Method for extracting DNA from animal tissue efficiently

An animal tissue, high-efficiency technology, applied in the field of DNA extraction, can solve the problems of DNA extraction, such as influence, loss, complicated operation steps, etc., to achieve the effect of reducing health damage, strong applicability, and simple operation

Inactive Publication Date: 2015-03-25
ANIMAL SCI RES INST GUANGDONG ACADEMY OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the composition of animal tissues is complex, including lipids, proteins, carbohydrates, vitamins, mineral elements, etc., which have an impact on the extraction of DNA, especially lipids, proteins and DNA are intertwined, which is difficult to remove
In addition, DNA is lost at every step of DNA extraction after cell lysis, so the more steps there are, the greater the chance of loss
In addition, most of the methods currently used involve the application of phenol / chloroform reagents, which are likely to cause environmental pollution and are harmful to the human health of experimental operators.
Moreover, the operation steps are complicated, the DNA yield is low, and it is difficult to quickly extract a large number of samples
[0004] Therefore, it is necessary to study a convenient, fast and practical method suitable for animal tissue DNA extraction, to solve the problem of low yield of DNA samples obtained by using conventional methods, and the operation steps cumbersome and other issues

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 DNA Extraction from Pig Muscle Tissue

[0035] (1) Cut 100 mg of pig muscle tissue sample, add 750 μL of buffer A, 37.5 μL of proteinase K solution with a mass concentration of 10 mg / mL, wherein buffer A is composed of 30 mM Tris, 50 mM EDTA, 50 mM NaCl, 1% (mass volume % content) Sodium dodecylsulfonate SDS composition, its pH is 7.5~8.0;

[0036] (2) Place the above mixture in a 55~58°C incubator (water bath or small electronic incubation equipment) for 3 hours to obtain a sample suspension;

[0037] (3) Shake for about 1 minute, add 250 μL of 6 mol L to the sample suspension -1 sodium chloride solution, mix well, and centrifuge at 14,000 rpm for 10 minutes at 4°C;

[0038] (4) Transfer 750 μL of the supernatant to a new centrifuge tube;

[0039] (5) Add 500 μL of isopropanol to the supernatant, mix vigorously (vortex for 1 minute), centrifuge at 14,000 rpm for 20 minutes at 4°C, remove the supernatant, and obtain a precipitate;

[0040] (6) Add 1 mL ...

Embodiment 2

[0046] Example 2 Extraction of Pig Liver Tissue DNA

[0047] (1) Cut 50 mg of pig liver tissue sample, add 750 μL of buffer A and 21 μL of proteinase K solution with a mass concentration of 20 mg / mL to obtain a mixture, in which buffer A is composed of 50 mM Tris, 100 mM EDTA, 100 mM NaCl, 2% Composed of sodium dodecylsulfonate SDS, its pH is 7.5~8.0;

[0048] (2) Place the above mixture in a 55~58°C incubator (water bath or small electronic incubation equipment) for 3 hours to obtain a sample suspension;

[0049] (3) Shake for about 1 minute, add 250 μL of 3mol L to the sample suspension -1 sodium chloride solution, mix well, and centrifuge at 14,000 rpm for 15 minutes at 4°C;

[0050] (4) Transfer 750 μL of the supernatant to a new centrifuge tube;

[0051] (5) Add 500 μL of isopropanol to the supernatant, mix vigorously (vortex for 1 minute), centrifuge at 14,000 rpm for 15 minutes at 4°C, remove the supernatant, and obtain a precipitate;

[0052] (6) Add 1 mL of 75% et...

Embodiment 3

[0058] Example 3 Extraction of pig adipose tissue DNA

[0059] (1) Cut out 100 mg of porcine adipose tissue sample, add 750 μL of buffer A, 30 μL of proteinase K solution with a mass concentration of 15 mg / mL, wherein buffer A consists of 80 mM Tris, 200 mM EDTA, 200 mM NaCl, 3% dodecyl Composed of sodium sulfonate SDS, its pH is 7.5~8.0;

[0060] (2) Place the above mixture in a 55~58°C incubator (water bath or small electronic incubation equipment) for 3 hours to obtain a sample suspension;

[0061] (3) Shake for about 1 minute, add 250 μL of 4mol L to the sample suspension -1 sodium chloride solution, mix well, and centrifuge at 14,000 rpm for 15 minutes at 4°C;

[0062] (4) Transfer 750 μL of the supernatant to a new centrifuge tube;

[0063] (5) Add 500 μL of isopropanol to the supernatant, mix vigorously (vortex for 1 minute), centrifuge at 13,000 rpm for 20 minutes at 4°C, remove the supernatant, and obtain a precipitate;

[0064] (6) Add 1 mL of 75% ethanol aqueo...

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PUM

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Abstract

The invention discloses a method for extracting DNA from an animal tissue efficiently. The method comprises the following steps of (1) selecting an animal tissue sample, and adding a buffer solution A and proteinase K solution into the animal tissue sample to obtain a mixture; (2) putting the mixture into incubation equipment, adjusting the temperature of the incubation equipment to 55-58 DEG C, and incubating the mixture for 2-5 hours to obtain a sample suspension; (3) oscillating the sample suspension, adding sodium chloride solution, mixing the sample suspension with the sodium chloride solution uniformly, and centrifuging to obtain a supernatant; (4) adding isopropanol into the supernatant, mixing the isopropanol and the supernatant uniformly, centrifuging, and removing the supernatant to obtain a precipitate; and (5) removing the residual isopropanol in the precipitate by washing with aqueous solution of ethanol, centrifuging, and standing to remove ethanol to obtain a precipitate which is the DNA of the animal tissue. The yield of DNA extracted from the animal tissue sample by adopting the method is high, and the method is simple in step, is harmless to the human body, can not cause pollution to environment and can be used for extracting a large amount of DNAs from the animal tissue samples rapidly.

Description

technical field [0001] The invention belongs to the technical field of DNA extraction, and in particular relates to a method for efficiently extracting animal tissue DNA. Background technique [0002] Animal tissues contain a large amount of DNA, which is the main chemical component of chromosomes and the material that makes up genes. The function of DNA molecules is to store all the genetic information of almost all proteins and RNA molecules that determine the traits of a species, to encode and design biological organisms to transcribe genes and express proteins in a certain time and space to complete all programs of directional development, and to preliminarily determine the biological Unique traits and personalities and all stress responses when interacting with the environment. Therefore, through the study of DNA, such as DNA methylation, base deletion, mutation and other conditions, we can indirectly understand or evaluate the physiological state of the animal body an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 蒋宗勇马现永胡友军
Owner ANIMAL SCI RES INST GUANGDONG ACADEMY OF AGRI SCI
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