A kit for purifying DNA by ultra-micro agarose gel and a method for purifying DNA

Abstract

This invention provides an ultra-micro agarose gel DNA purification kit and a method for purifying DNA, relating to the field of biotechnology. The kit includes: a sol solution, a purification solution, a washing solution, an elution buffer, an ion adsorption column, and a collection tube. The purification solution comprises 4-6M urea, 2-3M NaCl, and 70%-80% anhydrous ethanol. The ion adsorption column uses a double-layer nanomembrane, with the upper nanomembrane being a polycarbonate membrane and the lower nanomembrane being a polyamide membrane. The kit is low-cost and has high recovery efficiency. This invention also discloses a method for purifying DNA, using a unique buffer system and centrifugal adsorption column method, including steps such as sol-gel, purification, washing, and elution, for the recovery of DNA and PCR products from agarose gel. The method is simple, fast, has high recovery efficiency, and is safe and environmentally friendly.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to an ultra-micro agarose gel purification kit and a method for purifying DNA. Background Technology

[0002] DNA purification and analysis are widely used in molecular biology. In gene cloning, products of polymerase chain reaction (PCR) amplification are typically recovered, purified, and sequenced. Alternatively, linearized plasmid DNA can be recovered and purified by digesting circular plasmids with enzymes for subsequent molecular experiments. Common methods for recovering DNA fragments from agarose gels include DEAE-cellulose membrane filtration, low-melting-point agarose gel extraction, dialysis bag electrowashing, and perforation. However, due to the cumbersome experimental procedures, low DNA recovery efficiency, and high cost of these methods, column-based DNA recovery remains the most widely used and efficient method. However, column-based DNA recovery also has drawbacks, such as low recovery volume. Recovering large amounts of nucleic acids requires more adsorption columns, and existing adsorption columns primarily use silica hydroxyl groups, which is the main reason for the high cost of column-based DNA recovery. Summary of the Invention

[0003] The purpose of this invention is to provide an ultra-micro agarose gel purification kit and a method for purifying DNA, thereby solving the aforementioned problems. To achieve the above objective, the technical solution adopted by this invention is as follows:

[0004] This application provides an ultra-micro agarose gel purification DNA kit, characterized in that it comprises: a sol, a purification solution, a rinsing solution, an elution buffer, an ion adsorption column, and a collection tube; the purification solution comprises 4-6M urea, 2-3M NaCl, and 70%-80% anhydrous ethanol; the ion adsorption column employs a double-layer nanomembrane, the nanomembrane having a certain degree of flexibility and rigidity, possessing multiple ionic groups, and being resistant to strong acids, strong alkalis, high salts, and ethanol solutions.

[0005] Furthermore, the upper nanomembrane of the ion adsorption column is a polycarbonate membrane with a micropore diameter of less than 5 nm, used to filter urea and NaCl; the lower nanomembrane is a polyamide membrane containing hydrophilic amide groups, which can form hydrogen bonds with DNA to intercept it. The stronger the hydrogen bond, the stronger the DNA binding ability, thus achieving the separation of salt solution and DNA. Finally, at a pH higher than that of the rinsing solution, the purified DNA can be eluted from the nanomembrane.

[0006] Furthermore, the concentration of the impurity removal solution is 5-5.5M urea, 2.2M NaCl, and 70% anhydrous ethanol. The urea denatures non-specific proteins on the DNA, effectively protecting the structural stability of nucleic acids. The NaCl solution dissolves the DNA, which is then separated from the proteins by centrifugation. The 70% anhydrous ethanol washes away salt components from the aqueous phase of the DNA.

[0007] Furthermore, the sol solution comprises 50-100 mM Tris-HCl buffer and 7% phenol red solution, wherein the Tris-HCl buffer has a pH of 8.0 and is used to protect the stability of DNA.

[0008] Furthermore, the 7% phenol red solution is prepared as follows: Weigh 0.1g of phenol red into a mortar, add 5.7ml of 50mM NaOH solution and an appropriate amount of water, grind the phenol red, and dilute to 250ml. Store at 4℃. The phenol red solution acts as an acid-base indicator, turning yellow when the pH is below 6.4. Utilizing the acid-base indicator properties of phenol red solution allows for determining whether the sol solution has deteriorated, observing the sol effect, and monitoring pH changes, thereby achieving optimal binding and improving recovery efficiency.

