阅读说明：本技术 一种快速提取高纯度质粒dna的方法 (Method for rapidly extracting high-purity plasmid DNA ) 是由 樊菲 杨召军 薛帮凯 王硕 王立东 田艳维 于 2021-09-30 设计创作，主要内容包括：本发明提供一种快速提取高纯度质粒DNA的方法,包括1)细菌的培养、2)细菌的裂解、3)质粒DNA的分离、4)质粒DNA的沉淀,其中,在步骤3)中使用的离心柱为硅膜吸附柱,步骤4)中的清洗液A为5M异硫氰酸胍、20mM Tris-cl与浓度为37.5％乙醇的混合液,其pH为6.0。本发明的方法提取的质粒DNA的含量高、纯度高,且在制备过程中没有使用有害试剂,安全无隐患,不会伤害人员健康。(The invention provides a method for quickly extracting high-purity plasmid DNA, which comprises 1) culturing bacteria, 2) cracking the bacteria, 3) separating the plasmid DNA and 4) precipitating the plasmid DNA, wherein a centrifugal column used in the step 3) is a silicon membrane adsorption column, a cleaning solution A in the step 4) is a mixed solution of 5M guanidinium isothiocyanate, 20mM Tris-cl and 37.5% ethanol, and the pH value of the mixed solution is 6.0. The plasmid DNA extracted by the method has high content and high purity, no harmful reagent is used in the preparation process, the safety is safe, no hidden danger exists, and the health of people is not injured.)

1. A method for rapidly extracting high-purity plasmid DNA is characterized in that: the method comprises the following steps:

1) and (3) culturing bacteria: selecting a single bacterial colony to perform amplification culture in a liquid culture medium, then sucking the cultured bacterial liquid to be aseptically packaged in two centrifuge tubes, centrifuging, and sucking the supernatant liquid;

2) lysis of bacteria: adding the solution I into a centrifuge tube, and fully suspending the bacterial sediment; then adding the solution II, slowly turning over for a plurality of times, and fully and uniformly mixing; then adding the solution III, and slowly turning over for a plurality of times until a white polymer is formed;

3) isolation of plasmid DNA: centrifuging the solution obtained in the step 2), taking supernatant and placing the supernatant in a centrifugal column in a collection tube;

4) precipitation of plasmid DNA: after centrifugation, waste liquid in the collecting tube is discarded, and a centrifugal column is continuously loaded;

adding a cleaning solution A into the centrifugal column, centrifuging and then removing filtrate;

then adding a cleaning solution B into the centrifugal column, centrifuging, removing the filtrate, and repeating the step for 4 times;

placing the centrifugal column into a sterile EP tube of a sterile workbench, adding the preheated TE buffer solution, and standing for 1 min;

after centrifugation, removing the centrifugal column, obtaining the target DNA in a sterile EP tube, and storing at-20 ℃ or drying and storing at normal temperature;

the centrifugal column is a silicon film adsorption column, the cleaning solution A is a mixed solution of 5M guanidinium isothiocyanate, 20mM Tris-cl and 37.5% ethanol, and the pH value of the mixed solution is 6.0.

2. The method for rapidly extracting high-purity plasmid DNA according to claim 1, wherein: the method specifically comprises the following steps:

1) and (3) culturing bacteria: selecting a single bacterial colony to carry out amplification culture in a liquid culture medium, culturing for 12-16 hours at 35-40 ℃, sucking 3.0ml of cultured bacterial liquid, aseptically packaging in two centrifugal tubes of 1.5ml, centrifuging for 2min at 4 ℃ at 10000-12000 rpm, and sucking out supernatant;

2) lysis of bacteria: adding 150 mu L of the solution I into each centrifuge tube, and fully suspending the bacterial sediment; then adding 200 μ L of solution II, slowly turning over for several times to mix them thoroughly, and cracking for 3-5 min; then adding 250 mu L of solution III, slowly turning over for several times and gently oscillating for 10s until a white polymer is formed, and standing for 10min at room temperature;

3) isolation of plasmid DNA: centrifuging the solution obtained in step 2) at 4 ℃ at 10000-12000 rpm for 15min, and then taking the supernatant and placing the supernatant in a centrifugal column in a collection tube;

