CN102329791A - Mouse-tail DNA (deoxyribose nucleic acid) extraction kit applicable to the genotype of laboratory mouse and application thereof - Google Patents

Abstract

The invention discloses a simple, economical and reliable mouse tail DNA extraction kit, which is suitable for accurate identification of experimental mouse genotypes. The kit includes solution A and solution B, wherein solution A is composed of NaOH solution with a final concentration of 0.025N and EDTA solution with a final concentration of 2mM, and solution B is a Tris solution with a final concentration of 40mM, pH7.6. The operation steps are as follows: Take 0.2-0.5cm tissue from the tail tip of the mouse and place it in a 1.5ml EP tube; add 180μl of A solution, 100°C for 30min; then ice-bath for 2min, then add 180μl of B solution, and mix well to obtain tissue DNA. Can be used for genotype identification in the next step. The preparation method of the kit is simple, the components of liquid A and liquid B used are conventional biochemical reagents, the whole process does not require the participation of reagents such as proteinase K and phenol/chloroform, and there are no cumbersome steps such as multiple centrifugation, and the operation time does not exceed 1 hour. Economical and cost-effective, it is especially suitable for long-term, large-scale, and frequent laboratory genotype identification of experimental mice.

Description

Rat tail DNA extraction kit suitable for genotyping of experimental mice and its application

technical field

The invention relates to a mouse tail DNA extraction kit and application thereof, which is simple, economical and reliable, and is especially suitable for accurate identification of experimental mouse genotypes.

Background technique

The use of genetics to study various gene mutant mice has become one of the most powerful methods in life science and medical research, and transgenic mice (mainly including gene knock-in, knock-out and immunodeficiency mice, etc.) The strains and numbers are also increasing. This phenomenon not only effectively improves the scientific research platform and level, but also makes the genotyping of mice a long-term, large-scale and frequent routine work.

At present, the commonly used method involving tissue DNA extraction in genotyping is the extraction of rat tail DNA, and its traditional methods include phenol/chloroform extraction and high-salt extraction. Although these methods can obtain mouse tail DNA with higher purity and yield, they all have some common shortcomings: one is that the steps are more complicated, such as involving centrifugation many times; the other is that there are too many reagents involved, such as using Chloroform, ethanol, proteinase K and other reagents; third, the time is relatively long, for example, there is an overnight experiment step. Even for other methods reported in the literature, it takes at least 2 hours. The above characteristics have virtually increased the cost of experiments (including reagent costs and labor costs), which is not conducive to laboratories with tight funds and limited manpower to carry out related genotyping experiments.

Contents of the invention

In view of the deficiencies in the prior art, based on the actual needs of long-term, large-scale, and frequent laboratory work for genotyping experimental mice, the present invention provides a gene-typing method suitable for experimental mice that is easy to operate, low in cost, and reliable in results. Typing Mouse Tail DNA Extraction Kit.

The DNA extraction kit provided by the present invention includes liquid A and liquid B.

The solution A is composed of NaOH with a final concentration of 0.025N and EDTA with a final concentration of 2mM; each 40ml reagent contains 100μl 10N NaOH solution, 160μl 0.5M EDTA solution and 39.74ml ddH ₂ O.

The solution B is a Tris solution with a final concentration of 40mM; every 40ml of reagent contains 1.6ml of 1M Tris solution and 38.4ml of ddH ₂ O.

The present invention provides a rapid kit for extracting rat tail DNA suitable for genotype identification, which is carried out according to the following steps:

(1) Take 0.2-0.5cm of mouse tail tip tissue and place it in a 1.5ml EP tube;

(2) Add 180μl solution A;

(3) Put it in a boiling water bath for 30 minutes, or put it in a micro thermostat set at 100°C for 30 minutes, so that the nucleic acid components in the tissue cells can be released by lysis under the action of solution A and high temperature;

(4) Take out the EP tube and bathe in ice for 2 minutes;

(5) Add 180μl solution B and mix well;

(6) Store at 4°C and use for genotype identification.

