CN111676215A - Endotoxin-removing plasmid extraction kit and plasmid extraction method - Google Patents

Endotoxin-removing plasmid extraction kit and plasmid extraction method Download PDF

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CN111676215A
CN111676215A CN202010559313.6A CN202010559313A CN111676215A CN 111676215 A CN111676215 A CN 111676215A CN 202010559313 A CN202010559313 A CN 202010559313A CN 111676215 A CN111676215 A CN 111676215A
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endotoxin
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关红卫
李婧
魏洪运
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Shenyang Deyu Biotechnology Co ltd
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Abstract

An endotoxin-removing plasmid extraction kit and a plasmid extraction method, wherein the endotoxin-removing plasmid extraction kit at least comprises a thallus cracking reagent, an impurity removal reagent, a plasmid recovery reagent and an endotoxin removal reagent; according to the volume ratio, the volume ratio of each reagent is as follows: reagent one of the bacteria lysis reagent: reagent II of thallus lysis reagent: impurity removal reagent: plasmid recovery reagent: reagent a of endotoxin-removing reagent: the endotoxin removal reagent (10-30) is 20:20:30:7, and the reagent (10-30) is a reagent (B) for removing endotoxin. Also relates to a plasmid extraction method. The endotoxin-removing plasmid extraction kit has the advantages of simple operation for removing endotoxin, short operation time, high plasmid extraction efficiency, high endotoxin removal efficiency of over 99 percent, no limit on the extraction bacterial amount of the endotoxin-removing plasmid extraction kit, capability of meeting all experimental requirements of no endotoxin, biological research experiment and related pharmaceutical experiment comparison.

Description

Endotoxin-removing plasmid extraction kit and plasmid extraction method
Technical Field
The invention relates to the field of molecular biology, in particular to an endotoxin-removing plasmid extraction kit and a plasmid extraction method.
Background
Plasmid extraction and purification are important steps in experiments in many molecular biology studies. An extraction method which can obtain a large amount of high-purity endotoxin-free plasmids has important significance for molecular biology.
In many researches, especially in the aspects of diagnosing and treating cancers, monogenetic genetic diseases, cardiovascular diseases and the like by gene therapy and DNA vaccines, plasmid extraction shows outstanding advantages. At present, plasmids are becoming important gene delivery tools for gene therapy, and a target DNA fragment is delivered into a recipient cell for propagation and expression by gene recombination technology, and bacterial plasmids are one of the most commonly used vectors in the recombination technology. The plasmid also has specific genetic information that gives the host cell some genetic traits such as marker genes, which are very practical and valuable for genetic engineering research and its downstream applications.
However, in many studies related to molecular biology and pharmacy, the plasmids used cannot contain endotoxin components, because endotoxin has important influence on the DNA transfection process of primary cells and sensitive cultured cells, and the increase of endotoxin level causes the sharp reduction of the transfection efficiency, so that the extracted plasmids need to be subjected to endotoxin removal operation. However, the currently sold endotoxin removal kit has the defects of long endotoxin removal operation time, poor endotoxin removal effect and the like, and is designed into small-extract, medium-extract and large-extract kits without endotoxin plasmids according to the volume of the extracted bacteria, so that the design is complicated. Therefore, a new endotoxin-free plasmid extraction kit, and a plasmid extraction method are required.
Disclosure of Invention
The invention provides an endotoxin-removing plasmid extraction kit and a plasmid extraction method, aiming at the problems of long operation time, unsatisfactory endotoxin removal effect, complex kit types and the like of the currently marketed endotoxin-removing plasmid extraction kit. The endotoxin-removing plasmid extraction kit is developed based on an alkaline lysis method, is simple in endotoxin-removing operation, short in operation time, high in plasmid extraction efficiency, and capable of achieving the endotoxin removal efficiency of more than 99%, has no limit on the extraction bacterial quantity of the endotoxin-removing plasmid extraction kit, can meet the requirements of all endotoxin-free experiments, and is a biological research experiment and a related pharmaceutical experiment.
The invention relates to a de-endotoxin plasmid extraction kit, which at least comprises a thallus cracking reagent, an impurity removal reagent, a plasmid recovery reagent and an endotoxin removal reagent;
wherein, the thallus cracking reagent is divided into a reagent I and a reagent II, and the endotoxin removing reagent is divided into a reagent A and a reagent B; according to the volume ratio, the volume ratio of each reagent is as follows: reagent one of the bacteria lysis reagent: reagent II of thallus lysis reagent: impurity removal reagent: plasmid recovery reagent: reagent a of endotoxin-removing reagent: the endotoxin removal reagent (10-30) is 20:20:30:7, and the reagent (10-30) is a reagent (B) for removing endotoxin.
