阅读说明：本技术 一种提取细菌质粒dna的菌体裂解液、试剂盒及方法 (Thallus lysate, kit and method for extracting bacterial plasmid DNA ) 是由 赵斯斯 薛茜 陈辉 芦丽亚 万军飞 于 2021-08-10 设计创作，主要内容包括：本申请提供一种提取细菌质粒DNA的菌体裂解液、试剂盒及方法,所述菌体裂解液的组成为：4-5M蛋白质变性剂、100-500mM氯化钠、2-3wt％表面活性剂、38-62mM pH缓冲剂和余量的水；所述蛋白质变性剂包括异硫氰酸胍和盐酸胍中的至少一种。本申请主要利用表面活性剂和蛋白质变性剂(异硫氰酸胍和盐酸胍中的至少一种)协同作用对菌体细胞进行裂解；通过pH缓冲剂和氯化钠能够较好调节菌体裂解液的pH；该菌体裂解液的组成成分简单,成本低,不含有溶菌酶和RNaseA酶,可在室温下长期稳定保存且对后续实验无影响；使用该菌体裂解液的试剂盒提取质粒DNA的提取效率高,速度快,可以满足多种生物实验需求。(The application provides a thallus lysate, a kit and a method for extracting bacterial plasmid DNA, wherein the thallus lysate comprises the following components: 4-5M protein denaturant, 100-500mM sodium chloride, 2-3 wt% surfactant, 38-62mM pH buffer and balance water; the protein denaturant includes at least one of guanidinium isothiocyanate and guanidinium hydrochloride. The method mainly utilizes the synergistic effect of a surfactant and a protein denaturant (at least one of guanidine isothiocyanate and guanidine hydrochloride) to crack the somatic cells; the pH of the thallus lysate can be well adjusted through a pH buffering agent and sodium chloride; the thallus lysate has simple components and low cost, does not contain lysozyme and RNaseA enzyme, can be stably stored at room temperature for a long time and has no influence on subsequent experiments; the kit for extracting plasmid DNA by using the thallus lysate has high extraction efficiency and high speed, and can meet the requirements of various biological experiments.)

1. A thallus lysate for extracting bacterial plasmid DNA is characterized in that the thallus lysate comprises the following components: 4-5M protein denaturant, 100-500mM sodium chloride, 2-3 wt% surfactant, 38-62mM pH buffer and balance water; the protein denaturant includes at least one of guanidinium isothiocyanate and guanidinium hydrochloride.

2. The lysate for extracting plasmid DNA from bacteria of claim 1, wherein the surfactant comprises at least one of Triton X-100 and Tween-20;

preferably, the pH buffering agent comprises at least one of citric acid, sodium citrate, acetic acid and sodium acetate.

3. A kit for extracting bacterial plasmid DNA is characterized by comprising a thallus lysate, a washing solution and an eluent; the lysate is the lysate according to claim 1 or 2.

4. The kit for extracting bacterial plasmid DNA according to claim 3, wherein the washing solution comprises washing solution A and washing solution B;

the washing solution A comprises the following components: 0.5-3M guanidine hydrochloride, 30-70 wt% isopropanol and balance water;

the washing solution B comprises the following components: 40-80mM Tris-HCl and 40-80 wt% ethanol, and the pH of the washing solution B is 6.5-8.5.

5. The kit for extracting bacterial plasmid DNA according to claim 3, wherein the eluent comprises the following components: 2-10mM Tris-HCl; the pH of the eluent is 6.5-8.5.

6. The kit for extracting bacterial plasmid DNA according to any one of claims 3 to 5, wherein the kit further comprises a silica gel membrane adsorption column.

7. A method for extracting bacterial plasmid DNA, which is characterized in that the bacterial plasmid DNA is extracted by using the kit for extracting bacterial plasmid DNA according to any one of claims 3-6.

8. The method of extracting bacterial plasmid DNA as claimed in claim 7, wherein said method comprises:

carrying out amplification culture on the bacterial strain with the plasmid to obtain bacterial liquid, carrying out centrifugal separation on the bacterial liquid, and collecting bacterial precipitates;

performing thallus lysis on the thallus precipitate by using the thallus lysate to obtain a thallus lysate;

and separating and purifying the thallus lysate by using the washing solution and the eluent.

9. The method for extracting bacterial plasmid DNA as claimed in claim 8, wherein the amplification culture process comprises: inoculating a strain with plasmids in an LB culture medium containing antibiotics for culture;

preferably, the temperature of the culture is 30-37 ℃, and the time is 12-16 h;

preferably, the step of lysing the cell precipitate using the cell lysate includes: and uniformly mixing the thallus precipitate and the thallus lysate, and then standing.

