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Rapid extraction method of black sea bream genome DNA

An extraction method and genome technology, applied in the field of rapid extraction of black sea bream genomic DNA, can solve the problems of inapplicable genomic DNA extraction, heavy workload of genomic DNA extraction, and injury to experimental operators, achieving good application prospects and convenient extraction , The effect of low reagent cost

Inactive Publication Date: 2017-01-04
INST OF OCEANOLOGY & MARINE FISHERIES JIANGSU
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Problems solved by technology

[0004] The phenol / chloroform extraction method is the most commonly used method in fish genome extraction. This method is relatively mature and the extraction effect is ideal, but the operation is cumbersome and time-consuming, and most of the extraction reagents are toxic, which is easy to cause harm to experimental operators. harm
However, the cost of using a kit to extract is relatively high, and it is not suitable for large-scale extraction of genomic DNA.
At present, many biotechnology methods, such as the identification of families in genetic breeding, the analysis of population genetic diversity, and the evaluation of the effect of proliferation and release using molecular markers, etc., require a large number of individuals to analyze or screen, and the workload of genomic DNA extraction is relatively large.

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  • Rapid extraction method of black sea bream genome DNA

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Experimental program
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Effect test

Embodiment 1

[0025] DNA was extracted from ethanol-preserved black sea bream dorsal fin ray samples and used for PCR amplification of mitochondrial COI gene. The implementation steps are as follows:

[0026] (1) Cut 20 μg of absolute ethanol and soak the black sea bream fin ray sample in a 1.5mL centrifuge tube, and fully grind the sample on the tube wall with a glass grinding rod;

[0027] (2) Add 200 μL of extraction buffer (1% TritonX-100+100mM Tris-HCl (pH=8.0)+300mMNaAc+500mM NaCl), shake for 1min and mix thoroughly;

[0028] (3) Place the metal bath or constant temperature water bath at 65°C for 30 minutes, and mix it occasionally;

[0029] (4) Take out the sample, cool it, and centrifuge it at 13000r / min for 3min in a centrifuge;

[0030] (5) Aspirate the supernatant into another new tube for the next step of PCR amplification. The black sea bream genome DNA extracted by the method is relatively complete and the effect is ideal. The agarose gel electrophoresis picture of the bla...

Embodiment 2

[0032] (1) Cut 20 μg of black sea bream fresh muscle sample into a 1.5mL centrifuge tube, and fully grind the sample on the tube wall with a glass grinding rod;

[0033] (2) Add 300 μL of extraction buffer (1% TritonX-100+100mM Tris-HCl (pH=8.0)+300mMNaAc+500mM NaCl), shake for 1min and mix well to obtain black sea bream tissue extract;

[0034] (3) Place the constant temperature water bath at 65°C for 35 minutes, and mix it occasionally;

[0035] (4) Take out the sample, cool it, and let it stand for 5 minutes;

[0036] (5) The supernatant is the obtained black sea bream genomic DNA, which is used for the next step of PCR amplification.

[0037] (6) Use a pair of black sea bream mitochondrial COI gene primers to carry out PCR amplification on the above-mentioned extracted DNA, and the primer sequence is:

[0038] Upstream primer: 5′-TACTCCTTACAGACCGAAAT-3′;

[0039] Downstream primer: 5′-TTATGAAGAAAGTGGCAGA-3′;

[0040] Use 25μL amplification system: 2.5μL 10×PCR buffer, ...

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Abstract

The invention relates to a rapid extraction method of black sea bream genome DNA. The method includes the steps of grinding or shearing a black sea bream fine ray or muscle sample, adding the material to an extraction buffer solution to be vibrated and evenly mixed to obtain a black sea bream tissue extraction solution, putting the solution at 65 DEG C for 20-40 min, taking out a sample, and conducting cooling, standing still or centrifuging to obtain supernate to obtain the black sea bream genome DNA. The method is simple in extraction step, rapid and low in requirements for experiment instruments, the extraction of the genome DNA can be completed within 1 hour, and the method is more rapid and has good application prospects compared with an extraction method using a kit.

Description

technical field [0001] The invention belongs to the field of genome DNA extraction, in particular to a method for rapidly extracting black sea bream genome DNA. Background technique [0002] With the large-scale application of molecular biology methods in fish research, the extraction of genomic DNA has become one of the most commonly used techniques, and it is the premise and guarantee for the success of various researches. [0003] Phenol / chloroform extraction is a commonly used method for DNA extraction. It removes impurities such as proteins, polysaccharides, and phenols through organic solvent extraction. Usually, the processed samples are added to cell lysate, proteinase K, phenol, and The mixture of chloroform and isoamyl alcohol is processed step by step, and the nucleic acid can be separated by adding ethanol for precipitation. Phenol can denature proteins and inhibit the degradation of DNase. After adding phenol, since the link between protein and DNA has been br...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003C12Q2527/125C12Q2523/32
Inventor 贾超峰张志勇陈淑吟刘海林许津张志伟祝斐
Owner INST OF OCEANOLOGY & MARINE FISHERIES JIANGSU
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