The thallus cracking reagent comprises a first reagent and a second reagent, wherein the first reagent comprises the following components in volume ratio: reagent two ═ 1: 1; the reagent I is an aqueous solution containing Tris-HCl and EDTA, wherein the molar concentration of Tris-HCl is 40-60 mmol/L, and the molar concentration of EDTA is 8-15 mmol/L; the second reagent is a mixed aqueous solution containing sodium hydroxide and SDS; in the second reagent, the molar concentration of sodium hydroxide is 0.1-0.3 mol/L, and the mass concentration of SDS is 0.5-2%.
The impurity removal reagent is an aqueous solution containing acetic acid, potassium acetate, calcium chloride, manganese chloride and guanidine hydrochloride, wherein in the impurity removal reagent, the molar concentration of acetic acid is 1.8mol/L, the molar concentration of potassium acetate is 0.42mol/L, the molar concentration of calcium chloride is 0.42mol/L, the molar concentration of manganese chloride is 0.66mol/L, and the molar concentration of guanidine hydrochloride is 0.52 mol/L.
The plasmid recovery reagent is an aqueous solution containing polyethylene glycol 8000 and dimethyl sulfoxide, wherein in the plasmid recovery reagent, the mass concentration of the polyethylene glycol 8000 is 20-30%, and the volume concentration of the dimethyl sulfoxide is 25-30%.
The endotoxin removing reagent comprises a reagent A and a reagent B, wherein the reagent A is an aqueous solution of sodium chloride, and the reagent B is an aqueous solution of sodium deoxycholate; wherein, in the sodium chloride aqueous solution, the molar concentration of the sodium chloride is 1-1.5 mol/L, and the mass percentage concentration of the sodium taurodeoxycholate aqueous solution is 15-20%.
Wherein, the plasmid recovery reagent is stored at 4 ℃, and other reagents are stored at room temperature.
The method for extracting the plasmid adopts the endotoxin-removing plasmid extraction kit, and comprises the following steps of:
step 1: inoculating and culturing gram-negative bacteria containing target plasmids to obtain a gram-negative bacteria culture solution, centrifuging, and removing supernatant to obtain gram-negative bacteria precipitates;
step 2: adding a thallus cracking reagent into the gram-negative thallus precipitate for thallus cracking to obtain a semitransparent lysate, and standing at room temperature for 2-3 min;
and step 3: adding 40-60 mu L of impurity removal reagent into every 1mL of gram-negative bacteria culture solution, fully mixing the semitransparent lysate and the impurity removal reagent, centrifuging at a high speed, and taking supernatant bacteria solution for later use;
and 4, step 4: adding 40-60 mu L of plasmid recovery reagent into every 1mL of gram-negative bacteria culture solution, fully mixing the supernatant liquid and the plasmid recovery reagent, performing vortex oscillation for 1-3 min, performing high-speed centrifugation, and removing the supernatant to obtain recovered plasmids with endotoxin;
and 5: adding 70-75% ethanol solution into the recovered plasmid with endotoxin, wherein the volume of the ethanol solution is 1mL when the gram-negative bacteria culture solution is 2-5 mL; when the gram-negative bacterium culture solution is 40-200mL, 5mL of ethanol solution with the volume concentration of 70-75%; vortex oscillation, then high-speed centrifugation, thoroughly removing supernatant after centrifugation, and obtaining a mixture of endotoxin and plasmid;
step 6: adding an aqueous solution of sodium chloride in an endotoxin removal reagent into a mixture of endotoxin and plasmid, wherein the addition volume is 0.21mL when the gram-negative bacterium culture solution is 2-5 mL; when the gram-negative bacteria culture solution is 40-200mL, the adding amount of the sodium chloride aqueous solution is 2.1 mL; dissolving the plasmid by vortex oscillation, and adding 0.04mL of sodium deoxycholate aqueous solution into the aqueous solution of sodium deoxycholate in the endotoxin removal reagent, wherein the volume of the aqueous solution is that when the gram-negative bacterium culture solution is 2-5 mL; when the gram-negative bacteria culture solution is 40-200mL, the addition amount of the aqueous solution of sodium taurocholate is 0.4 mL; performing vortex oscillation and high-speed centrifugation, sucking supernatant, and placing the supernatant in a centrifugal tube A without endotoxin and nucleic acid;
