阅读说明：本技术 一种具有多功能试剂组的无毒型质粒提取试剂盒及质粒提取方法 (Non-toxic plasmid extraction kit with multifunctional reagent group and plasmid extraction method ) 是由 赵宇翔 于 2021-09-14 设计创作，主要内容包括：本发明公开了一种具有多功能试剂组的无毒型质粒提取试剂盒及质粒提取方法,该试剂盒及质粒提取方法旨在解决现有技术下试剂盒内试剂组的功能单一,只能裂解某一类型的细胞,且提取的质粒浓度较低,并且样品最终的内毒素水平较高的技术问题。该试剂盒包括裂解试剂组、杂质去除试剂组、内毒素去除试剂组、质粒回收试剂和内毒素检测试剂,所述裂解试剂组用于裂解细胞,所述裂解试剂组包括试剂一和试剂二。该发明的具有多功能试剂组的无毒型质粒提取试剂盒及质粒提取方法,将多种试剂集中在一个试剂盒内,便于质粒的提取操作,便于对内毒素水平进行检测,确保对内毒素的去除效果,适用于不同类型的细胞裂解,采用该方法提取的质粒浓度较高。(The invention discloses a non-toxic plasmid extraction kit with a multifunctional reagent group and a plasmid extraction method, and aims to solve the technical problems that the reagent group in the reagent kit has a single function, can only crack a certain type of cells, the concentration of extracted plasmids is low, and the final endotoxin level of a sample is high in the prior art. The kit comprises a lysis reagent group, an impurity removal reagent group, an endotoxin removal reagent group, a plasmid recovery reagent and an endotoxin detection reagent, wherein the lysis reagent group is used for lysing cells, and comprises a first reagent and a second reagent. According to the non-toxic plasmid extraction kit with the multifunctional reagent set and the plasmid extraction method, multiple reagents are concentrated in one reagent kit, so that the extraction operation of plasmids is facilitated, the detection of endotoxin levels is facilitated, the removal effect of endotoxin is ensured, the kit is suitable for cell lysis of different types, and the concentration of plasmids extracted by the method is high.)

1. A non-toxic plasmid extraction kit with a multifunctional reagent group comprises a cracking reagent group, an impurity removal reagent group, an endotoxin removal reagent group, a plasmid recovery reagent and an endotoxin detection reagent; it is characterized in that the preparation method is characterized in that,

the Lysis reagent group is used for lysing cells and comprises a first reagent and a second reagent, wherein the first reagent comprises 1% of triton X-100 and a phenylmethylsulfonyl fluoride solution, the volume ratio of the triton X-100 to the phenylmethylsulfonyl fluoride solution is 100:1.5, the second reagent comprises NP-40 lysine Buffer and the phenylmethylsulfonyl fluoride solution, and the volume ratio of the NP-40 lysine Buffer to the phenylmethylsulfonyl fluoride solution is 100: 1.5;

the impurity removal reagent group is used for obtaining a plasmid crude extract from a cell disruption solution, and comprises a reagent III and a reagent IV;

the plasmid recovery reagent is used for separating plasmids from bacterial genome DNA and removing proteins and other impurities so as to obtain relatively pure plasmids;

the endotoxin removal reagent group is used for removing endotoxin in a solution, and comprises a reagent five, a reagent six and a reagent seven, wherein the volume ratio of the reagent five to the reagent six to the reagent seven is 1.5: 125: 125;

the endotoxin detection reagent is used for detecting the content of endotoxin in the obtained plasmid solution.

2. The non-toxic plasmid extraction kit with multifunctional reagent set of claim 1, wherein the reagent three comprises 6-8M guanidine hydrochloride, 20mM Tris-HCl, ethanol and water, and the reagent four comprises ethanol and water.

