CN119061003A

CN119061003A - A transfection-grade plasmid extraction kit formula and extraction method thereof

Info

Publication number: CN119061003A
Application number: CN202411353558.8A
Authority: CN
Inventors: 李红良; 张鑫; 胡宇峰; 姚新欣; 程旭; 操蓉波
Original assignee: Gannan Institute Of Innovation And Transformation Medicine
Current assignee: Gannan Institute Of Innovation And Transformation Medicine
Priority date: 2024-09-26
Filing date: 2024-09-26
Publication date: 2024-12-03

Abstract

The invention discloses a transfection-grade plasmid extraction kit formula and an extraction method thereof, belonging to the technical field of plasmid DNA extraction. The kit comprises a cracking reagent set, an eluting reagent set and at least one silicon substrate adsorption column, wherein the cracking reagent set comprises a heavy suspension S1, a cracking solution S2 and a neutralizing solution S3, the eluting reagent set comprises a rinsing solution E1 and an eluting solution E2, and the volume ratio of each reagent of the cracking reagent set and the eluting reagent set is S1:S2:S3:E1:E2=30:30:30:80:10. The plasmid extraction kit formula provided by the invention has the advantages of simple and convenient operation, low cost, high plasmid purity, high extraction efficiency and the like, and can meet the experimental and research requirements of cell transfection, genetic engineering and the like.

Description

Transfection-grade plasmid extraction kit formula and extraction method thereof

Technical Field

The invention relates to the technical field of plasmid DNA extraction, in particular to a transfection-grade plasmid extraction kit formula and an extraction method thereof.

Background

A plasmid is a small, circular DNA molecule that is typically found in bacteria and is capable of replication independent of the chromosomal DNA of the bacteria. Plasmids generally carry genes which are advantageous for the survival of the bacteria, such as antibiotic resistance genes or metabolic genes. Therefore, plasmid extraction and purification are important links in modern molecular biology research experiments, and are generally used for extracting plasmid DNA from bacteria so as to perform subsequent cloning, sequencing or transfection experiments.

The transfection-grade plasmid is high-quality plasmid DNA which is suitable for cell transfection experiments after optimization and purification. These plasmids generally have the following characteristics:

high purity transfection-grade plasmid DNA requires removal of cell residues, proteins and other contaminants to ensure that cell growth and gene expression are not affected during transfection.

High concentration in order to increase the transfection efficiency, transfection-grade plasmids generally need to be used at higher concentrations.

Integrity plasmid DNA should maintain an intact circular structure to ensure efficient replication and expression within the cell.

Functional transfection-grade plasmids generally contain the functional elements required for expression of the gene of interest, such as promoters, enhancers, selectable markers, and the like.

However, the existing kit for extracting and purifying the transfection-grade plasmid has the problems of long operation time, various types of reagents and the like due to the multi-step flow and complex reagent composition.

Therefore, there is a need for a kit and a plasmid extraction method that can extract a large amount of transfection-grade plasmids with simple operation, short operation time, high plasmid extraction efficiency and high plasmid quality, which are of great significance to molecular biology research.

Disclosure of Invention

Aiming at the problems of long operation time, various reagent types and the like of the existing plasmid extraction kit, the invention provides a transfection-grade plasmid extraction kit and a plasmid extraction method. The kit is based on an alkaline lysis method and has the advantages of simple operation, short operation time, high plasmid extraction efficiency and high plasmid quality.

In order to achieve the above purpose of the invention, the following technical scheme is adopted:

In a first aspect, the invention provides a transfection-grade plasmid extraction kit formulation comprising a lysis reagent set, an elution reagent set and at least one silicon substrate adsorption column, wherein,

The lysis reagent group comprises a resuspension S1, a lysis solution S2 and a neutralization solution S3;

the eluting reagent group comprises a rinsing liquid E1 and an eluting liquid E2;

the volume ratio of each reagent of the lysis reagent group and the elution reagent group is S1:S2:S3:E1:E2=30:30:30:80:10.

Preferably, the heavy suspension S1 comprises 50 to 100mM Tris, 0mM to 10mM EDTA, 0mM to 50mM glucose and 300. Mu.g/mL to 500. Mu.g/mL RNase A.