10. The method for extracting bacterial plasmid DNA according to claim 8 or 9, wherein the process of separation and purification treatment comprises: transferring the thallus lysate to a silica gel membrane adsorption column, standing, performing centrifugal separation, discarding liquid, adding a washing solution, standing, performing centrifugal separation, and discarding liquid; then adding the eluent, standing and then carrying out centrifugal separation to obtain a plasmid DNA solution.

Technical Field

The invention relates to the technical field of biology, in particular to a thallus lysate, a kit and a method for extracting bacterial plasmid DNA.

Background

Plasmids are extrachromosomally self-replicating genetic units comprising eukaryotic organelles and DNA molecules outside the bacterial chromosome. Most bacterial plasmid DNA is a double-stranded closed circular DNA molecule, often carrying antibiotic resistance genes, such as ampicillin resistance and chloramphenicol resistance.

Plasmid DNA is a gene delivery vehicle, is widely applied to molecular biology research, and relates to various fields of gene cloning, gene sequence analysis, gene vaccines, gene editing and the like. The extraction of plasmid DNA molecules from bacteria is the most fundamental procedure in molecular biology experiments. Meanwhile, the efficiency, purity and quality of plasmid DNA molecule extraction are directly related to the subsequent performance of various molecular biological experiments, such as the success or failure of enzyme digestion connection, transformation, transfection, amplification, sequencing and other experiments. The main steps of plasmid DNA extraction include the culture of bacteria, the collection and lysis of bacteria, and the isolation and purification of plasmid DNA. In terms of the cracking mode, common plasmid extraction methods include an alkaline cracking method and a boiling method. The alkaline lysis process is currently the most common and classical process in the laboratory, where the alkaline lysis step requires three solution treatments: the main components of the solution I are Tris-HCl (Tris hydroxymethyl aminomethane hydrochloride) and EDTA (ethylene diamine tetraacetic acid) which are used for fully suspending thalli sediments; the main components of the solution II are NaOH and SDS (sodium dodecyl sulfate), the main functions are cell lysis and cell membrane structure destruction, and simultaneously, the hydrogen bonds of the chromosome DNA are broken, the SDS can enhance alkalinity, and simultaneously the SDS binds protein to generate precipitation; the main components of the solution III are acetic acid and potassium acetate, the effect is to neutralize sodium hydroxide in the solution II, the solution system is restored to be neutral, the renaturation of two complementary chains of plasmid DNA is rapid and accurate, the chromosome DNA is difficult to renaturate to form a winding net structure, and the chromosome DNA, protein-SDS compound and other impurities are precipitated by centrifugation. The plasmid extracted by the alkaline cracking method has high purity and high yield, but the method has relatively complex operation and longer required operation time. The boiling method is relatively traditional and extensive, the cracking is required to be carried out at the temperature of 100 ℃, the conditions are too violent, the plasmid is easy to break, and the control is difficult in experimental operation.

Most of the available plasmid DNA extracting kits are based on alkaline lysis method and combined with purifying column to separate and purify plasmid DNA. In the prior art, a method for extracting plasmids by a one-step method is reported, but the components of a cracking reagent of the used kit are more, lysozyme is required to synergistically increase the cracking effect, and RNaseA enzyme participates in removing the interference of RNA on plasmid extraction. However, the addition of lysozyme and RNaseA increases the cost of the reagent, and also has high requirements on the storage and transportation conditions of the reagent.

Disclosure of Invention

The invention aims to provide a thallus lysate, a kit and a method for extracting bacterial plasmid DNA, the method realizes one-step thallus lysis, namely column loading, has the advantages of simple operation, short operation time, simple components of the thallus lysate and low cost, and the kit using the thallus lysate has high extraction efficiency of extracting the plasmid DNA and has certain advantages in biological research experiments.

In order to achieve the purpose, the following technical scheme is adopted in the application:

a thallus lysate for extracting bacterial plasmid DNA, which comprises the following components: 4-5M protein denaturant, 100-500mM sodium chloride, 2-3 wt% surfactant, 38-62mM pH buffer and balance water; the protein denaturant includes at least one of guanidinium isothiocyanate and guanidinium hydrochloride.

In some embodiments, the surfactant comprises at least one of Triton X-100 and Tween-20;

in some embodiments, the pH buffering agent comprises at least one of citric acid, sodium citrate, acetic acid, and sodium acetate.

The application also provides a kit for extracting the bacterial plasmid DNA, wherein the kit comprises a thallus lysate, a washing solution and an eluent; the thallus lysate is the thallus lysate.

In some embodiments, the wash solution comprises wash solution a and wash solution B;

the washing solution A comprises the following components: 0.5-3M guanidine hydrochloride, 30-70 wt% isopropanol and balance water;

the washing solution B comprises the following components: 40-80mM Tris-HCl and 40-80 wt% ethanol, and the pH of the washing solution B is 6.5-8.5.