3. The non-toxic plasmid extraction kit having a multifunctional reagent set according to claim 1, wherein the reagent five is a 1.5ml affinity resin pre-packed column, the reagent six is 125ml regeneration buffer comprising 1M Tris-HCl, 0.5M EDTA and 1M glucose, the reagent seven is 125ml equilibration buffer comprising 2M NaOH and 10% SDS, and the pH of the reagent six and the reagent seven is 8.

4. The non-toxic plasmid extraction kit with multifunctional reagent set as claimed in claim 1, wherein the plasmid recovery reagent is an aqueous solution containing polyethylene glycol and dimethyl sulfoxide.

5. The nontoxic plasmid extraction kit of claim 1 wherein the endotoxin detection reagent is an aqueous solution containing 5-20% Triton X-114.

6. A method for extracting plasmid, which comprises the non-toxic plasmid extraction kit with multifunctional reagent set according to claims 1-5, and comprises the following steps:

the method comprises the following steps: obtaining a cell disruption solution:

a) according to the requirement, selecting a reagent I or a reagent II, dissolving and uniformly mixing 1% triton X-100 or NP-40 lysine Buffer at room temperature, and then adding a phenylmethylsulfonyl fluoride solution to ensure that the final concentration of the solution reaches 1mM;

b) centrifuging the culture solution to be treated at low speed for 1-3min, removing the supernatant, and collecting the precipitate;

c) adding the solution obtained in the step one into the precipitate, gently blowing and beating the precipitate by using a pipette to ensure that the solution is fully contacted with cells, centrifuging the solution, and taking supernatant liquid which is cell disruption liquid;

step two: obtaining a plasmid crude extract:

a) loading the cell disruption solution on a plasmid adsorption column, centrifuging and filtering out the liquid on the plasmid adsorption column;

b) washing the plasmid adsorption column with reagent III, centrifuging and filtering out the liquid in the plasmid adsorption column;

c) washing the plasmid adsorption column with reagent IV, centrifuging, filtering out the liquid of the plasmid adsorption column, and repeating the operation twice to obtain filtrate, namely the crude plasmid extract;

step three: adding a plasmid recovery reagent into the crude plasmid extraction solution in a ratio of 1000: 40-60, oscillating for 1-3min, centrifuging at high speed, and removing supernatant to obtain a recovered plasmid solution with endotoxin;

step four: removing endotoxin:

a) the method comprises the following steps Adjusting the pH value of the recovered plasmid solution to be 6-9, placing an affinity resin prepacked column on an iron support, vertically fixing, removing the top cover of the affinity resin prepacked column, allowing a protective solution to run dry under the action of gravity, adding 5ml of precooled reagent six into the affinity resin prepacked column, keeping the flow rate at 0.25ml/min, adding 5ml of reagent six again after the reagent six runs dry, and repeating the operation twice;

b) the method comprises the following steps Adding 6ml of precooled reagent seven, enabling the flow rate to be not higher than 0.25ml/min, receiving the recovered plasmid solution with endotoxin by using a pyrogen-free receiving tube after the volume of the effluent liquid reaches 1.5ml, adding 1.5-3ml of precooled reagent seven after the solution is drained, and collecting the plasmid solution;

step five: detecting the endotoxin content of the plasmid solution by using a plasmid solution endotoxin detection reagent, and repeating the operation of the fourth step if the endotoxin content of the plasmid solution cannot reach an expected value;

step six: the plasmid solution was centrifuged at high speed for 1 minute and 30 seconds, and the operation was repeated 5 times to completely remove the residual liquid and obtain plasmid, which was stored at-20 ℃.

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a non-toxic plasmid extraction kit with a multifunctional reagent group and a plasmid extraction method.

Background

The plasmid is widely existed in the biological world, has the autonomous replication ability, can keep constant copy number in progeny cells and express carried genetic information, the bacterial plasmid is a commonly used vector in DNA recombination technology, most plasmid vectors have some multipurpose auxiliary sequences, and the purposes comprise visual identification of recombinant clone by a histochemical method, generation of single-stranded DNA for sequence determination, in-vitro transcription of exogenous DNA sequence, identification of the insertion direction of a fragment, mass expression of exogenous genes and the like.