[0009] Furthermore, the rinsing solution comprises 70%-80% anhydrous ethanol, pH=7.5. 70%-80% anhydrous ethanol can effectively remove salt solutions and other impurities, and precipitate DNA.

[0010] Furthermore, the elution buffer comprises 1 mM-3 mM Tris-HCl, pH = 8.0-9.0, for eluting pure DNA from the nanomembrane, preferably at a concentration of 1 mM.

[0011] A method for purifying DNA, comprising the steps of:

[0012] Gel cutting: Under long-wave ultraviolet light, cut the target DNA band from the agarose gel, removing as much gel without DNA as possible;

[0013] Add sol: Place the target DNA band into a 1.5ml centrifuge tube and add 400-500μL of sol;

[0014] Sol: Place the centrifuge tube in a water bath at 50℃-65℃ until the target DNA band is completely dissolved. Mix every 2-3 minutes during the water bath.

[0015] Adjusting the sol solution: The pH of the sol solution is adjusted by titration with NaOH solution. The titration is stopped when the sol solution turns purple-red. This process is to stabilize the DNA environment and to activate the nanofilm on the centrifuge column to adsorb DNA in the subsequent use of the centrifuge column.

[0016] Impurity removal: Add 500 μL of impurity removal solution to the mixture and mix well, then centrifuge at 12,000 rpm for 1 min and keep the supernatant.

[0017] Column loading: Add the supernatant solution to the ion adsorption column located in the collection tube, then let it stand at room temperature for 1 min, and finally centrifuge at 12,000 rpm for 30 s and discard the waste liquid;

[0018] Rinsing: Add 600 μL of rinsing solution to the ion adsorption column, centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and repeat the rinsing once more.

[0019] Remove the rinsing solution: Centrifuge the ion adsorption column at 12,000 rpm for 2 min, discard the waste liquid, and remove as much rinsing solution as possible to avoid residual ethanol in the rinsing solution inhibiting the downstream reaction;

[0020] Elution: Remove the ion adsorption column from the collection tube and place it into a new centrifuge tube. Add 50 μL of elution buffer to the middle of the ion adsorption column, incubate at room temperature for 2 min, and centrifuge at 12,000 rpm for 1 min.

[0021] Furthermore, when the agarose gel contains PCR products, the process includes the following steps:

[0022] Gel cutting: Under long-wave ultraviolet light, cut the target PCR band from the agarose gel;

[0023] Add sol buffer: Place the target PCR band into a 1.5ml centrifuge tube, add water to make up to 100μL of PCR reaction solution or enzyme digestion reaction solution, and add 300μL of sol buffer and mix well;

[0024] Adjusting the pH of the sol solution: Titrate with NaOH solution to adjust the pH of the sol solution, and stop titrating when the sol solution turns purple-red;

[0025] Impurity removal: Add 500 μL of impurity removal solution to the mixture and mix well, then centrifuge at 12,000 rpm for 1 min and keep the supernatant.

[0026] Column loading: Add the supernatant solution to the ion adsorption column located in the collection tube, then let it stand at room temperature for 1 min, and finally centrifuge at 12,000 rpm for 30 s and discard the waste liquid;

[0027] Rinsing: Add 600 μL of rinsing solution to the ion adsorption column, centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and repeat the rinsing once more.

[0028] Remove rinsing solution: Centrifuge the ion adsorption column at 12,000 rpm for 2 min and discard the waste liquid;

[0029] Elution: Remove the ion adsorption column from the collection tube and place it into a new centrifuge tube. Add 50 μL of elution buffer to the middle of the ion adsorption column, incubate at room temperature for 2 min, and centrifuge at 12,000 rpm for 1 min.

[0030] The beneficial effects of this invention are as follows:

[0031] This invention utilizes a unique buffer system and centrifugal adsorption column method for the recovery of DNA and PCR products from agarose gels. Urea is used for impurity removal, which is far less toxic and less expensive than guanidine salts, exhibiting high specificity and denaturing non-specific proteins on DNA, effectively protecting the structural stability of nucleic acids. This invention also proposes a novel adsorption membrane for an ion adsorption column, which is low-cost and effectively adsorbs DNA, solving the problem of high cost associated with existing ion adsorption columns. Therefore, this invention has significant advantages such as simple and rapid operation, high recovery efficiency, safety, environmental friendliness, and low cost.