and 7: adding an aqueous solution of sodium taurocholate into the supernatant of the centrifuge tube A without endotoxin and nucleic acid, wherein the addition amount of the aqueous solution of sodium taurocholate is 0.01mL when the gram-negative bacteria culture solution is 2-5 mL; when the gram-negative bacteria culture solution is 40-200mL, the addition amount of the aqueous solution of sodium taurocholate is 0.1 mL; performing vortex oscillation and high-speed centrifugation, and sucking supernatant; putting the mixture into another centrifugal tube B without endotoxin and nucleic acid, repeating the step 7, and putting the obtained supernatant into a centrifugal tube C without endotoxin and nucleic acid;
and 8: adding isopropanol with the same volume as the supernatant into the supernatant in a centrifuge tube C without endotoxin and nucleic acid, performing vortex oscillation and uniform mixing, performing high-speed centrifugation, and removing the supernatant;
and step 9: adding ethanol with the volume concentration of 70-75% into the residual product in the centrifuge tube C without endotoxin and nucleic acid in the step 8, wherein the volume of the ethanol solution is 1mL when the gram-negative bacteria culture solution is 2-5 mL; when the gram-negative bacterium culture solution is 40-200mL, 5mL of ethanol solution with the volume concentration of 70-75%; vortex oscillation mixing, high speed centrifugation for many times, thoroughly removing residual liquid, and obtaining plasmid;
step 10: adding endotoxin-free water or 10mmol/L endotoxin-free Tris-HCl8.0 buffer solution to dissolve plasmid, extracting the obtained plasmid, and storing at-20 deg.C.
In the step 1, the centrifugation is carried out at a centrifugation speed of 6000-8000 rpm for 1-3 min.
In the step 2, the specific steps of adding the thallus cracking reagent are as follows:
(1) adding 40-60 mu L of reagent I into every 1mL of gram-negative bacterium culture solution, uniformly mixing gram-negative bacterium sediment and the reagent I in the bacterium cracking reagent, and performing vortex oscillation until a bacterium suspension is obtained;
(2) and uniformly mixing the thallus suspension and the reagent II in the thallus cracking reagent according to the proportion that 40-60 mu L of reagent II is added into every 1mL of gram-negative bacteria culture liquid until semitransparent lysate is obtained.
In the step 2(2), the mixing is performed gently to avoid the plasmid destruction.
In the step 3, the centrifugation speed of the high-speed centrifugation is 10000-13000 rpm, and the centrifugation time is 10-15 min.
In the step 4, the centrifugation speed of the high-speed centrifugation is 10000-13000 rpm, and the centrifugation time is 5-10 min.
In the step 5, the centrifugation speed of the high-speed centrifugation is 10000-13000 rpm, and the centrifugation time is 5-15 min.
In the step 6, the vortex oscillation time is 2-3 min, the centrifugation speed of high-speed centrifugation is 10000-13000 rpm, and the centrifugation time is 2 min.
In the step 7, the vortex oscillation time is 2-3 min, the centrifugation speed of the high-speed centrifugation is 10000-13000 rpm, and the centrifugation time is 2 min.
In the step 8, the vortex oscillation time is 1-2 min, the centrifugation speed of high-speed centrifugation is 10000-13000 rpm, and the centrifugation time is 10 min.
In the step 9, the centrifugal rate of the high-speed centrifugation is 10000-13000 rpm.
In the step 10, the dosage of endotoxin-free water or 10mmol/L endotoxin-free Tris-HCl8.0 buffer solution is as follows: 10 mul of endotoxin-free water or 10mmol/L of endotoxin-free Tris-HCl8.0 buffer solution is added into each 1mL of gram-negative bacteria culture solution.
The endotoxin-removing plasmid extraction kit and the plasmid extraction method have the beneficial effects that:
1. the reagent I used in the invention does not contain RNA enzyme, and has no influence on subsequent experiments.
2. The endotoxin-removing plasmid extraction kit and the plasmid extraction method are simple in components and high in plasmid extraction efficiency, the endotoxin removal efficiency can reach more than 99%, the endotoxin-free experiment requirements can be met, and the endotoxin-removing plasmid extraction kit is a biological research experiment and has advantages compared with related pharmaceutical experiments.
3. The endotoxin-removing plasmid extraction kit has no limit on the quantity of extracted bacteria, does not need to produce products with various specifications, can meet the requirement of customers on extracting bacteria liquid with different quantities, and can achieve the effect of one product with multiple purposes.
