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Sperm DNA extraction method

An extraction method and sperm technology, which is applied in the field of sperm DNA extraction, can solve the problems of difficult to meet the high requirements of molecular biology, the inability to separate genomic DNA, and the difficulty in releasing sperm DNA, so as to achieve uniform lysate, sufficient and complete lysis, and short extraction time Effect

Inactive Publication Date: 2021-01-05
GUANGDONG HAID GROUP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, conventional methods cannot effectively separate the genomic DNA and protamine of sperm, resulting in difficulties in the release of sperm DNA during digestion, low extraction yields, and low purity, making it difficult to meet the high requirements of molecular biology.

Method used

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  • Sperm DNA extraction method
  • Sperm DNA extraction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The identification of embodiment 1 Nile tilapia male individual

[0044] (1) Wrap the fish body with a clean towel, one person holds the fish with the left hand, and puts the belly of the fish upwards (that is, the abdomen faces the sky), and gently squeezes the abdomen with the right hand, and the genital opening (that is, the hole behind the anus) is in the shape of The papillary one is the male fish, and the slit-shaped reproductive hole is the female fish.

[0045] (2) When a male fish is found, another person scans the belly of the fish with a code scanner, reads and records the chip code and sex information of the male fish, so as to count the final number and source of male fish.

Embodiment 2

[0046] Example 2 Collection of Nile Tilapia Male Semen

[0047] (1) Take a sterile 1.5mL centrifuge tube, and mark the sample serial number on the top cover of the centrifuge tube according to the number of individual male fish (if there are 20 male fish in total, mark it as: 1, 2, 3...18, 19, 20) . Put the numbered centrifuge tubes in the centrifuge tube rack in order, and put the centrifuge tube rack on ice for later use.

[0048] (2) Wrap the fish body with a clean towel. One person holds the fish with his left hand, turns the belly of the fish up (that is, the belly faces the sky), and gently squeezes the belly with his right hand. When semen flows out of the genital opening, the other Use a 200μL micropipette to quickly absorb semen and transfer 1.5mL to a centrifuge tube with a designated number. At the same time, use a scanner to scan the abdomen of the fish, read and record the chip code of the male fish, and establish a one-to-one correspondence with the centrifuge t...

Embodiment 3

[0050] The extraction of embodiment 3 Nile tilapia sperm DNA

[0051] (1) Put 30 μL of semen into a 1.5 mL centrifuge tube, add 300 μL of washing buffer, mix gently by inversion, centrifuge at 800 g for 10 min, and discard the supernatant.

[0052] (2) Add 300 μL of washing buffer to wash the semen again, the method is the same as above, centrifuge at 800 g for 10 min, and discard the supernatant.

[0053] (3) Add 600 μL of lysis buffer to the precipitate obtained in the above step, and incubate in a 56° C. water bath for 1 h until the precipitation is completely digested to obtain a uniform and clear solution.

[0054] (4) After the incubation, take the centrifuge tube out of the water bath, place it at room temperature to bring it back to room temperature, then add 480 μL of pre-cooled isopropanol, and mix it upside down gently until obvious DNA precipitation can be seen until. Usually, the formation of DNA precipitate (white floc) can be found by gently inverting up and d...

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Abstract

The invention relates to the technical field of biology, and particularly relates to a sperm DNA extraction method. The method comprises the following steps of: 1) taking a semen sample, cleaning thesemen sample, and performinglysis on the semen sample with a lysis buffer to obtain a lysate; 2) uniformly mixing the lysate with an alcohol solvent, and collecting a precipitate; and 3) washing the precipitate obtained in the step 2) with a sodium citrate solution, and collecting a precipitate, wherein the lysis buffer is prepared from 4.03-4.45M of guanidine thiocyanate, 0.095-0.105M of sodium chloride, 0.95-1.05% of sodium dodecyl sarcosinate, 0.1425-0.1575M of dithiothreitol and 0.19-0.21mg / mL of protease K, and is prepared by using sterile water. The sperm DNA extraction method provided by the invention is short in extraction time, large in total amount and high in quality, and can meet the requirements of various subsequent molecular genetic experiments.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a sperm DNA extraction method. Background technique [0002] Genomic DNA is the carrier of animal genetic material, which is widely used in genetic engineering, environmental testing, environmental purification, agriculture, animal husbandry, food industry, medicine and gene therapy. With the development of molecular biology and molecular breeding, the extraction and preservation of genomic DNA has become more and more important in the process of animal science experiments and breeding production. [0003] Nile tilapia belongs to the cichlid family. It has the characteristics of short generation cycle, fast growth, no intermuscular spines, etc. It has become the main economic fish in aquaculture worldwide. Nile tilapia can be bred or farmed throughout the year in areas with suitable or controlled water temperature. Therefore, it not only has important commercial and economic value, ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 郭向召王爱波付涛于孟兰李雪柳钱雪桥
Owner GUANGDONG HAID GROUP
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