Preferably, the lysate S2 comprises 0.2M to 0.4M NaOH and 1% to 2% SDS.

Preferably, the neutralization solution S3 comprises a mixture of 1.32M to 5M acetic acid and salts thereof, at a pH of 4.8 to 5.5.

Preferably, the rinse E1 comprises 10mM to 50mM Tris and 0.1mM to 0.5mM EDTA and 70% ethanol, or 10mM to 50mM Tris and 70% ethanol, or 10mM to 50mM MOPS (3- (N-morpholino) propanesulfonic acid) and 70% ethanol.

Preferably, the eluent E2 comprises sterile double distilled water.

Preferably, the heavy suspension comprises 50mM Tris, 10mM EDTA and 500. Mu.g/mL RNase A.

Preferably, the lysate comprises 0.2M NaOH and 2% sds.

Preferably, the neutralization solution comprises 5M potassium acetate at pH 4.8, and the rinsing solution comprises 10mM Tris, 0.1mM EDTA and 70% ethanol.

In a second aspect, the present invention also provides a method for extracting a transfection-grade plasmid, using the formulation of the above-described transfection-grade plasmid extraction kit, the method comprising the steps of:

1) Re-suspending, namely adding the re-suspension into a bacterial sample to suspend thalli;

2) Cracking, namely adding a cracking solution, uniformly mixing and cracking the thalli to release plasmid DNA;

3) Neutralizing by adding neutralizing solution, mixing until white floccules appear, centrifuging the mixture, and collecting supernatant;

4) Loading onto column, filtering supernatant with filter column, collecting filtrate, adding 0.3 times volume of isopropanol, mixing, adding into adsorption column, centrifuging, and removing liquid phase;

5) Rinsing, namely adding rinsing liquid into an adsorption column, centrifuging, discarding waste liquid and airing;

6) Eluting, namely adding eluent into the adsorption column rinsed by impurities, centrifuging, and collecting plasmid DNA in a new sample tube.

Compared with the prior art, the invention has the beneficial effects that:

1. The plasmid extraction kit formula provided by the invention has high plasmid extraction purity and high quality, can meet the experimental and research requirements of cell transfection, genetic engineering and the like, and can effectively protect the stability of DNA, remove RNA and improve the purity of the extracted plasmid by reasonably configuring the components of the heavy suspension S1. By reasonably configuring the concentration of NaOH and SDS in the lysate S2, the lysis efficiency and the purity of DNA can be improved, and then high-quality plasmid DNA can be obtained. By reasonably configuring the concentration of acetic acid and salts thereof in the neutralization solution S3 and controlling the pH value, the recovery rate and purity of DNA can be improved, and high-quality plasmid DNA can be obtained. By reasonably configuring the concentrations of Tris, EDTA and ethanol in the rinse E1 and controlling the washing steps, the recovery rate and purity of DNA can be improved, and high-quality plasmid DNA can be obtained. By using sterile double distilled water, it is possible to ensure that the finally obtained DNA has high purity and to obtain a high quality DNA sample.

2. The formula of the plasmid extraction kit provided by the invention is developed based on an alkaline cracking method, and has the advantages of simplicity and convenience in operation, low cost, high plasmid purity, high extraction efficiency and the like. The method for extracting the transfection-grade plasmid can efficiently and rapidly extract high-purity plasmid DNA by combining a series of simple steps and using the special kit formula, and is suitable for subsequent transfection and molecular biology experiments.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.

In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, and it will be obvious to a person skilled in the art that other drawings can be obtained from these drawings without inventive effort.

FIG. 1 is a schematic diagram showing the result of electrophoresis of plasmid DNA extracted from EGFP sample of example 3 of the present invention;

FIG. 2 is a schematic diagram showing the result of electrophoresis of plasmid DNA extracted from IL6 sample in example 3 of the present invention.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

It should be noted that all directional indicators (such as up, down, left, right, front, and rear are used in the embodiments of the present invention) are merely for explaining the relative positional relationship, movement conditions, and the like between the components in a certain specific posture (as shown in the drawings), and if the specific posture is changed, the directional indicators are changed accordingly.