[0032] Other features and advantages of the invention will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing embodiments of the invention. The objects and other advantages of the invention may be realized and obtained by means of the structures particularly pointed out in the written description, claims, and drawings. Attached Figure Description

[0033] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 These are electrophoresis images of Embodiments 4, 5, and 6 of the present invention. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0036] It should be noted that similar reference numerals and letters in the following figures indicate similar items; therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures. Furthermore, in the description of this invention, terms such as "first," "second," etc., are used only to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0037] Example 1:

[0038] This embodiment provides an ultra-micro agarose gel purification kit for DNA, comprising a sol, a purification buffer, a washing buffer, an elution buffer, an ion adsorption column, and a collection tube. The sol comprises 50-100 mM Tris-HCl buffer and 7% phenol red solution, with the Tris-HCl buffer pH = 8.0; the purification buffer comprises 5-5.5 M urea, 2.2 M NaCl, and 70% anhydrous ethanol; the washing buffer comprises 70%-80% anhydrous ethanol, pH = 7.5; the elution buffer comprises 1 mM-3 mM Tris-HCl, pH = 8.0-9.0; the ion adsorption column employs a double-layer nanomembrane structure, with the upper nanomembrane being a polycarbonate membrane and the lower nanomembrane being a polyamide membrane.

[0039] The polycarbonate membrane has a micropore diameter of less than 5 nm.

[0040] The 7% phenol red solution is prepared as follows: Weigh 0.1g of phenol red into a mortar, add 5.7ml of 50mM NaOH solution and an appropriate amount of water, grind the phenol red, and make up to 250ml. Store at 4℃.

[0041] Example 2:

[0042] A method for purifying DNA requires the following instruments and reagents: centrifuge, 1.5 ml sterile centrifuge tubes, anhydrous ethanol, 150 mM NaOH, pipettes, and a NanoDrop spectrophotometer (ND-1000, Thermo Fisher Scientific, USA). Amplification was performed using the high-fidelity enzyme GXL DNA Polymerase from Baori Biotechnology Co., Ltd., and the primers listed below. The polymerase chain reaction program was: 98℃ for 30 s; 98℃ for 10 s, 58℃ for 15 s, 68℃ for 1 min / 3 min, with 30 cycles; 68℃ for 10 min.

[0043] Including the following steps:

[0044] Gel cutting: Under long-wave ultraviolet light, cut the target DNA band from the agarose gel, removing as much gel without DNA as possible;

[0045] Add sol: Place the target DNA band into a 1.5ml centrifuge tube and add 400-500μL of sol;

[0046] Sol: Place the centrifuge tube in a water bath at 50℃-65℃ until the target DNA band is completely dissolved. Mix every 2-3 minutes during the water bath.

[0047] Adjusting the sol solution: The pH of the sol solution is adjusted by titration with NaOH solution. The titration is stopped when the sol solution turns purple-red. This process is to stabilize the DNA environment and activate the nanofilm on the centrifugal adsorption column to adsorb DNA.

[0048] Impurity removal: Add 500 μL of impurity removal solution to the mixture and mix well, then centrifuge at 12,000 rpm for 1 min and keep the supernatant.

[0049] Column loading: Add the supernatant solution to the ion adsorption column located in the collection tube, then let it stand at room temperature for 1 min, and finally centrifuge at 12,000 rpm for 30 s and discard the waste liquid;

[0050] Rinsing: Add 600 μL of rinsing solution to the ion adsorption column, centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and repeat the rinsing once more.

[0051] Remove the rinsing solution: Centrifuge the ion adsorption column at 12,000 rpm for 2 min, discard the waste liquid, and remove as much rinsing solution as possible to avoid residual ethanol in the rinsing solution inhibiting the downstream reaction;

[0052] Elution: Remove the ion adsorption column from the collection tube and place it into a new centrifuge tube. Add 50 μL of eluent to the middle of the ion adsorption column, let it stand at room temperature for 2 min, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and obtain the recovered product.

[0053] Example 3:

[0054] When the agarose gel contains PCR products, the steps include:

[0055] Gel cutting: Under long-wave ultraviolet light, cut the target PCR band from the agarose gel;

[0056] Add sol buffer: Place the target PCR band into a 1.5ml centrifuge tube, add water to make up to 100μL of PCR reaction solution or enzyme digestion reaction solution, and add 300μL of sol buffer and mix well;

[0057] Adjusting the pH of the sol solution: Titrate with NaOH solution to adjust the pH of the sol solution, and stop titrating when the sol solution turns purple-red;

[0058] Impurity removal: Add 500 μL of impurity removal solution to the mixture and mix well, then centrifuge at 12,000 rpm for 1 min and keep the supernatant.

