Kit and method for plasmid extraction

Abstract

Provided is a kit for plasmid extraction, comprising: A. a resuspension solution comprising 0.1 M to 0.3 M glucose, 10 mM to 20 mM ethylenediaminetetraacetic acid, 15 mM to 30 mM tris(hydroxymethyl)aminomethane, and water; B. a lysis solution comprising 0.1 M to 0.5 M sodium hydroxide, 0.15 M to 0.4 M arginine, 0.01 M to 0.05 M sodium dodecyl sulfate, and water; C. a neutralizing solution; D. a deproteinizing rinsing solution WB1; E. a desalting rinsing solution WB2; and F. optionally, an eluent. The resuspension solution and the lysis solution are used in a volume ratio of 1:0.8 to 1:1.2, preferably in a volume ratio of 1:1. Also provided is a method for plasmid extraction using the kit.

Description

A kit and method for plasmid extraction Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a kit and method for extracting plasmids. Background Technology

[0002] Escherichia coli is a common prokaryotic vector cell used for gene cloning and protein expression experiments. Typically, the target gene is transformed into E. coli, and as positive bacteria multiply, plasmids are extracted for subsequent experiments. Efficiently and massively preparing plasmids using E. coli has always been a fundamental requirement in the field of biotechnology. Furthermore, the outermost layer of the E. coli cell wall membrane is composed of lipopolysaccharide (LPS), which is released during plasmid extraction (also known as endotoxin), significantly impacting subsequent transfection efficiency. Therefore, obtaining large quantities of plasmids and further reducing the endotoxin content remain problems that need to be solved in this field.

[0003] Summary of the Invention

[0004] To address the aforementioned technical problems in the prior art, on the one hand, to obtain plasmids in large quantities and efficiently, the present invention provides a kit for plasmid extraction, comprising:

[0005] A. A resuspension comprising 0.1M to 0.3M glucose, 10mM to 20mM ethylenediaminetetraacetic acid, 15mM to 30mM tris(hydroxymethyl)aminomethane, and water; preferably, the concentration of glucose is 0.15M; preferably, the concentration of ethylenediaminetetraacetic acid is 15mM; preferably, the concentration of tris(hydroxymethyl)aminomethane is 20mM; preferably, the pH of the resuspension is 8.0 to 9.0, more preferably 8.0;

[0006] B. A lysis buffer comprising 0.1 M to 0.5 M sodium hydroxide, 0.15 M to 0.4 M arginine, 0.01 M to 0.05 M sodium dodecyl sulfate, and water; preferably, the concentration of sodium hydroxide is 0.2 M; preferably, the concentration of arginine is 0.2 M; preferably, the concentration of sodium dodecyl sulfate is 0.03 M; preferably, the pH of the lysis buffer is 10 to 12.1, more preferably 11;

[0007] C. Neutralizing solution;

[0008] D. Protein-removing rinsing solution WB1;

[0009] E. Desalination rinse solution WB2;

[0010] F. Optional elution solution.

[0011] The resuspension and the lysis buffer are used in a volume ratio of 1:0.8 to 1:1.2, preferably in a volume ratio of 1:1.

[0012] In some preferred embodiments, to further reduce the endotoxin content in the plasmid, the kit further includes an endotoxin-removing solution comprising:

[0013] a. Triton X-114 comprising 5% to 10% of the total volume of the endotoxin solution, preferably 8% of the total volume of the endotoxin solution;

[0014] b. An organic solvent comprising 8% to 15% of the total volume of the endotoxin solution, wherein the organic solvent is selected from at least one of chloroform, ethyl acetate, isopropanol, and ethanol; preferably, the organic solvent comprises 10% of the total volume of the endotoxin solution, and preferably, the organic solvent is ethanol;

[0015] c. 3 mM to 5 mM of tris(hydroxymethyl)aminomethane, preferably, the concentration of said tris(hydroxymethyl)aminomethane is 4 mM;

[0016] d. Sodium chloride from 0.08M to 0.2M, preferably, the concentration of the sodium chloride is 0.1M;

[0017] e. Water;

[0018] Preferably, the endotoxin solution and the supernatant obtained by centrifugation after adding the neutralizing solution are used at a volume ratio of 8% to 12%, more preferably at a volume ratio of 10%.