Furthermore, the description of "first," "second," etc. in this disclosure is for descriptive purposes only and is not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In addition, the technical solutions of the embodiments may be combined with each other, but it is necessary to base that the technical solutions can be realized by those skilled in the art, and when the technical solutions are contradictory or cannot be realized, the combination of the technical solutions should be considered to be absent and not within the scope of protection claimed in the present invention.

Aiming at the problems of long operation time, various reagent types and the like of the existing plasmid extraction kit, the invention provides a transfection-grade plasmid extraction kit formula and an extraction method thereof, and has the advantages of simple operation, short operation time, high plasmid extraction efficiency and high plasmid quality.

Specific embodiments of the present invention will be described in detail below:

The first aspect of the invention provides a transfection-grade plasmid extraction kit formulation comprising a lysis reagent set, an elution reagent set and at least one silicon substrate adsorption column.

the volume ratio of each reagent of the lysis reagent group and the elution reagent group is S1:S2:S3:E1:

E2=30:30:30:80:10。

In particular, the method comprises the steps of,

Heavy suspension S1, comprising 50 to 100mM Tris, 0mM to 10mM EDTA, 0mM to 50mM glucose and 300. Mu.g/mL to 500. Mu.g/mL RNase A (an enzyme capable of degrading RNA).

Lysate S2, which comprises 0.2M to 0.4M NaOH and 1% to 2% SDS (sodium dodecyl sulfate).

A neutralization solution S3 comprising a mixture of 1.32M to 5M acetic acid and salts thereof, having a pH of 4.8 to 5.5;

A rinse E1 comprising 10mM to 50mM Tris and 0.1mM to 0.5mM EDTA and 70% ethanol, or 10mM to 50mM Tris and 70% ethanol, or 10mM to 50mM MOPS (3- (N-morpholino) propanesulfonic acid) and 70% ethanol;

Eluent E2, which comprises sterile double distilled water.

Among them, resuspension S1 is an important buffer in plasmid extraction, in particular Tris (aminomethane) is a common buffer capable of maintaining the pH of the solution stable, this concentration being suitable for most biological experiments, usually maintained in the pH range 7.5-8.5, helping to preserve the stability of the DNA. EDTA (ethylenediamine tetraacetic acid) is a chelating agent capable of binding to metal ions (such as Mg ² + and Ca ² +) to inhibit the activity of enzymes such as DNase and prevent DNA degradation, and the concentration of EDTA can be adjusted to balance the effects of protection and the intracellular environment. Glucose can act as an osmotic agent to help maintain the osmotic pressure of the cells and prevent excessive expansion or contraction of the cells during lysis. The addition of RNase A effectively removes RNA from the sample, ensures the purity of the extracted plasmid DNA, and particularly when downstream applications (e.g., PCR, cloning, etc.) are required, the presence of RNA may interfere with the experimental results.

The lysate S2 is a key buffer solution for cell lysis in the plasmid extraction process, and specifically NaOH is a strong base, and can effectively destroy cell membranes and cell walls and promote cell lysis. High concentrations of NaOH denature the DNA to form single stranded DNA, facilitating subsequent isolation and purification processes. In the invention, the concentration of NaOH is controlled between 0.2M and 0.4M in plasmid extraction so as to ensure effective cell lysis and avoid excessive degradation of DNA.

The neutralization solution S3 plays an important role in the plasmid extraction process, and is mainly used for neutralizing the high pH value of the lysate S2 and promoting the precipitation of DNA. Specifically, acetic acid is a weak acid that is effective in neutralizing NaOH in the lysate, lowering the pH to a range suitable for DNA precipitation (typically pH 4.8 to 5.5). In this pH range, DNA precipitates from solution, facilitating subsequent isolation and purification. Acetate salts (e.g., sodium acetate, etc.) can help stabilize the neutralization reaction and provide adequate ionic strength.