At present, the invention patent with patent number cn201710352847.x discloses a plasmid extraction kit and an extraction method, which comprises a lysis reagent group for lysing cells, a washing reagent group for removing impurities, a plasmid dissolution reagent and an endotoxin removal reagent. Preferably, the lysis reagent set consists of lysates I, II and III, wherein lysate I is an aqueous solution comprising 10mM EDTA, 0.1067mg/ml RNase A and 25mM Tris-HCl, and the pH of lysate I is 8.0; the lysis solution II is an aqueous solution containing 0.2M NaOH and 1% SDS by mass fraction; the lysis solution III is an aqueous solution containing 2-6M guanidine hydrochloride and 0.5M potassium acetate, and the pH value of the lysis solution III is 4.2. Preferably, the washing reagent group consists of a washing liquid A and a washing liquid B, wherein the washing liquid A is an aqueous solution containing 6-8M guanidine hydrochloride, 20mM Tris-HCl and 30-50% ethanol by volume fraction, and the pH of the washing liquid A is 6.0; the washing liquid B is ethanol water solution with the volume fraction of 75-90%. Preferably, the endotoxin-removing reagent is an aqueous solution comprising 5-20% TritonX-114. The components are simple and convenient to obtain, but the function of the reagent group in the kit is single, only certain types of cells can be cracked, the concentration of extracted plasmids is low, and the final endotoxin level of a sample is high.

Therefore, in order to solve the problem of the above-mentioned single function of the kit, it is necessary to solve the problem so as to improve the use scenario of the kit.

Disclosure of Invention

(1) Technical problem to be solved

Aiming at the defects of the prior art, the invention aims to provide a non-toxic plasmid extraction kit with a multifunctional reagent set and a plasmid extraction method, and the kit and the plasmid extraction method aim to solve the technical problems that the reagent set in the reagent kit has single function, can only crack a certain type of cells, the concentration of extracted plasmids is low, and the final endotoxin level of a sample is high in the prior art.

(2) Technical scheme

In order to solve the technical problems, the invention provides a non-toxic plasmid extraction kit with a multifunctional reagent group and a plasmid extraction method, the kit comprises a cracking reagent group, an impurity removal reagent group, an endotoxin removal reagent group, a plasmid recovery reagent and an endotoxin detection reagent,

the Lysis reagent group is used for lysing cells and comprises a first reagent and a second reagent, wherein the first reagent comprises 1% of triton X-100 and a phenylmethylsulfonyl fluoride solution, the volume ratio of the triton X-100 to the phenylmethylsulfonyl fluoride solution is 100:1.5, the second reagent comprises NP-40 lysine Buffer and the phenylmethylsulfonyl fluoride solution, and the volume ratio of the NP-40 lysine Buffer to the phenylmethylsulfonyl fluoride solution is 100: 1.5;

the impurity removal reagent group is used for obtaining a plasmid crude extract from a cell disruption solution, and comprises a reagent III and a reagent IV;

the plasmid recovery reagent is used for separating plasmids from bacterial genome DNA and removing proteins and other impurities so as to obtain relatively pure plasmids;

the endotoxin removal reagent group is used for removing endotoxin in a solution, and comprises a reagent five, a reagent six and a reagent seven, wherein the volume ratio of the reagent five to the reagent six to the reagent seven is 1.5: 125: 125;

the endotoxin detection reagent is used for detecting the content of endotoxin in the obtained plasmid solution.

Further, the reagent III comprises 6-8M guanidine hydrochloride, 20mM Tris-HCl, ethanol and water, and the reagent IV comprises ethanol and water.