4. In the endotoxin-removing plasmid extraction kit, the impurity removal reagent adopts specific substances and specific concentration, and the pH value is fixed, so that the purity of the extracted plasmid can be obviously improved, and the plasmid is prevented from generating impurities.
Drawings
FIG. 1 shows the result of the TAE agarose gel electrophoresis of 1.5% of the plasmid extracted in example 2 of the present invention;
FIG. 2 shows the results of concentration and purity measurements of the plasmid extracted in example 2-1 of the present invention using KAIAO K5600 micro-spectrophotometer.
FIG. 3 shows the results of concentration and purity measurements of the plasmid extracted in example 2-2 of the present invention using KAIAO K5600 micro-spectrophotometer.
FIG. 4 shows the results of concentration and purity measurements of plasmids extracted in examples 2 to 3 of the present invention using KAIAO K5600 micro-spectrophotometer.
FIG. 5 shows the results of concentration and purity measurements of the extracted plasmid in example 3-1 of the present invention using KAIAO K5600 micro-spectrophotometer.
FIG. 6 shows the results of concentration and purity measurements of the extracted plasmid in example 3-2 of the present invention using KAIAO K5600 micro-spectrophotometer.
FIG. 7 shows the results of concentration and purity measurements with KAIAO K5600 micro-spectrophotometer in example 3-3 of the present invention.
Detailed Description
The following examples are included to describe the principles and operation of the present invention, but are not intended to limit the scope of the invention.
In the following example, in the step 1, when the volume of the escherichia coli bacterial liquid is 40-200mL, the escherichia coli bacterial liquid needs to be collected into a 50mL centrifuge tube; when the volume of the escherichia coli liquid is 1-5 mL, the escherichia coli liquid needs to be collected into a 1.5mL centrifuge tube.
In the step 3, when the volume of the escherichia coli bacterial liquid in the step 1 is 1-5 mL, the inversion conditions are as follows: and (4) slightly reversing the centrifuge tube 6-15 times until a semitransparent lysate is obtained. In the step 1, when the volume of the escherichia coli liquid is 40-200mL, the conditions are reversed: the tube was gently inverted by motion until a translucent lysate was obtained.
EXAMPLE 1 construction of plasmid extraction kit
The endotoxin-removing plasmid extraction kit comprises a thallus cracking reagent, an impurity removal reagent, a plasmid recovery reagent and an endotoxin removal reagent, wherein the thallus cracking reagent is a reagent I, the reagent II, the impurity removal reagent is a reagent III, the plasmid recovery reagent is a reagent IV, and the endotoxin removal reagent is a reagent V and a reagent VI. The reagent I is a mixed aqueous solution of 50mmol/L of LTris-HCl and 10mmol/L of EDTA, the reagent II is a mixed aqueous solution of 0.2mol/L of sodium hydroxide and 1% of SDS (sodium dodecyl sulfate), the reagent III is a mixed aqueous solution consisting of 1.8mol/L of acetic acid, 0.42mol/L of potassium acetate, 0.42mol/L of calcium chloride, 0.66mol/L of manganese chloride and 0.52mol/L of guanidine hydrochloride, the reagent IV is a mixed aqueous solution of 25% of polyethylene glycol 8000 and 25% of dimethyl sulfoxide by volume, the reagent V is an aqueous solution of 1.2mol/L of sodium chloride, and the reagent VI is an aqueous solution of 20% of sodium deoxycholate by mass.
All reagents are prepared by double distilled water, the used chemical reagents are more than analytical grade, wherein the reagent IV needs to be stored at 4 ℃, and other reagents are stored at room temperature.
Example 2 plasmid miniprep procedure
1. In a 50mL culture tube, the E.coli strain with plasmid was inoculated into 10mL 2 XYT culture solution containing appropriate antibiotics, and cultured at 37 ℃ for 12-16h with shaking at 240rpm to obtain E.coli culture solution.
2. 5mL of the above E.coli culture solution was put into a 1.5mL centrifuge tube, centrifuged at 8000rpm (. about.6,000 Xg) for 1min, and E.coli cell pellets were collected and the supernatant was thoroughly aspirated.
3. Adding 200 mu L of reagent I into the escherichia coli thallus sediment, and performing vortex oscillation until thallus suspension is obtained.
4. And adding 200 mu L of reagent II into the thallus turbid liquid, gently inverting the centrifuge tube for 6-15 times until semitransparent lysate is obtained, and standing for 2-3 min at room temperature.
5. Adding 50 mu L of reagent III into each 1mL of escherichia coli culture solution, adding the reagent III into the semitransparent lysis solution, violently reversing for 8-12 times, centrifuging at 12000rpm for 10min, and transferring the supernatant bacterial solution into a new centrifugal tube.
6. Adding 200 mu L of reagent IV into the supernatant liquid, carrying out vortex oscillation for 1-3 min, then centrifuging at 12000rpm for 10min, and removing the supernatant to obtain the recovered plasmid with endotoxin.
7. To the recovery plasmid with endotoxin centrifuge tube, add 1mL volume concentration of 75% ethanol solution, vortex, then 12000rpm centrifugation for 5min, completely remove the supernatant.
8. Adding 200 mu L of reagent V into a centrifuge tube, carrying out vortex oscillation to dissolve plasmids, adding 40 mu L of reagent VI, carrying out vortex oscillation for 2-3 min, centrifuging at 10000rpm for 2min, absorbing supernate, and putting the supernate into another new centrifuge tube without endotoxin and nucleic acid, wherein consumables used in the subsequent steps are endotoxin-free and nucleic acid-free.
9. Adding 10 mu L of reagent six into the supernatant, carrying out vortex oscillation for 2-3 min, centrifuging at 10000rpm for 2min, sucking the supernatant, and putting the supernatant into another new endotoxin-free and nucleic acid-free centrifugal tube.
10. Repeating the step (9) once.
11. Adding isopropanol with the volume being 1 time of that of the supernatant obtained in the step (10), carrying out vortex oscillation for 1-2 min, mixing uniformly, centrifuging at 12000rpm for 10min, and absorbing and removing the supernatant.
12. Adding 1mL of ethanol with the volume concentration of 70% into a centrifuge tube, uniformly mixing by vortex oscillation, centrifuging at 12000rpm for 3min, pouring off supernatant, centrifuging at 12000rpm for 30s again, and completely sucking out residual liquid.
13. Add 50 u L volume of endotoxin free water or 10mmol/L endotoxin free Tris-HCl8.0 buffer solution to dissolve plasmid, store at-20 ℃.
14. The plasmid miniprep procedure described above was repeated three times and labeled as example 2-1, example 2-2, and example 2-3, respectively. Then, the sample was subjected to 1.5% TAE agarose gel electrophoresis in the order of example 2-1, example 2-2 and example 2-3. The electrophoresis results are shown in FIG. 1.
The results of concentration and purity measurements were carried out using a K5600 micro-spectrophotometer by KAIAO and are shown in FIGS. 2, 3 and 4, respectively.
TABLE 1 detection of the concentration and purity of the small extracted plasmids
Examples of the experiments 260/230 260/280 Plasmid concentration (ng/. mu.L)
Example 2-1 2.285 1.843 246.4
Examples 2 to 2 2.238 1.84 225.6
Examples 2 to 3 2.095 1.82 233.5
EXAMPLE 3 plasmid Mass extraction procedure
1. Coli strain with plasmid was inoculated into 200mL of a medium containing an appropriate antibiotic (2 XYT is recommended for the antibiotic medium, and other medium may be used, 2 XYT is used in this example) in a 1000mL conical flask, and cultured at 37 ℃ and 240rpm for 12-16 hours with shaking to obtain a cultured E.coli solution.
2. 160mL of the above overnight-cultured Escherichia coli culture solution was added to a centrifuge tube, centrifuged at 8000rpm (6,000 Xg) for 1min to collect the precipitate of Escherichia coli cells, and the supernatant was completely removed.
3. Adding a reagent I into a centrifugal tube, adding 1/20 with the proportion being the volume of the collected escherichia coli culture solution, fully and uniformly mixing by using a vortex oscillator, and suspending escherichia coli thalli precipitates to obtain a thalli suspension.
4. Adding the reagent II into a centrifugal tube filled with the thallus suspension, adding the reagent II into the centrifugal tube with the volume the same as that of the reagent I, gently rotating the centrifugal tube to fully mix and crack the thallus until the solution becomes clear and viscous to obtain a semitransparent lysate, and standing at room temperature for 3 min.
5. Adding a third reagent into a centrifuge tube filled with semitransparent lysate, adding the volume of the third reagent into the centrifuge tube to be the same as that of the first reagent, gently rotating and uniformly mixing the third reagent and the reagent for a plurality of times to form white precipitates, then violently shaking the mixture to fully mix the white precipitates (at the moment, the white precipitates are in a tiny flocculent shape), and centrifuging the mixture for 10min at full speed (more than or equal to 12,000 rpm).