[0059] Column loading: Add the supernatant solution to the ion adsorption column located in the collection tube, then let it stand at room temperature for 1 min, and finally centrifuge at 12,000 rpm for 30 s and discard the waste liquid;

[0060] Rinsing: Add 600 μL of rinsing solution to the ion adsorption column, centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and repeat the rinsing once more.

[0061] Remove rinsing solution: Centrifuge the ion adsorption column at 12,000 rpm for 2 min and discard the waste liquid;

[0062] Elution: Remove the ion adsorption column from the collection tube and place it into a new centrifuge tube. Add 50 μL of eluent to the middle of the ion adsorption column, let it stand at room temperature for 2 min, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and obtain the recovered product.

[0063] Example 4:

[0064] Using the kit of the present invention, the efficiency of DNA recovery from PCR products was studied, including the following steps:

[0065] Fragment amplification: Target DNA of 600 bp was obtained by designing specific primers;

[0066] Direct PCR product recovery: Follow the steps in Example 3. The reaction volume for each experimental group was 50 μL. During elution, 50 μL of elution buffer was added to elute the DNA in order to calculate the DNA recovery rate.

[0067] DNA recovery efficiency was verified using a spectrophotometer and 1% agarose gel electrophoresis (10 μL for each recovered sample). Three biological replicate experiments were performed, designated A1, A2, and A3. The results are shown in Table 1 and... Figure 1 .

[0068] Table 1: Efficiency of DNA recovery from PCR products

[0069]

[0070] Example 5:

[0071] Using the kit of this invention, the efficiency of DNA recovery from agarose gel was studied, including the following steps:

[0072] Fragment amplification: By designing specific primers, the target DNA, with a size of 600 bp, is obtained.

[0073] Agarose gel electrophoresis: Prepare agarose gels using 1×TAE buffer. Separate the target DNA bands by performing 1% agarose gel electrophoresis on the PCR products. Load 50 μL of sample into each well, and elute with 50 μL of elution buffer to calculate the DNA recovery rate.

[0074] Gel extraction and recovery of target DNA: The procedure is performed according to the steps in Example 3 of this invention. During gel extraction and recovery, ultraviolet light observation can damage DNA fragments; therefore, low-energy, long-wave ultraviolet light should be used whenever possible, and the treatment time under ultraviolet light should be minimized.

[0075] DNA recovery efficiency was verified using a spectrophotometer and 1% agarose gel electrophoresis (10 μL for each recovered sample). Three biological replicate experiments were performed, designated as experimental groups B1, B2, and B3. The results are shown in Table 2. Figure 1 .

[0076] Table 2: Efficiency of DNA recovery from agarose gel

[0077]

[0078] The experimental results of Examples 3 and 4 show that the kit of the present invention can achieve a relatively stable recovery efficiency of about 85%, whether the DNA is recovered directly from PCR products or by agarose gel electrophoresis.

[0079] Example 6:

[0080] Using the kit of this invention, the recovery efficiency of DNA fragments of different sizes was studied, including the following steps:

[0081] Obtaining target DNA of different sizes: 2kbp, 3kbp and 5kbp target DNA bands were obtained by polymerase chain reaction.

[0082] Agarose gel electrophoresis: Prepare agarose gels using 1×TAE buffer. Perform electrophoresis using a 1% agarose gel with 1×TAE buffer as the electrophoresis buffer. Load 50 μL of sample into each gel well, and elute with 50 μL of elution buffer to calculate DNA recovery.

[0083] Gel extraction and recovery of target DNA: The procedure is performed according to the steps in Example 2 of this invention. During gel extraction and recovery, ultraviolet light observation can damage DNA fragments; therefore, low-energy, long-wave ultraviolet light should be used as much as possible, and the treatment time under ultraviolet light should be minimized.

[0084] DNA recovery efficiency was verified using a spectrophotometer and 1% agarose gel electrophoresis (10 μL for each recovered sample). Three biological replicate experiments were performed, with experimental groups C1-C9. The results are shown in Table 3. Figure 1 .