Rinse E1 is an important step in plasmid extraction, which is used to wash the precipitated DNA during plasmid extraction, to remove impurities and salts, and to ensure that the final obtained DNA is of high purity. In particular, tris (aminomethane) is a commonly used buffer capable of maintaining a suitable pH, typically between pH 7.5 and 8.5, during DNA washing, which protects DNA from degradation. EDTA (ethylenediamine tetraacetic acid) is a chelating agent that is effective in removing metal ions (such as Mg ² + and Ca ² +), which may promote degradation of DNA, and by binding to metal ions, EDTA helps to preserve the integrity of DNA. Ethanol is used to precipitate DNA and remove residual salts and impurities during rinsing, and 70% ethanol concentration is effective to wash DNA while reducing its solubility and helping to maintain it in a precipitated state.

Eluent E2 is typically used in the last step of the plasmid extraction or DNA isolation process in order to elute the purified DNA from the precipitate. The double distilled water is distilled and deionized water with extremely high purity, and is suitable for biological experiment, and the use of sterile double distilled water can avoid exogenous pollution effectively and ensure that eluted DNA is not polluted.

The reagent kit formula of the invention can effectively protect the stability of DNA and remove RNA by reasonably configuring the components of the heavy suspension S1, thereby improving the purity of the extracted plasmid. By reasonably configuring the concentration of NaOH and SDS in the lysate S2, the lysis efficiency and the purity of DNA can be improved, and then high-quality plasmid DNA can be obtained. By reasonably configuring the concentration of acetic acid and salts thereof in the neutralization solution S3 and controlling the pH value, the recovery rate and purity of DNA can be improved, and high-quality plasmid DNA can be obtained. By reasonably configuring the concentrations of Tris, EDTA and ethanol in the rinse E1 and controlling the washing steps, the recovery rate and purity of DNA can be improved, and high-quality plasmid DNA can be obtained. By using sterile double distilled water, it is possible to ensure that the finally obtained DNA has high purity and to obtain a high quality DNA sample.

In some preferred embodiments of the invention, the heavy suspension comprises 50mM Tris, 10mM EDTA and 500. Mu.g/mL RNase A.

In some preferred embodiments of the invention, the lysate comprises 0.2M NaOH and 2% sds.

In some preferred embodiments of the invention, the neutralization solution comprises 5M potassium acetate, pH 4.8.

In some preferred embodiments of the invention, the rinse solution comprises 10mM Tris and 0.1mM EDTA and 70% ethanol.

The second aspect of the invention also provides a method for extracting transfection-grade plasmid, which is based on the formula of the transfection-grade plasmid extraction kit and comprises the following steps:

1) And (3) re-suspending, namely adding re-suspension into the bacterial sample to suspend the bacterial cells, and ensuring that the bacterial cells are fully re-suspended in the step so as to improve the subsequent lysis efficiency.

2) And (3) cracking, namely adding a cracking solution, uniformly mixing and cracking the thalli to release plasmid DNA, wherein in the step, the next step is carried out immediately after the cracking to prevent DNA degradation.

3) Neutralizing, adding neutralizing solution, mixing until white floccule appears, centrifuging the mixture, and collecting supernatant, wherein in this step, sufficient mixing is required to obtain good precipitation effect.

4) Filtering the supernatant with a filter column, adding 0.3 times of isopropanol, mixing, adding into an adsorption column, centrifuging, and removing liquid phase, wherein the addition of isopropanol can help to adsorb plasmid DNA, and ensure proper centrifuging time and rotation speed.

5) Rinsing, namely adding rinsing liquid into the adsorption column, centrifuging, discarding waste liquid, and airing, wherein the rinsing step is helpful for removing impurities, and ensures that the column is completely dried so as to improve the eluting efficiency.

6) Eluting, namely adding eluent into the adsorption column rinsed by impurities, centrifuging, and collecting plasmid DNA in a new sample tube. In this step, the volume and temperature of the eluent can affect the elution efficiency of the DNA, and warm eluent (e.g., 60 ℃) can be used during the experiment to increase the yield.

It should be noted that each step in the above extraction method of the present invention maintains a sterile operation, ensuring that the extracted plasmid DNA is not contaminated.