6. Pouring the supernatant obtained after centrifugation into another clean centrifugal tube, adding a reagent IV into the centrifugal tube, wherein the volume of the added reagent IV is the same as that of the reagent I, performing brief vortex oscillation to fully mix the mixture, and centrifuging the mixture for 10min at full speed (more than or equal to 12,000 rpm); the supernatant was decanted, centrifuged at ≥ 12,000rpm for 30s, and the supernatant was aspirated off thoroughly using a pipette.
7. Adding 1mL of reagent V into a centrifuge tube, adding 2mL of absolute ethyl alcohol, performing vortex oscillation to fully mix the mixture, and centrifuging the mixture for 10min at full speed (more than or equal to 12,000 rpm); the supernatant was decanted, centrifuged at ≥ 12,000rpm for 30s, and the supernatant was aspirated off thoroughly using a pipette. The purpose is to clean the plasmid extracted primarily and remove impurities.
8. Adding 2mL of reagent V into the EP tube, performing vortex oscillation to dissolve plasmids, adding 0.40mL of reagent VI, mixing uniformly, centrifuging at the maximum revolution for 2min, sucking the supernatant, and putting the supernatant into another clean EP tube;
9. continuously adding 0.1mL of reagent six into the supernatant, uniformly mixing the mixture, centrifuging the mixture for 2min at full speed (more than or equal to 12,000rpm), and sucking the supernatant into another clean EP tube;
10. repeating the operation of the 9 th step once;
11. adding isopropanol with the same volume into the supernatant, mixing uniformly, centrifuging at full speed (more than or equal to 12,000rpm) for 10min, and removing the supernatant; adding 5mL of 70% ethanol, performing vortex oscillation, centrifuging again at full speed (more than or equal to 12,000rpm) for 3min, and completely removing supernatant;
12. 1.6mL of endotoxin-free water or 10mmol/L of endotoxin-free Tris-HCl8.0 buffer was added to the tube to dissolve the plasmid, and the extracted plasmid was stored at-20 ℃.
13. The plasmid mass extraction procedure described above was repeated three times and labeled as example 3-1, example 3-2, and example 3-3, respectively. The results of concentration and purity measurements using a K5600 micro spectrophotometer by KAIAO are shown in FIGS. 5, 6 and 7, respectively.
TABLE 2 detection of plasmid concentration and purity by mass extraction
Examples of the experiments 260/230 260/280 Plasmid concentration (ng/. mu.L)
Example 3-1 2.24 1.82 217.6
Examples 3 to 2 2.2 1.81 209.7
Examples 3 to 3 2.24 1.81 215.3
Example of detection
Endotoxin detection
Reagent materials: the sensitivity was 0.125EU/mL limulus reagent, bacterial endotoxin working standard and bacterial endotoxin test water (all available from Limulus amebocyte reagents Biotech Ltd.).
The contact reagent used in the test and the vessel used for the test article must be heat-source-free and free of interference factors.
Procedure for the preparation of the
1. According to the 'instruction for using bacterial endotoxin working standard substance', positive endotoxin standard solutions of 0.5EU/mL, 0.25EU/mL, 0.125EU/mL and 0.06EU/mL are prepared.
2. Negative control solution: namely, water for bacterial endotoxin test.
3. Positive control solution: namely, positive endotoxin standard solutions of 0.5EU/mL, 0.25EU/mL, 0.125EU/mL and 0.06 EU/mL.
4. Add endotoxin test water to the limulus reagent in the amount indicated, and gently shake to completely dissolve the limulus reagent. Care was taken not to cause air bubbles and the reagent was used within 10min after dissolution.
5. 0.1mL of negative control, positive endotoxin standard control solution, and 2 ng/. mu.L, 3 ng/. mu.L, 4 ng/. mu.L, 5 ng/. mu.L, 6 ng/. mu.L of plasmid test samples were added to each reaction tube.
6. The tube mouth is slightly and evenly shaken, and the tube is vertically placed into a thermostat with the temperature of 37 ℃ for incubation for 60 minutes, and vibration is avoided during the incubation period.
7. Result judgment
The tube was gently removed from the thermostat and slowly inverted 180 degrees. If the content in the pavilion is solid gel, the gel is not deformed and does not slip off from the tube wall, and the gel is positive; negative for not being gel or for not remaining intact and slipping off the tube wall despite the production of gel.