In some embodiments, the composition of the eluent is: 2-10mM Tris-HCl; the pH of the eluent is 6.5-8.5.

In some embodiments, the kit further comprises a silica gel membrane adsorption column.

The application also provides a method for extracting the bacterial plasmid DNA, which is obtained by adopting the kit for extracting the bacterial plasmid DNA.

In some embodiments, the method comprises:

carrying out amplification culture on the bacterial strain with the plasmid to obtain bacterial liquid, carrying out centrifugal separation on the bacterial liquid, and collecting bacterial precipitates;

performing thallus lysis on the thallus precipitate by using the thallus lysate to obtain a thallus lysate;

and separating and purifying the thallus lysate by using the washing solution and the eluent.

In some embodiments, the process of expanding the culture comprises: inoculating a strain with plasmids in an LB culture medium containing antibiotics for culture;

preferably, the temperature of the culture is 30-37 ℃, and the time is 12-16 h;

in some embodiments, the step of lysing the bacterial pellet using the bacterial lysate comprises: and uniformly mixing the thallus precipitate and the thallus lysate, and then standing.

In some embodiments, the process of separating and purifying comprises: transferring the thallus lysate to a silica gel membrane adsorption column, standing, performing centrifugal separation, discarding liquid, adding a washing solution, standing, performing centrifugal separation, and discarding liquid; then adding the eluent, standing and then carrying out centrifugal separation to obtain a plasmid DNA solution.

The invention has the beneficial effects that:

the components of the thallus lysate for extracting the plasmid DNA of the bacteria comprise a protein denaturant, sodium chloride, a surfactant and a pH buffer, and the synergistic effect of the surfactant and the protein denaturant (at least one of guanidinium isothiocyanate and guanidinium hydrochloride) is mainly utilized to crack thallus cells; the pH of the thallus lysate is adjusted to be between 3 and 4 by a pH buffering agent and sodium chloride, gDNA, plasmid DNA and RNA can be well distinguished, and meanwhile, the plasmid DNA and a silica gel adsorption film can be better combined under the condition; the thallus lysate has simple components and low cost, does not contain lysozyme and RNaseA enzyme, can be stably stored at room temperature for a long time and has no influence on subsequent experiments; the kit for extracting plasmid DNA by using the thallus lysate has high extraction efficiency and high speed, and can meet the requirements of various biological experiments.

Drawings

To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, and it should be understood that the following drawings only illustrate some embodiments of the present invention, and therefore should not be considered as limiting the scope of the present invention.

FIG. 1 is a graph showing the results of agarose gel electrophoresis of high copy plasmid DNA (pUC57) extracted by the kit of examples 1-3;

FIG. 2 is a graph showing the results of agarose gel electrophoresis of high copy plasmid DNA (pUC57) extracted by the kits of example 1, example 4 and example 5;

FIG. 3 is a graph showing the results of agarose gel electrophoresis of example 5 using the kit and a commercially available kit to extract high copy plasmid DNA (pUC 57);

FIG. 4 is a graph showing the results of agarose gel electrophoresis of high copy plasmid DNA (pUC57) extracted by the kits of example 5 and comparative example 1;

FIG. 5 is a graph showing agarose gel electrophoresis results of high copy plasmid DNA (pUC57) extracted by the kits of comparative example 2, comparative example 3, and example 5;

FIG. 6 is a diagram showing the results of agarose gel electrophoresis of the low copy plasmid DNA (8K) extracted by the kit of example 5.

Detailed Description

The terms as used herein:

"prepared from … …" is synonymous with "comprising". The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

The conjunction "consisting of … …" excludes any unspecified elements, steps or components. If used in a claim, the phrase is intended to claim as closed, meaning that it does not contain materials other than those described, except for the conventional impurities associated therewith. When the phrase "consisting of … …" appears in a clause of the subject matter of the claims rather than immediately after the subject matter, it defines only the elements described in the clause; other elements are not excluded from the claims as a whole.

When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as a range of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when the range "1 ~ 5" is disclosed, the ranges described should be construed to include the ranges "1 ~ 4", "1 ~ 3", "1 ~ 2 and 4 ~ 5", "1 ~ 3 and 5", and the like. When a range of values is described herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range.

In these examples, the parts and percentages are by mass unless otherwise indicated.

"part by mass" means a basic unit of measure indicating a mass ratio of a plurality of components, and 1 part may represent any unit mass, for example, 1g or 2.689 g. If we say that the part by mass of the component A is a part by mass and the part by mass of the component B is B part by mass, the ratio of the part by mass of the component A to the part by mass of the component B is a: b. alternatively, the mass of the A component is aK and the mass of the B component is bK (K is an arbitrary number, and represents a multiple factor). It is unmistakable that, unlike the parts by mass, the sum of the parts by mass of all the components is not limited to 100 parts.

