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Extraction method for aniseed genome DNA (Deoxyribonucleic Acid)

An extraction method and genome technology, applied in the field of molecular biology, can solve the problems of high anise secondary organism content and difficulty in extracting high-quality DNA, and achieve the effect of improving purity and high purity

Inactive Publication Date: 2012-08-22
YUNNAN ACAD OF FORESTRY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problem of difficult extraction of high-quality DNA caused by the high content of anise secondary organisms, the present invention improves the commonly used CTAB method, and at the same time combines certain steps of kit extraction to obtain an effective method for the extraction of anise genome DNA

Method used

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  • Extraction method for aniseed genome DNA (Deoxyribonucleic Acid)
  • Extraction method for aniseed genome DNA (Deoxyribonucleic Acid)
  • Extraction method for aniseed genome DNA (Deoxyribonucleic Acid)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] (1) Add liquid nitrogen to 0.1 g octagonal leaves (dry sample) and grind into powder;

[0032] (2) Add 6 mL of CTAB extract at 65 ℃ to the octagonal leaf powder of step (1), mix well, keep it in a 65 ℃ water bath for 40 minutes, shake 4 times in the meantime to make it fully mixed to obtain a mixture; The CTAB extract is: the mother liquor is a mixed solution containing 1.4 mol / L NaCI, 100 mmol / L Tris-HCI, and 20 mmol / L EDTA. The preparation is as follows: Weigh 16.363 g of NaCI, 2.428 g of Tris- Base, 1.489 g of EDTA, adjust the pH to 8.0 with HCl, and then dilute to 200 mL, and subject to routine sterilization; add 3 g of CTAB, 4 g of PVP and 4 ml of β-mercaptoethanol to 200 ml of mother liquor during use;

[0033] (3) Centrifuge the mixture obtained in step (2) at 12000 rpm for 10 minutes to obtain supernatant A;

[0034] (4) Add an equal volume of chloroform and isoamyl alcohol solution to the supernatant A of step (3), and the volume ratio of chloroform:isoamyl alcohol i...

Embodiment 2

[0043] (1) Add liquid nitrogen to 0.1 g octagonal leaves (dry sample) and grind into powder;

[0044] (2) Add 8 mL of CTAB extract solution at 50 ℃ to the octagonal leaf powder of step (1), mix well, keep it in a water bath at 50 ℃ for 60 minutes, shake 3 times during which it is fully mixed to obtain a mixture; where CTAB The extract is the same as in Example 1;

[0045] (3) Centrifuge the mixture of step (2) at 8000 rpm for 8 minutes to obtain supernatant A;

[0046] (4) Add an equal volume of chloroform:isoamyl alcohol solution to the supernatant A obtained in step (3), where the volume ratio of chloroform:isoamyl alcohol is 24:1, mix well and let stand at room temperature for 10 minutes, Centrifuge at 8000 rpm for 10 minutes, and take out the supernatant; add an equal volume of chloroform: isoamyl alcohol solution (24:1) to the supernatant again, mix well, stand at room temperature and centrifuge to obtain the supernatant B;

[0047] (5) According to 2.5 times the volume of supe...

Embodiment 3

[0055] (1) Add liquid nitrogen to 0.1 g octagonal horn sample (dry sample) and grind into powder;

[0056] (2) Add 7 mL of CTAB extract solution preheated at 55 ℃ to the star anise powder in step (1), mix well, keep it in a 55 ℃ water bath for 30 minutes, shake 3 times during which it is fully mixed to obtain a mixture; The CTAB extract is the same as in Example 1;

[0057] (3) Centrifuge the mixture of step (2) at 10,000 rpm for 8 minutes to obtain supernatant A;

[0058] (4) Add an equal volume of chloroform:isoamyl alcohol solution to the supernatant A obtained in step (3), where the volume ratio of chloroform:isoamyl alcohol is 24:1, mix well and let stand at room temperature for 8 minutes, Centrifuge at 10,000 rpm for 8 minutes, and then take out the supernatant; add an equal volume of chloroform: isoamyl alcohol solution (24:1) to the supernatant again, mix well, stand at room temperature and centrifuge to obtain the supernatant B;

[0059] (5) In the amount of 3.0 times the v...

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Abstract

The invention provides an extraction method for an aniseed genome DNA (Deoxyribonucleic Acid). The method comprises the following steps of: adding a CTAB (Cetyltrimethyl Ammonium Bromide) extract solution, uniformly mixing and centrifuging to obtain a supernatant A; adding a chloroform solution and an isoamyl alcohol solution, which are the same in volume, into the supernatant A, uniformly mixing, centrifuging, then adding the chloroform solution and the isoamyl alcohol solution, which are the same in volume, into the obtained supernatant, uniformly mixing, standing and performing centrifugal separation to obtain a supernatant B; adding absolute ethyl alcohol at the temperature of minus 30 DEG C into the supernatant B and centrifuging to obtain precipitates; purifying the precipitates again by utilizing an adsorption column used in a conventional kit, eluting, standing, centrifuging and returning the obtained eluent to the adsorption column; and centrifuging the obtain liquid, so as to obtain an aniseed genome DNA solution. The method provided by the invention can be used for finally obtained high-quality genome DNA of recalcitrant plant aniseed by virtue of integrating the characteristics of a CTAB method and a kit extraction method.

Description

Technical field [0001] The invention relates to a method for extracting genomic DNA of recalcitrant plants, in particular to a method for extracting octagonal genomic DNA, and belongs to the technical field of molecular biology. Background technique [0002] Recalcitrant plant taxa (Recalcitrant plant taxa) refers to those plants whose genomic DNA extraction is very difficult due to their special cell structure and high content of secondary products. These secondary products mainly include polysaccharides, polyphenols, proteins and other unknown extremely viscous compounds. When DNA is extracted, they are usually co-precipitated or bundled with nuclear genomic DNA, which seriously affects the quality of DNA and makes it difficult to carry out molecular operations such as amplification and restriction. There are many forest tree species belonging to this type of plant, and star anise is one of them. [0003] star anise( Illicium verum Hook. f.) belongs to the octagonal genus (Ill...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 陈海云陈少瑜宁德鲁吴涛耿树香谷丽萍白平李勇杰毛云玲张艳丽肖良俊马婷王洋贺娜
Owner YUNNAN ACAD OF FORESTRY