Method for extracting biomacromolecules in agarose clone formation experiment

Abstract

The invention relates to the technical field of biology, and particularly discloses a method for extracting biomacromolecules in an agarose clone formation experiment. The method can effectively extract nucleic acid (DNA and RNA) and protein in an agarose cloning formation experiment for molecular biological analysis, such as PCR, Q-RT-PCR and Western-blot analysis, and has the advantages that the efficiency is high, the operation is simple, the quality of extracted nucleic acid (DNA and RNA) and protein samples is good, the analysis requirement of bioinformatics is completely met, and the like. According to the method, a CTAC extracting solution is adopted to dissolve agarose wrapping cells, then nucleic acid precipitation is carried out in a chloroform extraction and isopropanol purification precipitation mode, and then DNA / RNA separation and purification are carried out on nucleic acid precipitation, so that the extraction efficiency is high, sample deviation is effectively avoided, and the detection accuracy is high. And the obtained DNA / RNA sample can completely meet the subsequent bioinformatics analysis. According to the method, PBS is adopted to wash and dissolve agarose coated cells, although a certain protein sample is lost, FBS pollution can be effectively reduced, finally, the protein is further concentrated through methanol, and the obtained protein sample completely meets the requirement of protein analysis.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for extracting biomacromolecules in an agarose clone formation experiment. Background Art

[0002] The agarose colony formation assay is an important technique widely used in cell biology and oncology research. The assay simulates the three-dimensional environment of cell growth in vivo by culturing cells in semi-solid agarose medium, allowing cells with colony formation ability to form independent clones. This experimental method can be used to evaluate the proliferation ability of cells, the degree of malignant transformation, and the sensitivity to drugs and other treatments.

[0003] In the agarose colony formation experiment, biomacromolecules such as nucleic acids (DNA and RNA) and proteins play a vital role. These biomacromolecules are involved in the life processes of cells such as growth, division, differentiation and apoptosis, and may undergo specific changes during the colony formation process. For example, the expression level of certain genes may change, and the modification state of proteins may affect the colony formation ability of cells. Therefore, the extraction and analysis of biomacromolecules in the agarose colony formation experiment is of great significance for a deeper understanding of the mechanism of colony formation and the development of new treatment strategies.

[0004] However, the existing RNA / DNA extraction kit cannot dissolve / remove the agarose encapsulating the cells, and can only extract non-agarose-encapsulated cells. When it is directly used to extract DNA\RNA in agarose cloning experiments, it not only has poor extraction efficiency, but also has certain sample deviations. In addition, the commonly used protein lysis buffer RIPA and the like are also difficult to dissolve agarose. When it is directly used to extract proteins in agarose cloning experiments, it not only has poor extraction efficiency, but also has certain sample deviations. At the same time, when agarose is cultured, there will also be some FBS, which is easy to cause contamination of the extracted protein and cause certain interference to the quantification of the protein. Summary of the invention

[0005] Based on this, the object of the present invention is to provide a method for extracting biomacromolecules in an agarose clone formation experiment, the method having the advantages of high extraction efficiency, simple operation, good quality of the extracted nucleic acid (DNA and RNA) and protein samples, and fully meeting the analysis requirements of bioinformatics. By using the method, nucleic acids (DNA and RNA) and proteins in an agarose clone formation experiment can be effectively extracted for molecular biological analysis, such as PCR, Q-RT-PCR, and Western-blot analysis.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a method for extracting RNA in an agarose colony formation experiment, the method comprising the following steps:

[0008] (1) Dissolving the agarose-cell mixture using CTAC extract. Preferably, the preparation method of the CTAC extract includes the following: mixing 2% w / v CTAC, 2% w / v PVP-40, 2M sodium chloride, 100mM Tris-HCl (pH 8.0), and 20mM EDTA in RNAse-free water, dissolving it after high temperature and high pressure treatment to prepare CTAC extract, and placing it at room temperature for use. Preferably, the method of dissolving the agarose-cell mixture with CTAC extract is: preheating the CTAC extract at 65°C in a dark environment, adding 2% v / v β-mercaptoethanol and mixing evenly, and adding it to the agarose-cell mixture after removing the upper culture medium of the agarose clone formation experiment at a volume ratio of 1:5 to dissolve the agarose-cell mixture.

