Endotoxin-removing plasmid extraction kit and application thereof

Abstract

The invention discloses a magnetic bead method endotoxin-free plasmid extraction kit, relates to the technical field of plasmid extraction and endotoxin removal, including: solution I, solution II, solution III, magnetic bead liquid I, magnetic bead liquid II, cleaning solution and eluent; solution I includes: sucrose, EDTA, Tris-HCl, NaCl; solution II includes: KO H, SDS, EDTA; solution III includes: potassium acetate, sodium acetate, trehalose, pH is 5-5.6; magnetic bead liquid I includes: NaCl, sodium citrate, magnetic beads I, pH is 5.0-5.5; magnetic bead liquid II includes: guanidine hydrochloride, urea, anhydrous ethanol, isopropanol, magnetic beads II, pH is 5.5-6.5. The present invention prepares an endotoxin-free plasmid extraction kit based on a double magnetic bead method, which has the advantages of convenience, low cost and good safety, can remove endotoxins in the plasmid extraction process, and can also be used to directly remove endotoxins in plasmids, and the obtained plasmids can meet the requirements of molecular biology experiments and cell experiments such as enzyme cutting, sequencing, transfection and virus packaging. At the same time, full automation of endotoxin-free plasmid extraction can be achieved.

Description

Technical Field

[0001] The invention relates to the technical field of plasmid extraction and endotoxin removal, and more specifically to an endotoxin-removing plasmid extraction kit and application thereof. Background Art

[0002] Plasmid is a circular double-stranded small molecule DNA that can replicate independently and be stably inherited. It is also the most commonly used genetic engineering vector. The extraction and purification of plasmid DNA is an important prerequisite for genetic engineering. Therefore, efficient and rapid purification of plasmids is of great significance. The methods of plasmid separation widely used in laboratories at present mainly include silicon matrix membrane adsorption separation method and magnetic bead adsorption method. The plasmid extracted by silicon matrix membrane adsorption separation method has high purity, but it needs to be centrifuged many times and the amount of plasmid extracted is small. The magnetic bead method is a new separation and purification technology developed based on magnetic nanomaterials. It can efficiently adsorb DNA, is simple and fast to operate, and is suitable for large-scale extraction of DNA samples. The matching automated nucleic acid extractor can realize automation and high throughput of nucleic acid extraction.

[0003] Endotoxin, also known as lipopolysaccharide, is a component of the cell membrane of Gram-negative bacteria. Its molecular weight, charge and hydrophobicity are similar to those of plasmid DNA. When Gram-negative bacterial cells are lysed, the endotoxin on the cell wall surface will be released and enter the solution. When the plasmid is extracted, it will be mixed with the plasmid and extracted. Endotoxin will reduce the transfection efficiency of primary cells and sensitive cultured cells and interfere with the in vitro transfection of immune cells.

[0004] Endotoxin has extremely strong thermal stability and chemical stability. To completely inactivate endotoxin, it needs to be dry-baked at 250°C for 1 hour or soaked in strong acid or strong alkali for more than 4 hours. In the medical field, distillation + high temperature inactivation is usually used. However, the above technical means will cause the plasmid to denature and inactivate. Other reported methods for removing endotoxin include ultracentrifugation, ethanol graded precipitation, chromatography, activated carbon adsorption, resin adsorption, TritonX-114 extraction, etc. At present, the commonly used methods for removing endotoxin from plasmids are TritonX-114 fog point method and chromatography method. The TritonX-114 fog point method is time-consuming, the concentration of the obtained plasmid is low, and toxic TritonX-114 will remain; the level of endotoxin removal by chromatography can reach the clinical level, but the cost is high and the plasmid concentration is also low. Therefore, it is necessary to improve and improve the existing technology.