"and/or" is used to indicate that one or both of the illustrated conditions may occur, e.g., a and/or B includes (a and B) and (a or B).

The application provides a thallus lysate for extracting bacterial plasmid DNA, which comprises the following components: 4-5M protein denaturant, 100-500mM sodium chloride, 2-3 wt% surfactant, 38-62mM pH buffer and balance water.

The protein denaturant includes at least one of guanidinium isothiocyanate and guanidinium hydrochloride; the protein denaturant can rapidly dissolve protein, so that cell structure is broken, and nucleoprotein is rapidly separated from nucleic acid due to disappearance of secondary structure damage; the protein denaturant is preferably guanidine isothiocyanate, and can convert most proteins into random coil state, and can rapidly break cells.

In some embodiments, the surfactant comprises at least one of Triton X-100 and Tween-20; the surfactant can dissolve lipid on cell membrane, nuclear membrane and organelle membrane, and increase permeability of cell membrane; can regulate the solubility of DNA in water without destroying its structure.

In some embodiments, the pH buffering agent comprises at least one of citric acid, sodium citrate, acetic acid, and sodium acetate; preferably, the pH buffer is citric acid and sodium citrate according to (27-39): (11-23). The sodium citrate is a weak acid strong alkali salt, can form a strong pH buffering agent by being compatible with citric acid, and can adjust the pH within a small range; in addition, the sodium citrate also has excellent retarding performance and stability.

The components of the thallus lysate for extracting the plasmid DNA of the bacteria comprise a protein denaturant, sodium chloride, a surfactant and a pH buffer, and the synergistic effect of the surfactant and the protein denaturant (at least one of guanidinium isothiocyanate and guanidinium hydrochloride) is mainly utilized to crack thallus cells; the pH of the lysate is adjusted to be between 3 and 4 by a pH buffering agent and sodium chloride, gDNA, plasmid DNA and RNA can be well distinguished, and meanwhile, the plasmid DNA and a silica gel adsorption film can be better combined under the condition; the thallus lysate has simple components and low cost, does not contain lysozyme and RNaseA enzyme, can be stably stored at room temperature for a long time, and has no influence on subsequent experiments.

The application also provides a kit for extracting the bacterial plasmid DNA, wherein the kit comprises a thallus lysate, a washing solution and an eluent; the thallus lysate is the thallus lysate; the kit using the thallus lysate is convenient to prepare, and the extraction efficiency of extracting plasmid DNA by using the kit is high, the speed is high, and the requirements of various biological experiments can be met.

In some embodiments, the wash solution comprises wash solution a and wash solution B; the washing solution A comprises the following components: 0.5-3M guanidine hydrochloride, 30-70 wt% isopropanol and balance water; the washing solution B comprises the following components: 40-80mM Tris-HCl and 40-80 wt% ethanol, and the pH of the washing solution B is 6.5-8.5.

It should be noted that the washing solution a mainly functions to remove proteins, and the washing solution B mainly functions to remove salt ion impurities.

In some embodiments, the composition of the eluent is: 2-10mM Tris-HCl; the pH of the eluent is 6.5-8.5.

In some embodiments, the kit further comprises a silica gel membrane adsorption column.

The application also provides a method for extracting the bacterial plasmid DNA, which is obtained by adopting the kit for extracting the bacterial plasmid DNA.

In some embodiments, the method comprises:

s10, carrying out amplification culture on the bacterial strain with the plasmid to obtain bacterial liquid, carrying out centrifugal separation on the bacterial liquid, and collecting bacterial precipitates.

In some embodiments, the process of expanding the culture comprises: inoculating a strain with plasmids in an LB culture medium containing antibiotics for culture; preferably, the strain is cultured in a volume ratio to the LB medium of 1: (100-200) in the LB medium; the culture temperature is 30-37 deg.C, and the culture time is 12-16 h.

It should be noted that the selection of the above antibiotics is related to the plasmid DNA to be extracted, and if the extracted plasmid DNA is ampicillin resistant, ampicillin is added to LB medium; if the extracted plasmid DNA is kanamycin-resistant, kanamycin is added to the LB medium.

Preferably, the rotation speed of the centrifugal separation is 10000-12000rpm, and the time is 2-3 min.

And S20, performing thallus lysis on the thallus precipitate by using the thallus lysate to obtain a thallus lysate.

In some embodiments, the step of lysing the bacterial pellet with the bacterial lysate includes: uniformly mixing the thallus precipitate and the thallus lysate, and then standing; the dosage of the thallus lysate is (500-; the standing time is about 5-15 min.

And S30, separating and purifying the thallus lysate by using the washing solution and the eluent.

