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Construction method of clupanodom punchtatus microsatellite enrichment library

A construction method and microsatellite technology, applied in the field of molecular biology, can solve the problems of low acquisition purity of target DNA and low PCR reaction sensitivity, and achieve the effects of reducing construction cost, improving PCR amplification effect and improving efficiency.

Inactive Publication Date: 2019-02-15
ZHEJIANG OCEAN UNIV
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Problems solved by technology

[0005] Existing technologies, such as the Chinese invention patent with the authorized announcement number CN 102174714 B, disclose a method for constructing a microsatellite enrichment library of Panonychus citrus, using the magnetic bead enrichment method to integrate the reported microsatellite site screening strategy , through a series of steps including genomic DNA extraction, enzyme digestion, adapter ligation, magnetic bead enrichment, and library construction, a microsatellite-enriched library of Panonychus citrus was established; however, this method is limitedly suitable for the development of microscopic insects and mite Class microsatellite markers, and the purity of the target DNA is low, and the reaction sensitivity of PCR amplification is not high

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  • Construction method of clupanodom punchtatus microsatellite enrichment library

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Embodiment 1

[0024] A method for constructing a microsatellite enrichment library of chrysalis, comprising the following steps:

[0025] 1) Enzyme digestion: Genomic DNA was extracted from the muscle or fin ray tissue of the catfish, and the genomic DNA was digested with the restriction endonuclease MseI. The enzyme digestion reaction system was: 1.5 μL NEB 10× ligation buffer, 0.1 μL 100×BSA, 0.1 μL 5M NaCl solution, 1 μL restriction enzyme MseI, 10 μL genomic DNA at a concentration of 100 ng / μL;

[0026]The steps of extracting genomic DNA are as follows: cut up the muscle or fin ray tissue samples of the spotted catfish, and place them in extraction solution I: 70ml of Tris-HCl and disodium hydrogen phosphate with a concentration of 120mM, 0.3g of fatty alcohol ether ammonium sulfate, and a concentration of 120mM in 30ml Add 0.5 μg plasmid DNA and 0.1 μg chitin, freeze and thaw 2-3 times, the freezing temperature is -126 ° C, the melting temperature is 45 ° C, centrifuge, collect the sup...

Embodiment 2

[0041] A method for constructing a microsatellite enrichment library of chrysalis, comprising the following steps:

[0042] 1) Enzyme digestion: Genomic DNA was extracted from the muscle or fin ray tissue of the catfish, and the genomic DNA was digested with the restriction endonuclease MseI. The enzyme digestion reaction system was: 2.5 μL NEB 10× ligation buffer, 0.25 μL 100×BSA, 0.25 μL 5M NaCl solution, 1 μL restriction endonuclease MseI, 20 μL genomic DNA with a concentration of 100 ng / μL;

[0043] The steps of extracting genomic DNA are as follows: cut up the muscle or fin ray tissue samples of the spotted catfish, and place them in extraction solution I: 70ml of Tris-HCl and disodium hydrogen phosphate with a concentration of 120mM, 0.3g of fatty alcohol ether ammonium sulfate, and a concentration of 120mM in 30ml Add 0.5 μg plasmid DNA and 0.1 μg chitin, freeze and thaw 2-3 times, the freezing temperature is -166 ° C, the melting temperature is 50 ° C, centrifuge, coll...

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Abstract

The invention discloses a construction method of a clupanodom punchtatus microsatellite enrichment library. The method comprises the steps of digestion, preparation of a joint solution, connection ofthe joint, PCR amplification of a connection product, enrichment of magnetic balls and construction of the library; circular DNA and chitin are added during a process of extraction of clupanodom punchtatus genome DNA, and thus the clupanodom punchtatus microsatellite marking and screening accuracy and obtaining efficiency can be effectively improved; chitin liquid is added to a PCR amplification reaction solution to improve the reaction sensitivity of PCR amplification, and thus a complete clupanodom punchtatus microsatellite mark can be obtained. The method has the beneficial effects of beingsimple, efficient, low in cost, high in microsatellite marking and screening accuracy and obtaining efficiency, and high in PCR reaction success rate.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for constructing a microsatellite enrichment library of chrysalis. technical background [0002] Konosirus punctatus belongs to Clupeiformes, Clupeidae, Dorosomatinae, and Konosirus. It is a coastal warm-water pelagic fish widely distributed in China, North Korea, The coasts of South Korea and Japan are produced in my country's Bohai Sea, Yellow Sea, East China Sea, and South China Sea. They are important fishing targets for offshore fisheries in China, Japan, and the Korean Peninsula. Catfish is of great significance to the sediment resuspension in coastal ecosystems and the material exchange at the water-bottom interface; at the same time, it is the prey object of a variety of high-trophic fish, and plays a key role in connecting the preceding and the following in the structure of the marine food web. At the same time, because of its high meat content, tender...

Claims

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Application Information

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IPC IPC(8): C12N15/10C40B50/06
CPCC12N15/1013C40B50/06
Inventor 刘炳舰龚理柳意樊吕振明刘立芹江丽华段雯朱科桦张伟男刘珠
Owner ZHEJIANG OCEAN UNIV
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