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Method for efficiently extracting DNA in animal blood

An animal and blood technology, applied in the field of high-efficiency extraction of animal DNA, can solve the problems of affecting the DNA yield, complicated operation, and a large number of reagents, and achieve the effect of improving the DNA yield

Inactive Publication Date: 2020-08-07
YUNNAN ANIMAL SCI & VETERINARY INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current optimization studies are all focused on improving the formulations of various liquid preparations in the kit, but there is no literature report on whether the existing technology will affect the DNA yield by improving the liquid addition sequence and digestion incubation time. In addition, the extraction process of the existing technology Involving multiple transfers of liquid, the operation is complicated, time-consuming, prone to pollution, low in efficiency, and requires a large amount of reagents and other consumables

Method used

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  • Method for efficiently extracting DNA in animal blood
  • Method for efficiently extracting DNA in animal blood
  • Method for efficiently extracting DNA in animal blood

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Extract goat blood DNA by commercially available OMEGA blood DNA extraction kit and supporting conventional methods

[0032] According to the common commercially available OMEGA blood DNA extraction kit and supporting conventional methods, DNA was extracted from 145 frozen blood samples of goats, and the operation method was as follows:

[0033] 1. Take 250μl whole blood to a centrifuge tube, if the blood sample is not enough for 250μl, make up with DNA eluent;

[0034] 2. Add 25 μl protein digestion enzyme and 250 μl cell lysate. Vortex at high speed for 15 seconds to mix thoroughly; to remove RNA, add 5μl RNase (50mg / ml);

[0035] 3. Incubate and digest at 65 degrees for 10 minutes, and vortex briefly during the incubation;

[0036] 4. Add 260 μl of absolute ethanol to the digestion solution, vortex at high speed for 20 seconds to mix thoroughly, and briefly centrifuge to remove the liquid on the cap into the tube;

[0037] 5. Put the column equipped in t...

Embodiment 2

[0047] Embodiment two extracts goat blood DNA by the method of the present invention

[0048] According to the method and steps of the present invention, use the cell lysate, protein digestion enzyme, protein washing solution, rinsing solution stock solution, and DNA eluent in the OMEGA blood DNA extraction kit to extract DNA from frozen blood of 531 goats. Methods as below:

[0049] 1) Take 25 μl of protein digestion enzyme to the bottom of a 1.5ml centrifuge tube, then add 250 μl of cell lysate;

[0050] 2) Take 250 μl goat whole blood anticoagulant blood and add it to the centrifuge tube in step 1). When the blood sample is less than 250 μl, make up with DNA eluent, and vortex at 3000 rpm for at least 15 seconds immediately after adding;

[0051] 3) Incubate the centrifuge tube of the mixed solution obtained in step 2) in a 65-degree water bath for 20 minutes, take out the centrifuge tube after 10 minutes of incubation, vortex and shake at 3000rpm for 5 seconds, then put i...

Embodiment 3

[0065] Embodiment three determines that the method of the present invention extracts the liquid addition order of goat blood DNA and the comparative test of digestion incubation time

[0066] Three frozen-preserved goat anticoagulated whole blood samples were used for comparative experiments, and were divided into 6 experimental groups according to Table 2, each group included 3 samples and 2 replicates, each replicate took 250 μl blood samples, and finally washed with 100 μl DNA Dehydrate to dissolve the DNA. The DNA extraction method of group 1 is the method of the present invention, the DNA extraction method of group 2 is a conventional method, the DNA extraction method of group 3 is a conventional method and only prolongs the incubation time to 20 minutes, and the DNA extraction method of group 4 is a conventional method and only prolongs the incubation time to 15 minutes The method for extracting DNA in group five is a conventional method, only extending the incubation ti...

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Abstract

The invention relates to a method for efficiently extracting DNA in animal blood, which comprises the following steps: adding a protein digestive enzyme to the bottom of a centrifuge tube, adding a cell lysis solution, adding a blood sample, carrying out high-speed vortex oscillation, incubating in a 65-DEG C water bath for 20 minutes, adding absolute ethyl alcohol, sufficiently and uniformly mixing, centrifuging to obtain a blood DNA digestion lysis solution, rinsing and eluting by using a silica gel membrane column to finally obtain a centrifugate which is an animal blood DNA extracting solution, and storing at the temperature of -20 DEG C. The method comprises the following steps: adding the protein digestive enzyme to the bottom of the centrifuge tube, adding a cell lysate and adding ablood sample so that the digestion time of protein digestive enzymes is prolonged to 20 minutes, the yield of obtained DNA can be increased by 159%, and pipette tips for detecting every 24 samples inone batch are reduced by 23, and the extraction cost of animal blood unit DNA can be remarkably reduced on the basis of using commercially available conventional kits.

Description

technical field [0001] The invention relates to a method for efficiently extracting animal DNA from blood, in particular to a method for efficiently extracting DNA in animal blood. [0002] technical background [0003] Without killing animals, extracting DNA from blood for subsequent detection, sequencing, genetics and molecular biology research is a commonly used biological research method. The principle is to use protein digestion enzymes and lysis reagents to lyse samples to release nucleic acids , then add an organic solvent to precipitate the nucleic acid or adsorb it on a solid medium, then absorb the supernatant (waste liquid), wash the nucleic acid with washing liquid several times, and finally dissolve the precipitated nucleic acid with a dissolving solution or remove the nucleic acid from the solid medium elutes down. Currently on the market, nucleic acid purification kits can be roughly divided into three categories in terms of purification methods: centrifugal p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 兰蓉朱兰洪琼花欧阳依娜邵庆勇
Owner YUNNAN ANIMAL SCI & VETERINARY INST
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