Further, reagent five is 1.5ml of an affinity resin pre-packed column, reagent six is 125ml of a regeneration buffer comprising 1M Tris-HCl, 0.5M EDTA and 1M glucose, reagent seven is 125ml of an equilibration buffer comprising 2M NaOH and 10% SDS, and reagent six and reagent seven have a pH of 8.

Further, the plasmid recovery reagent is an aqueous solution containing polyethylene glycol and dimethyl sulfoxide.

Further, the endotoxin detection reagent is an aqueous solution containing 5-20% TritonX-114.

A plasmid extraction method comprises the above nontoxic plasmid extraction kit with a multifunctional reagent group, and comprises the following steps:

the method comprises the following steps: obtaining a cell disruption solution:

a) according to the requirement, selecting a reagent I or a reagent II, dissolving and uniformly mixing 1% triton X-100 or NP-40 lysine Buffer at room temperature, and then adding a phenylmethylsulfonyl fluoride solution to ensure that the final concentration of the solution reaches 1mM;

b) centrifuging the culture solution to be treated at low speed for 1-3min, removing the supernatant, and collecting the precipitate;

c) adding the solution obtained in the step one into the precipitate, gently blowing and beating the precipitate by using a pipette to ensure that the solution is fully contacted with cells, centrifuging the solution, and taking supernatant liquid which is cell disruption liquid;

step two: obtaining a plasmid crude extract:

a) loading the cell disruption solution on a plasmid adsorption column, centrifuging and filtering out the liquid on the plasmid adsorption column;

b) washing the plasmid adsorption column with reagent III, centrifuging and filtering out the liquid in the plasmid adsorption column;

c) washing the plasmid adsorption column with reagent IV, centrifuging, filtering out the liquid of the plasmid adsorption column, and repeating the operation twice to obtain filtrate, namely the crude plasmid extract;

step three: adding a plasmid recovery reagent into the crude plasmid extraction solution in a ratio of 1000: 40-60, oscillating for 1-3min, centrifuging at high speed, and removing supernatant to obtain a recovered plasmid solution with endotoxin;

step four: removing endotoxin:

a) the method comprises the following steps Adjusting the pH value of the recovered plasmid solution to be 6-9, placing an affinity resin prepacked column on an iron support, vertically fixing, removing the top cover of the affinity resin prepacked column, allowing a protective solution to run dry under the action of gravity, adding 5ml of precooled reagent six into the affinity resin prepacked column, keeping the flow rate at 0.25ml/min, adding 5ml of reagent six again after the reagent six runs dry, and repeating the operation twice;

b) the method comprises the following steps Adding 6ml of precooled reagent seven, enabling the flow rate to be not higher than 0.25ml/min, receiving the recovered plasmid solution with endotoxin by using a pyrogen-free receiving tube after the volume of the effluent liquid reaches 1.5ml, adding 1.5-3ml of precooled reagent seven after the solution is drained, and collecting the plasmid solution;

step five: and (4) detecting the endotoxin content of the plasmid solution by using the plasmid solution endotoxin detection reagent, and repeating the operation of the fourth step if the endotoxin content of the plasmid solution cannot reach the expected value.

Step six: the plasmid solution was centrifuged at high speed for 1 minute and 30 seconds, and the operation was repeated 5 times to completely remove the residual liquid and obtain plasmid, which was stored at-20 ℃.

(3) Advantageous effects

Compared with the prior art, the invention has the beneficial effects that: the invention relates to a non-toxic plasmid extraction kit with a multifunctional reagent group and a plasmid extraction method, wherein a plurality of reagents are concentrated in one reagent box, so that the extraction operation of plasmids is facilitated, an endotoxin detection reagent is added in the reagent box, so that the endotoxin level can be conveniently detected, the endotoxin removal reagent group can be repeatedly used, the removal effect of endotoxin is ensured, and the endotoxin level is ensured to meet the requirement.