The method for extracting the transfection-grade plasmid can efficiently and rapidly extract high-purity plasmid DNA by combining a series of simple steps and using the special kit formula, and is suitable for subsequent transfection and molecular biology experiments.

In some specific embodiments of the invention, the plasmid is a low copy plasmid or a high copy plasmid. Wherein "low copy plasmid" refers to a plasmid having only 1-2 copies in a cell, and "high copy plasmid" refers to a plasmid having 10-200 copies in a cell. The person skilled in the art is able to formulate formulations suitable for low-copy plasmid and high-copy plasmid extraction based on the disclosure of the present invention, which is not described in any more detail herein, for example for low-copy formulations the bacterial sample bacterial load can be suitably increased and the amount of suspension, lysate and neutralization solution used suitably.

Example 1

Exemplary kit compositions of the invention and method steps for extracting transfection-grade plasmid DNA

TABLE 1 composition of exemplary kits of the invention

All reagents were double distilled water configuration and all chemical purity was above analytical purity, with lysis reagent solution 1 stored at 4 ℃ and other reagents stored at room temperature.

The following steps of extraction of transfection-grade plasmid DNA were performed according to the ratios of the respective reagents given in table 1 above, wherein adsorption columns, collection tubes, centrifuge tubes, bench centrifuges, pipettes, vortex shakers, water baths or metal baths, absolute ethanol, isopropanol, etc. were purchased from a conventional reagent store. Specifically, the step of extracting the transfection-grade plasmid DNA comprises:

1. column equilibration by adding 2.5mL of the column equilibration solution to the column CP6 (the column was placed in a 50mL collection tube), centrifuging at 8,000rpm (8,228 Xg) for 2min, pouring out the waste liquid from the collection tube, and returning the column to the collection tube.

2. And (3) re-suspending, namely adding 400mL of the overnight cultured bacterial liquid into a centrifuge tube, centrifuging at 8,000rpm (8,228 multiplied by g) at room temperature for 10min, collecting bacteria, and sucking the supernatant as much as possible. 8mL of the solution heavy suspension is added into a centrifuge tube with the bacterial cell sediment left, and a pipettor or a vortex oscillator is used for thoroughly suspending the bacterial cell sediment (note: bacterial sediment needs to be thoroughly suspended, if bacterial blocks which are not thoroughly mixed are not uniformly mixed, the cracking effect is affected, and the extraction amount and the purity are low).

3.8 ML of lysate is added into a centrifuge tube, and the centrifuge tube is gently turned up and down for 6-8 times, so that thalli are fully lysed, and the centrifuge tube is left at room temperature for 5min (note: gently mixing, and not shaking vigorously, so as not to pollute DNA).

4. And (3) neutralizing, namely adding 8mL of neutralizing solution into the centrifuge tube, immediately and gently turning over the centrifuge tube up and down for 6-8 times, and fully and uniformly mixing until white dispersion flocculent precipitate appears in the solution. Placing at room temperature for about 10min, centrifuging at 8,000rpm (8,228 ×g) for 10min, separating the white precipitate to the bottom of the tube, and filtering to obtain filtrate (note: for low copy plasmid, thallus dosage can be increased and dosage of heavy suspension, lysate and neutralization solution can be proportionally increased at the same time).

4. And (3) loading the column, namely calculating the volume of filtrate, adding isopropanol with the volume which is 0.3 times of the volume of the filtrate into the filtrate, uniformly mixing the mixture upside down, transferring the mixture into an adsorption column CP6, and placing the adsorption column into a 50mL collecting tube. Centrifuging at room temperature for 2min at 8,000rpm (8,228 Xg), pouring out the waste liquid in the collecting pipe, and putting the adsorbing column CP6 back into the collecting pipe.

5. Rinsing, namely adding 10mL of rinsing liquid into the adsorption column CP6, centrifuging at 8,000rpm (8,228 Xg) for 2min, discarding the waste liquid in the collecting pipe, and putting the adsorption column back into the collecting pipe. This step may be repeated 1-2 times. Then, the column CP6 was replaced in the collection tube, and centrifuged at 8,000rpm (8,228 Xg) for 5 minutes to remove the residual rinse liquid in the column.