[0009] (2) Add chloroform for extraction. Preferably, the specific extraction method is: after the liquid is evenly mixed, chloroform is added in a volume ratio of 1:1 and mixed again, and the operation is repeated once.

[0010] (3) adding isopropanol to the extract to purify and precipitate the nucleic acid precipitate. Preferably, the specific purification precipitation method is: centrifuge at 15,000g for 5 minutes at room temperature, mix the supernatant with isopropanol at a volume ratio of 1:1, centrifuge again at 15,000g for 15 minutes at room temperature, remove the supernatant at room temperature, wash the nucleic acid precipitate twice with 70% alcohol and air-dry.

[0011] (4) RNA isolation and purification To obtain RNA samples: Add RNase-free water to the dried nucleic acid precipitate, then add DNase I enzyme, control the temperature to digest DNA impurities, and use the RNA extraction kit FavorPrep TM Tissue Total RNA MiniKit is used to extract RNA. FARB Buffer and β-mercaptoethanol are added to the RNA precipitate-water mixed suspension, and the mixture is shaken and mixed until the RNA precipitate is dissolved; then RNase-free alcohol is added and mixed evenly, and the mixture is filtered through a FARB Mini Column, washed with WashBuffer 2, and RNase-free water is added to elute RNA on the FARB Mini Column membrane to obtain an RNA sample for qPCR experimental analysis. Preferably, the specific method is: 50ul of RNase-free water is added to the dried nucleic acid precipitate, and then 1ul of DNase I enzyme is added to digest DNA impurities at 37°C for 30 minutes, and then, the RNA extraction kit FavorPrep TMTissue Total RNA Mini Kit was used to extract RNA. To every 50 μl of RNA precipitate-water mixed suspension, 350 μl of FARB Buffer and 3.5 μl of β-mercaptoethanol were added. The mixture was shaken and mixed until the RNA precipitate was dissolved. Then, an equal volume of 70% RNase-free alcohol was added and mixed evenly. The mixture was filtered through a FARB Mini Column, and washed four times with 750 μl of Wash Buffer 2. Then, 40-100 μl of RNase-free water was added to elute RNA from the FARB Mini Column membrane to obtain RNA samples for RNA analysis.

[0012] The present invention provides a method for extracting DNA in an agarose colony formation experiment, the method comprising the following steps:

[0013] (1) Dissolving the agarose-cell mixture using CTAC extract. Preferably, the preparation method of the CTAC extract includes the following: mixing 2% w / v CTAC, 2% w / v PVP-40, 2M sodium chloride, 100mM Tris-HCl (pH 8.0), and 20mM EDTA in RNAse-free water, dissolving it after high temperature and high pressure treatment to prepare CTAC extract, and placing it at room temperature for use. Preferably, the method of dissolving the agarose-cell mixture with CTAC extract is: preheating the CTAC extract at 65°C in a dark environment, adding 2% v / v β-mercaptoethanol and mixing evenly, and adding it to the agarose-cell mixture after removing the upper culture medium of the agarose clone formation experiment at a volume ratio of 1:5 to dissolve the agarose-cell mixture.

[0014] (2) Add chloroform for extraction. Preferably, the specific extraction method is: after the liquid is evenly mixed, chloroform is added in a volume ratio of 1:1 and mixed again, and the operation is repeated once.

[0015] (3) adding isopropanol to the extract to purify and precipitate the nucleic acid precipitate. Preferably, the specific purification precipitation method is: centrifuge at 15,000g for 5 minutes at room temperature, mix the supernatant with isopropanol at a volume ratio of 1:1, centrifuge again at 15,000g for 15 minutes at room temperature, remove the supernatant at room temperature, wash the nucleic acid precipitate twice with 70% alcohol and air-dry.

[0016] (4) DNA separation and purification: DNA samples were obtained by adding RNase-free water to the dried nucleic acid precipitate, then adding RNase A, controlling the temperature to digest RNA impurities, and using the Blood and Tissue Kit to extract gDNA for PCR analysis. Preferably, the specific method of DNA separation and purification is: add 50ul of RNase-free water to the dried nucleic acid precipitate, then add 1ul of RNase A, digest RNA impurities at 37°C for 30 minutes, and use the Blood and Tissue Kit was used to extract gDNA and obtain gDNA samples for subsequent DNA analysis.

[0017] The present invention also provides a method for extracting protein in an agarose colony formation experiment, the method comprising the following steps:

[0018] (1) Wash the agarose-cell mixture with PBS. Preferably, the specific method is as follows: add 2 ml of PBS to a 24-well plate, and mix the agarose-cell mixture after removing the upper culture medium of the agarose clone formation experiment with PBS. Pool the PBS-agarose-cell mixtures from three 24-well plates, transfer them into a 15 ml centrifuge tube, add about 13 ml of PBS, and centrifuge at 700 g for 2 minutes. Remove 10 ml of the upper supernatant, add 10 ml of PBS for a second wash, remove 13 ml of the supernatant, and quickly freeze the washed agarose-cell mixture with liquid nitrogen.

[0019] (2) Dissolution with RIPA lysis buffer. Preferably, the specific method is: add 500 μl of RIPA lysis buffer + protease inhibitor + phosphatase inhibitor + 2% β-mercaptoethanol to each 1 ml of agarose-cell mixture, and then heat it at ≥95°C for 10 minutes.

[0020] (3) Centrifugal cooling and separation. Preferably, the specific method is: cool on ice for 1 hour, wait for the agarose-cell mixture to semi-solidify, centrifuge at the highest speed for 30 minutes in a 4°C centrifuge, transfer the supernatant to a new EP tube, add 1:1 volume of 100% methanol and 1 / 5 volume of chloroform, mix well, and centrifuge at 15,000g for 5 minutes. Remove the supernatant and retain the white separation layer.

[0021] (4) Purification and precipitation of proteins with methanol. Preferably, the specific method is: add 100% methanol in a volume ratio of 1:1, mix well, centrifuge at 15,000 g for 5 minutes, remove the supernatant, and retain the white precipitate EB.

[0022] (5) Redissolving the protein. Preferably, the specific method is: after air drying, use an appropriate amount of RIPA lysis buffer to re-dissolve the protein precipitate to obtain a protein sample for protein analysis.

[0023] The beneficial effects of the present invention are: the method of the present invention can effectively extract nucleic acids (DNA and RNA) and proteins in agarose clone formation experiments for molecular biological analysis, such as PCR, Q-RT-PCR, Western-blot analysis, with high efficiency, simple operation, good quality of nucleic acids (DNA and RNA) and protein samples extracted, and fully meet the analysis needs of bioinformatics. The method of the present invention uses CTAC extraction solution to dissolve the agarose encapsulated cells, and then uses chloroform extraction and isopropanol purification precipitation to precipitate nucleic acids, and then separates and purifies the nucleic acid precipitate for DNA / RNA. It not only has high extraction efficiency, but also effectively avoids sample bias, and the DNA / RNA samples obtained can fully meet the subsequent bioinformatics analysis. The method of the present invention uses PBS to wash and dissolve the cells encapsulated in agarose. Although a certain amount of protein samples will be lost, FBS contamination can be effectively reduced. Finally, the protein is further concentrated by methanol, and the obtained protein sample fully meets the needs of protein analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Provides a technical roadmap for the extraction of biomacromolecules for agarose colony formation experiments.

[0025] Figure 2 RNA extracted from HMEC cells in agarose colony formation experiments.

[0026] Figure 3 These are the results of qPCR validation of HMEC cell RNA extracted from the agarose clone formation experiment; 3A is the relative expression result of ST, and 3B is the relative expression result of TP53.

[0027] Figure 4 The experimental results were verified by PCR of gDNA extracted from HMEC cells in agarose colony formation experiment.

[0028] Figure 5 The results of Western-blot experiments are shown for proteins extracted from HMEC cells in agarose colony formation experiments. DETAILED DESCRIPTION

[0029] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary illustrations and explanations of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope that the present invention is intended to protect. It should be noted that the experimental materials whose sources are not indicated in the embodiments of the present invention can all be obtained commercially, and the experimental methods in the embodiments of the present invention whose specific conditions are not indicated are usually carried out according to conventional experimental methods in the art or according to the methods recommended by the experimental material manufacturers.

[0030] The present invention provides a method for extracting biomacromolecules in an agarose colony formation experiment, and the extraction technology roadmap is as follows: Figure 1 The following is a detailed description of the specific embodiments.

[0031] Example 1 Extraction of Cellular RNA in Agarose Colony Formation Experiment

[0032] (1) Prepare CTAC extract: Mix 2% w / v CTAC (292737, CTAC, Sigma Aldrich, St. Louis, MI, USA), 2% w / v PVP-40 (PVP-40, Sigma Aldrich, St. Louis, MI, USA), 2 M sodium chloride, 100 mM Tris-HCl (pH 8.0), and 20 mM EDTA (EDTA, Sigma Aldrich, St. Louis, MI, USA) in RNAse-free water, dissolve it after high temperature and high pressure treatment to make CTAC extract, and keep it at room temperature for use.

[0033] (2) Dissolving the agarose-cell mixture with CTAC extract: preheat the CTAC extract at 65°C in a dark environment, add 2% v / v β-mercaptoethanol and mix evenly, and add the mixture in a volume ratio of 1:5 to the agarose-cell mixture after removing the upper culture medium of the agarose colony formation experiment to dissolve the agarose-cell mixture.

[0034] (3) Chloroform extraction: After the mixture is evenly mixed, chloroform (Sigma Aldrich, St. Louis, MI, USA) is added in a 1:1 volume ratio and mixed again, and this step is repeated once.

[0035] (4) Isopropanol purification precipitation: After centrifugation at 15,000 g for 5 minutes at room temperature, the supernatant was mixed with isopropanol (Sigma Aldrich, St. Louis, MI, USA) at a volume ratio of 1:1, and centrifuged again at 15,000 g for 15 minutes at room temperature. The supernatant was removed at room temperature, and the nucleic acid precipitate was washed twice with 70% alcohol and air-dried.

[0036] (5) RNA isolation and purification: Add 50ul of RNase-free water to the dried nucleic acid precipitate, and then add 1ul of DNase I enzyme (Fermentas / Thermo Fisher, Waltham, MA, USA) to digest DNA impurities at 37°C for 30 minutes. Then, use the RNA extraction kit FavorPrep TM RNA was extracted using Tissue Total RNA Mini Kit (FATRK 001-2, Favorgen Biotech Corporation, Ping-Tung, Taiwan). 350 μl of FARB Buffer and 3.5 μl of β-mercaptoethanol were added to each 50 μl of RNA precipitate-water suspension, and the mixture was shaken until the RNA precipitate was dissolved. Then an equal volume of 70% RNase-free alcohol was added and mixed evenly. The mixture was filtered through a FARB Mini Column, washed four times with 750 μl of Wash Buffer 2, and then 40-100 μl of RNase-free water was added to elute RNA on the FARB Mini Column membrane for subsequent qPCR verification experiments.

[0037] The above method was used to extract HMEC cell RNA from 3D agarose colony formation experiments. The results showed that the HMEC cell RNA extracted from 2D adherent traditional culture had the same quality and no gDNA contamination ( Figure 2 ) and subjected them to qPCR experiments. The results showed that the gene expression can be accurately measured ( Figure 3 AB).

[0038] Example 2 Extraction of cell DNA in agarose colony formation experiment

[0039] (1) Prepare CTAC extract: Mix 2% w / v CTAC (292737, CTAC, Sigma Aldrich, St. Louis, MI, USA), 2% w / v PVP-40 (PVP-40, Sigma Aldrich, St. Louis, MI, USA), 2 M sodium chloride, 100 mM Tris-HCl (pH 8.0), and 20 mM EDTA (EDTA, Sigma Aldrich, St. Louis, MI, USA) in RNAse-free water, dissolve it after high temperature and high pressure treatment to make CTAC extract, and keep it at room temperature for use.

[0040] (2) Dissolving the agarose-cell mixture with CTAC extract: preheat the CTAC extract at 65°C in a dark environment, add 2% v / v β-mercaptoethanol and mix evenly, and add the mixture in a volume ratio of 1:5 to the agarose-cell mixture after removing the upper culture medium of the agarose colony formation experiment to dissolve the agarose-cell mixture.

[0041] (3) Chloroform extraction: After the mixture is evenly mixed, chloroform (Sigma Aldrich, St. Louis, MI, USA) is added in a 1:1 volume ratio and mixed again, and this step is repeated once.

[0042] (4) Isopropanol purification precipitation: After centrifugation at 15,000 g for 5 minutes at room temperature, the supernatant was mixed with isopropanol (Sigma Aldrich, St. Louis, MI, USA) at a volume ratio of 1:1, and centrifuged again at 15,000 g for 15 minutes at room temperature. The supernatant was removed at room temperature, and the nucleic acid precipitate was washed twice with 70% alcohol and air-dried.

[0043] (5) DNA separation and purification: Add 50ul of RNase-free water to the dried nucleic acid precipitate, and then add 1ul of RNase A (19101, Qiagen, Venlo, The Netherlands) to digest RNA impurities at 37°C for 30 minutes. gDNA was extracted using Blood and Tissue Kit (69504, Qiagen, Venlo, The Netherlands) and used for subsequent PCR verification experiments.

[0044] HMEC-hTERT-ST-shp53 cells expressing pbev control and pb-NRAS were mixed at a ratio of 0%, 5%, 20%, and 100%, and gDNA was extracted using the above method. PCR was used to detect the expression of exogenous pb-NRAS. The results showed that gDNA extracted from the 3D agarose clone formation experiment could be accurately used in the PCR experiment ( Figure 4 ).

[0045] Example 3 Extraction of cell proteins in agarose colony formation experiment

[0046] Add 2 ml of PBS to one 24-well plate, and mix the agarose-cell mixture after removing the upper culture medium of the agarose colony formation experiment with PBS. Pool the PBS-agarose-cell mixture from three 24-well plates, transfer to a 15 ml centrifuge tube, add about 13 ml of PBS, and centrifuge at 700 g for 2 minutes. Remove 10 ml of the upper supernatant, add 10 ml of PBS for a second wash, remove 13 ml of the supernatant, and freeze the washed agarose-cell mixture in liquid nitrogen. Add 500 μl of RIPA lysis buffer (ThermoFisher, Waltham, MA, USA) + protease inhibitors + phosphatase inhibitors (Roche) + 2% β-mercaptoethanol to each 1 ml of agarose-cell mixture, and then heat it at ≥95°C for 10 minutes. Then cool on ice for 1 hour. After the agarose-cell mixture is semi-solidified, centrifuge at the highest speed for 30 minutes in a 4°C centrifuge. Transfer the supernatant to a new EP tube, add 1:1 volume 100% methanol and 1 / 5 volume chloroform, mix well, and centrifuge at 15,000g for 5 minutes. Remove the supernatant and retain the white separation layer. Add 1:1 volume 100% methanol again, mix well, and centrifuge at 15,000g for 5 minutes. Remove the supernatant, retain the white precipitate, air-dry, and then use an appropriate amount of RIPA lysis buffer to re-dissolve the protein precipitate.