Detailed Description

In order to make the technical means, the original characteristics, the achieved purposes and the effects of the invention easily understood and obvious, the technical solutions in the embodiments of the present invention are clearly and completely described below to further illustrate the invention, and obviously, the described embodiments are only a part of the embodiments of the present invention, but not all the embodiments.

Example 1

The specific embodiment is a non-toxic plasmid extraction kit with a multifunctional reagent group and a plasmid extraction method, the kit comprises a cracking reagent group, an impurity removal reagent group, an endotoxin removal reagent group, a plasmid recovery reagent and an endotoxin detection reagent,

the Lysis reagent group is used for lysing cells and comprises a first reagent and a second reagent, wherein the first reagent comprises 1% of triton X-100 and phenylmethylsulfonyl fluoride solution, the volume ratio of the 1% of triton X-100 to the phenylmethylsulfonyl fluoride solution is 100:1.5, the second reagent comprises NP-40 lysine Buffer and phenylmethylsulfonyl fluoride solution, and the volume ratio of the NP-40 lysine Buffer to the phenylmethylsulfonyl fluoride solution is 100: 1.5;

the impurity removal reagent group is used for obtaining a plasmid crude extract from the cell disruption solution, and comprises a reagent III and a reagent IV;

the plasmid recovery reagent is used for separating plasmids from bacterial genome DNA and removing proteins and other impurities so as to obtain relatively pure plasmids;

the endotoxin removal reagent group is used for removing endotoxin in the solution, and comprises a reagent five, a reagent six and a reagent seven, wherein the volume ratio of the reagent five to the reagent six to the reagent seven is 1.5: 125: 125;

the endotoxin detection reagent is used for detecting the content of endotoxin in the obtained plasmid solution.

Further, reagent three contained 6-8M guanidine hydrochloride, 20mM Tris-HCl, ethanol and water, and reagent four contained ethanol and water.

Further, reagent five was 1.5ml of the affinity resin pre-packed column, reagent six was 125ml of a regeneration buffer containing 1M Tris-HCl, 0.5M EDTA and 1M glucose, reagent seven was 125ml of an equilibration buffer containing 2M NaOH and 10% SDS, and reagent six and reagent seven had a pH of 8.

Further, the plasmid recovery reagent is an aqueous solution containing polyethylene glycol and dimethyl sulfoxide.

Further, the endotoxin detection reagent is an aqueous solution containing 5-20% TritonX-114.

A plasmid extraction method comprises the above nontoxic plasmid extraction kit with a multifunctional reagent group, and comprises the following steps:

the method comprises the following steps: obtaining a cell disruption solution:

a) according to the requirement, selecting a reagent I or a reagent II, dissolving and uniformly mixing 1% triton X-100 or NP-40 lysine Buffer at room temperature, and then adding a phenylmethylsulfonyl fluoride solution to ensure that the final concentration of the solution reaches 1mM;

b) centrifuging the culture solution to be treated at low speed for 1-3min, removing the supernatant, and collecting the precipitate;

c) adding the solution obtained in the step one into the precipitate, gently blowing and beating the precipitate by using a pipette to ensure that the solution is fully contacted with cells, centrifuging the solution, and taking supernatant liquid which is cell disruption liquid;

step two: obtaining a plasmid crude extract:

a) loading the cell disruption solution on a plasmid adsorption column, centrifuging and filtering out the liquid on the plasmid adsorption column;

b) washing the plasmid adsorption column with reagent III, centrifuging and filtering out the liquid in the plasmid adsorption column;

c) washing the plasmid adsorption column with reagent IV, centrifuging, filtering out the liquid of the plasmid adsorption column, and repeating the operation twice to obtain filtrate, namely the crude plasmid extract;

step three: adding a plasmid recovery reagent into the crude plasmid extract according to the proportion of 1000:40, oscillating for 1-3min, centrifuging at high speed, and removing supernatant to obtain recovered plasmid solution with endotoxin;

step four: removing endotoxin:

a) the method comprises the following steps Adjusting the pH value of the recovered plasmid solution to enable the pH value of the recovered plasmid solution to be less than 6.8, placing an affinity resin prepacked column on an iron support, vertically fixing, removing the top cover of the affinity resin prepacked column, enabling a protective solution to run dry under the action of gravity, adding 5ml of precooled reagent six into the affinity resin prepacked column, keeping the flow rate at 0.25ml/min, adding 5ml of reagent six again after the reagent six runs dry, and repeating the operation twice;

b) the method comprises the following steps Adding 6ml of precooled reagent seven, enabling the flow rate to be not higher than 0.25ml/min, receiving the recovered plasmid solution with endotoxin by using a pyrogen-free receiving tube after the volume of the effluent liquid reaches 1.5ml, adding 1.5-3ml of precooled reagent seven after the solution is drained, and collecting the plasmid solution;

step five: and (4) detecting the endotoxin content of the plasmid solution by using the plasmid solution endotoxin detection reagent, and repeating the operation of the fourth step if the endotoxin content of the plasmid solution cannot reach the expected value.

Step six: the plasmid solution was centrifuged at high speed for 1 minute and 30 seconds, and the operation was repeated 5 times to completely remove the residual liquid and obtain plasmid, which was stored at-20 ℃.

Example 2

The specific embodiment is a non-toxic plasmid extraction kit with a multifunctional reagent group and a plasmid extraction method, the kit comprises a cracking reagent group, an impurity removal reagent group, an endotoxin removal reagent group, a plasmid recovery reagent and an endotoxin detection reagent,

the Lysis reagent group is used for lysing cells and comprises a first reagent and a second reagent, wherein the first reagent comprises 1% of triton X-100 and phenylmethylsulfonyl fluoride solution, the volume ratio of the 1% of triton X-100 to the phenylmethylsulfonyl fluoride solution is 100:1.5, the second reagent comprises NP-40 lysine Buffer and phenylmethylsulfonyl fluoride solution, and the volume ratio of the NP-40 lysine Buffer to the phenylmethylsulfonyl fluoride solution is 100: 1.5;

the impurity removal reagent group is used for obtaining a plasmid crude extract from the cell disruption solution, and comprises a reagent III and a reagent IV;

the plasmid recovery reagent is used for separating plasmids from bacterial genome DNA and removing proteins and other impurities so as to obtain relatively pure plasmids;

the endotoxin removal reagent group is used for removing endotoxin in the solution, and comprises a reagent five, a reagent six and a reagent seven, wherein the volume ratio of the reagent five to the reagent six to the reagent seven is 1.5: 125: 125;

the endotoxin detection reagent is used for detecting the content of endotoxin in the obtained plasmid solution.

Further, reagent three contained 6-8M guanidine hydrochloride, 20mM Tris-HCl, ethanol and water, and reagent four contained ethanol and water.

Further, reagent five was 1.5ml of the affinity resin pre-packed column, reagent six was 125ml of a regeneration buffer containing 1M Tris-HCl, 0.5M EDTA and 1M glucose, reagent seven was 125ml of an equilibration buffer containing 2M NaOH and 10% SDS, and reagent six and reagent seven had a pH of 8.

Further, the plasmid recovery reagent is an aqueous solution containing polyethylene glycol and dimethyl sulfoxide.

Further, the endotoxin detection reagent is an aqueous solution containing 5-20% TritonX-114.