6. Air-drying, namely, the residue of ethanol in the rinsing liquid can influence the subsequent enzymatic reaction (such as enzyme digestion, PCR and the like) experiments. In order to ensure that the downstream experiments are not affected by residual ethanol, the adsorption column CP6 may be uncapped and left at room temperature for several minutes to thoroughly dry the residual rinse solution in the adsorbent material.

7. And (3) eluting, namely placing the adsorption column CP6 in a clean 50mL collecting pipe, suspending and dripping 1mL of eluent into the middle part of the adsorption film, standing for 5min at room temperature, and centrifuging for 2min at room temperature at 8,000rpm (8,228 Xg). The eluate from the 50mL centrifuge tube was transferred in its entirety to a clean 1.5mL centrifuge tube and stored at-20 ℃.

In order to increase the plasmid DNA recovery efficiency, can be added to the adsorption column, repeat step 7.

The inventors of the present invention found that the amount of bacterial liquid can be 400mL to 500mL when plasmid DNA extraction is performed by the above method in combination with the kit shown in Table 1.

Example 2:

embodiments of transfection-grade plasmid miniprep based on exemplary kit compositions of the present invention (see table 1), specifically, the extraction steps of transfection-grade plasmid miniprep include:

1. Column equilibration by adding 500. Mu.L of the column equilibration solution to the adsorption column (the adsorption column was placed in a 2mL collection tube), centrifuging at 10,000rpm for 1min, pouring off the waste liquid from the collection tube, and returning the adsorption column to the collection tube.

2. Resuspension, namely, 35mL of the overnight cultured bacterial liquid is added into a centrifuge tube, and the bacteria are collected by centrifugation at 10,000rpm for 10min at room temperature, and the supernatant is sucked as much as possible. Add 500. Mu.L of the solution resuspension to the centrifuge tube with the bacterial pellet left, use a pipette or vortex shaker to thoroughly suspend the bacterial pellet (note: bacterial pellet is required to be thoroughly suspended, if not thoroughly mixed with bacterial cake, the lysis effect is affected, resulting in lower extraction and purity).

3. And (3) cracking, namely adding 500 mu L of cracking liquid into a centrifugal tube, and immediately and gently overturning up and down for 6-8 times to fully crack the thalli, and standing for 5min at room temperature (note: gently mixing, and not shaking vigorously so as to prevent DNA pollution).

4. And (3) neutralizing, namely adding 500 mu L of neutralizing solution into a centrifuge tube, immediately and gently turning over the centrifuge tube up and down for 6-8 times, and fully and uniformly mixing until white dispersion flocculent precipitation appears in the solution. Placing at room temperature for about 10min, centrifuging at 10,000rpm for 10min to separate white precipitate from the bottom of the tube, and filtering to obtain filtrate (note: for low copy plasmid, thallus dosage can be increased and dosage of heavy suspension, lysate and neutralization solution can be proportionally increased simultaneously).

4. And (3) loading the mixture on a column, namely calculating the volume of filtrate, adding isopropanol with the volume which is 0.3 times of the volume of the filtrate into the filtrate, uniformly mixing the mixture upside down, transferring the mixture into an adsorption column, and placing the adsorption column into a 2mL collecting pipe. Centrifuging at 10,000rpm for 2min at room temperature, pouring out the waste liquid in the collecting pipe, and putting the adsorption column back into the collecting pipe.

5. Rinsing, namely adding 600 mu L of rinsing liquid into the adsorption column CP6, centrifuging for 2min at 10,000rpm, discarding the waste liquid in the collecting pipe, and putting the adsorption column back into the collecting pipe. This step may be repeated 1-2 times. Then, the adsorption column CP6 was replaced in the collection tube, and centrifuged at 10,000rpm for 2 minutes to remove the residual rinse liquid in the adsorption column.

6. Air-drying, namely, the residue of ethanol in the rinsing liquid can influence the subsequent enzymatic reaction (such as enzyme digestion, PCR and the like) experiments. In order to ensure that the downstream experiments are not affected by residual ethanol, the adsorption column can be uncapped and left at room temperature for several minutes to thoroughly dry the residual rinse solution in the adsorption material.