In some embodiments, the separation and purification process comprises:

s301, transferring all the thallus lysate onto a silica gel membrane adsorption column, then placing the silica gel membrane adsorption column into a collecting pipe, standing for 1-2min at room temperature, centrifuging for 1-2min at the rotating speed of 10000-;

s302: adding a washing solution A into the silica gel membrane adsorption column, centrifuging for 1-2min at the rotating speed of 10000-12000rpm, taking out the silica gel membrane adsorption column, pouring out liquid in the collecting pipe, and then putting the silica gel membrane adsorption column into the collecting pipe; adding a washing solution A into the silica gel membrane adsorption column for centrifugal washing once, wherein the dosage of the washing solution A is (500-;

s303: adding 500 plus 700ul of washing solution B into the silica gel membrane adsorption column, centrifuging for 1-2min at the rotating speed of 10000 plus 12000rpm, taking out the silica gel membrane adsorption column, pouring out the liquid in the collection pipe, and then putting the silica gel membrane adsorption column into the collection pipe; adding a washing solution B into the silica gel membrane adsorption column for centrifugal washing once, wherein the dosage of the washing solution B is (500-;

s305: placing the collecting pipe into a centrifuge, centrifuging for 2min at the rotating speed of 10000-12000rpm to remove the residual washing liquid on the silica gel membrane adsorption column, particularly further removing the residual ethanol on the silica gel membrane adsorption column, and avoiding the influence on the subsequent elution and purification steps;

s306: when the ethanol is completely volatilized, putting the silica gel membrane adsorption column into a clean centrifugal tube, adding eluent into the silica gel membrane adsorption column, standing for 2-3min at room temperature, centrifuging for 1-2min at the rotating speed of 10000-12000rpm, taking out the silica gel membrane adsorption column, and collecting the supernatant in the centrifugal tube, namely the plasmid DNA solution; the dosage of the eluent is (50-100) ul/(0.5-1.5) ml of thallus lysate.

Embodiments of the present invention will be described in detail below with reference to specific examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1

A kit for extracting bacterial plasmid DNA comprises a thallus lysate, a washing solution A, a washing solution B, an eluent and a silica gel membrane adsorption column; wherein:

the composition of the thallus lysate is as follows: 33mM citric acid, 17mM sodium citrate, 250mM sodium chloride, 3% Triton X-100, and 4M guanidinium isothiocyanate;

the washing solution A comprises the following components: 1M guanidine hydrochloride and 50% isopropanol;

the washing solution B comprises the following components: 50mM Tris-HCl and 80% ethanol, pH 7.0;

the eluent comprises the following components: 2.5mM Tris-HCl, pH 8.0;

adsorption column: nucleic acid purification adsorption columns (including collection tubes) from Biotechnology engineering (Shanghai) Ltd.

Example 2

A kit for extracting bacterial plasmid DNA comprises a thallus lysate, a washing solution A, a washing solution B, an eluent and a silica gel membrane adsorption column; wherein:

the composition of the thallus lysate is as follows: 27mM citric acid, 23mM sodium citrate, 250mM sodium chloride, 2% Triton X-100, and 4M guanidinium isothiocyanate;

the washing solution A comprises the following components: 1M guanidine hydrochloride and 50% isopropanol;

the washing solution B comprises the following components: 50mM Tris-HCl and 80% ethanol, pH 7.0;

the eluent comprises the following components: 2.5mM Tris-HCl, pH 8.0;

adsorption column: nucleic acid purification adsorption columns (including collection tubes) from Biotechnology engineering (Shanghai) Ltd.

Example 3

A kit for extracting bacterial plasmid DNA comprises a thallus lysate, a washing solution A, a washing solution B, an eluent and a silica gel membrane adsorption column; wherein:

the composition of the thallus lysate is as follows: 39mM citric acid, 11mM sodium citrate, 250mM sodium chloride, 2% Triton X-100, and 4M guanidinium isothiocyanate;

the washing solution A comprises the following components: 1M guanidine hydrochloride and 50% isopropanol;

the washing solution B comprises the following components: 50mM Tris-HCl and 80% ethanol, pH 7.0;

the eluent comprises the following components: 2.5mM Tris-HCl, pH 8.0;

adsorption column: nucleic acid purification adsorption columns (including collection tubes) from Biotechnology engineering (Shanghai) Ltd.

Example 4

A kit for extracting bacterial plasmid DNA comprises a thallus lysate, a washing solution A, a washing solution B, an eluent and a silica gel membrane adsorption column; wherein:

the composition of the thallus lysate is as follows: 33mM citric acid, 17mM sodium citrate, 250mM sodium chloride, 3% Triton X-100, and 4.5M guanidinium isothiocyanate;

the washing solution A comprises the following components: 1M guanidine hydrochloride and 50% isopropanol;

the washing solution B comprises the following components: 50mM Tris-HCl and 80% ethanol, pH 7.0;

the eluent comprises the following components: 2.5mM Tris-HCl, pH 8.0;

adsorption column: nucleic acid purification adsorption columns (including collection tubes) from Biotechnology engineering (Shanghai) Ltd.