4) precipitation of plasmid DNA: centrifuging at 10000-12000 rpm for 1min, discarding the waste liquid in the collecting tube, and continuously loading the centrifugal column;

adding 400 mu L of cleaning solution A into the centrifugal column, centrifuging at 10000-12000 rpm for 30s, and removing the filtrate;

then 580 mul of cleaning solution B is added into the centrifugal column, 10000rpm-12000rpm are used for centrifugation for 30s, the filtrate is discarded, and the step is repeated for 4 times;

placing the column into a 1.5ml sterile EP tube on a sterile workbench, adding preheated TE buffer solution 60-100 μ L, and standing for 1 min;

centrifuging at 10000-12000 rpm for 1min, removing the centrifugal column, and obtaining the target DNA in a sterile EP tube, and storing at-20 ℃ or drying at normal temperature.

3. The method for rapidly extracting high purity plasmid DNA according to claim 1 or 2, wherein: solution I was autoclaved with 25mM Tris-cl and 10mM EDTA and obtained by adding 2.5mg/ml RNaseA, and its pH was 8.0.

4. The method for rapidly extracting high purity plasmid DNA according to claim 1 or 2, wherein: solution II was 0.2M NaOH with 1% SDS.

5. The method for rapidly extracting high purity plasmid DNA according to claim 1 or 2, wherein: solution III was 5M guanidine hydrochloride with 4M potassium acetate at pH 4.8.

6. The method for rapidly extracting high purity plasmid DNA according to claim 1 or 2, wherein: the cleaning liquid B is ethanol with the concentration of 60-80%.

7. The method for rapidly extracting high purity plasmid DNA according to claim 1 or 2, wherein: the TE buffer was 10mM Tris-HCl, pH8.0 and 1mM EDTA, pH 8.0.

Technical Field

The invention belongs to the technical field of nucleic acid extraction and purification, and particularly relates to a method for quickly extracting high-purity plasmid DNA.

Background

Plasmid is circular double-stranded DNA molecule which exists outside the bacterial chromosome and can be independently replicated and stably inherited, and plasmid DNA is a common carrier for genetic engineering and can carry exogenous genes into bacteria, animal cells or plants for amplification and expression. The efficiency and quality of plasmid DNA extraction are directly related to the success of subsequent experiments (enzyme digestion, PCR amplification, etc.), so that the method has important significance in quickly and efficiently extracting and transmitting plasmids from bacterial cells.

The alkaline cracking method is the most widely used method for preparing plasmid DNA, and the alkaline deformation extraction of plasmid DNA achieves the separation purpose based on the difference between the deformation and the renaturation of the chromosome DNA and the plasmid DNA. Under alkaline conditions at pH up to 12.6, the hydrogen bonds of chromosomal DNA break and the double helix structure unravels and deforms. Most of the hydrogen bonds of plasmid DNA are broken, but the two complementary strands of supercoiled covalently closed circular form are not completely separated, when pH is adjusted to neutral by NaAc/KAc high salt buffer solution with pH4.8, the denatured plasmid DNA returns to its original configuration and is stored in solution, while the chromosomal DNA cannot renaturate to form an entangled network structure, and the chromosomal DNA is removed by centrifugation, precipitating together with unstable macromolecular RNA, protein-SDS complex, etc.

However, the existing alkaline cracking method has the following defects:

1. in the experimental process, the impurity protein and RNA can not be removed completely, so that the purity of the finally extracted plasmid DNA is not high.

2. The extracted DNA content is low.

3. Harmful reagents such as phenol/chloroform/isoamylol are used in the experimental process, which is not good for the health of people.

Disclosure of Invention

The invention aims to solve the problem of providing a method for quickly extracting high-purity plasmid DNA, the plasmid DNA extracted by the method has high content and high purity, no harmful reagent is used in the preparation process, the method is safe and free of hidden danger, and the method does not harm the health of personnel.

