Tissue lysis solution and method for extracting animal tissue DNA

Abstract

The invention discloses a tissue lysis solution and a method for extracting animal tissue DNA, the tissue lysis solution is composed of tris-HCl, KCl, NP40, Tween 20 and pure water, and the extraction method comprises the following steps: adding protease K into the tissue lysis solution, uniformly mixing, adding into the animal tissue, carrying out cell lysis for 15-30 minutes at 56 DEG C, inactivating for 5 minutes at 95 DEG C, centrifuging for 2 minutes at 12000 rpm, and taking the supernatant, namely the animal tissue DNA. The tissue lysis solution is simple in component and low in production cost, DNA extraction is rapid and convenient, the obtained DNA is high in purity and good in stability, and the requirements of experiments such as PCR can be completely met.

Description

Technical Field

[0001] The invention relates to the technical field of DNA extraction, and in particular to a tissue lysate and a method for extracting DNA from animal tissues. Background Art

[0002] The extraction of DNA from animal tissues generally requires two steps: cell lysis and DNA purification. Lysis is the process of freeing the nucleic acids in the animal tissues in the lysis system, and purification is the process of removing other impurities such as proteins, salts, sugars, etc. Currently, the more commonly used DNA extraction methods have some problems. For example, although the high-salt method for extracting DNA is simple and fast, the purity and stability of the obtained DNA are poor, which affects the later PCR reaction; the phenol-chloroform extraction method requires a long digestion time and the extraction steps are cumbersome, and it may also introduce phenol contamination, affecting the experimental process. Summary of the invention

[0003] The purpose of the present invention is to provide a tissue lysis solution and a method for extracting animal tissue DNA in view of the above problems in the prior art, which can simply and quickly lyse animal tissue cells to release DNA, and the extraction is convenient and time-saving.

[0004] The technical solution of the present invention is described in detail as follows: In a first aspect, the present invention provides a tissue lysis solution, comprising the following components: 100mM tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride), 500mM KCl (potassium chloride), 0.45%(wt%) NP40 (ethylphenyl polyethylene glycol), 0.45%(wt%) Tween 20 (polyoxyethylene sorbitan monolaurate), The balance is pure water.

[0005] Optionally or preferably, the pH value of the tissue lysis solution is 8.3.

[0006] In a second aspect, the present invention provides a method for extracting DNA from animal tissues, comprising the following steps: (1) Add the above tissue lysis buffer and 1% proteinase K to animal tissues, lyse cells at 56°C for 15-30 minutes, and inactivate at 95°C for 5 minutes; (2) Centrifuge at 12000 rpm for 2 minutes and take the supernatant, which is the animal tissue DNA.

[0007] Among them, 1% proteinase K was prepared by dissolving 1g proteinase K in 100mL pure water.

[0008] Since proteinase K is easily degraded, if it is used in a short period of time, the tissue lysis buffer and proteinase K can be mixed and placed at 4°C and used together directly; but for long periods of time, proteinase K needs to be stored at -20°C, dissolved in water when used, and then added to the tissue lysis buffer or directly added to the animal tissue to be treated.

[0009] Optionally or preferably, in the above method, every 100 μL of tissue lysis solution is used to process 2-10 mg of animal tissue.

[0010] Optionally or preferably, in the above method, the time for lysing cells is 15 minutes.

[0011] Optionally or preferably, in the above method, the amount of tissue lysis solution added is 100 μL.

[0012] Optionally or preferably, in the above method, 1 μL of the supernatant is taken.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The tissue lysate of the present invention has simple ingredients and low cost. It only has four ingredients: tris-HCl, KCl, NP40 and Tween 20. It does not contain organic solvents such as chloroform, isopropanol, ethanol, phenol, etc., and will not cause organic solvent pollution. The formula does not contain Mg used to separate DNA from other molecules such as RNA or protein. 2+ , high-purity and high-stability DNA can still be obtained, and 1μL of extraction solution can meet the PCR requirements for DNA purity and stability.

[0014] The concentration of tris-HCl in the lysate of the present invention is increased to 100 mM (the conventional concentration is between 10-50 mM), and the concentration of KCl is increased to 500 mM (the conventional concentration is 2.7-50 mM). Increasing the concentrations of Tris-HCl and KCl has the following effects on extracting DNA from animal tissues: Tris-HCl Tris-HCl is a buffer, and increasing the concentration to 100mM helps maintain a more stable pH value. During the DNA extraction process, the activity of many enzymes is sensitive to pH. The appropriate pH helps to ensure the activity of enzymes such as proteinase K, so that it can better decompose proteins in tissues and release DNA more fully. In addition, in the subsequent extraction steps, a stable pH environment can prevent the chemical structure of DNA from being destroyed.

[0015] KCl High concentration of KCl (500mM) is conducive to destroying cell structure. It can change the permeability of cell membranes, making it easier for DNA in cells to be released into the lysis solution. At the same time, KCl can also precipitate some proteins in a high-salt environment through salt precipitation, reduce protein contamination of DNA, and help improve the purity of extracted DNA.

