Plasmid extraction kit

Abstract

The invention discloses a plasmid extraction kit which comprises a resuspension buffer solution P1, a lysis buffer solution P2 and a neutralization buffer solution P3, the resuspension buffer solution P1 comprises Tris-HCl, EDTA (Ethylene Diamine Tetraacetic Acid), a carbohydrate compound, RNase A and a first indicator; the cracking buffer solution P2 comprises NaOH and a surfactant; the neutralization buffer solution P3 is prepared from guanidine hydrochloride, potassium acetate, acetic acid, urea, tetradecane amide propyl hydroxypropyl sulfobetaine and N-lauroyl sodium amino acid. According to the extraction kit, components in a preparation are improved, so that the concentration and quality of plasmids are improved, meanwhile, the occurrence of endotoxin in the plasmid extraction process is reduced, and visual monitoring is also carried out.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to a plasmid extraction kit. Background Art

[0002] Molecular cloning procedures are complex and involve many steps, so methods are needed to reliably isolate large amounts of highly pure plasmid DNA. Contaminants are a persistent problem, most of which are produced by the initial step of bacterial lysis by clearing the lysate. In addition, neutralization and clearing steps are performed. These steps are often nodes where the desired plasmid DNA or nucleic acid is significantly lost. Conventional methods for isolating plasmid DNA include collecting bacterial cells and obtaining plasmid DNA. Unwanted contaminating media and cell components are often referred to as clearing bacteria or cell lysates. Cell lysis can be performed in a variety of ways, including mechanical sonication or mixing, enzymatic digestion, and traditional chemical alkaline hydrolysis. Alkaline lysis remains the basis of many plasmid purification methods, although other methods are also used, such as boiling lysis, chemical reagents (such as Triton) lysis, etc. Whether in terms of the ease of plasmid purification operation or the purity and concentration obtained from plasmid purification, alkaline lysis is a better choice.

[0003] Currently, the reagents in the common test kits are transparent and colorless, which can easily cause confusion during use and lead to an increase in human errors in experiments. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a plasmid extraction kit, which improves the components in the preparation to increase the concentration and quality of the plasmid, while reducing the occurrence of endotoxins during the plasmid extraction process, and can also perform visual monitoring.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] Plasmid extraction kit, including resuspension buffer P1, lysis buffer P2, neutralization buffer P3;

[0007] The resuspension buffer P1 includes Tris-HCl, EDTA, a carbohydrate compound, RNaseA and a first indicator; wherein the concentration of the Tris-HCl is 50-150 mM; the concentration of the EDTA is 10-100 mM; the concentration of the carbohydrate compound is 10-100 mM; the concentration of the RNaseA is 0.1-0.5 mg / ml; the first indicator includes phenol red or reactive red, and its concentration is 0.03-0.1 mM;

[0008] The lysis buffer P2 includes NaOH and a surfactant; wherein the concentration of the NaOH is 100-250 mM; the surfactant includes sodium dodecyl sulfate or lithium dodecyl sulfonate, and the concentration thereof is 1-5%;

[0009] The neutralization buffer P3 includes guanidine hydrochloride, potassium acetate, acetic acid, urea, tetradecanoylamidopropyl hydroxypropyl sulfobetaine and sodium N-lauroyl aminate; wherein the concentration of the guanidine hydrochloride is 1.0-3.5M; the concentration of the potassium acetate is 0.5-1.0M; the concentration of the acetic acid is 2.0-3.6M; the concentration of the urea is 0.5-5.0M; the concentration of the tetradecanoylamidopropyl hydroxypropyl sulfobetaine is 0.5-5.0%; and the concentration of the sodium N-lauroyl aminate is 0.10-0.50%.

[0010] In summary, the present invention has the following beneficial effects:

[0011] The present invention discloses a plasmid rapid extraction kit, which not only greatly shortens the time of plasmid extraction (controls the extraction time within 15 minutes), but also reduces the generation of endotoxins, reduces the contamination of plasmid DNA by impurities such as RNA and genomic DNA, and ensures purity while increasing yield. Most importantly, the reagents in this kit are dyed, which greatly helps users to monitor the experimental process visually. The reagents in similar kits commonly found on the market are colorless and transparent, and the operating steps required for plasmid extraction cannot be effectively and specifically observed. The operation based on feeling alone inevitably increases the error caused by human operation. The present invention effectively helps users avoid such human errors. In addition to dyeing, the present invention specifically adds urea to enhance the final concentration of plasmid extraction on the basis of traditional plasmid extraction reagents, and also adds tetradecanoyl amide propyl hydroxypropyl sulfobetaine to remove foreign proteins to improve the purity of the plasmid. The combination of the two improves the concentration and purity of the extracted plasmid. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the agarose gel electrophoresis result of plasmid DNA extracted from 5 ml bacterial solution;

[0013] Figure 2 The kit is mainly composed of three buffers with different functions, which show different colors after being treated with dyes;

[0014] Figure 3 This is the first step of plasmid DNA extraction process;

[0015] Figure 4 The second step is the extraction process of plasmid DNA. DETAILED DESCRIPTION

[0016] The present invention will be further described with reference to the accompanying drawings.

[0017] The present invention adds dye or other indicator in nucleic acid separation buffer, and the amount of dye and inherent characteristics make it provide enough color for monitoring, but do not change cell lysate, plasmid DNA, purified nucleic acid or retain after final chromatographic purification.Due to the presence of component dye, the color of cell lysate is easy to transition of color by eyes.Component dye makes the process visualization of final purification of plasmid DNA or nucleic acid from the lysate cleared by the present invention.There are many commercially available candidate dyes or indicators for consideration on the market.The property of the indicator selected by the present invention is that the change of color is related to the pH value of the solution, to monitor the state of the cell lysate from which the nucleic acid is purified.Due to the presence of component dye, the color of cell lysate is easy to transition of simple color by eyes.

[0018] The present invention relates to a column extraction method for separating pure plasmid DNA from Escherichia coli and other organisms. The plasmid extraction kit of the present invention comprises bacterial resuspension solution, bacterial lysis solution, plasmid renaturation solution, elution solution, and silica gel adsorption column. Compared with other prior arts, the present invention adds an indicator dye to the alkaline lysis buffer, which can conveniently and quickly perform visual monitoring of the resuspension, lysis and neutralization steps. On the basis of visualization, the filler material, density and composition ratio of each component reagent of the adsorption column are further optimized. The plasmid rapid extraction kit of the present invention not only increases the plasmid yield, improves the plasmid purity, and reduces endotoxin, but also reduces the error caused by actual operation due to simplified steps.

[0019] The present invention uses phenol red, a pH indicator dye. Phenol red is yellow when the pH is about 6.8 and red when the pH is about 8.4, and its transition range is between pH 6.8 and 8.4. Phenol red is added to a suspension buffer P1 (neutral pH 7.0-8.0, red in color) and a neutralization buffer P3 (acidic, yellow in color); active blue or other blue-green active dyes are added to a lysis buffer P2, and P2 is blue. The selected dye will not affect the result of extracting the plasmid. When each buffer is added in sequence, the user can clearly observe the changes in the lysate, thereby effectively reducing the probability of error.

[0020] The present invention preferably uses low-toxic or non-toxic dyes, which can be used alone or in combination, including but not limited to methyl red, methyl green, bromophenol blue, bromophenol green, reactive red, reactive orange, reactive blue, etc., so as to provide obvious visual or spectrophotometric monitoring of each solution during the lysis process.

[0021] As described above, the present invention provides a plasmid rapid extraction kit, the contents of which are as follows:

[0022] (1) Resuspension buffer P1:

[0023] Tris-HCl, with a concentration of 50-150 mM, pH 8.0;

[0024] EDTA, the concentration is 10-100 mM;

[0025] A carbohydrate compound, the concentration of which is 10 to 100 mM; wherein the carbohydrate compound includes glucose, sucrose or trehalose;

[0026] RNaseA (RNA enzyme, stored at 4°C), the concentration is 0.1-0.5 mg / ml;

[0027] The first indicator has a concentration of 0.03-0.1 mM; the first indicator includes phenol red or reactive red.

[0028] Preferably, resuspension buffer P1:

[0029] The concentration of Tris-HCl is 100-120 mM;

[0030] The concentration of EDTA is 10-50 mM;

[0031] The concentration of sugar compounds is 50-100 mM;

[0032] The concentration of RNaseA is 0.1-0.2 mg / ml;

[0033] The first indicator includes phenol red or reactive red, and the concentration thereof is 0.03-0.05 mM.

[0034] (2) Lysis buffer P2:

[0035] NaOH, the concentration is 100-250 mM;

[0036] A surfactant, the concentration of which is 1 to 5% m / v; wherein the surfactant comprises sodium dodecyl sulfate (SDS) or lithium dodecyl sulfonate (LDS);

[0037] A second indicator having a concentration of 0.01 to 0.2 mM; wherein the second indicator comprises reactive blue or bromophenol blue;

[0038] Preferably, lysis buffer P2:

[0039] The concentration of NaOH is 100-250 mM;

[0040] The concentration of the surfactant is 1 to 5%;

[0041] The concentration of the second indicator is 0.05-0.10 mM.

[0042] (3) Neutralization buffer P3:

[0043] Guanidine hydrochloride, the concentration of which is 1.0 to 3.5 M;

[0044] Potassium acetate, the concentration is 0.5~1.0M

[0045] Acetic acid, the concentration is 2.0~3.6M

[0046] Urea, the concentration is 0.5~5.0M

[0047] Tetradecanoylamidopropyl hydroxypropyl sulfobetaine, at a concentration of 0.5 to 5.0% m / v

[0048] Sodium N-lauroylamino acid, the concentration of which is 0.10-0.50% m / v;

[0049] A third indicator, whose concentration is 0.01 to 0.10 mM; wherein the third indicator comprises phenol red or reactive red;

[0050] Preferably, the neutralization buffer P3:

[0051] The concentration of guanidine hydrochloride is 2.0-2.5M;

[0052] The concentration of potassium acetate is 0.65-0.7M;

[0053] The concentration of acetic acid is 2.7-3.2M;

[0054] The concentration of urea is 1.0 to 3.0 M;

[0055] The concentration of tetradecanoylamidopropyl hydroxypropyl sulfobetaine is 1-2%;

[0056] The concentration of sodium N-lauroylamino acid is 0.15-0.35%;

[0057] The concentration of the third indicator is 0.01-0.05 mM.

[0058] (4) Deproteinized liquid PD:

[0059] Anhydrous ethanol solution, the concentration of which is 20-30% v / v ; Preferably, the concentration of the anhydrous ethanol solution is 30%;

[0060] Guanidine hydrochloride, the concentration of which is 2.0 to 4.0 M; preferably, the concentration of guanidine hydrochloride is 3 M;

[0061] Tris-HCl, with a concentration of 25-50 mM, pH 8.0; preferably, the concentration of Tris-HCl is 25 mM;

[0062] KCl, the concentration of which is 100-200 mM; preferably, the concentration of KCl is 100 mM;

[0063] (5) Rinse liquid PB:

[0064] Anhydrous ethanol solution, the concentration of which is 70-85% v / v ; Preferably, the concentration of the anhydrous ethanol solution is 80%;

[0065] Potassium acetate, with a concentration of 0.15 to 0.20 M; preferably, the concentration of potassium acetate is 0.18 M;

[0066] (6) Eluent TE:

[0067] Tris-HCl, with a concentration of 10 mM, pH 8.5;

[0068] EDTA, the concentration of which is 100 mM. Specific embodiment:

[0070] Embodiment 1:

[0071] (1) Resuspension buffer P1:

[0072] Tris-HCl, with a concentration of 100 mM, pH 8.0;

[0073] EDTA, the concentration of which is 50 mM;

[0074] Trehalose, at a concentration of 50 mM;

[0075] RNaseA (RNA enzyme, stored at 4°C), the concentration is 0.15 mg / ml;

[0076] Phenol red, at a concentration of 0.05 mM.

[0077] (2) Lysis buffer P2:

[0078] NaOH, the concentration of which is 200 mM;

[0079] Lithium dodecyl sulfate (LDS), at a concentration of 5%;

[0080] Reactive blue, its concentration is 0.10mM.

[0081] Compared with SDS reagent, lithium dodecyl sulfonate (LDS) is used because it is less likely to foam and can reduce experimental operation errors.

[0082] (3) Neutralization buffer P3:

[0083] Guanidine hydrochloride, the concentration of which is 2.0 M;

[0084] Potassium acetate, its concentration is 0.7M

[0085] Acetic acid, with a concentration of 3.1 M

[0086] Urea, its concentration is 2.0M;

[0087] Tetradecanoylamidopropyl hydroxypropyl sulfobetaine at a concentration of 1.5% w / v;

[0088] Sodium N-lauroylamino acid, at a concentration of 0.3% w / v;

[0089] Phenol red, at a concentration of 0.05 mM.

[0090] (4) Deproteinized liquid PD:

[0091] Anhydrous ethanol solution, the concentration of which is 30%;

[0092] Guanidine hydrochloride, at a concentration of 3 M;

[0093] Tris-HCl, with a concentration of 25 mM, pH 8.0;

[0094] KCl, the concentration of which is 100 mM;

[0095] (5) Rinse liquid PB:

[0096] Anhydrous ethanol solution, the concentration of which is 80%;

[0097] Potassium acetate, its concentration is 0.18M;

[0098] (6) Eluent TE:

[0099] Tris-HCl, with a concentration of 10 mM, pH 8.5;

[0100] EDTA, the concentration of which is 100 mM.

[0101] Except for the silica-based column, other reagents and equipment (such as anhydrous ethanol, 1.5 ml centrifuge tubes, pipettes, etc.) can be prepared by yourself. According to the ratio of the above reagents, the following plasmid DNA extraction steps are carried out, such as Figure 3 and Figure 4 As shown:

[0102] 1. Resuspension: Take 1.5-5mL of overnight cultured bacterial solution and add it to a 1.5mL centrifuge tube. Centrifuge at room temperature for 30s at 13,000rpm (~9,500×g) to collect bacteria and remove the supernatant as much as possible. Add 150μL of solution resuspension solution P1 to the centrifuge tube with bacterial pellets, use a pipette to mix or vortex to thoroughly suspend the pellet (Note: This step requires thorough suspension of the bacterial pellet, otherwise it will affect the lysis effect, resulting in low plasmid concentration and purity). The solution is visibly red.

[0103] 2. Lysis: Add 150 μL of lysis buffer P2 to the centrifuge tube and mix gently immediately to fully lyse the bacteria. There is no need to leave it at room temperature (Note: Mix gently until the color is uniform. Do not shake violently to avoid genomic DNA breakage and contamination of plasmid DNA. At this time, the bacterial solution should become clear and viscous. If it does not become clear, it may be due to too many bacteria and incomplete lysis. The amount of bacteria should be reduced).

[0104] 3. Neutralization: Add 300μL of neutralizing solution P3 to the centrifuge tube, and immediately and gently turn it upside down 10 times to mix thoroughly. The solution will be uniform in color. Cell fragments and complexes of metal ions and proteins, i.e. white flocculent precipitates, will form in the solution. No need to leave it at room temperature. Centrifuge at 13,000rpm (~9,500×g) for 3~5min, discard the white precipitate, and keep the supernatant. Try to avoid aspirating the white precipitate to avoid contaminating the plasmid. (Note: For low-copy plasmids, you can increase the amount of bacteria and increase the amount of resuspension solution, lysis solution and neutralization solution in proportion).

[0105] 4. Column loading: Transfer the supernatant to the adsorption column and place the adsorption column in the collection tube. Centrifuge at room temperature at 13,000 rpm (~9,500 × g) for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.

[0106] 5. Protein removal and rinsing: Add 500 μL of protein removal rinsing solution PD to the adsorption column, centrifuge at 13,000 rpm (~9,500×g) for 30 s, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0107] 6. Desalting and rinsing: Add 500 μL of desalting rinsing solution PB to the adsorption column, centrifuge at 13,000 rpm (~9,500 × g) for 30 seconds, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube. This step is repeated twice in total. Then, put the adsorption column back into the collection tube, centrifuge at 13,000 rpm (~9,500 × g) for 60 seconds to remove as much excess ethanol as possible.

[0108] 7. Drying: The residual ethanol in the rinse solution will affect the subsequent enzymatic reaction (such as enzyme digestion, PCR, etc.) experiments. To ensure that the downstream experiments are not affected by the residual ethanol, the adsorption column can be opened and placed at room temperature for 2 to 3 minutes to completely remove the residual ethanol.

[0109] 8. Elution: Place the adsorption column in a clean 1.5mL collection tube, add 25-50μL of elution buffer TE to the middle part of the adsorption film, leave it at room temperature for 1-2 minutes, and then centrifuge it at room temperature at 13,000rpm (~9,500×g) for 60s. Transfer all the eluate in the 1.5mL centrifuge tube to a clean 1.5mL centrifuge tube and store it at -20℃.

[0110] (Note: To improve the recovery efficiency, the obtained TE eluate can be added back into the adsorption column and step 8 can be repeated).

[0111] In this example, the inter-batch repeatability of three batches of samples was tested using different plasmids, different strains, and different bacterial liquid volumes. Specifically, based on the above exemplary kit and plasmid DNA extraction steps of the present invention, plasmid DNA was extracted from three batches of samples of 1.5 mL and 5 mL bacterial liquid of DH5-PET32, TOP10-PET28, and Rossetta-GEM35 strains, respectively, and the extraction results were compared with other similar kits available on the market (referred to as prior art 1 and 2). The results are shown in Tables 1-3 below:

[0112] Table 1: Extraction results of plasmid DNA from DHS-PET32 strains of different bacterial concentrations and batches

[0113]

[0114]

[0115] Table 2: Extraction results of plasmid DNA from TOP10-PET28 strains of different bacterial concentrations and batches

[0116]

[0117] Table 3: Extraction results of plasmid DNA from Rossetta-GEM35 strains of different bacterial concentrations and batches

[0118]

[0119] As shown in Tables 1-3, the average inter-batch CV of three batches of samples of DH5α-PET32, TOP10-PET28, and Rossetta-GEM35 strains with different bacterial liquid amounts is less than 10%, showing good inter-batch repeatability, that is, stable and reliable. As shown in the data, the kit of the present invention is superior to the prior art in terms of both the final average concentration measurement value and the value of 260 / 230, which means that the obtained plasmid DNA concentration and purity are higher.

[0120] The results of agarose gel electrophoresis of plasmid DNA extracted from 5 ml of bacterial solution are as follows: Figure 1 As shown. Compared with two prior art kits of other companies (i.e., Thermo Scientific GeneJET K0503 and QIAGEN-tip10000), the gel run results of the plasmid DNA extracted by the column-type rapid extraction kit of the present invention show that the concentration and purity are significantly better than those of the prior art kits. The concentration measurement values ​​of the prior art kits are average, and the gel run results are also different from those of the present invention. The influencing factors may be the large number of small genomic fragments and RNA residues.

[0121] The main differences between Examples 2 to 5 and Example 1 are the urea concentration, the tetradecanoylamidopropyl hydroxypropyl sulfobetaine concentration and the guanidine hydrochloride concentration in the neutralization buffer P3. The extraction results are shown in Table 4.

[0122] The difference between Examples 6 to 9 and Example 1 lies in the concentration of urea, myristyl amide propyl hydroxypropyl sulfobetaine and guanidine hydrochloride in the neutralization buffer P3. The extraction results are shown in Table 5.

[0123] The difference between Examples 10 to 13 and Example 1 lies in the concentration of urea, myristyl amide propyl hydroxypropyl sulfobetaine and guanidine hydrochloride in the neutralization buffer P3. The extraction results are shown in Table 6.

[0124] The present invention specifically adds urea and tetradecanoylamidopropyl hydroxypropyl sulfobetaine on the basis of the traditional plasmid extraction neutralization solution. Urea and guanidine hydrochloride have a solubilizing effect on hydrophobic amino acid residues. Because urea and guanidine hydrochloride both have the ability to form hydrogen bonds, when they are in high concentrations, they can destroy the hydrogen bond structure of water, resulting in urea and guanidine hydrochloride becoming better solvents for non-polar residues, so that the hydrophobic residues inside the protein molecules are stretched and the solubility is increased. The denaturation caused by urea and guanidine hydrochloride is usually reversible. Tetradecanoylamidopropyl hydroxypropyl sulfobetaine is an amphoteric surfactant and also a denatured zwitterionic detergent, which can be used as a denatured zwitterionic detergent for membrane biochemistry. In the present invention, it is used to separate and purify and encapsulate the membrane protein after separation and purification, which can effectively increase the dissolution of membrane protein, less cause the inactivation of membrane protein, and is used for removing protein in the neutralization solution. As shown in Table 4, urea can significantly improve the concentration and purity of plasmid extraction without adding tetradecanoylamidopropyl hydroxypropyl sulfobetaine. As shown in Table 5, 1.5% tetradecanoylamidopropyl hydroxypropyl sulfobetaine can stably enhance the purity of plasmid extraction based on 4M urea.

[0125] Table 4: Extraction results of plasmid DNA from the same strain PET285ml at different urea contents

[0126]

[0127]

[0128] Table 5: Extraction results of plasmid DNA from the same strain PET285ml at different tetradecanoylamidopropyl hydroxypropyl sulfobetaine contents

[0129]

[0130] Table 6: Extraction results of plasmid DNA from the same strain PET285ml at the same concentration of tetradecanoylamidopropyl hydroxypropyl sulfobetaine and urea and different guanidine hydrochloride contents

[0131]

[0132]

[0133] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A plasmid extraction kit, characterized in that: It includes resuspension buffer P1, lysis buffer P2, and neutralization buffer P3; The resuspension buffer P1 includes Tris-HCl, EDTA, a carbohydrate compound, RNaseA and a first indicator; wherein the concentration of the Tris-HCl is 50-150 mM; the concentration of the EDTA is 10-100 mM; the concentration of the carbohydrate compound is 10-100 mM; the concentration of the RNaseA is 0.1-0.5 mg / ml; the first indicator includes phenol red or reactive red, and its concentration is 0.03-0.1 mM; The lysis buffer P2 includes NaOH and a surfactant; wherein the concentration of the NaOH is 100-250 mM; the surfactant includes sodium dodecyl sulfate or lithium dodecyl sulfonate, and the concentration thereof is 1-5%; The neutralization buffer P3 includes guanidine hydrochloride, potassium acetate, acetic acid, urea, tetradecanoylamidopropyl hydroxypropyl sulfobetaine and sodium N-lauroyl aminate; wherein the concentration of the guanidine hydrochloride is 1.0-3.5M; the concentration of the potassium acetate is 0.5-1.0M; the concentration of the acetic acid is 2.0-3.6M; the concentration of the urea is 0.5-5.0M; the concentration of the tetradecanoylamidopropyl hydroxypropyl sulfobetaine is 0.5-5.0%; and the concentration of the sodium N-lauroyl aminate is 0.10-0.50%.

2. The plasmid extraction kit according to claim 1, characterized in that: In the resuspension buffer P1, the sugar compound includes glucose, sucrose or trehalose.

3. The plasmid extraction kit according to claim 1 or 2, wherein In the resuspension buffer P, the concentration of Tris-HCl is 100-120 mM; the concentration of EDTA is 10-50 mM; the concentration of the carbohydrate compound is 50-100 mM; the concentration of RNaseA is 0.1-0.2 mg / ml; and the first indicator includes phenol red or reactive red, and its concentration is 0.03-0.05 mM.

4. The plasmid extraction kit according to claim 3, wherein The lysis buffer P2 further comprises a second indicator, and the second indicator comprises reactive blue or bromophenol blue.

5. The plasmid extraction kit according to claim 4, characterized in that: The concentration of the second indicator is 0.01-0.2 mM.

6. The plasmid extraction kit according to claim 4 or 5, characterized in that In the lysis buffer P2, the concentration of NaOH is 100-250 mM; the surfactant includes sodium dodecyl sulfate or lithium dodecyl sulfonate, and the concentration thereof is 1-5%; and the concentration of the second indicator is 0.05-0.10 mM.

7. The plasmid extraction kit according to claim 6, wherein In the neutralization buffer P3, the neutralization buffer P3 further comprises a third indicator, and the third indicator comprises phenol red or reactive red.

8. The plasmid extraction kit according to claim 7, characterized in that: The concentration of the third indicator is 0.01-0.10 mM.

9. The plasmid extraction kit according to claim 7 or 8, characterized in that In the neutralization buffer P3, the concentration of the guanidine hydrochloride is 2.0-3.0M; the concentration of the potassium acetate is 0.65-0.7M; the concentration of the acetic acid is 2.7-3.2M; the concentration of the urea is 1.0-3.0M; the concentration of the tetradecanoylamidopropyl hydroxypropyl sulfobetaine is 1-2%; the concentration of the sodium N-lauroylamino acid is 0.15-0.35%; and the concentration of the third indicator is 0.01-0.05mM.

10. The plasmid extraction kit according to claim 9, wherein In the neutralization buffer P3, the concentration of the guanidine hydrochloride is 2.0-2.5M; the concentration of the urea is 2.0-3.0M.

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