[0005] Therefore, how to effectively remove endotoxins when extracting plasmids is an urgent problem that technicians in this field need to solve. Summary of the invention

[0006] In view of this, the present invention provides a double magnetic bead method endotoxin removal plasmid extraction kit and application.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A magnetic bead method endotoxin removal plasmid extraction kit, comprising: solution I, solution II, solution III, magnetic bead liquid I, magnetic bead liquid II, cleaning solution and eluent;

[0009] Wherein, solution I comprises: 1-2% sucrose, 40-75mmol / L EDTA, 25-50mmol / L Tris-HCl, 0.5-1.0mol / L NaCl, pH 8.0;

[0010] Solution II includes: 0.1mol / L-0.3mol / L KOH, 0.08%-0.15% by volume of S DS, and 0.02-0.1mol / L EDTA;

[0011] Solution III includes: 1-2 mol / L potassium acetate, 1-2 mol / L sodium acetate, 0.2-0.6 mol / L trehalose, pH 5-5.6;

[0012] Magnetic bead solution I includes: 0.5-1.0 mol / L NaCl, 0.05-0.1 mol / L sodium citrate, 30-50 mg / mL magnetic beads I, pH 5.0-5.5;

[0013] The magnetic bead solution II includes: 1.2-1.8 mol / L guanidine hydrochloride, 0.01-0.05 mol / L urea, 20-30% anhydrous ethanol by volume, 30-40% isopropanol by volume, 20-30 mg / mL magnetic bead II, and a pH value of 5.5-6.5.

[0014] In the above technical solution, the magnetic beads I are magnetic microspheres with nickel magnetic beads as carriers and polyhistidine as ligands, and the particle size is 0.5 μm to 1 μm.

[0015] In the above technical solution, the magnetic bead liquid II is monodisperse, superparamagnetic hydroxyl magnetic beads with a particle size of 200 to 500 nm.

[0016] In the above technical solution, the cleaning solution is an ethanol solution with a volume fraction of 60-75%; the eluent is 10-15 mmol / L Tris-HCl, pH 7.0-8.0.

[0017] As the same inventive concept as the present invention, the present invention also requests protection for the use of the kit in extracting plasmids and removing endotoxins.

[0018] As the same inventive concept as the present invention, the present invention also claims a method for extracting plasmids by removing endotoxins using a magnetic bead method, comprising the following steps:

[0019] (1) 1% plasmid-containing E. coli cells were cultured in LB medium at 37°C with shaking overnight;

[0020] (2) Take 1 mL of bacterial cells and add them to a centrifuge tube. Centrifuge at 12000 rpm for 30 seconds and discard the supernatant.

[0021] (3) The recovered bacterial cells were resuspended in 0.1 mL of Solution I and shaken vigorously to mix thoroughly.

[0022] (4) Add 0.2 mL of solution II and gently invert 5 to 8 times until the solution becomes transparent. Let stand at room temperature for 2 to 3 minutes.

[0023] (5) Add 0.15 mL of solution III and immediately mix by gently inverting for 5 to 10 times until no more white flocculent precipitate is formed. Let stand at room temperature for 1 min.

[0024] (6) Centrifuge at 12000 rpm for 5 min, take the supernatant and transfer it to a new centrifuge tube;

[0025] (7) Add 30 μL of magnetic bead solution I to the centrifuge tube containing the supernatant, mix well by pipetting, and place at room temperature for 2 to 3 minutes. Then place the centrifuge tube on a magnetic rack for 1 to 2 minutes and perform magnetic separation until the supernatant is completely clear. Take the supernatant and transfer it to a new centrifuge tube.

[0026] (9) Add 100 μL of magnetic bead solution II to the centrifuge tube, mix well by pipetting, and place at room temperature for 2 to 3 min. Then place the centrifuge tube on the magnetic rack. After the magnetic beads are completely absorbed to the tube wall, use a pipette to discard the supernatant and remove the centrifuge tube from the magnetic rack.

[0027] (10) Add 1 mL of 70% ethanol to the centrifuge tube and mix thoroughly by pipetting. Place the centrifuge tube on a magnetic rack for 1 min to allow the magnetic beads to be completely absorbed by the tube wall. Discard the supernatant and remove the centrifuge tube from the magnetic rack. Dry at room temperature for 5 to 8 min until no liquid remains in the centrifuge tube.

[0028] (11) Add 50-100 μL of TE Buffer to the centrifuge tube and mix gently with a pipette. Elute at room temperature for 5 min, then place on a magnetic rack for 1-2 min. After the magnetic beads are completely absorbed to the wall of the centrifuge tube, carefully pipette the supernatant into a new centrifuge tube to obtain the plasmid DNA product.

[0029] It can be seen from the above technical solutions that, compared with the prior art, the technical effects achieved by the present invention are:

[0030] 1) The magnetic beads I used in the present invention use nickel magnetic beads as carriers and polyhistidine as ligands. There are imidazole groups in the polyhistidine structural units on the magnetic beads I, which are all positively charged below the isoelectric point (pH6.5), and the endotoxin pKa value is about 1.3. In the buffer system provided by the magnetic bead liquid I, the polyhistidine magnetic beads can form a stable complex with the endotoxin molecules by electrostatic action, and the process of adsorbing endotoxin molecules is similar to the flocculation process, and does not adsorb plasmid DNA, and has good specificity. At the same time, the magnetic beads I have good biocompatibility, no toxic side effects on cells and human bodies, and have the advantages of low price and good safety. The present invention systematically investigates the effects of factors such as salt concentration, pH value, and magnetic bead dosage on the removal of endotoxins, optimizes the conditions for removing endotoxins, thereby achieving a good endotoxin removal effect, and can effectively remove endotoxins directly from the plasmid crude liquid.

[0031] 2) Solution III of the present invention is a reasonable compound system of potassium acetate, sodium acetate and trehalose, which can quickly neutralize the lysate, make the plasmid DNA quickly and effectively renature, and at the same time, it has excellent effects on the precipitation and separation of proteins, cell fragments and genomic DNA in the sample; trehalose is added to solution III, and alginic acid can not only effectively protect the plasmids in the supernatant (crude extract) obtained by centrifugation, but also prevent the magnetic bead solution I from adsorbing endotoxins while adsorbing plasmids, avoiding the loss of plasmids, thereby improving the plasmid yield, and realizing the removal of endotoxins in plasmids under mild conditions, without losing too much plasmids and without affecting the biological activity of the plasmids.

[0032] 3) The present invention adopts a double magnetic bead method to respectively realize the separation of endotoxin and the extraction of plasmid. The magnetic bead I can specifically adsorb endotoxin, thereby removing it; the magnetic bead liquid II contains magnetic beads II, which are used to bind plasmids, and cooperate with the unique buffer system of the present invention to prepare an endotoxin-free plasmid DNA extraction kit based on the double magnetic bead method, which has the advantages of convenience, low cost and good safety. The kit provided by the present invention can remove endotoxins during the plasmid extraction process, and can also be used to directly remove endotoxins in plasmids. The obtained plasmid can meet the requirements of molecular biology experiments and cell experiments such as enzyme cutting, sequencing, transfection and virus packaging. At the same time, full automation of endotoxin-free plasmid extraction can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0034] Figure 1The accompanying drawings are electrophoretic patterns of plasmid DNA extracted by the kit of the present invention; wherein lanes 1 and 2 are patterns of plasmid DNA extracted in Example 4; and lanes 3 and 4 are patterns of plasmid DNA extracted in Example 5;

[0035] Figure 2 The attached figure is the electrophoresis pattern of plasmid DNA of different concentrations extracted by the kit of Example 6; wherein 1, 2, and 3 are plasmids extracted from 0.5 mL, 1.5 mL, and 2.5 mL of bacteria, respectively. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Example 1

[0038] A magnetic bead method endotoxin removal plasmid DNA extraction kit comprises a solution I, a solution II, a solution III, a magnetic bead liquid I, a magnetic bead liquid II, a cleaning solution and an eluent.