Example 5

A kit for extracting bacterial plasmid DNA comprises a thallus lysate, a washing solution A, a washing solution B, an eluent and a silica gel membrane adsorption column; wherein:

the composition of the thallus lysate is as follows: 33mM citric acid, 17mM sodium citrate, 250mM sodium chloride, 3% Triton X-100, and 5M guanidinium isothiocyanate;

the washing solution A comprises the following components: 1M guanidine hydrochloride and 50% isopropanol;

the washing solution B comprises the following components: 50mM Tris-HCl and 80% ethanol, pH 7.0;

the eluent comprises the following components: 2.5mM Tris-HCl, pH 8.0;

adsorption column: nucleic acid purification adsorption columns (including collection tubes) from Biotechnology engineering (Shanghai) Ltd.

Example 6

A kit for extracting bacterial plasmid DNA comprises a thallus lysate, a washing solution A, a washing solution B, an eluent and a silica gel membrane adsorption column; wherein:

the composition of the thallus lysate is as follows: 33mM citric acid, 17mM sodium citrate, 500mM sodium chloride, 3% Triton X-100, and 4.5M guanidinium isothiocyanate;

the washing solution A comprises the following components: 0.5M guanidine hydrochloride and 70% isopropanol;

the washing solution B comprises the following components: 80mM Tris-HCl and 40% ethanol, pH 8.0;

the eluent comprises the following components: 10mM Tris-HCl, pH 6.5;

adsorption column: nucleic acid purification adsorption columns (including collection tubes) from Biotechnology engineering (Shanghai) Ltd.

Example 7

A kit for extracting bacterial plasmid DNA comprises a thallus lysate, a washing solution A, a washing solution B, an eluent and a silica gel membrane adsorption column; wherein:

the composition of the thallus lysate is as follows: 33mM citric acid, 17mM sodium citrate, 100mM sodium chloride, 3% Triton X-100, and 4.5M guanidinium isothiocyanate;

the washing solution A comprises the following components: 3M guanidine hydrochloride and 30% isopropanol;

the washing solution B comprises the following components: 40mM Tris-HCl and 80% ethanol, pH 6.5;

the eluent comprises the following components: 6mM Tris-HCl, pH 7.5;

adsorption column: nucleic acid purification adsorption columns (including collection tubes) from Biotechnology engineering (Shanghai) Ltd.

Comparative example 1

A kit for extracting bacterial plasmid DNA comprises a thallus lysate, a washing solution A, a washing solution B, an eluent and a silica gel membrane adsorption column; wherein:

the composition of the thallus lysate is as follows: 300mM sodium chloride, 3% Triton X-100 and 4M guanidinium isothiocyanate;

the washing solution A comprises the following components: 1M guanidine hydrochloride and 50% isopropanol;

the washing solution B comprises the following components: 50mM Tris-HCl and 80% ethanol, pH 7.0;

the eluent comprises the following components: 2.5mM Tris-HCl, pH 8.0;

adsorption column: nucleic acid purification adsorption columns (including collection tubes) from Biotechnology engineering (Shanghai) Ltd.

Comparative example 2

A kit for extracting bacterial plasmid DNA comprises a thallus lysate, a washing solution A, a washing solution B, an eluent and a silica gel membrane adsorption column; wherein:

the composition of the thallus lysate is as follows: 33mM citric acid, 17mM sodium citrate, 250mM sodium chloride, 3% Triton X-100;

the washing solution A comprises the following components: 1M guanidine hydrochloride and 50% isopropanol;

the washing solution B comprises the following components: 50mM Tris-HCl and 80% ethanol, pH 7.0;

the eluent comprises the following components: 2.5mM Tris-HCl, pH 8.0;

adsorption column: nucleic acid purification adsorption columns (including collection tubes) from Biotechnology engineering (Shanghai) Ltd.

Comparative example 3

A kit for extracting bacterial plasmid DNA comprises a thallus lysate, a washing solution A, a washing solution B, an eluent and a silica gel membrane adsorption column; wherein:

the composition of the thallus lysate is as follows: 33mM citric acid, 17mM sodium citrate, 250mM sodium chloride, 3% Triton X-100, and 2M guanidinium isothiocyanate;

the washing solution A comprises the following components: 1M guanidine hydrochloride and 50% isopropanol;

the washing solution B comprises the following components: 50mM Tris-HCl and 80% ethanol, pH 7.0;

the eluent comprises the following components: 2.5mM Tris-HCl, pH 8.0;

adsorption column: nucleic acid purification adsorption columns (including collection tubes) from Biotechnology engineering (Shanghai) Ltd.

Example 8

A method of extracting bacterial plasmid DNA comprising:

(1) inoculating E.coli strain with plasmid in LB culture medium containing appropriate antibiotic, and shake culturing at 37 deg.C for 15 hr; taking 1ml of the cultured bacterial liquid, centrifuging at 10000rpm for 3min, collecting thalli, pouring out or sucking dry the culture medium, and collecting thalli precipitates.

(2) And adding 700ul of thallus lysate into the collected thallus precipitate, uniformly mixing to suspend the thallus precipitate, and standing at room temperature for 5min to obtain the thallus lysate.

(3) Transferring the thallus lysate to a silica gel membrane adsorption column, standing at room temperature for 1min, centrifuging at 12000rpm for 2min, taking out the silica gel membrane adsorption column, pouring out the liquid in the collection tube, and putting the silica gel membrane adsorption column into the collection tube.

(4) Adding 500ul of washing solution A into the silica gel membrane adsorption column, centrifuging at 10000rpm for 2min, taking out the silica gel membrane adsorption column, pouring off the liquid in the collection tube, and putting the silica gel membrane adsorption column into the collection tube.

(5) Adding 500ul of washing solution B into the silica gel membrane adsorption column, centrifuging at 10000rpm for 2min, taking out the silica gel membrane adsorption column, pouring off the liquid in the collection tube, and putting the silica gel membrane adsorption column into the collection tube.

(6) Repeating the step (5) once.

(7) The collection tube was placed in a centrifuge and centrifuged at 12000rpm for 2 min.

(8) And after the ethanol is completely volatilized, putting the silica gel membrane adsorption column into a clean 1.5ml centrifuge tube, adding 50ul of eluent into the center of the silica gel membrane adsorption column membrane, standing at room temperature for 2min, centrifuging at 12000rpm for 2min, taking out the silica gel membrane adsorption column, collecting the supernatant in the centrifuge tube, namely plasmid DNA solution, and storing at-20 ℃ or using in subsequent experiments.

Test example 1

Selecting a strain: coli strain with high copy plasmid pUC57 was selected for the experiment.

Selecting a kit: extraction of bacterial plasmid DNA was performed using the kit of examples 1-3.

The extraction method of bacterial plasmid DNA comprises the following steps: extraction of bacterial plasmid DNA was performed using the method of example 8.

The plasmid DNAs extracted by the kits of examples 1 to 3 were subjected to agarose gel electrophoresis detection, and the results are shown in FIG. 1 for 3 groups (corresponding to the kit of example 1, the kit of example 2, and the kit of example 3, each group being 3 parallel), wherein the sample corresponding to Lane 1 is DNA molecular weight standard (5000 bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom), Lane 2-4 is the result of the plasmid DNA extracted by the kit of example 1, Lane 5-7 is the result of the plasmid DNA extracted by the kit of example 2, and Lane 8-10 is the result of the plasmid DNA extracted by the kit of example 3.

The results show that: the yield of plasmid DNA extracted by the kit of the embodiment 1-3 is high; compared with the yield of plasmid DNA extracted by the kit of example 2 and example 3, the yield of plasmid DNA extracted by the kit of example 1 is higher; in addition, the cracking process of extracting the plasmid pUC57 from the escherichia coli culture only takes 5min, so that the experimental time is greatly saved, and the experimental efficiency is improved.

Test example 2

Selecting a strain: coli strain with high copy plasmid pUC57 was selected for the experiment.

Selecting a kit: extraction of bacterial plasmid DNA was performed using the kits of example 1, example 4 and example 5.

Bacterial plasmid DNA extraction: extraction of bacterial plasmid DNA was performed using the method of example 8.

The plasmid DNAs extracted by the kits of example 1, example 4 and example 5 were subjected to agarose gel electrophoresis detection, and the results are shown in FIG. 2, wherein the samples in lane 1 correspond to DNA molecular weight standards (5000 bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom), lanes 2-4 are the results of the plasmid DNAs extracted by the kit of example 1, lanes 5-7 are the results of the plasmid DNAs extracted by the kit of example 4, and lanes 8-10 are the results of the plasmid DNAs extracted by the kit of example 5, respectively, and 3 groups (each group corresponds to the kit of example 1, the kit of example 4 and the kit of example 5, respectively).

The results show that: the yield of plasmid DNA extracted by the kit of the embodiment 4 and the embodiment 5 is higher, and the yield of the embodiment 4 is equivalent to that of the embodiment 5; meanwhile, the cracking process of extracting the plasmid pUC57 from the escherichia coli culture only takes 5min, so that the experimental time is greatly saved, and the experimental efficiency is improved.

In addition, the above-mentioned example 6 and example 7 kit extraction of plasmid DNA yield and example 4 kit extraction of plasmid DNA yield is equivalent.

Test example 3

Selecting a strain: coli strain with high copy plasmid pUC57 was selected for the experiment.