The present invention is based on the actual needs of genotyping experiments, simplifies the conventional extraction process, selects the NaOH solution in the A liquid and high temperature factors to directly crack the tissue, so that the DNA can be fully released, and at the same time uses the EDTA solution in the A liquid and the The integration of Mg ²⁺ reduces the activity of DNase, thereby further protecting the integrity of the DNA sample, while the Tris solution in solution B continues to provide a suitable lysis environment for the tissue. Since the genotype identification of experimental mice does not require too high DNA purity and yield, the present invention does not use reagents such as proteinase K and phenol/chloroform, nor does it use corresponding centrifugation methods, thereby greatly reducing the experimental steps. and time. At the same time, the present invention focuses on the control of the ratio of liquid A/sample, which can ensure that the obtained product can be used for accurate genotype identification. The inventor used the kit to carry out a large number of experiments in relevant laboratories of the Harvard Medical School and the Third Affiliated Hospital of the Third Military Medical University in the United States, and the results were reliable.

It can be seen that the preparation method of the kit is simple, the components of liquid A and liquid B used are conventional biochemical reagents, the whole process does not require the participation of reagents such as proteinase K and phenol/chloroform, and there are no tedious steps such as multiple centrifugation, and the operation time does not exceed 1h , fast, economical, and cost-effective, especially suitable for long-term, large-scale, and frequent identification of laboratory mouse genotypes.

Description of drawings

Figure 1 is the result of agarose gel electrophoresis for the genotype identification of Foxp3-GFPKI mice with a C57B/6 background: Lane M is DNA Marker (DL2000, the band molecular weight from top to bottom is 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp), lanes 1-9 are the expression results of Foxp3 in different mice, lane NC is the negative control. Its template is mouse tail DNA extracted according to the kit method provided by the present invention. The results show that, except for the negative control, all mouse bands are 470bp, indicating that these samples are all Foxp3-GFPKI homozygous mice.

Figure 2A and Figure 2B are the results of flow cytometric analysis of peripheral lymph nodes and splenocyte suspensions of Foxp3-GFPKI mice.

Detailed ways

The following examples are for better understanding of the present invention, but are not limited thereto. The experimental methods in the following examples, unless otherwise specified, are conventional methods; the experimental materials used in the following examples, unless otherwise specified, are conventional biochemical reagents.

Embodiment 1: kit preparation

Solution A 40ml: Take 100μl 10N NaOH solution and place it in a 50ml plastic container, then add 160μl 0.5M EDTA solution and 39.74ml ddH ₂ O respectively, and mix well.

Solution B 40ml: Take 1.6ml 1M Tris solution and place it in a 50ml glass container, add 38.4ml ddH ₂ O, mix well, pH 7.6.

Example 2: Genotyping of Foxp3-GFP knock-in mice with C57B/6 background (that is, knock-in the green fluorescent protein into the mouse so that it is linked to the Foxp3 gene, so that all cells expressing green fluorescence are Foxp3-positive cells)

Foxp3-GFP knock-in adult mice paired with C57B/6 background were donated by Harvard Medical School (purchased from Jackson Laboratory, USA), and placed in the Experimental Animal Center of the Third Affiliated Hospital of the Third Military Medical University according to the SPF animal standard. , Foxp3 gene identification was carried out after the young mice were weaned and separated into cages;

(1) Take 0.2-0.5cm tissue from the mouse tail tip and place it in a 1.5ml EP tube;

(2) Add 180μl solution A;

(3) 100°C for 30 minutes;

(4) Take out the EP tube and bathe in ice for 2 minutes;

(5) Add 180μl solution B and mix well;

(6) Store at 4°C for genotype identification:

PCR primer synthesis (Shanghai Sangong): Foxp3-1 (common)-CAA AAC CAA GAA AAG GTG GGC,