8. Results of the experiment
After incubation at 37 ℃ for 60 minutes, the content of the negative control is in a water state and does not form gel, the content of the positive control is in a gel state, the content of the; 2 ng/. mu.L, 3 ng/. mu.L, 4 ng/. mu.L of the plasmid test samples produced gels but did not remain intact, slipped off the tube wall, 5 ng/. mu.L, 6 ng/. mu.L did not slip off the tube wall.
Thus, according to the formula endotoxin content of 0.125 EU/mL/plasmid concentration (mg/mL), the plasmid endotoxin content extracted by the endotoxin-free plasmid extraction kit of the present invention was found to be between 25-31.25 EU/mg.

Claims (10)

1. The endotoxin-removing plasmid extraction kit is characterized by comprising a thallus cracking reagent, an impurity removal reagent, a plasmid recovery reagent and an endotoxin removal reagent;
wherein, the thallus cracking reagent is divided into a reagent I and a reagent II, and the endotoxin removing reagent is divided into a reagent A and a reagent B; according to the volume ratio, the volume ratio of each reagent is as follows: reagent one of the bacteria lysis reagent: reagent II of thallus lysis reagent: impurity removal reagent: plasmid recovery reagent: reagent a of endotoxin-removing reagent: the endotoxin removal reagent (10-30) is 20:20:30:7, and the reagent (10-30) is a reagent (B) for removing endotoxin.
2. The endotoxin-free plasmid extraction kit as claimed in claim 1, wherein the lysis reagent comprises a first reagent and a second reagent, wherein the first reagent: reagent two ═ 1: 1; the reagent I is an aqueous solution containing Tris-HCl and EDTA, wherein the molar concentration of Tris-HCl is 40-60 mmol/L, and the molar concentration of EDTA is 8-15 mmol/L; the second reagent is a mixed aqueous solution containing sodium hydroxide and SDS; in the second reagent, the molar concentration of sodium hydroxide is 0.1-0.3 mol/L, and the mass concentration of SDS is 0.5-2%.
3. The endotoxin-removing plasmid extraction kit of claim 1, wherein the impurity removal reagent is an aqueous solution containing acetic acid, potassium acetate, calcium chloride, manganese chloride and guanidine hydrochloride, wherein in the impurity removal reagent, the molar concentration of acetic acid is 1.8mol/L, the molar concentration of potassium acetate is 0.42mol/L, the molar concentration of calcium chloride is 0.42mol/L, the molar concentration of manganese chloride is 0.66mol/L, and the molar concentration of guanidine hydrochloride is 0.52 mol/L.
4. The endotoxin-removing plasmid extraction kit as claimed in claim 1, wherein the plasmid recovery reagent is an aqueous solution containing polyethylene glycol 8000 and dimethyl sulfoxide, wherein the mass concentration of the polyethylene glycol 8000 and the volume concentration of the dimethyl sulfoxide are 20-30% in the plasmid recovery reagent.
5. The endotoxin-removing plasmid extraction kit as claimed in claim 1, wherein the endotoxin-removing reagent comprises a reagent A and a reagent B, wherein the reagent A is an aqueous solution of sodium chloride, and the reagent B is an aqueous solution of sodium taurodeoxycholate; wherein, in the sodium chloride aqueous solution, the molar concentration of the sodium chloride is 1-1.5 mol/L, and the mass percentage concentration of the sodium taurodeoxycholate aqueous solution is 15-20%.
6. The endotoxin-free plasmid extraction kit of claim 1, wherein the plasmid endotoxin content extracted by the endotoxin-free plasmid extraction kit is 25-31.25 EU/mg.