[0047] The above method was used to extract HMEC cell proteins in 3D agarose clone formation experiments for Western-blot experiments. The results showed that the protein expression can be accurately measured in accordance with the HMEC cell proteins extracted from 2D adherent traditional culture ( Figure 5 ).

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. All modifications, equivalent substitutions, improvements, etc. within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for extracting RNA in an agarose colony formation experiment, characterized in that: The method comprises the following: (1) Dissolving the agarose-cell mixture using CTAC extraction solution; (2) Add chloroform to extract; (3) adding isopropanol to the extract for purification and precipitation to obtain a nucleic acid precipitate; (4) RNA isolation and purification To obtain RNA samples: Add RNase-free water to the dried nucleic acid precipitate, then add DNase I enzyme, control the temperature to digest DNA impurities, and use the RNA extraction kit FavorPrep TM RNA was extracted using the Tissue Total RNA Mini Kit. FARB Buffer and β-mercaptoethanol were added to the RNA precipitate-water mixed suspension and shaken until the RNA precipitate was dissolved. RNase-free alcohol was then added and mixed evenly. The mixture was filtered through a FARB Mini Column and washed with WashBuffer 2. RNase-free water was added to elute RNA on the FARB Mini Column membrane to obtain RNA samples for qPCR experimental analysis.

2. The method according to claim 1, characterized in that In step (1), the preparation method of the CTAC extract comprises the following steps: mixing 2% w / v CTAC, 2% w / v PVP-40, 2M sodium chloride, 100mM Tris-HCl (pH 8.0), and 20mM EDTA in RNAse-free water, dissolving the mixture after high temperature and high pressure treatment to prepare a CTAC extract, and placing the mixture at room temperature for later use.

3. The method according to claim 2, characterized in that In step (1), the specific method of dissolving the agarose-cell mixture with CTAC extract is: preheating the CTAC extract in a dark environment at 65°C, adding 2% v / v β-mercaptoethanol and mixing evenly, and adding it to the agarose-cell mixture after removing the upper culture medium of the agarose clone formation experiment at a volume ratio of 1:5 to dissolve the agarose-cell mixture.

4. The method according to claim 1, characterized in that: In step (2), the specific method of chloroform extraction is: after the liquid is evenly mixed, chloroform is added in a volume ratio of 1:1 and mixed again, and the operation is repeated once.

5. The method according to claim 1, characterized in that In step (3), the specific method of isopropanol purification precipitate is as follows: after centrifugation at a speed of 15,000g at room temperature for 5 minutes, the supernatant is mixed with isopropanol at a volume ratio of 1:1, and centrifugation at a speed of 15,000g at room temperature for 15 minutes again, the supernatant is removed at room temperature, and the nucleic acid precipitate is washed twice with 70% alcohol and then air-dried.

6. The method according to claim 1, characterized in that In step (4), the specific method of RNA isolation and purification is as follows: add 50ul of RNase-free water to the dried nucleic acid precipitate, then add 1ul of DNase I enzyme, digest DNA impurities at 37°C for 30 minutes, and then use RNA extraction kit FavorPrep TM Tissue Total RNA Mini Kit was used to extract RNA. 350 μl of FARB Buffer and 3.5 μl of β-mercaptoethanol were added to every 50 μl of RNA precipitate-water mixed suspension. The mixture was shaken and mixed until the RNA precipitate was dissolved. Then an equal volume of 70% RNase-free alcohol was added and mixed evenly. The mixture was filtered through a FARB Mini Column and washed four times with 750 μl of Wash Buffer 2. Then, 40-100 μl of RNase-free water was added to elute RNA from the FARB Mini Column membrane to obtain RNA samples for RNA analysis.

