Preparation method for procambarus clarkia simple sequence repeats (SSR) primer

A technology of Procambarus clarkii and products, applied in the field of genetic engineering

Active Publication Date: 2015-06-24
ZHEJIANG INST OF FRESH WATER FISHERIES
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Problems solved by technology

The disadvantage of SSR is that for organisms with unknown DNA se

Method used

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  • Preparation method for procambarus clarkia simple sequence repeats (SSR) primer
  • Preparation method for procambarus clarkia simple sequence repeats (SSR) primer
  • Preparation method for procambarus clarkia simple sequence repeats (SSR) primer

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Embodiment

[0038] A preparation method for Procambarus clarkii SSR primers, comprising the following steps:

[0039] 1. Extraction of Genomic DNA from Procambarus clarkii

[0040] STE buffer: NaCl 0.1 mol / L: 50 ml; Tris-Cl 0.05 mol / L (pH=8.0): 25 ml; EDTA 0.05 mol / L (pH=8.0): 50 ml; ddH 2 O: 50 ml; adjust the pH of the solution to 8.0, and sterilize it for later use.

[0041] Cut live Procambarus clarkii abdominal muscle 100±10mg into a 1.5 mL centrifuge tube, and cut the tissue into fine pieces with clean scissors. Add the following reagents in turn to the centrifuge tube: 500 μL STE buffer; 25 μL proteinase K (10 mg / mL) (merck); 75 μL 10% (mass volume ratio) SDS, mix well and digest overnight at 56 °C. Add an equal volume of water-saturated phenol solution, and gently invert and mix for more than 5 minutes. Centrifuge at 7000r / min for 5min, and carefully transfer the supernatant to another clean centrifuge tube. Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1, v...

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Abstract

The invention discloses a preparation method for a procambarus clarkia simple sequence repeats (SSR) primer. The preparation method comprises the steps: (1), extracting procambarus clarkia genome DNAs: extracting the genome DNAs from procambarus clarkia abdominal muscles; (2), hybridizing a probe and a target segment: hybridizing the biotin labeled probe and the target segment to obtain a hybridization product; (3), enriching magnetic beads: enriching the hybridization product by a magnetic bead enrichment method to obtain an enrichment product; (4), performing positive clone sequencing, and designing a primer amplification gene DNA according to a microsatellite flanking sequence; (5), performing SSR primer detection. The preparation method for the procambarus clarkia SSR primer has the advantages that a microsatellite sequence of the procambarus clarkia whole genome can be simply and quickly screened; a large number of SSR primers can be developed at a short time; technical measures are provided for germ plasma conservation and genetic improvement of the procambarus clarkia.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for preparing an SSR primer of Procambarus clarkii. Background technique [0002] Procambarus clarkii belongs to Arthropoda, Crustacea, Orgerdecapoda, Reptantia, Cambaridae, Procambarus . Procambarus clarkii is native to northeastern Mexico and the south-central United States. Due to human and environmental factors, it has spread to at least 15 states in the United States; it was introduced to Japan as a bullfrog bait in 1918, and it multiplied and spread in Japan on a large scale; in 1929 Introduced to my country from Japan, it is currently distributed in 21 provinces, municipalities, and autonomous regions in my country, and has become a dominant population in some lakes and ditches. At present, the genetic diversity of Procambarus clarkii is mostly studied by molecular techniques such as RAPD, mitochondrial cytochrome I (COI) and microsatellites. The app...

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12N15/10
Inventor 李喜莲李飞顾志敏贾永义黄鲜明
Owner ZHEJIANG INST OF FRESH WATER FISHERIES
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