[0016] The method for extracting DNA from animal tissue provided by the present invention adopts the above-mentioned tissue lysis solution. The whole operation process is simple and rapid. Lysis only takes 15 minutes, followed by inactivation for 5 minutes and centrifugation for 2 minutes to obtain a DNA supernatant that meets the PCR requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Electrophoresis results of PCR on bovine tissue DNA.

[0018] Figure 2 These are the electrophoresis results of PCR on mouse tissue DNA. DETAILED DESCRIPTION

[0019] In order to make the technical personnel in this field better understand the present application scheme, the present application will be clearly and completely described below in conjunction with the embodiments and drawings. Obviously, the described embodiments are only embodiments of a part of the present application, rather than all embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work should belong to the scope of protection of this application. The instruments and reagents used in the embodiments are all derived from commercial channels unless otherwise specified.

[0020] Example 1 Formulation of tissue lysis solution and DNA extraction method of the present invention 100mM tris-HCl 500mM KCl 0.45% NP40 0.45% Tween 20 Remaining pure water The percentages in the formula are by mass. Adjust the pH to 8.3 and store at 4°C. Add 1% proteinase K before use (1% proteinase K is prepared by dissolving 1g proteinase K in 100ml water).

[0021] DNA extraction method: (1) Take 2-10 mg of animal tissue, add 100 μL of the above tissue lysis buffer with proteinase K added, lyse the cells at 56°C for 15 minutes, and inactivate at 95°C for 5 minutes; (2) Centrifuge at 12000 rpm for 2 minutes, take 1 μL of the supernatant and perform PCR experiments.

[0022] Comparative Example 1 Existing tissue lysis solution formula 10 mM tris-HCl (pH 9.0) 50mM KCl 2.5 mM MgCl2 0.1% Triton X-100 0.5% NP40 0.5% Tween 20 0.1mg / mL Proteinase K Remaining amount of water. The percentages in the formula are by mass.

[0023] DNA extraction method: (1) Take 2-10 mg of animal tissue, add 100 μL of the above-mentioned existing tissue lysis buffer, lyse the cells at 56°C for 40 minutes, and inactivate at 100°C for 5 minutes; (2) Centrifuge at 12000 rpm for 2 minutes, take 1 μL of the supernatant and perform PCR experiments.

[0024] The tissue lysates of Example 1 and Comparative Example 1 and the corresponding DNA extraction methods were used to extract DNA from bovine and mouse tissue samples and perform PCR. The primer sequences used in PCR are as follows: Cattle ACTB primer sequences: F: 5'-GCACAATGAAGATCAAGATCATC-3', R: 5'-CTAACAGTCCGCCTAGAAGCA-3'.

[0025] Mouse ACTB Primer Sequences: F: 5'-GGCTGTATTCCCCTCCATCG-3', R: 5'-CCAGTTGGTAACAATGCCATGT-3'.

[0026] PCR procedure: 95°C for 5 minutes; 95°C for 30 seconds, 56°C for 30 seconds, 72°C for 20 seconds, repeat this step for 35 cycles; 72°C for 10 minutes.

[0027] The length of the amplified product obtained by PCR in bovine tissue is 173 bp, and the electrophoresis pattern is as follows Figure 1 As shown, the length of the amplified product obtained by PCR in mouse tissue is 154 bp, and the electrophoresis diagram is as follows Figure 2 In the two electrophoresis diagrams, the first column on the left is the marker, columns 2-4 are the DNA amplification products extracted in comparative example 1, and columns 5-7 are the DNA amplification products extracted in example 1.

[0028] It can be seen from the amplification results that the purity and stability of the DNA obtained by the lysis solution and DNA extraction method of the present invention are very good and can fully meet the requirements of PCR.

[0029] The invention concept is described in detail using specific examples herein, and the description of the above embodiments is only used to help understand the core idea of ​​the invention. It should be pointed out that any obvious modification, equivalent substitution or other improvement made by a person of ordinary skill in the art without departing from the invention concept should be included in the protection scope of the present invention.

Claims

1. A tissue lysis solution, characterized in that: It is composed of the following ingredients: 100mM tris-HCl, 500 mM KCl, 0.45%(wt%) NP40, 0.45%(wt%) Tween 20, The balance is pure water.

2. The tissue lysate according to claim 1, characterized in that The pH value is 8.

3.

3. A method for extracting DNA from animal tissue, characterized in that: The following steps are involved: (1) Add the tissue lysis solution described in claim 1 or 2 and 1% proteinase K to animal tissues, lyse the cells at 56°C for 15 to 30 minutes, and inactivate at 95°C for 5 minutes; (2) Centrifuge at 12000 rpm for 2 minutes and take the supernatant, which is the animal tissue DNA.

4. The method according to claim 3, characterized in that: Each 100 μL tissue lysis buffer is used to process 2-10 mg of animal tissue.

5. The method according to claim 3, characterized in that: The time for lysing cells was 15 minutes.

6. The method according to claim 3, characterized in that The amount of tissue lysis buffer added is 100 μL.

7. The method according to claim 3, characterized in that Take 1 μL of the supernatant.

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