Plasmid extraction kit and extraction method

Abstract

The invention discloses a plasmid extraction kit and an extraction method. The plasmid extraction kit comprises a resuspension solution, a lysis solution, a neutralizing solution, a cleaning solution and an eluent, the resuspension is a Tris-HCl aqueous solution containing EDTA (Ethylene Diamine Tetraacetic Acid), glucose, ribonuclease, lysozyme and polyethylene glycol hexadecyl ether. The invention also discloses a plasmid extraction method which comprises the following steps: centrifuging a bacterial liquid of gram-negative bacteria containing plasmids, collecting thalli, and discarding a culture medium; re-suspending: adding a re-suspending solution, fully re-suspending, uniformly mixing, and incubating; sequentially carrying out steps of splitting decomposition, neutralization, purification, cleaning, centrifugal idling and elution to obtain a clarified solution containing plasmids. According to the method disclosed by the invention, the polyethylene glycol hexadecyl ether is added into the resuspension, and incubation is performed, so that the plasmid extraction amount is greatly increased, and the loss of plasmid extraction in subsequent links is reduced.

Description

Technical Field

[0001] This invention relates to the field of plasmid extraction technology, and more specifically, to a plasmid extraction kit and extraction method. Background Technology

[0002] Plasmid DNA serves as a core carrier and tool in biotechnology fields such as genetic engineering, molecular cloning, gene therapy, and vaccine development. Its efficient, high-quality, and stable preparation is a crucial prerequisite for successful downstream applications. Plasmid extraction is a routine experimental step for isolating and purifying plasmid DNA from host cells (such as the common E. coli).

[0003] Currently, commercially available plasmid extraction kits based on the alkaline lysis method have become the primary means of obtaining plasmid DNA in laboratories due to their relatively simple operation, high throughput, and good purification effect. Typical commercial kits usually include lysis buffer (containing SDS and NaOH), neutralization buffer (containing high concentration of potassium acetate / sodium acetate), binding buffer, washing buffer, and elution buffer, and rely on solid-phase carriers such as silica gel membrane centrifuge columns or magnetic microspheres for DNA adsorption, washing, and elution.

[0004] However, existing mainstream plasmid extraction technologies, including widely used commercial kits, still suffer from significant plasmid DNA loss during plasmid extraction, resulting in insufficient plasmid DNA extraction. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a plasmid extraction kit and extraction method. By optimizing the composition of the plasmid extraction kit, the plasmid extraction yield is significantly increased, and plasmid loss is reduced.

[0006] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a plasmid extraction kit, comprising a resuspension, a lysis buffer, a neutralization buffer, a washing buffer, and an elution buffer; wherein the resuspension is a Tris-HCl aqueous solution comprising EDTA, glucose, ribonuclease, lysozyme, and polyethylene glycol hexadecyl ether.

[0007] As some specific embodiments of the present invention, in the resuspension, the concentration of EDTA is 10~100 mM, the concentration of glucose is 100~1000 mM, the concentration of ribonuclease is 5~10 mg / ml, the concentration of lysozyme is 1~5 mg / ml, the mass-volume fraction of polyethylene glycol hexadecyl ether is 0.5~1%, the concentration of Tris-HCl is 10~50 mM, and the pH of the resuspension is 8.0~8.5.

[0008] As some specific embodiments of the present invention, the lysis solution includes an alkali and SDS; the alkali is selected from NaOH or KOH, the concentration of the alkali is 10~100 mM, and the mass-volume fraction of the SDS is 0.1~1%.

[0009] As some specific embodiments of the present invention, the neutralizing solution includes guanidine hydrochloride and potassium acetate, wherein the concentration of guanidine hydrochloride is 1~4 M, the concentration of potassium acetate is 100~800 mM, and the pH value of the neutralizing solution is 4.5~5.0.

[0010] As some specific embodiments of the present invention, the cleaning solution is a mixed aqueous solution of ethanol and Tris-HCl, wherein the volume fraction of ethanol is 75-80%, the concentration of Tris-HCl is 10-20 mM, and the pH value of the cleaning solution is 7.5-8.0.

[0011] As some specific embodiments of the present invention, the eluent is a Tris-HCl aqueous solution containing EDTA, wherein the concentration of EDTA is 1~5 mM, the concentration of Tris-HCl is 10~50 mM, and the pH value of the eluent is 7.5~8.0.

[0012] As some specific embodiments of the present invention, the kit also includes a silica membrane centrifuge column.

[0013] Secondly, the present invention provides a plasmid extraction method, which uses the kit described in any one of the above claims to extract plasmids from Gram-negative bacteria, specifically including the following steps: S1. Centrifuge the bacterial culture containing plasmids of Gram-negative bacteria, collect the bacterial cells, and discard the culture medium; S2, Resuspension: Add resuspension solution, resuspend thoroughly and mix well, then incubate. S3, pyrolysis: Add pyrolysis solution, gently invert to mix, and let stand until the solution is clear and transparent; S4. Neutralization: Add neutralizing solution, invert and mix well until a white flocculent precipitate appears, centrifuge and take the supernatant. S5. Purification: Add the supernatant to a silica gel membrane centrifuge column, centrifuge, and discard the liquid in the centrifuge tube; S6. Cleaning: Inject the cleaning solution into the silica membrane centrifuge column, centrifuge, and discard the liquid in the centrifuge tube; S7. Centrifugal idling; S8. Elution: Inject elution buffer into the silica gel membrane centrifuge column, and centrifuge to obtain a clear solution containing plasmids.

[0014] As some specific embodiments of the present invention, in step S1, the Gram-negative bacteria are Gram-negative bacteria that have been transformed with plasmids and cultured for 16-20 hours.

[0015] As some specific embodiments of the present invention, in step S1, the centrifugal force is 12000-13000g and the time is 1-5 min.

[0016] As some specific embodiments of the present invention, in steps S4 and / or S5 and / or S6 and / or S7 and / or S8, the centrifugal force is 12000-13000 g and the time is 1-3 min.

[0017] As some specific embodiments of the present invention, in step S2, the incubation temperature is 37°C and the time is 30~45 min; in step S2, the resuspension and bacterial cells are mixed by vortex oscillation.

[0018] The resuspension is used to suspend bacterial cells and maintain pH and osmotic pressure. The resuspension of this invention includes polyethylene glycol hexadecyl ether Brij-58, which effectively dissolves cell and nuclear membranes while maintaining the activity and function of membrane proteins. Through incubation, the cell membrane is in its natural "fluid" state, and the physical properties of lipids and proteins are consistent with the in vivo environment. This allows Brij-58 to interact with the membrane in the most efficient and controllable manner.

[0019] This method employs an alkaline lysis technique. First, the bacterial cells are resuspended and incubated with a resuspension containing polyethylene glycol hexadecyl ether. Then, the bacterial cells are lysed using NaOH and SDS, causing denaturation of chromosomal DNA and proteins, while plasmid DNA is retained due to its structural stability. After neutralization, the chromosomal DNA and proteins form a precipitate, while the plasmid DNA remains in the supernatant. The supernatant is then passed through a silica gel centrifuge column. Under high-salt conditions, the plasmid DNA specifically adsorbs onto the membrane, impurities are washed away, and finally, the purified plasmid is eluted with a low-salt buffer.

[0020] Compared with the prior art, the present invention has the following beneficial effects: This invention improves upon existing plasmid extraction kits and methods by adding polyethylene glycol hexadecyl ether to the resuspension and incubating it, which greatly increases the plasmid extraction yield and reduces plasmid loss in subsequent steps. Attached Figure Description

[0021] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 The image shows agarose gel electrophoresis images of plasmid DNA extracted from the kits used in Examples 1 and Comparative Examples 1 to 4. Detailed Implementation

[0022] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0023] This method uses alkaline lysis to extract plasmid DNA. First, bacterial cells are resuspended in a resuspension solution and incubated. Then, the bacterial cells are lysed using NaOH and SDS, causing denaturation of chromosomal DNA and proteins, while plasmid DNA is retained due to its structural stability. After neutralization, chromosomal DNA and proteins form a precipitate, while plasmid DNA remains in the supernatant. Adsorption-elution is then performed, specifically passing the supernatant through a silica gel membrane centrifuge column. Under high-salt conditions, plasmid DNA specifically adsorbs onto the membrane, impurities are washed away, and finally, the purified plasmid is eluted with a low-salt buffer.

[0024] Example 1 1. This embodiment provides a plasmid extraction kit, comprising: (1) P1 resuspension: 10mM EDTA, 100mM glucose, 5mg / ml ribonuclease, 10mM Tris-HCl, 0.5% (w / v) Brij-58 (polyethylene glycol hexadecyl ether), 1mg / mL Lysozyme (lysozyme), pH 8.0.

[0025] (2) P2 lysis buffer: 100mM KOH, 1% (w / v) SDS.

[0026] (3) N3 neutralization solution: 4M guanidine hydrochloride, 800mM potassium acetate, pH 5.0.

[0027] (4) PB cleaning solution: 20mM Tris-HCl, 80% (v / v) ethanol, pH 7.5.

[0028] (5) EB elution buffer: 1 mM EDTA, 10 mM Tris-HCl, pH 7.5.

[0029] (6) The kit also includes a silica membrane centrifuge column.

[0030] 2. Plasmid extraction using the kit described above according to the present invention specifically includes the following steps: (1) Take 0.2 mL of Escherichia coli culture that has been transformed with pCCL-PGK-EGFP plasmid and cultured for 18 h, centrifuge at 12000 g for 3 min, collect the bacterial cells, and discard the culture medium; (2) Add 150 μL of P1 resuspension, vortex to mix, and incubate at 37°C for 30 minutes; (3) Add 150 μL of P2 lysis buffer, gently invert to mix, and let stand for 1-2 min until the solution is clear and transparent; (4) Add 350 μL of N3 neutralization solution, invert and mix well, a white flocculent precipitate appears, centrifuge at 13000g for 2 min and take the supernatant; (5) Inject the supernatant into a silica gel membrane centrifuge column and centrifuge at 13000g for 1 min, then discard the liquid in the centrifuge tube; (6) After injecting 500 μL of PB cleaning solution into the silica gel membrane centrifuge column, centrifuge at 13000g for 1 min and discard the liquid in the centrifuge tube; (7) Centrifuge at 13000g for 3 minutes; (8) After injecting 100 μL of EB elution buffer into the silica gel membrane centrifuge column, centrifuge at 13000g for 1 min. A clear solution containing plasmids will be obtained in the centrifuge tube.

[0031] Comparative Example 1 1. This comparative example provides a plasmid extraction kit, which specifically includes the following components: (1) P1 resuspension: 10mM EDTA, 50mM Tris-HCl, pH 8.0.

[0032] (2) P2 lysis buffer: 200mM NaOH, 1% (w / v) SDS.

[0033] (3) N3 neutralization solution: 4.2M guanidine hydrochloride (GuHCl), 0.9M potassium acetate, pH 4.8.

[0034] (4) PB cleaning solution: 5M guanidine hydrochloride, 30% (v / v) ethanol, pH 5.

[0035] (5) EB elution buffer: 10 mM Tris–HCl, 80% (v / v) ethanol, pH 7.5.

[0036] (6) The kit also includes a silica membrane centrifuge column.

[0037] 2. Plasmid extraction was performed on the same E. coli strain as in Example 1 using the kit of this comparative example, specifically including the following steps: (1) Take 0.2 mL of Escherichia coli culture that has been transformed into pCCL-PGK-EGFP plasmid and cultured for 18 h, centrifuge at 12000 g for 3 min, collect the bacterial cells, and discard the culture medium; (2) Add 250 μL of P1 resuspension and vortex to mix; (3) Add 250 μL of P2 lysis buffer, gently invert to mix, and let stand for 1-2 min until the solution is clear and transparent; (4) Add 350 μL of N3 neutralization solution, invert and mix well, a white flocculent precipitate appears, centrifuge at 13000g for 2 min and take the supernatant; (5) Add the supernatant to a silica gel membrane centrifuge column and centrifuge at 13000g for 1 min, then discard the liquid in the centrifuge tube; (6) Add 750 μL of PB cleaning solution to the silica gel membrane centrifuge column and centrifuge at 13000g for 1 min, then discard the liquid in the centrifuge tube; (7) Centrifuge at 13000g for 3 minutes; (8) Add 100 μL of EB elution buffer to the silica gel membrane centrifuge column and centrifuge at 13000g for 1 min. A clear solution containing plasmids will be obtained in the centrifuge tube.

[0038] Comparative Example 2 1. This comparative example provides a plasmid extraction kit, which specifically includes the following components: (1) P1 resuspension: 10mM EDTA, 50mM glucose, 100μg / ml RNase A, 25mM Tris-HCl, pH 8.0.

[0039] (2) P2 lysis buffer: 250mM NaOH, 1% (w / v) SDS.

[0040] (3) N3 neutralization solution: 3M potassium acetate, 5M acetic acid, pH 5.0.

[0041] (4) PB cleaning solution: 10mM Tris-HCl, 80% (v / v) ethanol, pH 7.5.

[0042] (5) EB elution buffer: 10 mM Tris–HCl, pH 7.5.

[0043] (6) The kit also includes a silica membrane centrifuge column.

[0044] 2. Plasmid extraction was performed on the same E. coli strain as in Example 1 using the kit of this comparative example, specifically including the following steps: (1) Take 0.2 mL of Escherichia coli culture that has been transformed into pCCL-PGK-EGFP plasmid and cultured for 18 h, centrifuge at 12000 g for 3 min, collect the bacterial cells, and discard the culture medium; (2) Add 250 μL of P1 resuspension and vortex to mix; (3) Add 250 μL of P2 lysis buffer, gently invert to mix, and let stand for 1-2 min until the solution is clear and transparent; (4) Add 350 μL of N3 neutralization solution, invert and mix well, a white flocculent precipitate appears, centrifuge at 13000g for 2 min and take the supernatant; (5) Add the supernatant to a silica gel membrane centrifuge column and centrifuge at 13000g for 1 min, then discard the liquid in the centrifuge tube; (6) Add 750 μL of PB cleaning solution to the silica gel membrane centrifuge column and centrifuge at 13000g for 1 min, then discard the liquid in the centrifuge tube; (7) Centrifuge at 13000g for 3 minutes; (8) Add 100 μL of EB elution buffer to the silica gel membrane centrifuge column and centrifuge at 13000g for 1 min. A clear solution containing plasmids will be obtained in the centrifuge tube.

[0045] Comparative Example 3 Compared with Example 1, this comparative example is identical to Example 1 except that Brij-58 is missing from the resuspension and no incubation is performed in the resuspension step.

[0046] 1. This comparative example provides a plasmid extraction kit, comprising: (1) P1 resuspension: 10mM EDTA, 100mM glucose, 5mg / ml ribonuclease, 10mM Tris-HCl, 1mg / mL Lysozyme, pH 8.0.

[0047] (2) P2 lysis buffer: 100 mM KOH, 1% (w / v) SDS.

[0048] (3) N3 neutralization solution: 4M guanidine hydrochloride, 800mM potassium acetate, pH 5.0.

[0049] (4) PB cleaning solution: 20mM Tris-HCl, 80% (v / v) ethanol, pH 7.5.

[0050] (5) EB elution buffer: 1 mM EDTA, 10 mM Tris-HCl, pH 7.5.

[0051] (6) The kit also includes a silica membrane centrifuge column.

[0052] 2. Plasmid extraction using the above-described kit of the present invention specifically includes the following steps: (1) Take 0.2 mL of Escherichia coli culture that has been transformed into pCCL-PGK-EGFP plasmid and cultured for 18 h, centrifuge at 12000g for 3 min, collect the bacterial cells, and discard the culture medium; (2) Add 150 μL of P1 resuspension and vortex to mix. (3) Add 150 μL of P2 lysis buffer, gently invert to mix, and let stand for 1-2 min until the solution is clear and transparent; (4) Add 350 μL of N3 neutralization solution, invert and mix well, a white flocculent precipitate appears, centrifuge at 13000g for 2 min and take the supernatant; (5) Inject the supernatant into a silica gel membrane centrifuge column and centrifuge at 13000g for 1 min, then discard the liquid in the centrifuge tube; (6) After injecting 500 μL of PB cleaning solution into the silica gel membrane centrifuge column, centrifuge at 13000g for 1 min and discard the liquid in the centrifuge tube; (7) Centrifuge at 13000g for 3 minutes; (8) After injecting 100 μL of EB elution buffer into the silica gel membrane centrifuge column, centrifuge at 13000g for 1 min. A clear solution containing plasmids will be obtained in the centrifuge tube.

[0053] Comparative Example 4 Compared with Example 1, this comparative example only omits incubation in the resuspension step; the kit components and other extraction steps are the same as in Example 1.

[0054] 1. This comparative example provides a plasmid extraction kit, comprising: (1) P1 resuspension: 10mM EDTA, 100mM glucose, 5mg / ml ribonuclease, 10mM Tris-HCl, 0.5% (w / v) Brij-58 (polyethylene glycol hexadecyl ether), 1mg / mL Lysozyme (lysozyme), pH 8.0.

[0055] (2) P2 lysis buffer: 100mM KOH, 1% (w / v) SDS.

[0056] (3) N3 neutralization solution: 4M guanidine hydrochloride, 800mM potassium acetate, pH 5.0.

[0057] (4) PB cleaning solution: 20mM Tris-HCl, 80% (v / v) ethanol, pH 7.5.

[0058] (5) EB elution buffer: 1 mM EDTA, 10 mM Tris-HCl, pH 7.5.

[0059] (6) The kit also includes a silica membrane centrifuge column.

[0060] 2. Plasmid extraction using the above-described kit of the present invention specifically includes the following steps: (1) Take 0.2 mL of Escherichia coli culture that has been transformed into pCCL-PGK-EGFP plasmid and cultured for 18 h, centrifuge at 12000g for 3 min, collect the bacterial cells, and discard the culture medium; (2) Add 150 μL of P1 resuspension and vortex to mix. (3) Add 150 μL of P2 lysis buffer, gently invert to mix, and let stand for 1-2 min until the solution is clear and transparent; (4) Add 350 μL of N3 neutralization solution, invert and mix well, a white flocculent precipitate appears, centrifuge at 13000g for 2 min and take the supernatant; (5) Inject the supernatant into a silica gel membrane centrifuge column and centrifuge at 13000g for 1 min, then discard the liquid in the centrifuge tube; (6) After injecting 500 μL of PB cleaning solution into the silica gel membrane centrifuge column, centrifuge at 13000g for 1 min and discard the liquid in the centrifuge tube; (7) Centrifuge at 13000g for 3 minutes; (8) After injecting 100 μL of EB elution buffer into the silica gel membrane centrifuge column, centrifuge at 13000g for 1 min. A clear solution containing plasmids will be obtained in the centrifuge tube.

[0061] Example 1 The plasmids extracted in Example 1 and Comparative Examples 1-4 were simultaneously analyzed by agarose gel electrophoresis, and the results are as follows: Figure 1 As shown. Figure 1 The six lanes in the middle, from left to right, are: Marker, Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4. The results showed that plasmids were successfully extracted from the examples and all comparative examples, with high plasmid purity and no obvious contamination.

[0062] Example 2 The plasmid extraction yields of the above embodiments and comparative examples were statistically analyzed, and the results are shown in Table 1 below: Table 1. Plasmid Extraction Amount

[0063] According to the results in the table above, it can be seen that the plasmid extraction yield of Example 1 is significantly higher than that of the other parallel examples, with higher unit yield and less plasmid loss during the extraction process.

[0064] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A plasmid extraction kit, characterized in that, It includes a resuspension, a lysis buffer, a neutralization buffer, a washing buffer, and an elution buffer; the resuspension is an aqueous solution of Tris-HCl containing EDTA, glucose, ribonuclease, lysozyme, and polyethylene glycol hexadecyl ether.

2. The plasmid extraction kit according to claim 1, characterized in that, In the resuspension, the concentration of EDTA is 10-100 mM, the concentration of glucose is 100-1000 mM, the concentration of ribonuclease is 5-10 mg / ml, the concentration of lysozyme is 1-5 mg / ml, the mass-volume fraction of polyethylene glycol hexadecyl ether is 0.5-1%, the concentration of Tris-HCl is 10-50 mM, and the pH of the resuspension is 8.0-8.

5.

3. The plasmid extraction kit according to claim 1, characterized in that, The lysis buffer comprises an alkali and SDS; the alkali is selected from NaOH or KOH, the concentration of the alkali is 10~100 mM, and the mass-volume fraction of the SDS is 0.1~1%.

4. The plasmid extraction kit according to claim 1, characterized in that, The neutralizing solution comprises guanidine hydrochloride and potassium acetate, wherein the concentration of guanidine hydrochloride is 1-4 M, the concentration of potassium acetate is 100-800 mM, and the pH value of the neutralizing solution is 4.5-5.

0.

5. The plasmid extraction kit according to claim 1, characterized in that, The cleaning solution is a mixed aqueous solution of ethanol and Tris-HCl, wherein the volume fraction of ethanol is 75-80%, the concentration of Tris-HCl is 10-20 mM, and the pH value of the cleaning solution is 7.5-8.

0.

6. The plasmid extraction kit according to claim 1, characterized in that, The eluent is an aqueous solution of Tris-HCl containing EDTA, wherein the concentration of EDTA is 1-5 mM, the concentration of Tris-HCl is 10-50 mM, and the pH of the eluent is 7.5-8.

0.

7. The plasmid extraction kit according to claim 1, characterized in that, The kit also includes a silica membrane centrifuge column.

8. A method for plasmid extraction, characterized in that, Plasmid extraction from Gram-negative bacteria using the kit described in any one of claims 1-7 includes the following steps: S1. Centrifuge the bacterial culture containing plasmids of Gram-negative bacteria, collect the bacterial cells, and discard the culture medium; S2, Resuspension: Add resuspension solution, resuspend thoroughly and mix well, then incubate. S3, pyrolysis: Add pyrolysis solution, gently invert to mix, and let stand until the solution is clear and transparent; S4. Neutralization: Add neutralizing solution, invert and mix well until a white flocculent precipitate appears, centrifuge and take the supernatant. S5. Purification: Add the supernatant to a silica gel membrane centrifuge column, centrifuge, and discard the liquid in the centrifuge tube; S6. Cleaning: Inject the cleaning solution into the silica membrane centrifuge column, centrifuge, and discard the liquid in the centrifuge tube; S7. Centrifugal idling; S8. Elution: Inject elution buffer into the silica gel membrane centrifuge column, and centrifuge to obtain a clear solution containing plasmids.

9. The plasmid extraction method according to claim 8, characterized in that, In step S1, the centrifugal force is 12000-13000 g and the time is 1-5 min; In steps S4 and / or S5 and / or S6 and / or S7 and / or S8, the centrifugal force is 12000-13000 g and the time is 1-3 min.

10. The plasmid extraction method according to claim 8, characterized in that, In step S2, the incubation temperature is 37°C and the incubation time is 30~45 min.

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