[0039] Solution I: 1% sucrose, 75mmol / L EDTA (pH8.0), 25mmol / L Tris-HCl (pH8.0), 0.75mol / L NaCl;

[0040] Solution II: 0.2 mol / L KOH; 0.15% by volume SDS, 0.05 mol / L EDTA;

[0041] Solution III: 1.5 mol / L potassium acetate, 1 mol / L sodium acetate, 0.2 mol / L trehalose, pH 5.0;

[0042] Magnetic bead solution I: NaCl concentration is 0.8 mol / L, sodium citrate concentration is 0.05 mol / L, solution pH is 5.0, magnetic beads I are suspended therein, and the concentration of magnetic beads I is 30 mg / mL; magnetic beads I are magnetic microspheres with nickel magnetic beads as carriers and polyhistidine as ligands, and the particle size is 0.5 μm to 1 μm;

[0043] Magnetic bead solution II: Guanidine hydrochloride concentration is 1.2 mol / L, urea concentration is 0.05 mol / L, anhydrous ethanol volume ratio is 20%, isopropanol volume ratio is 35%, and the solution pH is 6.5. Magnetic beads II are suspended therein, the particle size of magnetic beads II is 1 μm, and the concentration of magnetic beads II is 20 mg / mL; magnetic beads II are monodisperse, superparamagnetic hydroxy magnetic beads, and the particle size is 200-500 nm;

[0044] Cleaning solution: 70% ethanol solution by volume.

[0045] Eluent: 10 mmol / L Tris-HCl, pH 7.0-8.0.

[0046] Example 2

[0047] A magnetic bead method endotoxin removal plasmid DNA extraction kit comprises a solution I, a solution II, a solution III, a magnetic bead liquid I, a magnetic bead liquid II, a cleaning solution and an eluent.

[0048] Solution I: 1.5% sucrose, 40 mmol / L EDTA (pH 8.0), 40 mmol / L Tris-HCl (pH 8.0), 1.0 mol / L NaCl.

[0049] Solution II: 0.3 mol / L KOH; 0.08% by volume SDS, 0.1 mol / L EDTA.

[0050] Solution III: 1 mol / L potassium acetate, 1.2 mol / L sodium acetate, trehalose concentration of 0.6 mol / L, pH 5.2.

[0051] Magnetic bead solution I: NaCl concentration is 0.5 mol / L, sodium citrate concentration is 0.08 mol / L, solution pH is 5.2, magnetic beads I are suspended therein, magnetic beads I concentration is 50 mg / mL; magnetic beads I concentration is 30 mg / mL; magnetic beads I are magnetic microspheres with nickel magnetic beads as carriers and polyhistidine as ligands, with a particle size of 0.5-1 μm;

[0052] Magnetic bead solution II: Guanidine hydrochloride concentration is 1.8 mol / L, urea concentration is 0.03 mol / L, anhydrous ethanol volume ratio is 30%, isopropanol volume ratio is 40%, and the solution pH value is 5.0. Magnetic beads II are suspended therein, the particle size of magnetic beads II is 0.5 μm, and the concentration of magnetic beads II is 25 mg / mL; magnetic beads II are monodisperse, superparamagnetic hydroxy magnetic beads, and the particle size is 200-500 nm;

[0053] Cleaning solution: 60% ethanol solution by volume.

[0054] Eluent: 12 mmol / L Tris-HCl, pH 7.0-8.0.

[0055] Example 3

[0056] A magnetic bead method endotoxin removal plasmid DNA extraction kit comprises a solution I, a solution II, a solution III, a magnetic bead liquid I, a magnetic bead liquid II, a cleaning solution and an eluent.

[0057] Solution I: 2% sucrose, 60 mmol / L EDTA (pH 8.0), 50 mmol / L Tris-HCl (pH 8.0), and 0.5 mol / L NaCl.

[0058] Solution II: 0.1 mol / L KOH; 0.1% by volume SDS, 0.02 mol / L EDTA.

[0059] Solution III: 2 mol / L potassium acetate, 2 mol / L sodium acetate, trehalose concentration of 0.4 mol / L, pH 5..6.

[0060] Magnetic bead solution I: NaCl concentration is 1.0 mol / L, sodium citrate concentration is 0.1 mol / L, and the solution pH is 5.5. Magnetic beads I are suspended therein, and the concentration of magnetic beads I is 40 mg / mL; magnetic beads I are magnetic microspheres with nickel magnetic beads as carriers and polyhistidine as ligands, and the particle size is 0.5 μm to 1 μm;

[0061] Magnetic bead solution II: Guanidine hydrochloride concentration is 1.5 mol / L, urea concentration is 0.01 mol / L, anhydrous ethanol volume ratio is 25%, isopropanol volume ratio is 30%, and the solution pH is 5.5. Magnetic beads II are suspended therein, the particle size of magnetic beads II is 0.8 μm, and the concentration of magnetic beads II is 30 mg / mL; magnetic beads II are monodisperse, superparamagnetic hydroxy magnetic beads, and the particle size is 200-500 nm;

[0062] Cleaning solution: 75% ethanol solution.

[0063] Eluent: 15mmol / L Tris-HCl, pH 7.0-8.0.

[0064] The monodisperse, superparamagnetic silica nanoparticles described in the above embodiments may be the magnetic beads disclosed in Patent No.: ZL201510488579.5, entitled: "Silica-coated ferroferric oxide nanoclusters as magnetic marker materials and preparation methods thereof".

[0065] Example 4: Extracting plasmid DNA using the kit prepared in Example 1

[0066] (1) 1% plasmid-containing E. coli cells were cultured in LB medium at 37°C with shaking overnight;

[0067] (2) Take 1 mL of bacterial cells and add them to a centrifuge tube. Centrifuge at 12000 r / min for 30 seconds and discard the supernatant.

[0068] (3) The recovered bacterial cells were resuspended in 0.1 mL of Solution I and shaken vigorously to mix thoroughly.

[0069] (4) Add 0.2 mL of solution II and gently invert 5 to 8 times until the solution becomes transparent. Let stand at room temperature for 2 to 3 minutes.

[0070] (5) Add 0.15 mL of solution III and immediately mix by gently inverting for 5 to 10 times until no more white flocculent precipitate is formed. Let stand at room temperature for 1 min.

[0071] (6) Centrifuge at 12000 rpm for 5 min, carefully take the supernatant and transfer part of it to a new centrifuge tube;

[0072] (7) Add 30 μL of magnetic bead solution I to the centrifuge tube containing the supernatant, mix well by pipetting, and place at room temperature for 2 to 3 min. Then place the centrifuge tube on a magnetic rack for 1 to 2 min and perform magnetic separation until the supernatant is completely clear. Carefully take the supernatant and transfer it to a new centrifuge tube.

[0073] (9) Add 100 μL of magnetic bead solution II to the centrifuge tube. Mix well by pipetting, and leave at room temperature for 2 to 3 minutes. Then place the centrifuge tube on the magnetic rack. After the magnetic beads are completely absorbed to the tube wall, use a pipette to discard the supernatant and remove the centrifuge tube from the magnetic rack.

[0074] (10) Add 1 mL of 70% ethanol to the centrifuge tube and mix thoroughly by pipetting. Place the centrifuge tube on a magnetic rack for 1 min to allow the magnetic beads to be completely absorbed by the tube wall. Discard the supernatant and remove the centrifuge tube from the magnetic rack. Dry at room temperature for 5 to 8 min until no liquid remains in the centrifuge tube.

[0075] (11) Add 100 μL of TE Buffer to the centrifuge tube and mix gently with a pipette. Elute at room temperature for 5 min, then place on a magnetic stand for 2 min. After the magnetic beads are completely absorbed to the wall of the centrifuge tube, carefully pipette the supernatant into a new centrifuge tube to obtain the plasmid DNA product.