A plasmid extraction method comprises the above nontoxic plasmid extraction kit with a multifunctional reagent group, and comprises the following steps:

the method comprises the following steps: obtaining a cell disruption solution:

a) according to the requirement, selecting a reagent I or a reagent II, dissolving and uniformly mixing 1% triton X-100 or NP-40 lysine Buffer at room temperature, and then adding a phenylmethylsulfonyl fluoride solution to ensure that the final concentration of the solution reaches 1mM;

b) centrifuging the culture solution to be treated at low speed for 1-3min, removing the supernatant, and collecting the precipitate;

c) adding the solution obtained in the step one into the precipitate, gently blowing and beating the precipitate by using a pipette to ensure that the solution is fully contacted with cells, centrifuging the solution, and taking supernatant liquid which is cell disruption liquid;

step two: obtaining a plasmid crude extract:

a) loading the cell disruption solution on a plasmid adsorption column, centrifuging and filtering out the liquid on the plasmid adsorption column;

b) washing the plasmid adsorption column with reagent III, centrifuging and filtering out the liquid in the plasmid adsorption column;

c) washing the plasmid adsorption column with reagent IV, centrifuging, filtering out the liquid of the plasmid adsorption column, and repeating the operation twice to obtain filtrate, namely the crude plasmid extract;

step three: adding a plasmid recovery reagent into the crude plasmid extract according to the proportion of 1000:60, oscillating for 1-3min, centrifuging at high speed, and removing supernatant to obtain recovered plasmid solution with endotoxin;

step four: removing endotoxin:

a) the method comprises the following steps Adjusting the pH value of the recovered plasmid solution to enable the pH value of the recovered plasmid solution to be larger than 8.2, placing an affinity resin prepacked column on an iron support, vertically fixing, removing the top cover of the affinity resin prepacked column, enabling a protective solution to run dry under the action of gravity, adding 5ml of precooled reagent six into the affinity resin prepacked column, keeping the flow rate at 0.25ml/min, adding 5ml of reagent six again after the reagent six runs dry, and repeating the operation twice;

b) the method comprises the following steps Adding 6ml of precooled reagent seven, enabling the flow rate to be not higher than 0.25ml/min, receiving the recovered plasmid solution with endotoxin by using a pyrogen-free receiving tube after the volume of the effluent liquid reaches 1.5ml, adding 1.5-3ml of precooled reagent seven after the solution is drained, and collecting the plasmid solution;

step five: and (4) detecting the endotoxin content of the plasmid solution by using the plasmid solution endotoxin detection reagent, and repeating the operation of the fourth step if the endotoxin content of the plasmid solution cannot reach the expected value.

Step six: the plasmid solution was centrifuged at high speed for 1 minute and 30 seconds, and the operation was repeated 5 times to completely remove the residual liquid and obtain plasmid, which was stored at-20 ℃.

Example 3

The specific embodiment is a non-toxic plasmid extraction kit with a multifunctional reagent group and a plasmid extraction method, the kit comprises a cracking reagent group, an impurity removal reagent group, an endotoxin removal reagent group, a plasmid recovery reagent and an endotoxin detection reagent,

the Lysis reagent group is used for lysing cells and comprises a first reagent and a second reagent, wherein the first reagent comprises 1% of triton X-100 and phenylmethylsulfonyl fluoride solution, the volume ratio of the 1% of triton X-100 to the phenylmethylsulfonyl fluoride solution is 100:1.5, the second reagent comprises NP-40 lysine Buffer and phenylmethylsulfonyl fluoride solution, and the volume ratio of the NP-40 lysine Buffer to the phenylmethylsulfonyl fluoride solution is 100: 1.5;

the impurity removal reagent group is used for obtaining a plasmid crude extract from the cell disruption solution, and comprises a reagent III and a reagent IV;

the plasmid recovery reagent is used for separating plasmids from bacterial genome DNA and removing proteins and other impurities so as to obtain relatively pure plasmids;

the endotoxin removal reagent group is used for removing endotoxin in the solution, and comprises a reagent five, a reagent six and a reagent seven, wherein the volume ratio of the reagent five to the reagent six to the reagent seven is 1.5: 125: 125;

the endotoxin detection reagent is used for detecting the content of endotoxin in the obtained plasmid solution.

Further, reagent three contained 6-8M guanidine hydrochloride, 20mM Tris-HCl, ethanol and water, and reagent four contained ethanol and water.