[0085] Table 3: Recovery efficiency of DNA fragments of different sizes

[0086]

[0087]

[0088] The experimental results show that, compared with the recovery results of different molecular weights, the recovery rate of this method is relatively stable for fragments of different molecular weights, and the recovery rate does not decrease with the increase of molecular weight. Therefore, the kit of this invention is suitable for PCR products, enzyme digestion products, and DNA bands separated by agarose gel at different molecular weights. The DNA recovery efficiency is related to the amount of DNA recovered, the amount of starting DNA, the elution volume, and the size of the DNA fragment. Generally, for DNA fragments of 1-15 μg, 100bp-5kb, the recovery rate can be as high as 85%. In addition, the recovered products meet the requirements of subsequent experiments based on the band brightness.

[0089] Figure 1 In the image, bands "A1, A2, A3" are electrophoresis images of recovered and purified 600bp DNA molecules obtained through polymerase chain reaction; bands "B1, B2, B3" are electrophoresis images of recovered and purified 600bp DNA molecules obtained through agarose gel electrophoresis; bands "C1, C2, C3" are electrophoresis images of recovered and purified 2Kbp DNA molecules obtained through agarose gel electrophoresis; bands "C4, C5, C6" are electrophoresis images of recovered and purified 3Kbp DNA molecules obtained through agarose gel electrophoresis; bands "C7, C8, C9" are electrophoresis images of recovered and purified 5Kbp DNA molecules obtained through agarose gel electrophoresis; M: 5K DNA Marker.

[0090] Example 7:

[0091] The efficiency of DNA recovery using the DNA adsorption membrane of this invention was investigated.

[0092] Most commercially available centrifuge column-based DNA adsorption kits use silica hydroxyl groups as the main material, resulting in higher costs. The adsorption membrane of this invention uses a nanomembrane primarily composed of polyamide and polycarbonate, saving on experimental costs. This embodiment compares the DNA recovery efficiency of two DNA adsorption membranes made of different materials. The centrifuge column corresponding to the DNA adsorption membrane of this invention is denoted by N, while the centrifuge columns for the DNA adsorption membranes from the other two companies are denoted by D and E, respectively.

[0093] Using equal amounts of the same experimental samples, after electrophoresis on a 1% agarose gel, the target bands were cut off, and the size and weight of the cut strips were kept consistent. In this example, to compare DNA recovery efficiency, except for the centrifugation adsorption column, all other experimental reagents, such as sol-gel solution, impurity removal solution, rinsing solution, elution solution, and experimental methods, were performed according to the kit of this invention and the experimental steps of Example 2. The results are shown in Table 4.

[0094] Table 4: DNA concentration measured using different DNA adsorption membranes for the same sample.

[0095]

[0096] Experimental results show that, specifically, the DNA adsorption membrane of this invention exhibits higher DNA recovery efficiency than the other two DNA adsorption columns, both in terms of DNA concentration and purity. The centrifugal adsorption column D recovered the lowest DNA concentration; the DNA recovery efficiency of column E (OD260 / OD280 < 1.7) indicates protein contamination in the recovered DNA sample. This demonstrates that the DNA adsorption nanomembrane proposed in this invention has significant effects on DNA adsorption and elution, thereby improving DNA recovery efficiency.

[0097] Example 8:

[0098] Compare the effectiveness of different kits in recovering DNA.

[0099] This embodiment mainly compares the DNA recovery efficiency of different kits. The kit of this invention is named Kit M, and the other two kits are named Kit F and Kit H, respectively. Equal amounts of the same experimental samples were used, and after 1% agarose gel electrophoresis, the target bands were cut off, ensuring that the size and weight of the cut strips were consistent. The experimental methods were performed according to the instructions of each kit. The results are shown in Table 5.

[0100] Table 5: DNA concentration measured using different kits for the same sample.

[0101]

[0102] Note: DNA recovery rate (%) = DNA concentration after experiment / DNA concentration before experiment. The DNA concentration before experiment was 650-680 ng / uL.

[0103] Experimental results show that the kit of this invention recovers the highest concentration of DNA, with a recovery efficiency exceeding 85%. The other two kits recovered DNA with lower purity, exhibiting contamination from guanidine salts and proteins (OD260 / OD280 < 1.8; OD260 / OD230 < 2). The DNA purified by this kit was uncontaminated by salts or proteins, indicating that the DNA adsorption membrane and the purification solution of this invention work synergistically. The DNA adsorption membrane controls the channels of the nanomembrane through the connection of sulfur atoms and acidic groups, and then firmly adsorbs DNA through hydrogen bonding. The purification solution removes most of the proteins, while the rinsing solution removes the salt solution bound to the DNA surface. The synergistic effect of the reagents improves both the DNA recovery efficiency and purity.

[0104] The amounts of each component used in this invention are the optimal ratios obtained by the inventors through analysis and experimentation. For some reagents with clearly defined ranges, it is recommended to prepare and use them within the range of minimum and maximum values. Specific embodiments are intended to explain this invention and should not be construed as limiting it. Reagents or instruments used are not explicitly specified and can be purchased or used from conventional manufacturers.

[0105] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A kit for purifying DNA using ultra-micro agarose gel, characterized in that, include: The apparatus includes a sol, a purification solution, a rinsing solution, an elution buffer, an ion adsorption column, and a collection tube. The purification solution contains 5-5.5 M urea, 2-3 M NaCl, and 70%-80% anhydrous ethanol. The ion adsorption column employs a double-layer nanomembrane structure, with the upper nanomembrane being a polycarbonate membrane and the lower nanomembrane being a polyamide membrane. The micropore diameter of the polycarbonate membrane is less than 5 nm. Among them, 2.2M NaCl and 70% anhydrous ethanol; The sol solution comprises 50-100 mM Tris-HCl buffer and 7% phenol red solution, wherein the Tris-HCl buffer has a pH of 8.0; Wherein: the rinsing solution comprises 70%-80% anhydrous ethanol, pH=7.5; The elution buffer comprises 1mM-3mM Tris-HCl with pH = 8.0-9.

0.

2. The ultra-micro agarose gel purification DNA kit according to claim 1, characterized in that, The 7% phenol red solution is prepared as follows: Weigh 0.1g of phenol red into a mortar, add 5.7ml of 50mM NaOH solution and an appropriate amount of water, grind the phenol red, and make up the volume to 250ml. Store at 4℃.

3. A method for purifying DNA, using the ultra-micro agarose gel DNA purification kit as described in claim 1, characterized in that, Including the following steps: Gel cutting: Under long-wave ultraviolet light, the target DNA band is cut from the agarose gel; Add sol: Place the target DNA band into a 1.5ml centrifuge tube and add 400-500μL of sol; Sol: Place the centrifuge tube in a water bath at 50℃-65℃ until the target DNA band is completely dissolved. Mix every 2-3 minutes during the water bath. Adjusting the pH of the sol solution: Titrate with NaOH solution to adjust the pH of the sol solution, and stop titrating when the sol solution turns purple-red; Impurity removal: Add 500 μL of impurity removal solution to the mixture and mix well, then centrifuge at 12,000 rpm for 1 min and keep the supernatant. Column loading: Add the supernatant solution to the ion adsorption column located in the collection tube, then let it stand at room temperature for 1 min, and finally centrifuge at 12,000 rpm for 30 s and discard the waste liquid; Rinsing: Add 600 μL of rinsing solution to the ion adsorption column, centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and repeat the rinsing once more. Remove rinsing solution: Centrifuge the ion adsorption column at 12,000 rpm for 2 min and discard the waste liquid; Elution: Remove the ion adsorption column from the collection tube and place it into a new centrifuge tube. Add 50 μL of elution buffer to the middle of the ion adsorption column, incubate at room temperature for 2 min, and centrifuge at 12,000 rpm for 1 min.

4. The method for purifying DNA according to claim 3, characterized in that, When the agarose gel contains PCR products, the steps include: Gel cutting: Under long-wave ultraviolet light, cut the target PCR band from the agarose gel; Add sol buffer: Place the target PCR band into a 1.5ml centrifuge tube, add water to make up to 100μL of PCR reaction solution or enzyme digestion reaction solution, and add 300μL of sol buffer and mix well; Adjusting the pH of the sol solution: Titrate with NaOH solution to adjust the pH of the sol solution, and stop titrating when the sol solution turns purple-red; Impurity removal: Add 500 μL of impurity removal solution to the mixture and mix well. Then centrifuge at 12,000 rpm for 1 min and keep the supernatant. Column loading: Add the supernatant to the ion adsorption column located in the collection tube, let it stand at room temperature for 1 min, and finally centrifuge at 12,000 rpm for 30 s and discard the waste liquid. Rinsing: Add 600 μL of rinsing solution to the ion adsorption column, centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and repeat the rinsing once more. Remove rinsing solution: Centrifuge the ion adsorption column at 12,000 rpm for 2 min and discard the waste liquid; Elution: Remove the ion adsorption column from the collection tube and place it into a new centrifuge tube. Add 50 μL of elution buffer to the middle of the ion adsorption column, incubate at room temperature for 2 min, and centrifuge at 12,000 rpm for 1 min.

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