[0076] (12) Agarose gel electrophoresis detection: 5 μL of the extracted plasmid DNA samples were loaded and subjected to 1% agarose gel electrophoresis. Figure 1 The middle lanes 1 and 2 are the plasmid DNA maps extracted by the kit and method of this example.

[0077] (13) The concentration and content of the obtained plasmid DNA were detected by ultraviolet spectrometer, and the endotoxin in the sample was detected using the horseshoe crab reagent produced by Xiamen Horseshoe Crab Reagent Biotechnology Co., Ltd. The results are shown in Table 1.

[0078] Example 5: Extracting plasmid DNA using the kit prepared in Example 3

[0079] (1) 1% E. coli cells containing the plasmid were cultured in LB medium at 37°C with shaking overnight.

[0080] (2) Take 0.5 mL of bacterial cells and add them to a centrifuge tube. Centrifuge at 12,000 rpm for 30 seconds and discard the supernatant.

[0081] (3) Resuspend the recovered bacterial cells in 0.1 mL of Solution I and shake vigorously to mix them thoroughly.

[0082] (4) Add 0.2 mL of Solution II and immediately invert gently 5 to 8 times until the solution becomes transparent. Let stand at room temperature for 2 to 3 minutes.

[0083] (5) Add 0.15 mL of Solution III and immediately mix by gently inverting for 5 to 10 times until no more white flocculent precipitate is formed. Let stand at room temperature for 1 min.

[0084] (6) Centrifuge at 12000 r / min for 5 min, carefully remove the supernatant and transfer part of the supernatant to a new centrifuge tube.

[0085] (7) Add 30 μL of magnetic bead solution I to the centrifuge tube containing the supernatant, mix well by pipetting, and leave at room temperature for 2 to 3 min. Then place the centrifuge tube on a magnetic rack for 1 to 2 min and magnetically separate until completely clear. Carefully remove the supernatant and transfer it to a new centrifuge tube.

[0086] (9) Add 100 μL of magnetic bead solution II to the centrifuge tube. Mix well by pipetting, and leave at room temperature for 2 to 3 min. Then place the centrifuge tube on the magnetic rack. After the magnetic beads are completely absorbed to the tube wall, use a pipette to discard the supernatant and remove the centrifuge tube from the magnetic rack.

[0087] (10) Add 1 mL of 70% ethanol to the centrifuge tube and mix thoroughly by pipetting. Place the centrifuge tube on a magnetic rack for 1 min to allow the magnetic beads to be completely absorbed by the tube wall. Discard the supernatant and remove the centrifuge tube from the magnetic rack. Dry the tube at room temperature for 5–8 min until no liquid remains in the tube.

[0088] (11) Add 100 μL of TE Buffer to the centrifuge tube and mix gently with a pipette. Elute at room temperature for 5 min, then place on a magnetic rack for 2 min. After the magnetic beads are completely absorbed to the wall of the centrifuge tube, carefully pipette the supernatant into a new centrifuge tube to obtain the plasmid DNA product.

[0089] (12) Agarose gel electrophoresis detection: 5 μL of the extracted plasmid DNA samples were loaded and subjected to 1% agarose gel electrophoresis. Figure 1 Lanes 3 and 4 in the middle are the plasmid DNA maps extracted by the kit and method of this example.

[0090] (13) The concentration and content of the obtained plasmid DNA were detected by ultraviolet spectrometer, and the endotoxin in the sample was detected using the horseshoe crab reagent detection reagent produced by Xiamen Horseshoe Crab Reagent Biotechnology Co., Ltd. The results are shown in Table 1. It can be observed from the table that the extracted plasmid DNA has a high concentration, and there is basically no interference and contamination of carbohydrates (sugars), salts or organic solvents in the extracted DNA, and the purity is high.

[0091] The concentration and content of the plasmid DNA extracted in Example 4 and Example 5 are shown in Table 1;

[0092] Table 1 Concentration and endotoxin content of plasmid DNA extracted from Examples 4 and 5

[0093]

[0094] Example 6 Kit for extracting plasmid DNA of different concentrations

[0095] (1) 1% E. coli cells containing the plasmid were cultured in LB medium at 37°C with shaking overnight.

