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DNA extracting method and kit

A kit and formula technology, applied in the field of molecular biology, can solve problems such as poor control of test parameters, easy loss of fungal hyphae, and easy frostbite skin, etc., achieve low technical requirements, improve grinding and breaking efficiency, and shorten experiment time Effect

Inactive Publication Date: 2018-11-23
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Ordinary grinding method and freeze-thaw method are not easy to control the test parameters, liquid nitrogen grinding is easy to frostbite the skin, fungal hyphae are also easy to lose during the grinding process, and the efficiency is not high

Method used

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  • DNA extracting method and kit
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  • DNA extracting method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1, the ultra-fast extraction of fungal genomic DNA

[0075] (1) Extraction process

[0076] 1) Fragmentation and lysis of fungal cells:

[0077] Scrape fungal microorganisms on the surface of grapes, or pick fungal samples, or collect 100mg of fungal mycelium, put it in a 2ml centrifuge tube, add sterilized grinding beads, and use a grinder to quickly grind until the mycelium finally becomes smooth powder, or grind and shake for 2-3 minutes.

[0078] Put the powder in another sterilized 2ml centrifuge tube, add 500-1000μl lysis solution, mix well, and lyse for 3-5min; the formula of the lysis solution is: 0.2-0.4mol / L sodium chloride, 3- 4mol / L guanidine hydrochloride, 2-4% CTAB by mass volume ratio, 0.1-0.3mol / L Tris, 0.02-0.03mol / L EDTA, 0.1-0.3% β-mercaptoethanol by mass volume ratio, mass 1-2% Triton x-100 by volume.

[0079] 2) Adsorption and impurity removal of fungal genomic DNA:

[0080] Soak one end of the filter paper into the above lysate and let...

Embodiment 2

[0108] Example 2, DNA Extraction in Plant Samples

[0109] Take soybeans as an example

[0110] 1) Extraction process

[0111] 1) Fragmentation and lysis of soybean cells:

[0112] Weigh 100mg of soybean powder, place it in a 2ml centrifuge tube, add 500-1000μl lysate, oscillate, mix, and lyse for 3-5min; the formula of the lyse is: 0.2-0.4mol / L sodium chloride, 3 -4mol / L guanidine hydrochloride, mass volume ratio is 2-4% CTAB, 0.1-0.3mol / L Tris, 0.02-0.03mol / L EDTA, mass volume ratio is 0.1-0.3% β-mercaptoethanol, Triton x-100 with a mass volume ratio of 1-2%.

[0113] 2) Adsorption and impurity removal of soybean genomic DNA:

[0114] Soak one end of the filter paper into the above lysate and let stand for 30-60s;

[0115]Transfer the filter paper into 500-700 μl rinse solution A, and let it stand for 15-30s; the formula of the rinse solution A is: 0.5-0.7mol / L NaCl, 5-7mol / L guanidine hydrochloride;

[0116] Transfer the filter paper into 500-700μl rinse solution B an...

Embodiment 3

[0143] Example 3, DNA Extraction in Bacteria and Virus Samples

[0144] Take Escherichia coli as an example

[0145] 1) Extraction process

[0146] 1) Breakage and lysis of E. coli cells:

[0147] Take 1ml of Escherichia coli liquid, put it in a 2ml centrifuge tube, add 500-1000μl of lysate, oscillate, mix, and lyse for 3-5min; the formula of the lysate is: 0.2-0.4mol / L sodium chloride, 3-4mol / L guanidine hydrochloride, 2-4% CTAB by mass volume ratio, 0.1-0.3mol / L Tris, 0.02-0.03mol / L EDTA, 0.1-0.3% β-mercaptoethanol by mass volume ratio , Triton x-100 with a mass volume ratio of 1-2%.

[0148] 2) Adsorption and impurity removal of Escherichia coli genomic DNA:

[0149] Soak one end of the filter paper into the above lysate and let stand for 30-60s;

[0150] Transfer the filter paper into 500-700 μl rinse solution A, and let it stand for 15-30s; the formula of the rinse solution A is: 0.5-0.7mol / L NaCl, 5-7mol / L guanidine hydrochloride;

[0151] Transfer the filter paper...

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Abstract

The invention provides a DNA extracting method and a kit. The method provided by the invention comprises using filter paper to adsorb DNA. On the premise of guaranteeing the DNA quality, extracting time is greatly shortened, extracting steps are simplified and extracting cost is reduced, so that a novel efficient feasible technical way is provided for molecular biology study. Compared with other existing DNA extracting methods, the method provided by the invention is simple to operate, is high in practicability, is relatively low in technical requirements of an operator, and also can be operated by staff without professional training. The kit provided by the invention avoids use of toxic and organic reagents such as phenol, chloroform and alcohol in a conventional method, is environmentally friendly and efficient, does not need to use other reagent apparatuses such as a centrifugal machine and a silicon-based centrifugal column, and remarkably reduces the implementation cost while conveniently implemented. The method has universality for extracting DNA in various samples such as animals, plants and microorganisms, and effectively saves the resources. A wall breaking method reducesthe cost while improving the efficiency.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a DNA extraction method and a kit. Background technique [0002] At present, the DNA extraction methods widely used in research at home and abroad include: SDS method, CTAB method, and spin column adsorption method with silica gel matrix as filter membrane, etc. To achieve the purpose of separation and recovery. The whole process is time consuming and expensive. With the development of molecular biology techniques, it is necessary to find a simple and ultra-fast method to obtain fungal DNA. [0003] In addition, fungi are eukaryotes, and fungal DNA extraction is an important link in the study of fungal molecular biology. Since fungal cells contain cell walls with chitin as the main component, the composition and structure of the cell walls of different species are different, and the cell walls are tough, which increases DNA. extraction difficulty. There ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 罗云波许文涛黄昆仑王沛杜再慧贺晓云
Owner CHINA AGRI UNIV