In order to solve the technical problems, the invention adopts the technical scheme that: a method for rapidly extracting high-purity plasmid DNA comprises the following steps:

1) and (3) culturing bacteria: selecting a single bacterial colony to perform amplification culture in a liquid culture medium, then sucking the cultured bacterial liquid to be aseptically packaged in two centrifuge tubes, centrifuging, and sucking the supernatant liquid;

2) lysis of bacteria: adding the solution I into a centrifuge tube, and fully suspending the bacterial sediment; then adding the solution II, slowly turning over for a plurality of times, and fully and uniformly mixing; then adding the solution III, and slowly turning over for a plurality of times until a white polymer is formed;

3) isolation of plasmid DNA: centrifuging the solution obtained in the step 2), taking supernatant and placing the supernatant in a centrifugal column in a collection tube;

4) precipitation of plasmid DNA: after centrifugation, waste liquid in the collecting tube is discarded, and a centrifugal column is continuously loaded;

adding a cleaning solution A into the centrifugal column, centrifuging and then removing filtrate;

then adding a cleaning solution B into the centrifugal column, centrifuging, removing the filtrate, and repeating the step for 4 times;

placing the centrifugal column into a sterile EP tube of a sterile workbench, adding the preheated TE buffer solution, and standing for 1 min;

after centrifugation, removing the centrifugal column, obtaining the target DNA in a sterile EP tube, and storing at-20 ℃ or drying and storing at normal temperature;

wherein the centrifugal column is a silicon film adsorption column, the cleaning solution A is a mixed solution of 5M guanidinium isothiocyanate, 20mM Tris-cl and 37.5% ethanol, and the pH value is 6.0.

A method for rapidly extracting high-purity plasmid DNA comprises the following steps:

1) and (3) culturing bacteria: selecting a single bacterial colony to carry out amplification culture in a liquid culture medium, culturing for 12-16 hours at 35-40 ℃, sucking 3.0ml of cultured bacterial liquid, aseptically packaging in two centrifugal tubes of 1.5ml, centrifuging for 2min at 4 ℃ at 10000-12000 rpm, and sucking out supernatant;

2) lysis of bacteria: adding 150 mu L of the solution I into each centrifuge tube, and fully suspending the bacterial sediment; then adding 200 μ L of solution II, slowly turning over for several times to mix them thoroughly, and cracking for 3-5 min; then adding 250 mu L of solution III, slowly turning over for several times and gently oscillating for 10s until a white polymer is formed, and standing for 10min at room temperature;

3) isolation of plasmid DNA: centrifuging the solution obtained in step 2) at 4 ℃ at 10000-12000 rpm for 15min, and then taking the supernatant and placing the supernatant in a centrifugal column in a collection tube;

4) precipitation of plasmid DNA: centrifuging at 10000-12000 rpm for 1min, discarding the waste liquid in the collecting tube, and continuously loading the centrifugal column;

adding 400 mu L of cleaning solution A into the centrifugal column, centrifuging at 10000-12000 rpm for 30s, and removing the filtrate;

then 580 mul of cleaning solution B is added into the centrifugal column, 10000rpm-12000rpm are used for centrifugation for 30s, the filtrate is discarded, and the step is repeated for 4 times;

placing the column into a 1.5ml sterile EP tube on a sterile workbench, adding preheated TE buffer solution 60-100 μ L, and standing for 1 min;

centrifuging at 10000-12000 rpm for 1min, removing the centrifugal column, and obtaining the target DNA in a sterile EP tube, and storing at-20 ℃ or drying at normal temperature.

Further, solution I was autoclaved with 25mM Tris-cl and 10mM EDTA and obtained by adding 2.5mg/ml RNaseA, and its pH was 8.0.

Further, solution II was 0.2M NaOH with 1% SDS.

Further, solution III was 5M guanidine hydrochloride with 4M potassium acetate at pH 4.8.

Further, the cleaning liquid B is ethanol with the concentration of 60-80%.

Further, the TE buffer was 10mM Tris. HCl, pH8.0 and 1mM EDTA, pH 8.0.

The invention has the advantages and positive effects that:

1. the method for extracting the plasmid DNA has high purity of the extracted plasmid DNA, does not use harmful reagents in the extraction process, can ensure the safety of an experiment, and can adsorb the cracked DNA by using a silicon film in a collecting tube in the method, thereby being convenient for removing foreign proteins and RNA in the later period and ensuring that no redundant DNA loss exists.

2. In the method, the cleaning solution A is a mixed solution of 5M guanidinium isothiocyanate, 20mM Tris-cl and 37.5% ethanol, and redundant foreign proteins can be dissolved during cleaning, so that the foreign proteins and RNA are removed completely, and the purity and the content of plasmid DNA are improved.