[0096] (2) Take 0.5 mL, 1.5 mL, and 2.5 mL of bacterial cells respectively and add them to centrifuge tubes. Centrifuge at 12,000 rpm for 30 seconds and discard the supernatant.

[0097] (3) Resuspend the recovered bacterial cells in 0.1 mL of Solution I and shake vigorously to mix them thoroughly.

[0098] (4) Add 0.2 mL of Solution II and immediately invert gently 5 to 8 times until the solution becomes transparent. Let stand at room temperature for 2 to 3 minutes.

[0099] (5) Add 0.15 mL of Solution III and immediately mix by gently inverting for 5 to 10 times until no more white flocculent precipitate is formed. Let stand at room temperature for 1 min.

[0100] (6) Centrifuge at 12000 r / min for 5 min, carefully remove the supernatant and transfer part of the supernatant to a new centrifuge tube.

[0101] (7) Add 30 μL of magnetic bead solution I to the centrifuge tube containing the supernatant, mix well by pipetting, and leave at room temperature for 2 to 3 min. Then place the centrifuge tube on a magnetic rack for 1 to 2 min and magnetically separate until completely clear. Carefully remove the supernatant and transfer it to a new centrifuge tube.

[0102] (9) Add 100 μL of magnetic bead solution II to the centrifuge tube. Mix well by pipetting, and leave at room temperature for 2 to 3 min. Then place the centrifuge tube on the magnetic rack. After the magnetic beads are completely absorbed to the tube wall, use a pipette to discard the supernatant and remove the centrifuge tube from the magnetic rack.

[0103] (10) Add 1 mL of 70% ethanol to the centrifuge tube and mix thoroughly by pipetting. Place the centrifuge tube on a magnetic rack for 1 min to allow the magnetic beads to be completely absorbed by the tube wall. Discard the supernatant and remove the centrifuge tube from the magnetic rack. Dry the tube at room temperature for 5–8 min until no liquid remains in the tube.

[0104] (11) Add 60 μL of TE Buffer to the centrifuge tube and mix gently with a pipette. Elute at room temperature for 5 min, then place on a magnetic rack for 2 min. After the magnetic beads are completely absorbed to the wall of the centrifuge tube, carefully pipette the supernatant into a new centrifuge tube to obtain the plasmid DNA product.

[0105] (12) Agarose gel electrophoresis detection: 5 μL of the extracted plasmid DNA samples were loaded and subjected to 1% agarose gel electrophoresis. The results were as follows: Figure 2 shown.

[0106] Comparative Example 1

[0107] (1) 1% E. coli cells containing the plasmid were cultured in LB medium at 37°C with shaking overnight.

[0108] (2) Plasmid DNA was extracted using the kit of the present invention, the kit of Company A (ordinary plasmid mini-extraction kit (without endotoxin removal reagent)), and the kit of Company B (endotoxin-free plasmid extraction kit).

[0109] (3) The concentration and content of the obtained plasmid DNA were detected by ultraviolet spectrometer, and the endotoxin in the sample was detected by using the horseshoe crab reagent detection reagent produced by Xiamen Horseshoe Crab Reagent Biotechnology Co., Ltd. The results are shown in Table 2. The experimental results show that the concentration of the plasmid obtained by the present invention is higher than that of the commercially available kit, and the endotoxin content is the lowest.