7. A method for extracting plasmid, which comprises the steps of using the endotoxin-free plasmid extraction kit of any one of claims 1 to 6, and:
step 1: inoculating and culturing gram-negative bacteria containing target plasmids to obtain a gram-negative bacteria culture solution, centrifuging, and removing supernatant to obtain gram-negative bacteria precipitates;
step 2: adding a thallus cracking reagent into the gram-negative thallus precipitate for thallus cracking to obtain a semitransparent lysate, and standing at room temperature for 2-3 min;
and step 3: adding 40-60 mu L of impurity removal reagent into every 1mL of gram-negative bacteria culture solution, fully mixing the semitransparent lysate and the impurity removal reagent, centrifuging at a high speed, and taking supernatant bacteria solution for later use;
and 4, step 4: adding 40-60 mu L of plasmid recovery reagent into every 1mL of gram-negative bacteria culture solution, fully mixing the supernatant liquid and the plasmid recovery reagent, performing vortex oscillation for 1-3 min, performing high-speed centrifugation, and removing the supernatant to obtain recovered plasmids with endotoxin;
and 5: adding 70-75% ethanol solution into the recovered plasmid with endotoxin, wherein the volume of the ethanol solution is 1mL when the gram-negative bacteria culture solution is 2-5 mL; when the gram-negative bacterium culture solution is 40-200mL, 5mL of ethanol solution with the volume concentration of 70-75%; vortex oscillation, then high-speed centrifugation, thoroughly removing supernatant after centrifugation, and obtaining a mixture of endotoxin and plasmid;
step 6: adding an aqueous solution of sodium chloride in an endotoxin removal reagent into a mixture of endotoxin and plasmid, wherein the addition volume is 0.21mL when the gram-negative bacterium culture solution is 2-5 mL; when the gram-negative bacteria culture solution is 40-200mL, the adding amount of the sodium chloride aqueous solution is 2.1 mL; dissolving the plasmid by vortex oscillation, and adding 0.04mL of sodium deoxycholate aqueous solution into the aqueous solution of sodium deoxycholate in the endotoxin removal reagent, wherein the volume of the aqueous solution is that when the gram-negative bacterium culture solution is 2-5 mL; when the gram-negative bacteria culture solution is 40-200mL, the addition amount of the aqueous solution of sodium taurocholate is 0.4 mL; performing vortex oscillation and high-speed centrifugation, sucking supernatant, and placing the supernatant in a centrifugal tube A without endotoxin and nucleic acid;
and 7: adding an aqueous solution of sodium taurocholate into the supernatant of the centrifuge tube A without endotoxin and nucleic acid, wherein the addition amount of the aqueous solution of sodium taurocholate is 0.01mL when the gram-negative bacteria culture solution is 2-5 mL; when the gram-negative bacteria culture solution is 40-200mL, the addition amount of the aqueous solution of sodium taurocholate is 0.1 mL; performing vortex oscillation and high-speed centrifugation, and sucking supernatant; putting the mixture into another centrifugal tube B without endotoxin and nucleic acid, repeating the step 7, and putting the obtained supernatant into a centrifugal tube C without endotoxin and nucleic acid;
and 8: adding isopropanol with the same volume as the supernatant into the supernatant in a centrifuge tube C without endotoxin and nucleic acid, performing vortex oscillation and uniform mixing, performing high-speed centrifugation, and removing the supernatant;
and step 9: adding ethanol with the volume concentration of 70-75% into the residual product in the centrifuge tube C without endotoxin and nucleic acid in the step 8, wherein the volume of the ethanol solution is 1mL when the gram-negative bacteria culture solution is 2-5 mL; when the gram-negative bacterium culture solution is 40-200mL, 5mL of ethanol solution with the volume concentration of 70-75%; vortex oscillation mixing, high speed centrifugation for many times, thoroughly removing residual liquid, and obtaining plasmid;
step 10: adding endotoxin-free water or 10mmol/L endotoxin-free Tris-HCl8.0 buffer solution to dissolve plasmid, extracting the obtained plasmid, and storing at-20 deg.C.
8. The method for extracting plasmids of claim 6, wherein in step 1, the centrifugation is carried out at 6000-8000 rpm for 1-3 min; the centrifugal rate of the high-speed centrifugation is 10000-13000 rpm.
9. The method for extracting plasmids of claim 6, wherein in the step 2, the specific steps of adding the thallus cracking reagent are as follows:
(1) adding 40-60 mu L of reagent I into every 1mL of gram-negative bacterium culture solution, uniformly mixing gram-negative bacterium sediment and the reagent I in the bacterium cracking reagent, and performing vortex oscillation until a bacterium suspension is obtained;
(2) and uniformly mixing the thallus suspension and the reagent II in the thallus cracking reagent according to the proportion that 40-60 mu L of reagent II is added into every 1mL of gram-negative bacteria culture liquid until semitransparent lysate is obtained.
10. The method for extracting plasmid as claimed in claim 6, wherein in the step 10, the amount of endotoxin-free water or 10mmol/L endotoxin-free Tris-HCl8.0 buffer solution is: 10 mul of endotoxin-free water or 10mmol/L of endotoxin-free Tris-HCl8.0 buffer solution is added into each 1mL of gram-negative bacteria culture solution.
CN202010559313.6A 2020-06-18 2020-06-18 Endotoxin-removing plasmid extraction kit and plasmid extraction method Pending CN111676215A (en)

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