[0019] Preferably, the pH of the endotoxin-removing solution is 7.5 to 8.5, more preferably 7.5.

[0020] In some embodiments, the kit further includes a column equilibration solution comprising 0.1 M to 0.3 M sodium hydroxide, 0.2 M to 0.5 M sodium chloride, and water; preferably, the concentration of the sodium hydroxide is 0.2 M, and more preferably, the concentration of the sodium chloride is 0.3 M; the pH of the column equilibration solution is 11 to 13, more preferably 12.

[0021] In some embodiments, the kit satisfies at least one of the following:

[0022] The neutralizing solution comprises 2M to 4M sodium acetate, 3% to 8% acetic acid and water, preferably 3M sodium acetate, preferably 5% acetic acid, and preferably 5% acetic acid. The pH of the neutralizing solution is preferably 5.8 to 7.2, and more preferably 6.

[0023] The protein-removing rinsing solution WB1 comprises 2M to 4M guanidine hydrochloride, 10mM to 20mM tris(hydroxymethyl)aminomethane, and water; preferably, the concentration of guanidine hydrochloride is 3M; preferably, the concentration of tris(hydroxymethyl)aminomethane is 15mM; preferably, the pH of the protein-removing rinsing solution WB1 is 4.0 to 5.8, more preferably 5.0;

[0024] The desalting rinse solution WB2 comprises 20 mM to 40 mM of tris(hydroxymethyl)aminomethane, 2 mM to 4 mM of ethylenediaminetetraacetic acid (EDTA), and water; preferably, the concentration of tris(hydroxymethyl)aminomethane is 30 mM; preferably, the concentration of EDTA is 3 mM; preferably, the pH of the desalting rinse solution WB2 is 5.0 to 8.0, more preferably 6.0;

[0025] The eluent is DEPC water, preferably with a pH of 6.8 to 7.4, more preferably 7.0.

[0026] In some embodiments, the kit further includes: an anion exchange resin adsorption column, an alcohol selected from ethanol or isopropanol for column loading, a collection tube and / or a pipette tip; preferably, the alcohol for column loading is isopropanol; preferably, the collection tube is a pyrogen-free collection tube; preferably, the pipette tip is a pyrogen-free pipette tip.

[0027] On the other hand, the present invention also provides an endotoxin removal solution, comprising:

[0028] a. Triton X-114 comprising 5% to 10% of the total volume of the endotoxin solution, preferably 8% of the total volume of the endotoxin solution;

[0029] b. An organic solvent comprising 8% to 15% of the total volume of the endotoxin solution, wherein the organic solvent is selected from chloroform, ethyl acetate, isopropanol, and ethanol; preferably, the organic solvent comprises 10% of the total volume of the endotoxin solution, and preferably, the organic solvent is ethanol;

[0030] c. 3 mM to 5 mM of tris(hydroxymethyl)aminomethane, preferably, the concentration of said tris(hydroxymethyl)aminomethane is 4 mM;

[0031] d. Sodium chloride from 0.08M to 0.2M, preferably, the concentration of the sodium chloride is 0.1M;

[0032] e. Water;

[0033] Preferably, the pH of the endotoxin-removing solution is 7.5 to 8.5, more preferably 7.5.

[0034] On the other hand, the present invention also provides a method for extracting plasmids using the kit of the present invention, which includes the following steps performed sequentially:

[0035] 1) Centrifuge the bacterial culture to obtain a supernatant and precipitated bacterial cells. Discard the supernatant and add a resuspension solution to the precipitated bacterial cells to resuspend the bacterial cells. Preferably, the bacterial culture is an *Escherichia coli* culture. Preferably, the OD of the bacterial culture is... 600 The value is between 0.6 and 0.8. Preferably, the volume ratio of the bacterial solution to the resuspension is 8:1 to 12:1, more preferably 10:1. Preferably, the centrifugation speed is 7,000 to 9,000 rpm and the time is 7 to 15 min (more preferably 10 min).

[0036] 2) Add the lysis buffer and mix well; preferably, the volume ratio of the resuspension to the lysis buffer is 1:0.8 to 1:1.2, more preferably 1:1;

[0037] 3) Add neutralizing solution, mix well, centrifuge, and obtain supernatant; preferably, the volume ratio of the neutralizing solution to the total volume of the liquid obtained in step 2) is 1:1 to 1:2, more preferably 1:1.3; preferably, the centrifugation speed is 7,000 to 9,000 rpm, and the time is 7 to 15 min (more preferably 10 min); preferably, the liquid obtained after mixing is allowed to stand at room temperature for 3 to 10 min (more preferably 5 min) before centrifugation;

[0038] 4) Add isopropanol or ethanol to the supernatant obtained in step 3), mix well, and then transfer to an anion exchange resin adsorption column and centrifuge; the volume ratio of isopropanol or ethanol to supernatant is 15% to 30%, preferably 20%; preferably, the centrifugation speed is 7,000 to 9,000 rpm and the time is 45s to 90s (more preferably 60s).

[0039] 5) Add protein-removing wash solution WB1 to the anion exchange resin adsorption column and centrifuge. Then add salt-removing wash solution WB2 to the anion exchange resin adsorption column and centrifuge. Before use, ethanol is added to both protein-removing wash solution WB1 and salt-removing wash solution WB2. The volume ratio of ethanol to protein-removing wash solution WB1 is 2:3 to 1:1, preferably 9:11, and the volume ratio of ethanol to salt-removing wash solution WB2 is 2:3 to 3:2, preferably 1:1. Preferably, the total volume of protein-removing wash solution WB1 and ethanol or the total volume of salt-removing wash solution WB2 and ethanol added to the anion exchange resin adsorption column is 4 to 8 mL, more preferably 6 mL. Preferably, the centrifugation speed is 7,000 to 9,000 rpm, and the time is 45 to 90 seconds (more preferably 60 seconds). Optionally, the rinsing step of adding salt-removing wash solution WB2 is repeated 1 to 3 times.

[0040] 6) Add elution solution to the anion exchange resin adsorption column and centrifuge to obtain plasmids; preferably, the centrifugation speed is 7,000 to 9,000 rpm and the time is 45 s to 90 s (more preferably 60 s); preferably, after adding elution solution to the anion exchange resin adsorption column, let it stand at room temperature for 1 to 3 min.

[0041] Based on the purpose of this invention, the bacterial solution used in step 1) is a bacterial solution that has undergone plasmid transformation, that is, plasmid transformation is performed according to methods known in the art, positive clones are screened, and bacterial solution for plasmid extraction is obtained.

[0042] In this document, the plasmids applicable to the kits and methods of the present invention can be any plasmid capable of using Escherichia coli as a host, such as VunD1, pGBKT7-53, pKC7, pet-32a(+), etc.; the Escherichia coli used for plasmid transformation can be various Escherichia coli strains such as TOP10, DH5α, Trans1-T1, etc.

[0043] In some embodiments, the method further includes step 3-1) between step 3) and step 4):

[0044] Add the endotoxin-removing solution to the supernatant obtained in step 3), mix well, incubate on ice for 8 to 15 minutes (preferably 10 minutes), and centrifuge to obtain the supernatant; preferably, the volume ratio of the endotoxin-removing solution to the supernatant is 8% to 12%, more preferably 10%; preferably, the centrifugation speed is 7,000 to 9,000 rpm, and the time is 45 to 90 seconds (more preferably 60 seconds); optionally, repeat step 3-1) 1 to 3 times;

[0045] In some embodiments, the method further includes step 3-2) between step 3) and step 4):

[0046] Add the column equilibration solution to the anion exchange resin adsorption column and centrifuge; preferably, the volume of the added column equilibration solution is 1 to 2 ml; preferably, the centrifugation speed is 7,000 to 9,000 rpm and the time is 45 s to 90 s (more preferably 60 s); preferably, after adding the column equilibration solution to the anion exchange resin adsorption column, let it stand at room temperature for 1 to 3 min.

[0047] The kits and methods of this invention can be applied to both low-copy and high-copy plasmids. In particular, for low-copy plasmids, the extraction effect can be improved by appropriately increasing the bacterial culture volume or increasing the lysis time.

[0048] In this article, "low-copy plasmids" refer to plasmids with the following characteristics: the plasmid replicates once when the bacterial chromosome replicates, and each bacterium contains only 1 to 2 copies of the plasmid; for example, pAC-85b and pAC-BETAipi. "High-copy plasmids" refer to plasmids with the following characteristics: they continue to replicate even after bacterial chromosome replication has stopped, and each bacterium generally contains about 20 copies of the plasmid; for example, ColE1, pMB1, and pMB9.

[0049] The reagent kit and method of the present invention have the following advantages and beneficial effects compared with the prior art:

[0050] By utilizing the optimized kit and plasmid extraction method of this invention, and through the synergistic use of the components, high-quality, endotoxin-free plasmids can be obtained in large quantities. The obtained plasmids can be used for routine molecular biology experiments such as enzyme digestion, sequencing, transformation, and library construction, and are particularly suitable for sensitive applications such as primary cell transfection, gene therapy, and vaccine research. Specifically, on the one hand, this invention significantly improves plasmid yield in various host cells and plasmids through optimized design of the resuspension and lysis buffer formulations, particularly the combined use of arginine with other components. Specifically, the kit of this invention can obtain plasmids with concentrations greater than 340 ng / μL, A260 / A280 ratios of 1.8-2.0, and A260 / A230 ratios of 2.0-2.3. On the other hand, this invention significantly reduces the endotoxin content in the prepared plasmids through optimized design of the components of the endotoxin-free solution. Since plasmid transfection efficiency decreases with increasing endotoxin content, obtaining plasmids with low or even no endotoxin content is crucial for primary cell and stem cell transfection. Furthermore, because endotoxin-containing plasmids may activate non-specific responses in immune cells, leading to false positive results, plasmids with endotoxin levels less than 0.1 EU / μg must be used for experiments in primary cell transfection, gene therapy, and vaccine research. The plasmids prepared using the kit and method of this invention contain less than 0.01 EU / μg (e.g., 0.0029–0.0052 EU / μg), which is far below the requirement of 0.1 EU / μg, and can be directly used in the aforementioned related studies. Attached Figure Description

[0051] Figure 1 shows the standard curve of the endotoxin detection horseshoe crab reagent in one embodiment of the present invention. Detailed Implementation

[0052] In this document, unless otherwise stated, scientific and technical terms used have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0053] The present disclosure will now be described in further detail with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the disclosure. Experimental methods in the embodiments that do not specify specific conditions are performed under conventional conditions known in the art or under conditions recommended by the manufacturer.

[0054] In the following embodiments, plasmids VunD1-TOP10 (VunD1 plasmid transformed into TOP10 cells), pGBKT7-53-DH5α (pGBKT7-53 transformed into DH5α cells), pKC7-Trans1-T1 (pKC7 plasmid transformed into Trans1-T1 cells), and pet-32a(+)-BL21(DE3) (pet-32a(+) transformed into BL21 cells) were extracted respectively to compare their extraction effects. Among them, TOP10, DH5α, Trans1-T1, and BL21 cells were purchased from Shanghai Biostime Biotechnology Co., Ltd., with catalog numbers BR1378512, BR1000048, BR1510339, and BR1000039, respectively. Plasmids VunD1, pGBKT7-53, pKC7, and pet-32a(+) were all commercially available plasmids commonly used in this field. Specifically, plasmid VunD1 is shown at http: / / www.biofeng.com / zaiti / dachang / VunD1.html, plasmid pGBKT7-53 at http: / / www.biofeng.com / zaiti / jiaomu / pGBKT7-53.html, plasmid pKC7 at http: / / www.biofeng.com / zaiti / dachang / pKC7.html, and plasmid pet-32a(+) at http: / / www.biofeng.com / zaiti / dachang / pet32a.html.

[0055] Unless otherwise specified, the methods used in the examples are all routine experimental procedures. Unless otherwise specified, all reagents and consumables used in the examples can be purchased from biological reagent companies.

[0056] Examples 1-2 and Comparative Examples 1-5: Effect of Arginine Concentration on Extraction Efficiency

[0057] Table 1: Components of the kit of Example 1 of the present invention

[0058] The solvent for all solutions in Table 1 is water.

[0059] The kits for Examples 2 and Comparative Examples 1-5 are basically the same as those for Example 1, except that the concentration of arginine in the lysis buffer is different (as shown in Table 2 below).

[0060] Plasmid extraction was performed using the kits shown in Examples 1-2 and Comparative Examples 1-5 according to the following steps, and the results are shown in Table 2 below. The anion exchange resin adsorption column used was purchased from DouDian Biotechnology, catalog number DNAF051-16-20.

[0061] The plasmid extraction method is as follows:

[0062] 1. Centrifuge and resuspend: Take 50 mL OD 600 For bacterial suspensions with a pH between 0.6 and 0.8, centrifuge at 8,000 rpm for 10 min to obtain supernatant and precipitated bacterial cells. Discard the supernatant, add 5 mL of resuspension to the precipitated bacterial cells, and mix thoroughly until there is no obvious clumping (avoid clumping, which may affect subsequent bacterial lysis).

[0063] 2. Lysis: Add 5 mL of lysis buffer to the resuspension from step 1, and gently invert to mix. At this point, the bacterial solution should be clear and transparent (the inversion should be gentle and quick, and the time should not be too long to prevent excessive lysis or damage from external force).

[0064] 3. Neutralization: Add 7.5 mL of neutralizing solution to the solution from step 2, gently invert and mix. At this point, a white flocculent precipitate will appear in the solution. Let it stand at room temperature for 5 min, then centrifuge at 8,000 rpm for 10 min to allow the white precipitate to gather at the bottom (if there is still some precipitate in the supernatant, you can aspirate the supernatant with the precipitate and centrifuge again).

[0065] 4. Column equilibration: Add 2 mL of column equilibration solution to the anion exchange resin adsorption column, let stand at room temperature for 2 min, centrifuge at 8,000 rpm for 1 min, discard the waste liquid, and keep it for later use.

[0066] 5. Column loading: Take the supernatant from step 3 and place it in a new centrifuge tube. Add isopropanol (the volume ratio of isopropanol to supernatant is 20%), invert and mix well. Then transfer it to an anion exchange resin adsorption column, centrifuge at 8,000 rpm for 1 min, and discard the waste liquid.

[0067] 6. Rinsing: Before use, ethanol is added to the protein-removing rinsing solution WB1 and the desalting rinsing solution WB2 listed in Table 1. The volume ratio of ethanol to protein-removing rinsing solution WB1 is 45:55, and the volume ratio of ethanol to desalting rinsing solution WB2 is 1:1. Then, 6 mL of protein-removing rinsing solution WB1 with added ethanol is added to the anion exchange resin adsorption column, centrifuged at 8,000 rpm for 1 min, and the waste liquid is discarded. 6 mL of desalting rinsing solution WB2 with added ethanol is added, centrifuged at 8,000 rpm for 1 min, and the waste liquid is discarded. This operation is repeated once.

[0068] 7. Ethanol evaporation: Place the adsorption column open at room temperature for 5 minutes to allow the ethanol to evaporate (residual ethanol will affect subsequent enzyme digestion and other operations, so the placement time can be extended).

[0069] 8. Elution: Transfer the adsorption column to a new centrifuge tube, add 1 mL of eluent to the center of the adsorption column, let stand for 1 min, centrifuge at 8,000 rpm for 1 min, and transfer the eluent to a new centrifuge tube to obtain the endotoxin-free plasmid. Repeat the elution once to increase the plasmid elution rate.

[0070] 9. Detection: The absorbance of the plasmid solution obtained in step 8 at A260 / A280 and A260 / A230 was measured using an ultra-micro UV-Vis spectrophotometer (model: NanoDrop 2000, manufacturer: Thermo Fisher Scientific). The plasmid concentration was also measured as follows: the spectrophotometer was zeroed using the elution buffer, and then the plasmid elution buffer obtained in step 8 was tested on the spectrophotometer.

[0071] The effects of arginine concentrations in the lysis buffer of Examples 1-2 and Comparative Examples 1-5 on plasmid extraction efficiency were compared. The plasmid concentration and purity were specifically analyzed. The results showed that the plasmids of Examples 1-2 had better yields and purity. In particular, the plasmids of Example 1 with an arginine concentration of 0M, 0.1M, 0.2M, 0.4M, and 0.5M had significantly improved yields and purity. The results are shown in Table 2.

[0072] Table 2

[0073] Examples 1', 3 and Comparative Example 6: Effects of Endotoxin Removal Solution

[0074] Example 3 uses the kit and method of Example 1, and further uses an endotoxin removal solution for the endotoxin removal step. The formulation of the endotoxin removal solution is shown below:

[0075] 8 vol% Triton X-114, 4 mM tris(hydroxymethyl)aminomethane, 0.1 M sodium chloride, 10 vol% ethanol and water; pH 7.5.

[0076] The endotoxin removal step is performed between steps 3 and 5 of Example 1 (before or after step 4), as follows:

[0077] Endotoxin removal: Add endotoxin removal solution (10% by volume) to the supernatant obtained in step 3, mix well, incubate on ice for 10 minutes, and centrifuge at 8,000 rpm for 1 minute (the mixture of endotoxin removal solution and supernatant becomes clear after incubation on ice; this step can be repeated 2-3 times for better endotoxin removal).

[0078] Then, following the method of Example 1, the "supernatant from step 3" in step 5 was replaced with the supernatant obtained after endotoxin removal for subsequent processing.

[0079] The kit and method of Comparative Example 6 are basically the same as those of Example 3. The difference is that the kit of Comparative Example 6 uses a detoxification solution commonly used in the prior art. The detoxification solution was purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number B518162.

[0080] The kit and method of Example 1 are exactly the same as those in Example 1.

[0081] Plasmids were extracted for Examples 1', 3, and Comparative Example 6. The concentration and purity of the obtained plasmids were detected. An endotoxin detection kit for horseshoe crabs (purchased from Xiamen Horseshoe Crab Reagent Biotechnology Co., Ltd., catalog number EC64405; tube quantitative chromogenic matrix method) was used to detect the endotoxin content in the obtained plasmids according to the kit instructions. Specifically, the endotoxin concentration was calculated based on the absorbance data of the sample at 545 nm and the standard curve. The standard curve is shown in Figure 1, and the results are shown in Table 3 below.

[0082] Table 3

[0083] As shown in Table 3, Example 3, using the endotoxin-removing solution specified in this invention, exhibits excellent endotoxin removal performance, with the resulting plasmid containing endotoxins between 0.0029 and 0.0052 EU / μg. In contrast, Comparative Example 6, using a conventional endotoxin-removing solution, showed endotoxins between 0.1 and 0.2 EU / μg, while Example 1', without the addition of endotoxin-removing solution, had endotoxins exceeding 0.5 EU / μg. The endotoxin content of the plasmid obtained in Example 3 of this invention is significantly lower than that in Comparative Example 6. Furthermore, both Example 3 and Example 1' yielded similarly high plasmid concentrations, indicating that the endotoxin-removing solution used in Example 3 does not affect the plasmid yield. The kit in Example 3, including the endotoxin-removing solution, significantly reduces endotoxin content while obtaining a large quantity of plasmids efficiently.

[0084] Examples 3', 4-5 and Comparative Examples 7-9: The effect of different concentrations of Triton X-114 in endotoxin removal solutions on endotoxin removal

[0085] The kits used in Example 3' are exactly the same as those used in Example 3. The kits used in Examples 4-5 and Comparative Examples 7-9 are basically the same as those used in Example 3'. The only difference is the concentration of Triton X-114 in the endotoxin-free solution used, as shown in Table 4 below.

[0086] The VunD1-TOP10 plasmid was extracted according to the plasmid extraction method in Example 3, and the endotoxin concentration in the obtained VunD1-TOP10 plasmid was determined. The results are shown in Table 4 below:

[0087] Table 4

[0088] The results shown in Table 4 indicate that the endotoxin removal effect of Triton X-114 at a concentration of 5%–10% is significantly better than that of Triton X-114 at other concentrations.

[0089] All publications, patent applications, patents, nucleic acid and amino acid sequences, and other references mentioned in this disclosure are incorporated herein by reference in their entirety.

[0090] It should be noted that all technical features described in this invention can be freely combined or integrated in any manner, unless they contradict each other. Various modifications and variations can be made to this invention without departing from its scope, as will be apparent to those skilled in the art. For example, features shown or described as part of one embodiment can be used with another embodiment to produce yet another embodiment. Therefore, this invention is intended to cover these modifications that fall within the scope of the appended claims and their equivalents.

Claims

1. A kit for plasmid extraction, comprising: A. A resuspension comprising 0.1M to 0.3M glucose, 10mM to 20mM ethylenediaminetetraacetic acid, 15mM to 30mM tris(hydroxymethyl)aminomethane, and water; preferably, the concentration of glucose is 0.15M; preferably, the concentration of ethylenediaminetetraacetic acid is 15mM; preferably, the concentration of tris(hydroxymethyl)aminomethane is 20mM; preferably, the pH of the resuspension is 8.0 to 9.0, more preferably 8.0; B. A lysis buffer comprising 0.1 M to 0.5 M sodium hydroxide, 0.15 M to 0.4 M arginine, 0.01 M to 0.05 M sodium dodecyl sulfate, and water; preferably, the concentration of sodium hydroxide is 0.2 M; preferably, the concentration of arginine is 0.2 M; preferably, the concentration of sodium dodecyl sulfate is 0.03 M; preferably, the pH of the lysis buffer is 10.0 to 12.1, more preferably 11.0; C. Neutralizing solution; D. Protein-removing rinsing solution WB1; E. Desalination rinse solution WB2; F. Optional elution solution; The resuspension and the lysis buffer are used in a volume ratio of 1:0.8 to 1:1.2, preferably in a volume ratio of 1:

1.

2. The kit according to claim 1, wherein, The kit also includes an endotoxin removal solution, which comprises: a. Triton X-114 comprising 5% to 10% of the total volume of the endotoxin solution, preferably 8% of the total volume of the endotoxin solution; b. An organic solvent comprising 8% to 15% of the total volume of the endotoxin solution, wherein the organic solvent is selected from at least one of chloroform, ethyl acetate, isopropanol, and ethanol; preferably, the organic solvent comprises 10% of the total volume of the endotoxin solution, and preferably, the organic solvent is ethanol; c. 3 mM to 5 mM of tris(hydroxymethyl)aminomethane, preferably, the concentration of said tris(hydroxymethyl)aminomethane is 4 mM; d. Sodium chloride from 0.08M to 0.2M, preferably, the concentration of said sodium chloride is 0.1M; and e. Water; Preferably, the endotoxin solution and the supernatant obtained by centrifugation after adding the neutralizing solution are used at a volume ratio of 8% to 12%, more preferably at a volume ratio of 10%. Preferably, the pH of the endotoxin-removing solution is 7.5 to 8.5, more preferably 7.

5.

3. The kit according to claim 1 or 2, wherein, The kit also includes a column equilibration solution containing 0.1M to 0.3M sodium hydroxide, 0.2M to 0.5M sodium chloride, and water; preferably, the concentration of the sodium hydroxide is 0.2M, and more preferably, the concentration of the sodium chloride is 0.3M; the pH of the column equilibration solution is 11 to 13, and more preferably 12.

4. The kit according to any one of claims 1 to 3, wherein, The kit satisfies at least one of the following: The neutralizing solution comprises 2M to 4M sodium acetate, 3% to 8% acetic acid and water, preferably 3M sodium acetate, preferably 5% acetic acid, and preferably 5% acetic acid. The pH of the neutralizing solution is preferably 5.8 to 7.2, and more preferably 6.

0. The protein-removing rinsing solution WB1 comprises 2M to 4M guanidine hydrochloride, 10mM to 20mM tris(hydroxymethyl)aminomethane, and water; preferably, the concentration of guanidine hydrochloride is 3M; preferably, the concentration of tris(hydroxymethyl)aminomethane is 15mM; preferably, the pH of the protein-removing rinsing solution WB1 is 4.0 to 5.8, more preferably 5.0; The desalting rinse solution WB2 comprises 20 mM to 40 mM of tris(hydroxymethyl)aminomethane, 2 mM to 4 mM of ethylenediaminetetraacetic acid (EDTA), and water; preferably, the concentration of tris(hydroxymethyl)aminomethane is 30 mM; preferably, the concentration of EDTA is 3 mM; preferably, the pH of the desalting rinse solution WB2 is 5.0 to 8.0, more preferably 6.0; and The eluent is DEPC water, preferably with a pH of 6.8 to 7.4, more preferably 7.

0.

5. The kit according to any one of claims 1 to 3, wherein, The kit further includes: anion exchange resin adsorption column, an alcohol selected from ethanol or isopropanol for column loading, a collection tube and / or a pipette tip; preferably, the alcohol for column loading is isopropanol; preferably, the collection tube is a pyrogen-free collection tube; preferably, the pipette tip is a pyrogen-free pipette tip.

6. An endotoxin-removing solution, comprising: a. Triton X-114 comprising 5% to 10% of the total volume of the endotoxin solution, preferably 8% of the total volume of the endotoxin solution; b. An organic solvent comprising 8% to 15% of the total volume of the endotoxin solution, wherein the organic solvent is selected from chloroform, ethyl acetate, isopropanol, and ethanol; preferably, the organic solvent comprises 10% of the total volume of the endotoxin solution, and preferably, the organic solvent is ethanol; c. 3 mM to 5 mM of tris(hydroxymethyl)aminomethane, preferably, the concentration of said tris(hydroxymethyl)aminomethane is 4 mM; d. Sodium chloride from 0.08M to 0.2M, preferably, the concentration of said sodium chloride is 0.1M; and e. Water; in, Preferably, the pH of the endotoxin-removing solution is 7.5 to 8.5, more preferably 7.

5.

7. A method for extracting plasmids using the kit according to any one of claims 1 to 5, comprising the following steps performed sequentially: 1) Centrifuge the bacterial culture to obtain a supernatant and precipitated bacterial cells. Discard the supernatant and add a resuspension solution to the precipitated bacterial cells to resuspend the bacterial cells. Preferably, the bacterial culture is an *E. coli* culture. Preferably, the OD of the bacterial culture is... 600 The value is between 0.6 and 0.

8. Preferably, the volume ratio of the bacterial solution to the resuspension is 8:1 to 12:1, more preferably 10:

1. 2) Add the lysis buffer and mix well; preferably, the volume ratio of the resuspension to the lysis buffer is 1:0.8 to 1:1.2, more preferably 1:1; 3) Add neutralizing solution, mix well, centrifuge, and obtain supernatant; preferably, the volume ratio of the neutralizing solution to the total volume of the liquid obtained in step 2) is 1:1 to 1:2, more preferably 1:1.3; preferably, the liquid obtained after mixing is allowed to stand at room temperature for 3 to 10 minutes (more preferably 5 minutes) before centrifugation. 4) Add isopropanol or ethanol to the supernatant obtained in step 3), mix well, and then transfer to an anion exchange resin adsorption column and centrifuge; the volume ratio of isopropanol or ethanol to supernatant is 15% to 30%, preferably 20%; 5) Add protein-removing wash buffer WB1 to the anion exchange resin adsorption column, centrifuge, then add salt-removing wash buffer WB2 to the anion exchange resin adsorption column, and centrifuge; wherein, Before use, ethanol is added to protein-removing rinsing solution WB1 and desalting rinsing solution WB2 respectively. The volume ratio of ethanol to protein-removing rinsing solution WB1 is 2:3 to 1:1, preferably 9:11, and the volume ratio of ethanol to desalting rinsing solution WB2 is 2:3 to 3:2, preferably 1:

1. Preferably, the total volume of protein-removing rinsing solution WB1 and ethanol or the total volume of desalting rinsing solution WB2 and ethanol added to the anion exchange resin adsorption column is 4 to 8 mL, more preferably 6 mL. Optionally, the rinsing step of adding desalting rinsing solution WB2 is repeated 1 to 3 times. 6) Add elution buffer to the anion exchange resin adsorption column and centrifuge to obtain plasmid.

8. The method according to claim 7, wherein, The method further includes step 3-1) between step 3) and step 4): Add the endotoxin-removing solution to the supernatant obtained in step 3), mix well, incubate on ice for 8 to 15 minutes, and centrifuge to obtain the supernatant; preferably, the volume ratio of the endotoxin-removing solution to the supernatant is 8% to 12%, more preferably 10%; Optionally, repeat step 3-1) 1-3 times.

9. The method according to claim 7 or 8, wherein, The method further includes step 3-2) between step 3) and step 4): Add the column equilibration solution to the anion exchange resin adsorption column and centrifuge; preferably, the volume of the added column equilibration solution is 1 to 2 ml.

10. The method according to any one of claims 7 to 9, wherein, The resulting plasmid satisfies at least one of the following: The resulting plasmid can be either a low-copy plasmid or a high-copy plasmid; The endotoxin content in the obtained plasmid was less than 0.01 EU / μg.

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