7. Eluting by placing the adsorption column in a clean 1.5mL collecting tube, suspending and dripping 100 μl of eluent into the middle part of the adsorption membrane, standing at room temperature for 5min, and centrifuging at room temperature at 10,000rpm for 2min. The obtained filtrate was a plasmid DNA solution and was stored at-20 ℃.

The inventors of the present invention found that the amount of bacterial liquid can be 15mL to 40mL when plasmid DNA extraction is performed by the above method in combination with the kit shown in Table 1.

Example 3:

Inter-batch stability test

In this example, the batch-to-batch reproducibility of three batches of samples was tested using different proteins (EGFP and IL 6). Specifically, three batches of plasmid DNA were each extracted from DH 5. Alpha. Strain based on the above-described exemplary kit of the present invention and the step of extracting plasmid DNA, the bacterial liquid volume was 35mL, and the results were compared with the results of extraction of other types of kits existing on the market (abbreviated as Prior Art 1), and the results are shown in tables 2 and 3 below.

TABLE 2 extraction results of plasmid DNA of EGFP

TABLE 3 extraction results of plasmid DNA for IL6

As can be seen from tables 2 and 3, the three samples of different proteins (EGFP and IL 6) each had a CV value of less than 10% between batches, showing good reproducibility between batches. Although the prior art kit gave a higher average concentration measurement, it was found that the 260/280 of the plasmid DNA extracted from the prior art kit was of a higher value, meaning that the DNA obtained could be contaminated with proteins or phenols.

The result of electrophoresis of the extracted plasmid DNA is shown in FIGS. 1-2. Compared with the kit in the prior art, the running gel fruit of the DNA extracted by the kit in the invention shows purity superior to the kit in the prior art. The concentration measurement of the prior art kit is high, but the running glue is not as good as the invention, and the small fragments and RNA residues are probably more.

The foregoing is only a specific embodiment of the invention to enable those skilled in the art to understand or practice the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A transfection-grade plasmid extraction kit formula is characterized by comprising a lysis reagent group, an elution reagent group and at least one silicon substrate adsorption column, wherein,

2. A transfection-grade plasmid extraction kit formulation as claimed in claim 1, wherein the resuspension S1 comprises 50 to 100mM Tris, 0mM to 10mM EDTA, 0mM to 50mM glucose and 300 μg/mL to 500 μg/mL RNase A.

3. A transfection-grade plasmid extraction kit formulation as claimed in claim 1, wherein the lysate S2 comprises 0.2M to 0.4M NaOH and 1% to 2% SDS.

4. The transfection-grade plasmid extraction kit of claim 1 wherein the neutralization solution S3 comprises a mixture of 1.32M to 5M acetic acid and salts thereof, and has a pH of 4.8 to 5.5.

5. A transfection-grade plasmid extraction kit formulation as claimed in claim 1, wherein the rinse E1 comprises 10mM to 50mM Tris and 0.1mM to 0.5mM EDTA and 70% ethanol, or 10mM to 50mM Tris and 70% ethanol, or 10mM to 50mM MOPS (3- (N-morpholino) propanesulfonic acid) and 70% ethanol.

6. A transfection-grade plasmid extraction kit formulation as claimed in claim 1, wherein the eluent E2 comprises sterile double distilled water.

7. A transfection-grade plasmid extraction kit formulation as claimed in any one of claims 2-6, wherein the resuspension comprises 50mM Tris, 10mM EDTA and 500. Mu.g/mL RNase A.

8. A transfection-grade plasmid extraction kit formulation as claimed in any one of claims 3-6, wherein the lysate comprises 0.2M NaOH and 2% SDS.

9. A transfection-grade plasmid extraction kit formulation as claimed in any one of claims 4-6, wherein the neutralisation solution comprises 5M potassium acetate, pH 4.8, and the rinsing solution comprises 10mM Tris and 0.1mM EDTA and 70% ethanol.

10. A method for extracting a transfection-grade plasmid, using the formulation of the transfection-grade plasmid extraction kit as claimed in any one of claims 1 to 9, characterized in that the method comprises the steps of: