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Non-invasive extraction method of Haliotis discus hannai ino genome DNA

A technology of wrinkled abalone and extraction method, which is applied in the direction of recombinant DNA technology, DNA preparation, etc., can solve the problems of death and permanent damage, achieve disinfection and sterilization of exogenous DNA interference, reduce exogenous DNA interference, and protect tissue integrity Effect

Inactive Publication Date: 2012-10-24
DALIAN HAIBAO FISHERY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional animal genome extraction methods mainly use muscle tissue or peripheral blood cells as DNA extraction objects. These methods will inevitably lead to wrinkled abalone in a state of stress, causing permanent damage or even death, and cannot continue to use test objects in production.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] The non-damaging method for extracting genome DNA of abalone wrinkle comprises the following steps: firstly take fresh and alive abalone wrinkle, wash it three times repeatedly with sterile artificial sea water, make its ventral surface upward, and let it stand at room temperature for 5 minutes. Use a cotton ball dipped in 70% alcohol to disinfect and stimulate the mantle of the wrinkled abalone, and let the treated wrinkled abalone stand for 5 minutes with the belly side up. After the mucus is secreted, scrape 0.1g of mucus and epidermal tissue Put the mixture into a centrifuge tube, add 600 microliters of absolute ethanol to the centrifuge tube to fix the sample, put the fixed sample on ice for 8 minutes, separate it in a centrifuge, and take the precipitate;

[0016] Add 600 microliters of cell lysate to the obtained precipitate. The cell lysate here is recommended to use Tris-Cl 100 mM, EDTA 50 mM, 1% SDS, pH 8.0, and shake to mix;

[0017] Add 6 μl of proteinase K ...

Embodiment 2

[0022] The non-damaging method for extracting genome DNA of abalone wrinkle comprises the following steps: firstly take fresh and alive abalone wrinkle, wash it three times repeatedly with sterile artificial sea water, make its ventral surface upward, and let it stand at room temperature for 5 minutes. Use a cotton ball dipped in 72% alcohol to disinfect and stimulate the mantle of the wrinkled abalone, and let the treated wrinkled abalone stand for 5 minutes with the belly side up. After the mucus is secreted, scrape 0.15g of mucus and epidermal tissue Put the mixture into a centrifuge tube, add 600 microliters of absolute ethanol to the centrifuge tube to fix the sample, put the fixed sample on ice for 5 minutes, separate it in a centrifuge, and take the precipitate;

[0023] Add 600 microliters of cell lysate to the obtained precipitate. The cell lysate here is recommended to use Tris-Cl 100 mM, EDTA 50 mM, 1% SDS, pH 8.0, and shake to mix;

[0024] Add 12 μl of proteinase ...

Embodiment 3

[0029] The non-damaging method for extracting genome DNA of abalone wrinkle comprises the following steps: firstly take fresh and alive abalone wrinkle, wash it three times repeatedly with sterile artificial sea water, make its ventral surface upward, and let it stand at room temperature for 5 minutes. Use a cotton ball dipped in 75% alcohol to disinfect and stimulate the mantle of the wrinkled abalone, and let the treated wrinkled abalone stand for 5 minutes with the belly side up. After the mucus is secreted, scrape 0.2g of mucus and epidermal tissue Put the mixture into a centrifuge tube, add 600 microliters of absolute ethanol to the centrifuge tube to fix the sample, put the fixed sample on ice for 10 minutes, separate it in a centrifuge, and take the precipitate;

[0030] Add 600 microliters of cell lysate to the obtained precipitate. The cell lysate here is recommended to use Tris-Cl 100 mM, EDTA 50 mM, 1% SDS, pH 8.0, and shake to mix;

[0031] Add 20 μl of proteinase ...

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Abstract

The invention discloses a non-invasive extraction method of Haliotis discus hannai ino genome DNA, comprising the steps of using 75% of alcohol to sterilize and irritate pallium of Haliotis discus hannai ino; scraping to obtain a mixture of mucus and epidermal tissue fragments; treating the mixture, centrifugally separating the mixture to obtain precipitate; adding a cell lysate and protease K to the precipitate; after digesting and oscillating, cooling the mixture for a certain time; adding NH4Ac to the sample; centrifugally separating to collect supernate; adding isopropanol to the supernate; after oscillating and mixing uniformly, and then centrifugally separating to obtain precipitate; using ethanol to wash the precipitate; drying the precipitate at room temperature for 5 minutes to obtain the Haliotis discus hannai ino genome DNA. The non-invasive extraction method of the Haliotis discus hannai ino genome DNA is simple, efficient and convenient, does not damage the Haliotis discus hannai ino and does not influence survival ability and production performance.

Description

technical field [0001] The invention relates to a method for extracting genomic DNA, in particular to a method for extracting genomic DNA without damage. Background technique [0002] Haliotisdiscus hannai belongs to the mollusca phylum Gastropoda, Probranchia, and Primitive Gastropoda. It is the only species of Baalaceae shellfish distributed in northern my country, and it is also an important object of mariculture in Liaoning and Shandong coasts. Using modern molecular biology techniques, the population genetic structure analysis and genetic improvement of Pan abalone must be extracted from Genomic DNA. The traditional animal genome extraction methods mainly use muscle tissue or peripheral blood cells as DNA extraction objects. These methods will inevitably lead to wrinkled abalone in a state of stress, causing permanent damage or even death, making it impossible for the test object to continue to be used in production. At present, the Walk-back selection method based on ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 刘小林刘明泰常亚青李杨李丹许淑芬李旭光张艳
Owner DALIAN HAIBAO FISHERY
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