[0110] Table 2 Comparison of the effects of different kits in extracting plasmid DNA

[0111]

[0112] Document CN107034212 A discloses a plasmid extraction kit and extraction method for removing endotoxins, which are essentially different from the present invention in terms of plasmid extraction method and extraction efficiency. For comparison, see Table 3:

[0113] Table 3

[0114]

[0115]

[0116] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0117] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A magnetic bead method for removing endotoxin plasmid extraction kit, characterized in that: include: Solution I, solution II, solution III, magnetic bead solution I, magnetic bead solution II, cleaning solution and elution solution; Wherein, solution I comprises: 1-2% sucrose, 40-75mmol / L EDTA, 25-50mmol / L Tris-HCl, 0.5-1.0mol / L NaCl, pH 8.0; Solution II includes: 0.1mol / L~0.3mol / L KOH, 0.08%~0.15% SDS by volume, and 0.02~0.1mol / L EDTA; Solution III includes: 1~2 mol / L potassium acetate, 1~2 mol / L sodium acetate, 0.2~0.6 mol / L trehalose, pH 5~5.6; The magnetic bead solution I comprises: 0.5-1.0 mol / L NaCl, 0.05-0.1 mol / L sodium citrate, 30-50 mg / mL magnetic beads I, and a pH of 5.0-5.5; the magnetic beads I are magnetic microspheres with nickel magnetic beads as carriers and polyhistidine as ligands, and the particle size is 0.5 μm-1 μm; The magnetic bead solution II comprises: 1.2-1.8 mol / L guanidine hydrochloride, 0.01-0.05 mol / L urea, 20-30% anhydrous ethanol by volume, 30-40% isopropanol by volume, 20-30 mg / mL magnetic beads II, and a pH of 5.5-6.5; the magnetic bead solution II is monodisperse, superparamagnetic hydroxy magnetic beads with a particle size of 200-500 nm; The cleaning solution is an ethanol solution with a volume fraction of 60-75%; the eluting solution is 10-15mmol / L Tris-HCl with a pH of 7.0-8.

0.

2. Use of the kit according to claim 1 in extracting plasmids and removing endotoxins.

3. A method for extracting plasmid using the kit according to claim 1, characterized in that: The following steps are involved: (1) 1% plasmid-containing E. coli cells were cultured in LB medium at 37°C with shaking overnight; (2) Take 1 mL of bacterial cells and add them to a centrifuge tube. Centrifuge at 12000 rpm for 30 seconds and discard the supernatant. (3) Resuspend the recovered bacteria in 0.1 mL of solution I and shake vigorously to mix thoroughly; (4) Add 0.2 mL of solution II and gently invert 5 to 8 times until the solution becomes transparent. Let stand at room temperature for 2 to 3 minutes. (5) Add 0.15 mL of solution III and immediately mix by gently inverting for 5 to 10 times until no more white flocculent precipitate is formed. Let stand at room temperature for 1 min. (6) Centrifuge at 12000 r / min for 5 min, take the supernatant and transfer it to a new centrifuge tube; (7) Add 30 μL of magnetic bead solution I to the centrifuge tube containing the supernatant, mix well by pipetting, and place at room temperature for 2 to 3 minutes. Then place the centrifuge tube on a magnetic rack for 1 to 2 minutes and perform magnetic separation until the supernatant is completely clear. Take the supernatant and transfer it to a new centrifuge tube. (9) Add 100 μL of magnetic bead solution II to the centrifuge tube, mix well by pipetting, and place at room temperature for 2 to 3 minutes. Then place the centrifuge tube on the magnetic rack. After the magnetic beads are completely absorbed to the tube wall, use a pipette to discard the supernatant and remove the centrifuge tube from the magnetic rack. (10) Add 1 mL of 70% ethanol to the centrifuge tube and mix thoroughly by pipetting. Place the centrifuge tube on a magnetic rack for 1 min to allow the magnetic beads to be completely absorbed by the tube wall. Discard the supernatant and remove the centrifuge tube from the magnetic rack. Dry at room temperature for 3–5 min until there is no liquid left in the centrifuge tube. (11) Add 50-100 μL of TE Buffer to the centrifuge tube and mix gently with a pipette. Elute at room temperature for 5 min, then place on a magnetic rack for 1-2 min. After the magnetic beads are completely absorbed to the wall of the centrifuge tube, carefully pipette the supernatant into a new centrifuge tube to obtain the plasmid DNA product.

Images