Detailed Description

The present invention will be described in detail with reference to the following embodiments.

Example 1:

a method for rapidly extracting high-purity plasmid DNA comprises the following steps:

1) and (3) culturing bacteria: selecting a single bacterial colony to perform amplification culture in a liquid culture medium, culturing for 12 hours at 35 ℃, sucking 3.0ml of cultured bacterial liquid, aseptically packaging in two centrifugal tubes of 1.5ml, centrifuging for 2min at 10000rpm at 4 ℃, and sucking and drying supernatant;

2) lysis of bacteria: adding 150 mu L of the solution I into each centrifuge tube, and fully suspending the bacterial sediment; then adding 200 μ L of solution II, slowly turning over for several times to mix them thoroughly, and cracking for 3 min; then adding 250 mu L of solution III, slowly turning over for several times and gently oscillating for 10s until a white polymer is formed, and standing for 10min at room temperature;

3) isolation of plasmid DNA: centrifuging the solution obtained in step 2) at 4 ℃ at 10000rpm for 15min, and then taking the supernatant to place in a centrifugal column in a collection tube; the centrifugal column is a silicon film adsorption column;

4) precipitation of plasmid DNA: centrifuging at 10000rpm for 1min, discarding the waste liquid in the collecting tube, and continuously loading the centrifugal column;

adding 400 mu L of cleaning solution A into the centrifugal column, centrifuging at 10000rpm for 30s, and removing the filtrate;

then 580 mul of cleaning solution B is added into the centrifugal column, the centrifugal column is centrifuged for 30s at 10000rpm, the filtrate is discarded, and the step is repeated for 4 times;

placing the column into a 1.5ml sterile EP tube on a sterile workbench, adding preheated TE buffer solution 60, and standing for 1 min;

centrifuging at 10000rpm for 1min, removing the centrifugal column, and storing at-20 deg.C in sterile EP tube to obtain target DNA.

Wherein, in the embodiment, the solution I is obtained by adding 2.5mg/ml of RNaseA after autoclaving 25mM Tris-cl and 10mM EDTA, and the pH value is 8.0; solution II was 0.2M NaOH and 1% SDS; the solution III is 5M guanidine hydrochloride and 4M potassium acetate, and the pH value is 4.8; the cleaning solution A is a mixed solution of 5M guanidinium isothiocyanate, 20mM Tris-cl and 37.5% ethanol, and the pH value of the mixed solution is 6.0; the cleaning solution B is ethanol with the concentration of 60 percent; the TE buffer was 10mM Tris-HCl, pH8.0 and 1mM EDTA, pH 8.0.

The DNA content extracted by the method of this example was 896. mu.g/mL, A₂₆₀/A₂₈₀＝1.85。

Example 2:

a method for rapidly extracting high-purity plasmid DNA comprises the following steps:

1) and (3) culturing bacteria: selecting a single bacterial colony to perform amplification culture in a liquid culture medium, culturing for 16 hours at 40 ℃, sucking 3.0ml of cultured bacterial liquid, aseptically packaging in two centrifugal tubes of 1.5ml, centrifuging at 12000rpm at 4 ℃ for 2min, and sucking the supernatant;

2) lysis of bacteria: adding 150 mu L of the solution I into each centrifuge tube, and fully suspending the bacterial sediment; then adding 200 μ L of solution II, slowly turning over for several times to mix them thoroughly, and cracking for 5 min; then adding 250 mu L of solution III, slowly turning over for several times and gently oscillating for 10s until a white polymer is formed, and standing for 10min at room temperature;

3) isolation of plasmid DNA: centrifuging the solution obtained in step 2) at 12000rpm at 4 ℃ for 15min, and then taking the supernatant and placing the supernatant in a centrifugal column in a collection tube; the centrifugal column is a silicon film adsorption column;

4) precipitation of plasmid DNA: centrifuging at 12000rpm for 1min, discarding the waste liquid in the collecting tube, and continuously loading the centrifugal column;

adding 400 mu L of cleaning solution A into the centrifugal column, centrifuging at 12000rpm for 30s, and removing the filtrate;

then 580 microliter of cleaning solution B is added into the centrifugal column, the centrifugal column is centrifuged at 12000rpm for 30s, the filtrate is discarded, and the step is repeated for 4 times;

placing the column into a 1.5ml sterile EP tube on a sterile workbench, adding preheated TE buffer solution 100 μ L, and standing for 1 min;

centrifuging at 12000rpm for 1min, removing the centrifugal column, and storing in sterile EP tube at-20 deg.C.

