phenylazo)- Trisodium 5-pyrazolone-3-carboxylate, 100 mM Tris. When configuring, dissolve all reagents in distilled water according to the proportion, and mix well.

[0031] Method for extracting microbial DNA from mouse feces:

[0032] S1: Fully mix 0.05g of feces sample with 0.1ml of lysate, vortex and oscillate at a high speed, and let stand at room temperature for 1 minute to obtain a mixed solution;

[0033] S2: Add 0.2ml of equilibrium ...

Embodiment 2

[0035] Kits for extracting microbial DNA include:

[0036] Lysis buffer: 0.001% of 5-chloro-2-methyl-4-isothiazolin-3-one, 5% of 2-methyl-4-isothiazolin-3-one, 0.01mM RNaseA, 0.5% of Polyvinylpyrrolidone, 200 mM 2-(N-morpholino)ethanesulfonic acid, 0.1% bromophenol red, 0.5% xylene cyanol FF. When configuring, dissolve all reagents in distilled water according to the proportion, and mix well.

[0037] Equilibrium solution: 0.01% of 3-(2-aminoethylamino)propyltrimethoxysilane, 0.25% of 1-(4-sulfonic acid phenyl)-4-(4-sulfonic acid phenylazo)- Trisodium 5-pyrazolone-3-carboxylate, 200 mM Tris. When configuring, dissolve all reagents in distilled water according to the proportion, and mix well.

[0038] The method for extracting microbial DNA from mouse feces was the same as in Example 1.

Embodiment 3

[0040] Lysis buffer: 12% of 5-chloro-2-methyl-4-isothiazolin-3-one, 10% of 2-methyl-4-isothiazolin-3-one, 0.01mM RNaseA, 0.5% of Polyvinylpyrrolidone, 100 mM 2-(N-morpholino)ethanesulfonic acid, 0.5% bromophenol red, 0.01% xylene cyanol FF. When configuring, dissolve all reagents in distilled water according to the proportion, and mix well.

[0041] Equilibrium solution: 15% of 3-(2-aminoethylamino)propyltrimethoxysilane, 0.25% of 1-(4-sulfonic acid phenyl)-4-(4-sulfonic acid phenylazo)- Trisodium 5-pyrazolone-3-carboxylate, 50 mM Tris. When configuring, dissolve all reagents in distilled water according to the proportion, and mix well.

[0042] The method for extracting microbial DNA from mouse feces was the same as in Example 1.

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Abstract

The invention relates to the technical field of molecular biology, in particular to a kit and a method for extracting microbial DNA. The kit for extracting the microbial DNA comprises a lysate and anequilibrium liquid. The lysate comprises 5-chloro-2-methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one, RNaseA, polyvinylpyrrolidone, 2-(N-morpholino) ethanesulfonic acid, bromophenol red andxylene cyanol FF. The equilibrium liquid comprises 3-(2-aminoethylamino) propyl trimethoxysilane, tartrazine and trihydroxymethyl aminomethane. The invention provides the kit and the method for extracting microbial DNA, wherein the method can complete the operation at room temperature, the extraction is quick, the DNA extracted by the method does not contain the interference of RNA and has high flux; and the lysate and the equilibrium liquid in the kit can effectively inhibit the growth of microorganisms, and have great advantages for processing and transporting samples containing infectious sources.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a kit for extracting microbial DNA and a method thereof. Background technique [0002] The purification of total DNA of environmental microorganisms is well known in the art, and has extremely wide applications in ecology, diagnosis and pharmaceutical research. With the continuous advancement of molecular biology and ecological research, DNA purified from environmental samples can be directly used for microbial genome research, analysis of microbial diversity, and further for research on the occurrence and development of animal and plant diseases. In these applications, DNA needs to be purified first, followed by polymerase chain amplification reaction or DNA sequence determination to reveal the changes in microbial diversity in the sample, and finally used for microbial diversity research. [0003] However, it is unavoidable that environmental microbial samples come f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 孙梓健曾俊儒汪凯丁泽琴陈云慧曾陈娟
Owner 成都罗宁生物科技有限公司
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