Selecting a kit: bacterial plasmid DNA was extracted using the kit of example 5 in comparison to a commercially available kit for one-step extraction of bacterial plasmid DNA (Beijing Baiolaibobobobobobo BTN 70903).

Bacterial plasmid DNA extraction: extraction of bacterial plasmid DNA was performed using the method of example 8.

The plasmid DNAs extracted by the above-mentioned commercially available kit and the kit of example 5 were subjected to agarose gel electrophoresis, and the results are shown in FIG. 3, wherein the sample in lane 1 is DNA molecular weight standard (5000 bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom), the samples in lane 2-4 are the results of plasmid DNA extracted by the commercially available kit, and the samples in lane 5-7 are the results of plasmid DNA extracted by the kit of example 5, for 2 groups (each group corresponds to the commercially available kit and the kit of example 5, and each group is 3 parallel).

The results show that: the yield of the bacterial plasmid DNA extracted by the kit of example 4 is equivalent to that of the bacterial plasmid DNA extracted by the commercially available kit, but the purity of the bacterial plasmid DNA extracted by the kit of example 4 is better, and the lysis process takes shorter time.

Test example 4

Selecting a strain: coli strain with high copy plasmid pUC57 was selected for the experiment.

Selecting a kit: extraction of bacterial plasmid DNA was performed using the kits of example 5 and comparative example 1.

Bacterial plasmid DNA extraction: extraction of bacterial plasmid DNA was performed using the method of example 8.

The plasmid DNAs extracted by the kits of example 5 and comparative example 1 were subjected to agarose gel electrophoresis detection for 2 groups (corresponding to the kit of example 4 and the kit of comparative example 1, respectively, each group was 3 parallel), and the results are shown in FIG. 4, where the sample in lane 1 is DNA molecular weight standard (5000 bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom), lanes 2-4 are the results of the plasmid DNA extracted by the kit of example 5, and lanes 5-7 are the results of the plasmid DNA extracted by the kit of comparative example 1.

The results show that: the lysate lacks a pH buffer, and the pH condition of the lysate is not properly adjusted, so that plasmid DNA cannot be effectively extracted.

Test example 5

Selecting a strain: coli strain with high copy plasmid pUC57 was selected for the experiment.

Selecting a kit: extraction of bacterial plasmid DNA was performed using the kits of comparative example 2, comparative example 3 and example 5.

Bacterial plasmid DNA extraction: extraction of bacterial plasmid DNA was performed using the method of example 8.

The extracted plasmid DNAs were subjected to agarose gel electrophoresis detection for 3 groups (corresponding to the kits of comparative example 2, comparative example 3 and example 5, each group was 3 parallel), and the results are shown in FIG. 5, wherein the sample corresponding to lane 1 is DNA molecular weight standard (5000 bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom), lanes 2-4 are the results of the plasmid DNAs extracted using the kit of comparative example 2, lanes 5-7 are the results of the plasmid DNAs extracted using the kit of comparative example 3, and lanes 8-10 are the results of the plasmid DNAs extracted using the kit of example 5.

The results show that: the lysate lacking the guanidinium isothiocyanate component cannot effectively extract the plasmid; the lysate of guanidinium isothiocyanate with too low concentration (less than 4M) can not be fully cracked, and the yield of extracted plasmid DNA is obviously lower.

Test example 6

Selecting a strain: coli strain with low copy self-made high molecular weight 8Kb plasmid (designated 8K) was selected for the experiment.

Selecting a kit: extraction of bacterial plasmid DNA was performed using the kit of example 5.

Bacterial plasmid DNA extraction: extraction of bacterial plasmid DNA was performed using the method of example 8.

The result of agarose gel electrophoresis detection of the plasmid DNA extracted by the kit of example 5 is shown in FIG. 6, wherein the sample corresponding to lane 1 is DNA molecular weight standard (5000 bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom), and lanes 2-4 are the result of 8K plasmid DNA extracted by the kit of example 5.

The results show that: the extraction yield and purity of the low-copy and large-molecular-weight plasmid DNA by using the kit of example 5 are equivalent to the extraction yield and purity of the high-copy plasmid DNA by using the kit of example 5 and the time consumption of the cracking process, and the same effect is excellent, which indicates that the kit of the application is also suitable for the plasmid with large molecular weight and low copy number.

When the test was performed by selecting the e.coli strain carrying the high copy plasmid pUC57 and selecting the e.coli strain carrying the low copy self-made large molecular weight plasmid 8Kb (designated as 8K), ampicillin was selected for each antibiotic to be added to the LB medium, and the concentration of the antibiotic in the LB medium was 50 ug/ml.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Furthermore, those skilled in the art will appreciate that while some embodiments herein include some features included in other embodiments, rather than other features, combinations of features of different embodiments are meant to be within the scope of the invention and form different embodiments. For example, in the claims above, any of the claimed embodiments may be used in any combination. The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.