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Extraction method of wild ginseng genome DNA (deoxyribonucleic acid)

An extraction method and genome technology, applied in the field of molecular biology, can solve the problems of inapplicability, low yield, low DNA yield, etc., and achieve the effects of reducing phenol pollution, high purity and high content

Inactive Publication Date: 2016-11-23
BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When using the above methods to extract wild ginseng genomic DNA, the ground sample becomes very viscous after being added to the extraction solution. The extracted DNA sample contains a large amount of secondary organisms that are not easily soluble in TE buffer, and the sticky substances entangled in it cause Difficult loading and electrophoresis during DNA detection, low DNA yield
[0004] Luo Zhiyong et al. described a method for extracting ginseng genomic DNA in the isolation of high-quality plant genomic DNA [J], Journal of Hunan Medical University, 2001, 26(2), 178-180, which improved the traditional CTAB method, Including increasing the concentration of CTAB and β-mercaptoethanol to improve the quality and yield of the extracted genomic DNA, but the genomic DNA obtained is partially degraded and the yield is not high, so it is not suitable for wild ginseng samples that are rare and expensive. Genomic DNA extraction
[0005] In order to solve the problem of difficult extraction of high-quality DNA and low yield caused by high secondary organism content in wild ginseng, the present invention improves the commonly used CTAB method and obtains an effective method for extracting genomic DNA from wild ginseng

Method used

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  • Extraction method of wild ginseng genome DNA (deoxyribonucleic acid)
  • Extraction method of wild ginseng genome DNA (deoxyribonucleic acid)
  • Extraction method of wild ginseng genome DNA (deoxyribonucleic acid)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] (1), get 0.2g wild ginseng sample and put into mortar, fully grind into powder with liquid nitrogen, add the 1ml 2%CTAB extraction buffer solution of preheating and dissolve, 2%CTAB extraction solution is made up of 2%CTAB, 100mmol / L TrisHCl, 20mmol / L EDTA, 1.4mol / L NaCl and 2%PVP, the pH value of the TrisHCl is 8.0, and the total volume is about 1ml; put it in a water bath at 65°C for 1h, centrifuge at 13000g for 10min, Take the supernatant and put it into a new centrifuge tube;

[0027] (2), then add about 0.5ml of chloroform and isoamyl alcohol mixture and mix, the ratio of chloroform and isoamyl alcohol in the chloroform and isoamyl alcohol mixture is 24:1, centrifuge at 13000g for 10min; take the supernatant in a new centrifuge tube;

[0028] (3), add 0.5ml chloroform to the supernatant, mix upside down, centrifuge at 13000g for 10min; take the supernatant and put it in a new centrifuge tube;

[0029] (4) Add 0.8ml of pre-cooled isoamyl alcohol to the supernatant...

Embodiment 2

[0032] (1), get 0.2g wild ginseng sample and put into mortar, fully grind into powder with liquid nitrogen, add the 1ml 2%CTAB extraction buffer solution of preheating and dissolve, 2%CTAB extraction solution is made up of 2%CTAB, 100mmol / L TrisHCl, 20mmol / L EDTA, 1.4mol / L NaCl and 2%PVP, the pH value of the TrisHCl is 8.0; the total volume is about 1ml; placed in a water bath for 3h at 65°C, centrifuged at 13000g for 15min, Take the supernatant and place it in a new centrifuge tube;

[0033] (2), then add about 0.5ml of chloroform and isoamyl alcohol mixture and mix, the ratio of chloroform and isoamyl alcohol in the chloroform and isoamyl alcohol mixture is 24:1, centrifuged at 13000g for 15min; take the supernatant in a new centrifuge tube;

[0034] (3), add 0.5ml chloroform to the supernatant, mix it upside down, and centrifuge at 13000g for 15min; take the supernatant and put it in a new centrifuge tube;

[0035] (4) Add 1.6ml of pre-cooled isoamyl alcohol to the supern...

Embodiment 3

[0038] (1), get 0.2g wild ginseng sample and put into mortar, fully grind into powder with liquid nitrogen, add the 1ml 2%CTAB extraction buffer solution of preheating and dissolve, 2%CTAB extraction solution is made up of 2%CTAB, 100mmol / L TrisHCl, 20mmol / L EDTA, 1.4mol / L NaCl and 2%PVP, the pH value of the TrisHCl is 8.0; the total volume is about 1ml; placed in a water bath for 3h at 65°C, centrifuged at 13000g for 15min, Take the supernatant and put it into a new centrifuge tube;

[0039] (2), then add about 0.5ml of chloroform and isoamyl alcohol mixture and mix, the ratio of chloroform and isoamyl alcohol in the chloroform and isoamyl alcohol mixture is 24:1, centrifuge at 13000g for 10min; take the supernatant in a new centrifuge tube;

[0040] (3), add 0.5ml chloroform to the supernatant, mix upside down, centrifuge at 13000g for 10min; take the supernatant and put it in a new centrifuge tube;

[0041] (4) Add 1.2ml of pre-cooled isopropanol to the supernatant, mix i...

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Abstract

The invention provides an extraction method of wild ginseng genome DNA (deoxyribonucleic acid). The method comprises the following steps of grinding ginseng samples into powder by liquid nitrogen; adding 2-percent CTAB extraction buffer liquid; after the uniform shaking, performing water bath at 65 DEG C for 1 to 3h; performing centrifugation for 10 to 15 min at the speed being 13000r / min; taking supernate and putting the supernate into a novel centrifugation tube; adding a chloroform and isoamylol mixed solution into the obtained centrifugation; performing turnover uniform mixing; performing centrifugation for 10 to 15min; adding chloroform into the obtained supernate and uniformly mixing the mixture; performing centrifugation for 10 to 15 min; adding precooled isoamylol into the obtained supernate; performing overturn uniform mixing; putting the materials for 1 to 3h at -20 DEG C; performing centrifugation for 15 min; removing the supernate; performing cleaning and DNA sedimentation twice by 75-percent ethanol; performing drying at the room temperature for 3 min; performing dissolution by redistilled water; performing storage at -20 DEG C for use. Secondary products generated in the DNA extraction process can be effectively removed; the DNA content of the obtained wild ginseng is high; the purity is high.

Description

technical field [0001] The invention relates to a method for extracting plant DNA, in particular to a method for extracting genome DNA of wild ginseng, and belongs to the technical field of molecular biology. Background technique [0002] Ginseng is a perennial herb of Araliaceae, and it is a precious medicinal material commonly used in my country. Ginseng germplasm resources mainly include wild ginseng and cultivated ginseng, and wild ginseng is a national rare and endangered species. In recent years, ginseng germplasm resources have been severely damaged due to the decline of cultivated ginseng quality, variety confusion, ginseng land continuous cropping obstacles, production slowdown and other reasons. There is an urgent need to construct a wild ginseng germplasm gene bank to collect and organize the valuable germplasm resources of ginseng. Wild ginseng is rich in secondary organisms such as polysaccharides, proteins, saponins, etc., which makes it have high utilization...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 王绪敏殷金龙郭海燕刘贵明王国良
Owner BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION