Plasmid extraction method and kit based on efficient splitting decomposition and rapid column equilibrium

Abstract

The invention discloses a plasmid extraction method based on efficient splitting decomposition and rapid column equilibrium and a kit. The plasmid extraction method comprises the following steps: weighing a P1 solution, a P2 solution, a P3 solution, a BA solution and an ER solution in a volume ratio of 1: 1: 1: (0.3-0.5): (0.1-0.3); centrifuging the bacterial liquid, discarding the supernatant, adding a P1 solution, resuspending, enabling the solution to be in a turbid state, adding a P2 solution, cracking, standing, enabling the solution to become clear and transparent, and judging whether the cracking is sufficient or not according to the transparency and OD600 value of the solution; adding a P3 solution to neutralize, standing and centrifuging, adding a BA solution to carry out a column balance step, and directly passing through a column after the solution is centrifuged; then adding an ER solution to remove endotoxin with the endotoxin content being lower than 0.05 EU / [mu] g, and finally removing proteins, removing impurities and eluting. The column balancing step is moved to be after cracking and neutralizing, the BA solution is added for column balancing, the solution directly passes through the column after being centrifuged, the waiting time after column balancing is shortened, and the column adsorption efficiency is improved; by optimizing reagent components and changing the operation process, the plasmid extraction efficiency and purity are synergistically improved.

Description

Technical Field

[0001] This invention relates to the field of biological reagents and nucleic acid extraction technology, specifically to a plasmid extraction method and kit based on efficient lysis and rapid column equilibration. Background Technology

[0002] Plasmids are circular double-stranded DNA molecules outside of chromosomes in organisms such as bacteria and yeast. The extraction and purification of plasmid DNA is one of the core technologies in molecular biology research, and its extraction efficiency, purity, and endotoxin content directly affect subsequent experimental results. Currently, most mainstream plasmid extraction kits on the market are based on alkaline lysis combined with silica membrane centrifugation column adsorption. The traditional extraction steps are column equilibration → lysis → neutralization → adsorption → washing → elution.

[0003] Traditional techniques have several technical drawbacks: 1) The column equilibration step is performed prematurely. After equilibration, the silica gel membrane centrifuge column is prone to packing material sedimentation and column bed voids due to prolonged settling time. Furthermore, the equilibration solution is prone to volatilization and salt crystallization, leading to a decrease in column adsorption efficiency. 2) The lysis buffer uses only a NaOH+SDS binary system, resulting in slow lysis rates, poor uniformity of bacterial lysis, and the presence of unlysolved bacterial nuclei, affecting plasmid yield. 3) The reagent formulation is singular, lacking targeted synergistic design, resulting in low plasmid concentrations and difficulty in meeting endotoxin content standards (often exceeding 0.1 EU / μg). 4) The unreasonable order of reagent use in the operation process further reduces the overall extraction efficiency.

[0004] In summary, developing a plasmid large-scale extraction kit with optimized reagent formulation and reconstructed operation sequence to achieve a dual improvement in lysis efficiency and column adsorption efficiency while reducing plasmid endotoxin content has become a pressing technical problem to be solved in this field. Summary of the Invention

[0005] To address the technical problems of low extraction efficiency, poor plasmid purity, high endotoxin content, long waiting time after column equilibration, and low adsorption efficiency in traditional plasmid extraction kits, this invention provides a low-cost plasmid extraction kit with more complete lysis and higher column adsorption efficiency, as well as a plasmid extraction method using this kit that achieves a synergistic improvement in plasmid concentration and purity, and reduces endotoxin content to below 0.05 EU / μg.

[0006] This invention provides the following technical solution: This plasmid extraction kit includes solutions P1, P2, P3, BA, and ER, with a volume ratio of P1:P2:P3:BA:ER = 1:1:1:0.3-0.5:0.1-0.3. The components are as follows: P1 solution: 25-50 mM Tris-HCl (tris(hydroxymethyl)aminomethane) hydrochloride), 10-20 mM EDTA·2Na (ethylenediaminetetraacetic acid disodium salt), 100-150 μg / ml RNase A (ribonuclease A), 50-100 mM glucose); P2 solution: 200-250 mM NaOH, 1%-2% (W / V) SDS, 0.5-1 mM EDTA·2Na (disodium ethylenediaminetetraacetate), 0.3%-0.5% Laureth-9 (polyoxyethylene (9) lauryl ether), 25-50 mM guanidine isothiocyanate; P3 solution: 4-5 M guanidine hydrochloride, 0.5-1.0 mM potassium acetate, glacial acetic acid; BA solution: 25-50 mM Tris-HCl (tris(hydroxymethyl)aminomethane) hydrochloride), 100-150 mM NaCl (sodium chloride), 1-5 mM EDTA·2Na (disodium ethylenediaminetetraacetate), 0.01%-0.05% Laureth-9 (polyoxyethylene (9) lauryl ether), 0.5-1 M guanidine isothiocyanate; ER solution: 10-20 mM Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride), 10-20 mM NaCl (sodium chloride), 10%-20% Triton X-114, 0.01%-0.02% Alizarin Yellow R.

[0007] A plasmid extraction method based on efficient lysis and rapid column equilibration is proposed, which utilizes an extraction kit for plasmid extraction.

[0008] A plasmid extraction method based on efficient lysis and rapid column equilibration includes the following steps: S1. Weigh out solutions with a volume ratio of P1 solution:P2 solution:P3 solution:BA solution:ER solution = 1:1:1:0.3-0.5:0.1-0.3; S2. Centrifugation of bacterial culture: Place the sample bacterial culture in a centrifuge tube, centrifuge at room temperature, discard the supernatant, add P1 solution to fully resuspend the bacterial precipitate. The solution will be turbid. Then add P2 solution and gently invert to mix. Let it stand at room temperature to lyse until the solution is completely clear and transparent. The degree of lysis is judged by the transparency of the solution at this time. Finally, add P3 solution and gently invert to neutralize until a white flocculent precipitate appears. Centrifuge at room temperature. S3. Add BA solution to the silica gel membrane centrifuge column to achieve column equilibration, centrifuge and discard the filtrate, and pass the supernatant after the previous centrifugation directly through the column; S4. Then, ER solution is added to the silica membrane centrifuge column to remove endotoxins. The endotoxin content is less than 0.05 EU / μg. Finally, protein removal, impurity removal and elution are performed, and the plasmid is finally collected. The plasmid concentration is found to be above 200 ng / μL.

[0009] Furthermore, the addition of P2 solution for lysis is based on the solution's transparency and OD. 600 The value is used to determine whether the cleavage is sufficient.

[0010] In the above technical solution, the OD of the pyrolysis solution 600 The value dropped to below 0.1.

[0011] Furthermore, the protein removal process uses a PR solution composed of 4-5 M guanidine hydrochloride, 25-50 mM Tris-HCl (tris(hydroxymethyl)aminomethane) hydrochloride, and 20%-30% isopropanol (by volume).

[0012] This invention focuses on optimizing the composition of the lysis buffer (P2 solution) and column equilibration buffer (BA solution). Traditional lysis buffers mostly use a NaOH+SDS binary system. We introduce a novel biodegradable nonionic surfactant, Laureth-9 (polyoxyethylene (9) lauryl ether), which has stronger wetting power than traditional surfactants and can quickly penetrate the outer membrane of E. coli, reducing incomplete local lysis and bacterial residue. Moreover, it is mild and does not damage the plasmid, is non-denaturing and non-ionic, and is stable in a high-alkalinity (NaOH) + SDS system, without causing plasmid denaturation or breakage, and has a higher supercoiling ratio. More importantly, it is bio-based, biodegradable, and has no persistent environmental toxicity, making it suitable for plasmid transfection, in vivo experiments, and GMP applications.

[0013] During the column equilibration stage, the addition of Laureth-9 significantly reduces surface tension, allowing for instantaneous wetting of the silica membrane and faster degassing, shortening the equilibration time by more than 50%, and greatly improving the flow rate and overall experience. Therefore, by moving the column equilibration step to after pyrolysis and neutralization, this invention also avoids low adsorption efficiency caused by problems such as packing material sedimentation and evaporation of the equilibration solution. In addition, it is compatible with high-salt, guanidine salt, and isopropanol systems, does not compete for silica binding sites, and the plasmid binding efficiency and purity are not affected.

[0014] Compared with the prior art, the beneficial effects of the present invention are: 1. Based on the traditional NaOH+SDS, the P2 lysis buffer formulation was restructured by adding 0.3%-0.5% Laureth-9 (a nonionic surfactant that forms an anionic / nonionic composite surfactant system with SDS to synergistically solubilize the bacterial membrane) and 25-50 mM guanidine isothiocyanate (a weak dissociation agent that accelerates membrane protein denaturation and unfolding). At the same time, 0.5-1 mM EDTA was added to stabilize the system, achieving rapid, uniform, and thorough lysis of the bacterial colonies and reducing solution turbidity. 2. The BA column equilibration solution formulation was optimized by adding 0.5-1 M guanidine isothiocyanate and 0.01%-0.05% Laureth-9. Guanidine isothiocyanate activates the hydroxyl groups of the silica membrane and enhances the adsorption capacity of plasmids, while Laureth-9 reduces surface tension and accelerates the wetting of the equilibration solution, thereby achieving rapid equilibration and efficient activation of the silica membrane. 3. The revolutionary reconstruction of the process sequence of this invention achieves plasmid extraction in the order of bacterial centrifugation, bacterial resuspension, lysis, neutralization, column equilibration, endotoxin removal, protein removal, impurity removal and elution. The column equilibration step is moved from the first step of the traditional extraction to after lysis-neutralization, avoiding problems such as packing material sedimentation and equilibration solution volatilization, and greatly improving column adsorption efficiency. 4. Synergistic effect of reagent system: The formulation of P2 lysis buffer, BA column equilibration buffer and ER endotoxin removal solution forms a synergistic effect. While the lysis efficiency is improved, the column adsorption efficiency and endotoxin removal efficiency are improved at the same time, achieving a double improvement in plasmid yield and purity.

[0015] In summary, this invention optimizes key reagent components and changes the operating procedure to synergistically improve plasmid extraction efficiency (by 102%) and reduce endotoxin content (by 65%). Attached Figure Description

[0016] Figure 1 This is a flowchart of the present invention; Figure 2 This is a comparison chart of the transparency of solutions after pyrolysis using the traditional method and the improved method of this invention; Figure 3 The graph shows the NanoDrop detection results of plasmids extracted by the traditional method and the improved method of this invention; Figure 4 The images show the nucleic acid electrophoresis results of plasmid extraction using traditional methods and the improved method of this invention. Detailed Implementation

[0017] 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 embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example

[0018] This plasmid extraction kit includes solutions P1, P2, P3, BA, and ER, with a volume ratio of P1:P2:P3:BA:ER = 1:1:1:0.3:0.1. The components are as follows: P1 solution: 25 mM Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride), 10 mM EDTA·2Na (ethylenediaminetetraacetic acid disodium salt), 100 μg / ml RNase A (ribonuclease A), 50 mM glucose); P2 solution: 200 mM NaOH, 1% (W / V) SDS, 0.5 mM EDTA·2Na (disodium ethylenediaminetetraacetate), 0.3% Laureth-9 (polyoxyethylene (9) lauryl ether), 25 mM guanidine isothiocyanate; The 1% SDS here refers to the weight / volume ratio (W / V), where 1g of SDS is brought to a final volume of 100ml; the 0.3% Laureth-9 here refers to the weight / volume ratio (W / V), where 0.3g of Laureth-9 is brought to a final volume of 100ml.

[0019] P3 solution: 4 M guanidine hydrochloride, 0.5 mM potassium acetate, glacial acetic acid; BA solution: 25 mM Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride), 100 mM NaCl (sodium chloride), 1 mM EDTA·2Na (disodium ethylenediaminetetraacetate), 0.01% Laureth-9 (polyoxyethylene (9) lauryl ether), 0.5 M guanidine isothiocyanate; The 0.01% Laureth-9 mentioned here refers to the mass / volume percentage (w / v), meaning that 0.01 g of Laureth-9 is diluted to 100 mL of solution.

[0020] ER solution: 10 mM Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride), 10 mM NaCl (sodium chloride), 10% Triton X-114, 0.01% Alizarin Yellow R.

[0021] The 10% Triton X-114 mentioned here refers to the mass / volume percentage (w / v), meaning that 10 g of Triton X-114 is diluted to 100 mL of solution.

[0022] A plasmid extraction method based on efficient lysis and rapid column equilibration is proposed, which utilizes an extraction kit for plasmid extraction.

[0023] A plasmid extraction method based on efficient lysis and rapid column equilibration includes the following steps: S1. Weigh out each solution with a volume ratio of P1 solution:P2 solution:P3 solution:BA solution:ER solution = 1:1:1:0.3:0.1; S2. Centrifugation of bacterial culture: Place the sample bacterial culture in a centrifuge tube, centrifuge at room temperature, discard the supernatant, add P1 solution to fully resuspend the bacterial precipitate. The solution will be turbid. Then add P2 solution and gently invert to mix. Let it stand at room temperature to lyse until the solution is completely clear and transparent. The degree of lysis is judged by the transparency of the solution at this time. Finally, add P3 solution and gently invert to neutralize until a white flocculent precipitate appears. Centrifuge at room temperature. S3. Add BA solution to the silica gel membrane centrifuge column to achieve column equilibration, centrifuge and discard the filtrate, and pass the supernatant after the previous centrifugation directly through the column; S4. Then, ER solution is added to the silica membrane centrifuge column to remove endotoxins. The endotoxin content is less than 0.05 EU / μg. Finally, protein removal, impurity removal and elution are performed, and the plasmid is finally collected. The plasmid concentration is found to be above 200 ng / μL.

[0024] Protein removal was performed using a PR solution consisting of 4 M guanidine hydrochloride, 25 mM Tris-HCl (tris(hydroxymethyl)aminomethane) hydrochloride, and 20% isopropanol (v / v); impurity removal was performed using a solution of 20 mM Tris-HCl, 200 mM NaCl, and 70% anhydrous ethanol; and elution was performed using a solution of 10 mM Tris-HCl and 0.05 mM EDTA·2Na. Example

[0025] This plasmid extraction kit includes solutions P1, P2, P3, BA, and ER, with a volume ratio of P1:P2:P3:BA:ER = 1:1:1:0.4:0.2. The components are as follows: P1 solution: 37.5 mM Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride), 15 mM EDTA·2Na (ethylenediaminetetraacetic acid disodium salt), 125 μg / ml RNase A (ribonuclease A), 75 mM glucose); P2 solution: 225 mM NaOH, 1.5% (W / V) SDS, 0.75 mM EDTA·2Na (disodium ethylenediaminetetraacetate), 0.4% Laureth-9 (polyoxyethylene (9) lauryl ether), 37.5 mM guanidine isothiocyanate; P3 solution: 4.5M guanidine hydrochloride, 0.75 mM potassium acetate, glacial acetic acid; BA solution: 37.5 mM Tris-HCl (tris(hydroxymethyl)aminomethane) hydrochloride), 125 mM NaCl (sodium chloride), 3 mM EDTA·2Na (disodium ethylenediaminetetraacetate), 0.03% Laureth-9 (polyoxyethylene (9) lauryl ether), 0.75 M guanidine isothiocyanate; ER solution: 15 mM Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride), 15 mM NaCl (sodium chloride), 15% Triton X-114, 0.015% Alizarin Yellow R.

[0026] A plasmid extraction method based on efficient lysis and rapid column equilibration is proposed, which utilizes an extraction kit for plasmid extraction.

[0027] A plasmid extraction method based on efficient lysis and rapid column equilibration includes the following steps: S1. Weigh out each solution with a volume ratio of P1 solution:P2 solution:P3 solution:BA solution:ER solution = 1:1:1:0.4:0.2; S2. Centrifugation of bacterial culture: Place the sample bacterial culture in a centrifuge tube, centrifuge at room temperature, discard the supernatant, add P1 solution to fully resuspend the bacterial precipitate. The solution will be turbid. Then add P2 solution and gently invert to mix. Let it stand at room temperature to lyse until the solution is completely clear and transparent. The degree of lysis is judged by the transparency of the solution at this time. Finally, add P3 solution and gently invert to neutralize until a white flocculent precipitate appears. Centrifuge at room temperature. S3. Add BA solution to the silica gel membrane centrifuge column to achieve column equilibration, centrifuge and discard the filtrate, and pass the supernatant after the previous centrifugation directly through the column; S4. Then, ER solution is added to the silica membrane centrifuge column to remove endotoxins. The endotoxin content is less than 0.05 EU / μg. Finally, protein removal, impurity removal and elution are performed, and the plasmid is finally collected. The plasmid concentration is found to be above 200 ng / μL.

[0028] Protein removal was performed using a PR solution consisting of 4.5 M guanidine hydrochloride, 37 mM Tris-HCl (tris(hydroxymethyl)aminomethane) hydrochloride, and 25% isopropanol (v / v); impurity removal was performed using a solution of 22.5 mM Tris-HCl, 225 mM NaCl, and 75% anhydrous ethanol; and elution was performed using a solution of 15 mM Tris-HCl and 0.075% mM EDTA·2Na. Example

[0029] This plasmid extraction kit includes solutions P1, P2, P3, BA, and ER, with a volume ratio of P1:P2:P3:BA:ER = 1:1:1:0.5:0.3. The components are as follows: P1 solution: 50 mM Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride), 20 mM EDTA·2Na (ethylenediaminetetraacetic acid disodium salt), 150 μg / ml RNase A (ribonuclease A), 100 mM glucose); P2 solution: 250 mM NaOH, 2% (W / V) SDS, 1 mM EDTA·2Na (disodium ethylenediaminetetraacetate), 0.5% Laureth-9 (polyoxyethylene (9) lauryl ether), 50 mM guanidine isothiocyanate; P3 solution: 5 M guanidine hydrochloride, 1.0 mM potassium acetate, glacial acetic acid; BA solution: 50 mM Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride), 150 mM NaCl (sodium chloride), 5 mM EDTA·2Na (disodium ethylenediaminetetraacetate), 0.05% Laureth-9 (polyoxyethylene (9) lauryl ether), 1 M guanidine isothiocyanate; ER solution: 20 mM Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride), 20 mM NaCl (sodium chloride), 20% Triton X-114, 0.02% Alizarin Yellow R.

[0030] A plasmid extraction method based on efficient lysis and rapid column equilibration is proposed, which utilizes an extraction kit for plasmid extraction.

[0031] A plasmid extraction method based on efficient lysis and rapid column equilibration includes the following steps: S1. Weigh out each solution with a volume ratio of P1 solution:P2 solution:P3 solution:BA solution:ER solution = 1:1:1:0.5:0.3; S2. Centrifugation of bacterial culture: Place the sample bacterial culture in a centrifuge tube, centrifuge at room temperature, discard the supernatant, add P1 solution to fully resuspend the bacterial precipitate. The solution will be turbid. Then add P2 solution and gently invert to mix. Let it stand at room temperature to lyse until the solution is completely clear and transparent. The degree of lysis is judged by the transparency of the solution at this time. Finally, add P3 solution and gently invert to neutralize until a white flocculent precipitate appears. Centrifuge at room temperature. S3. Add BA solution to the silica gel membrane centrifuge column to achieve column equilibration, centrifuge and discard the filtrate, and pass the supernatant after the previous centrifugation directly through the column; S4. Then, ER solution is added to the silica membrane centrifuge column to remove endotoxins. The endotoxin content is less than 0.05 EU / μg. Finally, protein removal, impurity removal and elution are performed, and the plasmid is finally collected. The plasmid concentration is found to be above 200 ng / μL.

[0032] Protein removal was performed using a PR solution consisting of 5 M guanidine hydrochloride, 50 mM Tris-HCl (tris(hydroxymethyl)aminomethane) hydrochloride, and 30% isopropanol (v / v); impurity removal was performed using a solution of 25 mM Tris-HCl, 250 mM NaCl, and 80% anhydrous ethanol; and elution was performed using a solution of 20 mM Tris-HCl and 0.1% mM EDTA·2Na.

[0033] After resuspending the solution with solution P1, the solution was turbid. After adding solution P2 and allowing it to stand, the solution became clear and transparent. Observe the transparency of the solution before and after the improved method. Figure 2 Further determination of the OD of the solution 600 Values ​​are shown in Table 1. After the improved method, the solution has higher transparency and OD values. 600 A lower value indicates more complete pyrolysis and higher pyrolysis efficiency.

[0034] Table 1 Solution 0D 600 contrast

[0035] By modifying the existing solution formulation and operating procedures, the concentration of extracted plasmids was increased by more than 90% compared to existing technologies, as shown in Table 2. The endotoxin content of the extracted plasmids was reduced by 65% ​​compared to existing technologies, as shown in Table 3.

[0036] Table 2 Results of plasmid concentration determination

[0037] Note: Serial numbers 0, 1, and 2 represent the measurement results (three replicates) of plasmids extracted using the improved method; serial numbers 3, 4, and 5 represent the measurement results (three replicates) of plasmids extracted using the traditional method. A260 / 280 is used to assess whether the sample is contaminated with protein and phenol; the normal range is 1.80-2.00. A260 / 230 assesses contamination with salt, sugar, and organic solvents; the normal range is 2.00-2.20. Within the normal range, a higher A260 / 230 ratio indicates a purer sample.

[0038] Table 3. Results of Endotoxin Level Measurement

[0039] Figure 2 The solution states after P2 pyrolysis are shown in both the conventional method and the improved method of this invention. Note: The left side of the figure shows extraction using the traditional method; the right side shows extraction using the improved method of this invention.

[0040] Conclusion: Compared with the traditional method, the improved method of this invention has higher transparency after adding lysis buffer.

[0041] Figure 3 The images show the detection results of plasmid NaneDrop extracted by the conventional method and the improved method of this invention. Note: IDs 2, 3, and 4 in the figure represent the detection results of plasmids extracted using traditional methods; IDs 5, 6, and 7 represent the detection results of plasmids extracted using traditional methods. Conclusion: Compared with traditional methods, the plasmid concentration extracted by this invention is effectively improved, from about 101 ng / μL to about 204 ng / μL; the plasmid purity is also higher within the acceptable range.

[0042] Figure 4 The images show the nucleic acid electrophoresis results of plasmid extraction using traditional methods and the improved method of this invention.

[0043] Note: From left to right: Marker (100-500 bp), plasmid extracted by the improved method of this invention (3 parallels), plasmid extracted by the traditional method (3 parallels), Marker (100-500 bp).

[0044] Conclusion: With the same sample loading amount, the improved method produces brighter plasmid bands than the traditional method, which corresponds to the higher plasmid concentration obtained by the improved method using NanoDrop detection compared to the traditional method (Table 2).

[0045] Advantages of this invention: 1. Significantly improved lysis efficiency: The composite formulation of P2 lysis buffer allows for more complete lysis of bacterial colonies. From a morphological perspective, the optimized formulation results in a more transparent solution after lysis; numerically, the OD value of the lysis solution is higher. 600 The value decreased from 0.4 in the traditional method to 0.09, with no unlysed bacterial nuclei remaining, laying the foundation for high plasmid yield; 2. Significantly improved column adsorption efficiency: Time sequence reconstruction and BA solution formulation optimization avoid low adsorption efficiency caused by problems such as packing material sedimentation and equilibrium liquid volatilization, and the silica membrane is more fully activated, resulting in a significant improvement in plasmid adsorption efficiency compared to traditional methods; 3. Synergistic improvement in plasmid yield and purity: Compared with traditional methods, the plasmid concentration extracted by this invention is increased from about 101 ng / μL to over 204 ng / μL, an increase of 102%; plasmid A260 / 280 reaches 1.80-2.00 and A260 / 230 reaches 2.00-2.20, both within the optimal purity range, free from protein, salt, and organic solvent contamination; 4. Significantly reduced endotoxin content: The targeted formulation of ER endotoxin removal solution reduces the endotoxin content of extracted plasmids from the traditional 0.1445 EU / μg to below 0.0496 EU / μg, a reduction of 65%, meeting the low endotoxin requirements in fields such as nucleic acid vaccines and gene therapy; 5. Simple operation and controllable cost: No additional equipment is required. Performance is improved only through formula optimization and time sequence reconstruction. There are no redundant operation steps, making it suitable for large-scale and routine plasmid extraction.

[0046] This invention is applicable to the efficient extraction of high-purity, low-endotoxin plasmid DNA and can be applied to research fields with stringent requirements for plasmid quality, such as nucleic acid vaccines and gene therapy.

[0047] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A plasmid extraction kit, characterized in that: This kit includes solutions P1, P2, P3, BA, and ER, with a volume ratio of P1:P2:P3:BA:ER = 1:1:1:0.3-0.5:0.1-0.

3. The components are as follows: P1 solution: 25-50 mM Tris-HCl, 10-20 mM EDTA·2Na, 100-150 μg / ml RNase A, 50-100 mM glucose; P2 solution: 200-250 mM NaOH, 1%-2% SDS, 0.5-1 mM EDTA, 0.3%-0.5% Laureth-9, 25-50 mM guanidine isothiocyanate; P3 solution: 4-5 M guanidine hydrochloride, 0.5-1.0 mM potassium acetate, glacial acetic acid; BA solution: 25-50 mM Tris-HCl, 100-150 mM NaCl, 1-5 mM EDTA·2Na, 0.01%-0.05% Laureth-9, 0.5-1 M guanidine isothiocyanate; ER solution: 10-20 mM Tris-HCl, 10-20 mM NaCl, 10%-20% Triton X-114, 0.01%-0.02% Alizarin Yellow R.

2. A plasmid extraction method based on efficient lysis and rapid column equilibration, characterized in that, Plasmid extraction was performed using the kit described in claim 1.

3. The plasmid extraction method based on efficient lysis and rapid column equilibration according to claim 2, characterized in that, Includes the following steps: S1. Weigh out solutions with a volume ratio of P1 solution:P2 solution:P3 solution:BA solution:ER solution = 1:1:1:0.3-0.5:0.1-0.3; S2. Centrifugation of bacterial culture: Place the sample bacterial culture in a centrifuge tube, centrifuge at room temperature, discard the supernatant, add P1 solution to fully resuspend the bacterial precipitate. The solution will be turbid. Then add P2 solution and gently invert to mix. Let it stand at room temperature to lyse until the solution is completely clear and transparent. The degree of lysis is judged by the transparency of the solution at this time. Finally, add P3 solution and gently invert to neutralize until a white flocculent precipitate appears. Centrifuge at room temperature. S3. Add BA solution to the silica gel membrane centrifuge column to achieve column equilibration, centrifuge and discard the filtrate, and pass the supernatant after the previous centrifugation directly through the column; S4. Then, ER solution is added to the silica membrane centrifuge column to remove endotoxins. The endotoxin content is less than 0.05 EU / μg. Finally, protein removal, impurity removal and elution are performed, and the plasmid is finally collected. The plasmid concentration is found to be above 200 ng / μL.

4. The plasmid extraction method based on efficient lysis and rapid column equilibration according to claim 3, characterized in that: P1 solution: 25-50 mM Tris-HCl, 10-20 mM EDTA·2Na, 100-150 μg / ml RNase A, 50-100 mM glucose; P2 solution: 200-250 mM NaOH, 1%-2% SDS, 0.5-1 mM EDTA, 0.3%-0.5% Laureth-9, 25-50 mM guanidine isothiocyanate; P3 solution: 4-5 M guanidine hydrochloride, 0.5-1.0 mM potassium acetate, glacial acetic acid; BA solution: 25-50 mM Tris-HCl, 100-150 mM NaCl, 1-5 mM EDTA·2Na, 0.01%-0.05% Laureth-9, 0.5-1 M guanidine isothiocyanate; ER solution: 10-20 mM Tris-HCl, 10-20 mM NaCl, 10%-20% Triton X-114, 0.01%-0.02% Alizarin Yellow R.

5. The plasmid extraction method based on efficient lysis and rapid column equilibration according to claim 3, characterized in that: In step S2, the addition of P2 solution for lysis is based on the solution's transparency and OD. 600 The value is used to determine whether the cleavage is sufficient.

6. The plasmid extraction method based on efficient lysis and rapid column equilibration according to claim 5, characterized in that: OD of the solution after lysis 600 The value dropped to below 0.

1.

7. The plasmid extraction method based on efficient lysis and rapid column equilibration according to claim 3, characterized in that: The protein removal process uses a PR solution consisting of 4-5 M guanidine hydrochloride, 25-50 mM Tris-HCl, and 20%-30% isopropanol (by volume).

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