Wherein, in the embodiment, the solution I is obtained by adding 2.5mg/ml of RNaseA after autoclaving 25mM Tris-cl and 10mM EDTA, and the pH value is 8.0; solution II was 0.2M NaOH and 1% SDS; the solution III is 5M guanidine hydrochloride and 4M potassium acetate, and the pH value is 4.8; the cleaning solution A is a mixed solution of 5M guanidinium isothiocyanate, 20mM Tris-cl and 37.5% ethanol, and the pH value of the mixed solution is 6.0; the cleaning solution B is ethanol with the concentration of 80 percent; the TE buffer was 10mM Tris-HCl, pH8.0 and 1mM EDTA, pH 8.0.

The DNA content extracted by the method of this example was 842. mu.g/mL, A₂₆₀/A₂₈₀＝1.83。

Example 3:

a method for rapidly extracting high-purity plasmid DNA comprises the following steps:

1) and (3) culturing bacteria: selecting a single bacterial colony to carry out amplification culture in a liquid culture medium, culturing for 14 hours at 38 ℃, sucking 3.0ml of cultured bacterial liquid, aseptically packaging in two centrifugal tubes of 1.5ml, centrifuging for 2min at 11000rpm at 4 ℃, and sucking and drying supernatant;

2) lysis of bacteria: adding 150 mu L of the solution I into each centrifuge tube, and fully suspending the bacterial sediment; then adding 200 μ L of solution II, slowly turning over for several times to mix them thoroughly, and cracking for 4 min; then adding 250 mu L of solution III, slowly turning over for several times and gently oscillating for 10s until a white polymer is formed, and standing for 10min at room temperature;

3) isolation of plasmid DNA: centrifuging the solution obtained in step 2) at 4 ℃ at 11000rpm for 15min, and then taking the supernatant to place in a centrifugal column in a collection tube; the centrifugal column is a silicon film adsorption column;

4) precipitation of plasmid DNA: centrifuging at 11000rpm for 1min, discarding the waste liquid in the collecting tube, and continuously loading the centrifugal column;

adding 400 mu L of cleaning solution A into the centrifugal column, centrifuging at 11000rpm for 30s, and removing the filtrate;

then 580 muL of cleaning solution B is added into the centrifugal column, the centrifugal column is centrifuged for 30s at 11000rpm, the filtrate is discarded, and the step is repeated for 4 times;

placing the column into a 1.5ml sterile EP tube on a sterile workbench, adding 80 μ L of preheated TE buffer solution, and standing for 1 min;

centrifuging at 11000rpm for 1min, removing the centrifugal column, obtaining the target DNA in a sterile EP tube, and drying and storing at normal temperature.

Wherein, in the embodiment, the solution I is obtained by adding 2.5mg/ml of RNaseA after autoclaving 25mM Tris-cl and 10mM EDTA, and the pH value is 8.0; solution II was 0.2M NaOH and 1% SDS; the solution III is 5M guanidine hydrochloride and 4M potassium acetate, and the pH value is 4.8; the cleaning solution A is a mixed solution of 5M guanidinium isothiocyanate, 20mM Tris-cl and 37.5% ethanol, and the pH value of the mixed solution is 6.0; the cleaning solution B is ethanol with the concentration of 70 percent; the TE buffer was 10mM Tris-HCl, pH8.0 and 1mM EDTA, pH 8.0.

The DNA content extracted by the method of this example was 909. mu.g/mL, A₂₆₀/A₂₈₀＝1.85。

The method for extracting the plasmid DNA has high purity of the extracted plasmid DNA, does not use harmful reagents in the extraction process, can ensure the safety of an experiment, and can adsorb the cracked DNA by using a silicon film in a collecting tube in the method, thereby being convenient for removing foreign proteins and RNA in the later period and ensuring that no redundant DNA loss exists. A of the obtained plasmid DNA₂₆₀/A_280＞1.80, meeting the requirements of conventional molecular biology experiments.

The three embodiments of the present invention have been described in detail, but the description is only for the preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made within the scope of the present invention shall fall within the scope of the present invention.