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A method for extracting genomic dna from bull frozen semen

A genome and frozen semen technology, applied in the field of genomic DNA extraction, can solve the problems of difficult release of sperm DNA, difficult digestion of frozen semen, genome purification obstacles, etc., and achieve the effect of high purity, low cost and easy use

Inactive Publication Date: 2011-12-28
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has the following problems in practical application: long extraction time, cumbersome operation, residual organic solvents in the product affect subsequent experiments, and difficult digestion of frozen semen, which brings high-quality and high-quantity frozen semen genomic DNA to the laboratory. difficulty
It makes it difficult for sperm DNA to be released during digestion and the yield is low
In addition, the diluted protective agent added in frozen semen for the purpose of dilution, nutrition and protection of sperm cells, the carbohydrates, lipids and proteins contained in it also bring obstacles to the digestion of cells and the purification of genome

Method used

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  • A method for extracting genomic dna from bull frozen semen
  • A method for extracting genomic dna from bull frozen semen
  • A method for extracting genomic dna from bull frozen semen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0073] Example 2 Bull Frozen Semen Genomic DNA Extraction Kit

[0074] The kit for extracting genomic DNA from bull frozen semen of the present invention comprises:

[0075] Option One:

[0076] Reagent I: 0.01-0.0125 mol / L of EDTA with a pH value of 8.0, 0.01 mol / L of Tris Cl with a pH value of 8.0, SDS 1%, NaCl 2.9 g / L, and DTT 9.6 g / L, prepared with distilled water; and reagent II : saturated saline solution.

[0077] Option II:

[0078] Reagent I: 0.0125 mol / L of EDTA at pH 8.0, 0.0125 mol / L of Tris Cl at pH 8.0, SDS 1.25%, NaCl 3.625 g / L, DTT 19.3 g / L, prepared with distilled water; and reagent II: saturated Salt water.

[0079] third solution:

[0080] Reagent I: EDTA 0.01mol / L with a pH value of 8.0, Tris·Cl 0.0125mol / L with a pH value of 8.0, SDS 1.25%, NaCl 3.6g / L, DTT 19.3g / L, prepared with distilled water; and Reagent II: saturated Salt water.

[0081] Option four:

[0082] Reagent I: EDTA 0.0125mol / L with pH 8.0, Tris·Cl 0.01mol / L with pH 8.0, SDS 1.25%, Na...

experiment example

[0095] Experimental example The application of the bull's frozen semen genomic DNA extracted by the method of the present invention

[0096] PCR amplification of the target fragment of GHR gene:

[0097] The primer sequence for the amplification of the target fragment of the GHR gene is F: 5'-AATACTTGGGCTAGCAGTGACAATAT-3' and R: 5'-ACTGGGTTGATGAAACACTTCACTC-3', the product length is 175bp, and the reaction system and conditions are as follows.

[0098] PCR reaction system (20 μL): 2 μL of 10× amplification buffer, 1.6 μL of 4 kinds of dNTP mixture (2.5 mmol / L), 0.4 μL of primer F (20 pmol / μL), 0.4 μL of primer R (20 pmol / μL), template DNA 1 μL of frozen semen genomic DNA (100 ng / μL) of Holstein bull obtained in Example 1, 0.2 μL of Taq DNA polymerase (5U / μL), ddH 2 O 14 μL (buffer, dNTP, and polymerase were purchased from TaKaRa Company).

[0099] PCR reaction conditions: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30s, annealing at 58°C for 30s, extensi...

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Abstract

The invention provides a method for extracting the genomic DNA of the frozen semen of bull, which comprises the steps of washing of the semen of the bull, lysis and digestion of the semen and purification of the genomic DNA. In the method, the digestion yield of the DNA of the semen of the bull is increased by improving the components and mixing ratio of digestive lysis solution and determining proper digestion time; meanwhile, instead of the conventional method for purification by organic solvent extraction, a simpler high-salt method is used to purify DNA, and the high-salt method has the advantages of using easily prepared reagent, effectively removing impurities, obtaining high-yield and high-quality genomic DNA of frozen semen, along with low cost and suitability for high-efficiency and batch preparation of genomic DNA of frozen semen.

Description

technical field [0001] The invention relates to a method for extracting genomic DNA, in particular to a method for extracting genomic DNA from frozen semen of bulls. Background technique [0002] Holstein cows are an important breed of livestock that provide dairy products for humans. With the renewal and improvement of people's requirements for the quality of dairy products, in order to meet market demand, it is necessary to breed dairy cows and improve the milk production performance of dairy cow groups. With the realization of genetic breeding and artificial insemination technology, the semen of excellent bulls is used to make a large amount of frozen semen (thin tube frozen semen or granule frozen semen), which facilitates the spread of their excellent genetic properties and improves group performance. With the development of molecular biology and genetics, the selection and performance improvement of Holstein dairy cows from the perspective of molecular biology are the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 孙东晓谢岩范学华刘锐初芹张毅张沅张胜利
Owner CHINA AGRI UNIV
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