Extraction method of rosewood heartwood genome DNA (deoxyribonucleic acid)

An extraction method and genome technology are applied in the extraction field of mahogany heartwood genomic DNA, which can solve the problems of extracting high-quality genomic DNA, and achieve the effect of less pollution and high quality.

Inactive Publication Date: 2014-07-02
SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to provide a method for extracting genomic DNA from the heartwood of mahogany in view of the defect that the existing common DNA extraction method is difficult to extract high-quality genomic DNA from the heartwood of mahogany

Method used

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  • Extraction method of rosewood heartwood genome DNA (deoxyribonucleic acid)
  • Extraction method of rosewood heartwood genome DNA (deoxyribonucleic acid)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Extracting Genomic DNA from the Heartwood of Dalbergia cochinchinensis

[0030] Reagent preparation and use requirements for the extraction process are as follows:

[0031] The formula of CTAB lysate is: 2g CTAB, 5g PVP, 8.18g NaCl, 0.74g Na 2 EDTA.2H 2 O, 2 mL of β-mercaptoethanol, 10 mL of 1 mol / L Tris-HCl (pH 8.0), add water to make up to 100 mL.

[0032] Grinding Bowl: Washed and then autoclaved.

[0033] Mix phenol: chloroform: isoamyl alcohol in a volume ratio of 25:24:1 to prepare 10 ml of a solution.

[0034] The extraction method of rosewood Dalbergia cochinchinensis genomic DNA comprises the following steps:

[0035] (1) Weigh 300mg of rosewood Dalbergia cochinchinensis heartwood chips, put them into the weighed chips after being ground and cooled, and grind them into powder in liquid nitrogen.

[0036] (2) Add 700 μl of 65°C preheated CTAB lysate to the powder, place it at 65°C for 5 hours, and invert the EP tube every 30 minutes during this pe...

Embodiment 2

[0042] Example 2 The quality verification of Dalbergia codica chinensis genomic DNA

[0043] 1. The reagent preparation and process of the verification process are as follows:

[0044] 1% agarose: Weigh 0.1g of agarose and dissolve in 10ml of 1×TAE.

[0045] The formula of 50×TAE buffer solution is: Tris242g, Na 2 EDTA.2H 2 O37.2g, add 800ml of deionized water, stir and dissolve, then add 57.1ml of acetic acid.

[0046] According to the reported Dalbergia cochinchinensis ITS2 sequence, design PCR identification upstream amplification primer and downstream amplification primer, the sequence of amplification primer is respectively as shown in SEQ ID NO:1 and SEQ ID NO:2 in the sequence list, PCR amplification The size of the amplification product was 364bp.

[0047] Synthetic PCR identification primers: PCR identification upstream amplification primer sequence (as shown in SEQ ID NO: 1 in the sequence listing) is: 5'-CTTGCATCGATGAAGAACGTAG-3';

[0048] The downstream amplif...

Embodiment 3

[0058] (1) Weigh 200mg of rosewood Dalbergia cochinchinensis heartwood chips, put them into the weighed chips after being ground and cooled, and grind them into powder in liquid nitrogen.

[0059] (2) Add 700 μl of 65°C preheated CTAB lysate to the powder, place it at 65°C for 3 hours, and invert the EP tube every 30 minutes during this period.

[0060] (3) Add cellulase at a final concentration of 30 U / g, bathe in 50°C water for 1 hour, and invert the EP tube continuously during this period.

[0061] (4) After cooling at room temperature, centrifuge at 10,000 rpm for 5 minutes, carefully absorb the supernatant and measure the volume of the supernatant, add the same volume of phenol: chloroform: isoamyl alcohol mixed solution = 25:24:1, mix well and then centrifuge at 12,000 rpm for 5 minutes.

[0062] (5) Transfer all supernatants to DNA adsorption columns in batches, centrifuge at 12,000 rpm for 1 min and discard the supernatant.

[0063] (6) Wash off the protein on the ads...

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Abstract

The invention discloses an extraction method of rosewood heartwood genome DNA (deoxyribonucleic acid). The extraction method comprises the following steps: grinding the rosewood heartwood into powder in liquid nitrogen; adding CTAB (Cetyltrimethyl Ammonium Bromide) splitting liquid to the obtained powder for splitting; adding cellulase to a split product for continuous splitting, centrifuging the obtained split product, taking supernatant, adding a mixed solution of phenol, chloroform and isoamyl alcohol, centrifuging and taking supernatant; transferring the obtained supernatant to a DNA adsorption column, centrifuging, discarding lower liquid, washing the adsorption column by using ethyl alcohol, centrifuging and discarding lower liquid; adding a conventional reagent for dissolving the DNA to the DNA adsorption column, and centrifuging, thus obtaining the rosewood heartwood genome DNA. The extraction method of the rosewood heartwood genome DNA is used for solving the defect of low quality of the genome DNA extracted from the rosewood heartwood by using a conventional genome DNA extraction method, extracting the genome DNA from the rosewood heartwood and preparing the high-quality genome DNA; the obtained genome DNA has extremely low pollution and can be used for PCR (Polymerase Chain Reaction) amplification identification and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for extracting genomic DNA from mahogany heartwood. Background technique [0002] Mahogany is a general term for a class of high-quality hardwoods with a red appearance, and has always been favored by people all over the world. In the past ten years, with the increasing collection of Chinese classical furniture and woodcarving handicrafts, the value of mahogany has doubled again and again, and a large number of counterfeit mahogany have appeared in the market. Traditional mahogany identification methods mainly identify mahogany heartwood color, smell, tube hole chord diameter, air-dry density, rays and other indicators. High-resolution identification of rosewood 'species' and locality. The key premise of realizing gene identification technology is how to extract high-quality DNA from the heartwood part of mahogany species. [0003] The heartwood is composed of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 李瑶周佳海张颖寿勇明
Owner SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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