7. A method for extracting DNA in an agarose colony formation experiment, characterized in that: The method comprises the following: (1) using CTAC extract to dissolve the agarose-cell mixture; preferably, the preparation method of the CTAC extract includes the following: mixing 2% w / v CTAC, 2% w / v PVP-40, 2M sodium chloride, 100mM Tris-HCl (pH 8.0), and 20mM EDTA in RNAse-free water, dissolving it after high temperature and high pressure treatment to prepare CTAC extract, and placing it at room temperature for use; preferably, the specific method of using CTAC extract to dissolve the agarose-cell mixture is: preheating the CTAC extract at 65°C in a dark environment, adding 2% v / v β-mercaptoethanol and mixing evenly, and adding it to the agarose-cell mixture after removing the upper culture medium of the agarose clone formation experiment at a volume ratio of 1:5 to dissolve the agarose-cell mixture; (2) adding chloroform for extraction; preferably, the specific method of chloroform extraction is: adding chloroform to the mixed liquid at a volume ratio of 1:1 and mixing again, and repeating the operation once; (3) adding isopropanol to the extract to purify and precipitate to obtain a nucleic acid precipitate; preferably, the specific method of purifying the precipitate with isopropanol is as follows: centrifuging at 15,000 g for 5 minutes at room temperature, mixing the supernatant with isopropanol at a volume ratio of 1:1, centrifuging again at 15,000 g for 15 minutes at room temperature, removing the supernatant at room temperature, washing the nucleic acid precipitate twice with 70% alcohol and air-drying; (4) DNA separation and purification: DNA samples were obtained by adding RNase-free water to the dried nucleic acid precipitate, then adding RNase A, controlling the temperature to digest RNA impurities, and using the Blood and Tissue Kit to extract gDNA for PCR experimental analysis; preferably, the specific method of DNA separation and purification is: add 50ul of RNase-free water to the dried nucleic acid precipitate, then add 1ul of RNase A, digest RNA impurities at 37°C for 30 minutes, and use the Blood and Tissue Kit was used to extract gDNA and obtain gDNA samples for subsequent DNA analysis.

8. A method for extracting protein in an agarose colony formation experiment, characterized in that: The method comprises the following: (1) washing the agarose-cell mixture with PBS; preferably, the specific method is as follows: adding 2 ml of PBS to one 24-well plate, and evenly mixing the agarose-cell mixture after removing the upper culture medium of the agarose clone formation experiment with PBS; pooling the PBS-agarose-cell mixtures from three 24-well plates, transferring them into a 15 ml centrifuge tube, adding about 13 ml of PBS, and centrifuging at 700 g for 2 minutes; Remove 10 ml of the upper supernatant, add 10 ml of PBS for a second wash, remove 13 ml of the supernatant, and freeze the washed agarose-cell mixture quickly with liquid nitrogen; (2) dissolving with RIPA lysis buffer; preferably, the specific method is: adding 500 μl of RIPA lysis buffer + protease inhibitor + phosphatase inhibitor + 2% β-mercaptoethanol to each 1 ml of agarose-cell mixture, and then heating it at ≥95°C for 10 minutes; (3) centrifugal cooling and separation; preferably, the specific method is: cooling on ice for 1 hour, after the agarose-cell mixture is semi-solidified, centrifuging at the highest speed in a 4°C centrifuge for 30 minutes, transferring the supernatant to a new EP tube, adding 100% methanol and 1 / 5 volume of chloroform in a 1:1 volume ratio, mixing evenly, centrifuging at 15,000g for 5 minutes, removing the supernatant, and retaining the white separation layer; (4) Purifying and precipitating the protein with methanol; preferably, the specific method is: adding 100% methanol by volume in a ratio of 1:1, mixing evenly, centrifuging at 15,000 g for 5 minutes, removing the supernatant, and retaining the white precipitate EB; (5) Redissolving the protein; preferably, the specific method is: after air drying, use an appropriate amount of RIPA lysis buffer to re-dissolve the protein precipitate to obtain a protein sample for protein analysis.

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