Foxp3-2 (wild type reverse)-CAG TGC TGT TGC TGT GTA AGG GTC, Foxp3-3 (mutant reverse)-GGA ATG CTC GTC AAG AAG ACA GG;

Samples were taken as follows so that the total volume was 15 μl: template (obtained by the above kit method) 2 μl, 2×Tag Master Mix (Promega, M712B) 7.5 μl, primer Foxp3-1 0.6 μl, primer Foxp3-2 0.6 μl, primer Foxp3-3 0.6 μl, ddH ₂ O 3.7 μl;

Complete the PCR reaction according to the following PCR parameters (Jackson Laboratory): first, 94°C for 3 minutes, then 35 cycles at 94°C for 30sec, 64°C for 1min, and 72°C for 1min, then 72°C for 2min, and store the final product at -20°C;

2% agarose gel electrophoresis to detect PCR products (see Figure 1): 510bp single band is C57B/6 wild-type mouse, 470bp single band is Foxp3-GFP knock-in homozygous mouse with C57B/6 background, 470bp and 510bp double bands are Foxp3-GFPKI heterozygous mice;

Randomly select the Foxp3-GFP knock-in homozygous mice identified above, and kill them by cervical dislocation;

Peripheral lymph nodes (including inguinal, axillary, and cervical lymph nodes) and spleen are collected and ground into a cell suspension;

Centrifuge at 1500rpm for 5min, discard the supernatant;

Add 2ml red blood cell lysate (Tiangen), mix well and let stand at room temperature for 2min;

Move the liquid to RPMI1640 medium (Lanzhou Minhai) pre-added with 6ml of 10% FBS (Hyclone) for washing;

Centrifuge at 1500rpm for 5min, discard the supernatant;

Add 400 μl of RPMI1640 medium containing 10% FBS to suspend and filter;

Flow cytometry (BD FACS Calibur) detects the GFP signal and the proportion of cells containing the signal;

Comparing the difference between the genotyping results using the mouse tail DNA obtained in the present invention and the flow cytometric analysis results, the reliability of the former is inferred, see Fig. 2A and Fig. 2B. The sample in Figure 2A is a C57B/6 wild-type mouse (control), and the sample in Figure 2B is a mouse identified as Foxp3-GFPKI homozygous in Figure 1. The results showed that the results of flow cytometry analysis were consistent with the results in Figure 1, indicating that the mouse tail DNA extraction quick kit provided by the present invention can be used for genotype identification, and the results are reliable.

Claims (3)

1. one kind is applicable to the genotypic mouse tail of experiment mice DNA extraction test kit; It is characterized in that: comprise A liquid and B liquid; Wherein A liquid 40ml is the NaOH solution of 0.025N and the EDTA solution composition that final concentration is 2mM by final concentration, B liquid 40ml; For final concentration is the Tris solution of 40mM, pH 7.6.

2. the compound method of the described mouse tail of claim 1 DNA extraction test kit is characterized in that: get 100 μ l 10N NaOH solution and place the 50ml plastic containers, add 160 μ l 0.5M EDTA solution and 39.74ml ddH more respectively ₂O, mixing promptly obtains A liquid; Get 1.6ml 1M Tris solution and place the 50ml Glass Containers, add 38.4ml ddH ₂O, mixing promptly obtains B liquid.

3. the method for use of the described mouse tail of claim 1 DNA extraction test kit is characterized in that carrying out as follows:

Get mouse tail point 0.2-0.5cm tissue and place 1.5ml EP pipe;

Add 180 μ l A liquid;

Place boiling water bath 30min, or place temperature to be set at 100 ℃ micro-thermostatted 30min, the nucleic acid component in the histocyte is discharged through cracking under A liquid and action of high temperature;

Take out the EP pipe, ice bath 2min;

Add 180 μ l B liquid, fully mixing;

4 ℃ of preservations promptly can be used for genotype identification.

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