Further, reagent five was 1.5ml of the affinity resin pre-packed column, reagent six was 125ml of a regeneration buffer containing 1M Tris-HCl, 0.5M EDTA and 1M glucose, reagent seven was 125ml of an equilibration buffer containing 2M NaOH and 10% SDS, and reagent six and reagent seven had a pH of 8.

Further, the plasmid recovery reagent is an aqueous solution containing polyethylene glycol and dimethyl sulfoxide.

Further, the endotoxin detection reagent is an aqueous solution containing 5-20% TritonX-114.

A plasmid extraction method comprises the above nontoxic plasmid extraction kit with a multifunctional reagent group, and comprises the following steps:

the method comprises the following steps: obtaining a cell disruption solution:

a) according to the requirement, selecting a reagent I or a reagent II, dissolving and uniformly mixing 1% triton X-100 or NP-40 lysine Buffer at room temperature, and then adding a phenylmethylsulfonyl fluoride solution to ensure that the final concentration of the solution reaches 1mM;

b) centrifuging the culture solution to be treated at low speed for 1-3min, removing the supernatant, and collecting the precipitate;

c) adding the solution obtained in the step one into the precipitate, gently blowing and beating the precipitate by using a pipette to ensure that the solution is fully contacted with cells, centrifuging the solution, and taking supernatant liquid which is cell disruption liquid;

step two: obtaining a plasmid crude extract:

a) loading the cell disruption solution on a plasmid adsorption column, centrifuging and filtering out the liquid on the plasmid adsorption column;

b) washing the plasmid adsorption column with reagent III, centrifuging and filtering out the liquid in the plasmid adsorption column;

c) washing the plasmid adsorption column with reagent IV, centrifuging, filtering out the liquid of the plasmid adsorption column, and repeating the operation twice to obtain filtrate, namely the crude plasmid extract;

step three: adding a plasmid recovery reagent into the crude plasmid extract according to the proportion of 1000:50, oscillating for 1-3min, centrifuging at high speed, and removing supernatant to obtain recovered plasmid solution with endotoxin;

step four: removing endotoxin:

a) the method comprises the following steps Adjusting the pH value of the recovered plasmid solution to 7-8, placing an affinity resin prepacked column on an iron support, vertically fixing, removing the top cover of the affinity resin prepacked column, allowing the protective solution to run dry under the action of gravity, adding 5ml of precooled reagent six into the affinity resin prepacked column, keeping the flow rate at 0.25ml/min, adding 5ml of reagent six again after the reagent six runs dry, and repeating the operation twice;

b) the method comprises the following steps Adding 6ml of precooled reagent seven, enabling the flow rate to be not higher than 0.25ml/min, receiving the recovered plasmid solution with endotoxin by using a pyrogen-free receiving tube after the volume of the effluent liquid reaches 1.5ml, adding 1.5-3ml of precooled reagent seven after the solution is drained, and collecting the plasmid solution;

step five: and (4) detecting the endotoxin content of the plasmid solution by using the plasmid solution endotoxin detection reagent, and repeating the operation of the fourth step if the endotoxin content of the plasmid solution cannot reach the expected value.

Step six: the plasmid solution was centrifuged at high speed for 1 minute and 30 seconds, and the operation was repeated 5 times to completely remove the residual liquid and obtain plasmid, which was stored at-20 ℃.

When working with example 3, the samples had the lowest endotoxin levels and the highest concentration of extracted plasmid.

Having thus described the principal technical features and basic principles of the invention, and the advantages associated therewith, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

TABLE 1

Furthermore, it should be understood that although the present description is described in terms of various embodiments, not every embodiment includes only a single embodiment, and such descriptions are provided for clarity only, and those skilled in the art will recognize that the embodiments described herein can be combined as a whole to form other embodiments as would be understood by those skilled in the art.