CN106867994B - Rapid precipitation buffer solution applied to plasmid DNA extraction and application thereof - Google Patents
Rapid precipitation buffer solution applied to plasmid DNA extraction and application thereof Download PDFInfo
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Abstract
The invention relates to a rapid precipitation buffer solution applied to plasmid DNA extraction and application thereof. The buffer solution for rapid precipitation integrates the effects of chaotropic salt and a surfactant, can neutralize NaOH, improves the salt concentration in a system, enables proteins, genome DNA and RNA to be subjected to salting-out precipitation, and is added with the buffer solution for rapid precipitation applied to extraction of plasmid DNA after alkaline lysis, so that most of proteins, genome DNA, non-supercoiled plasmid DNA and RNA exist in an insoluble substance state, a tighter agglomerate is formed, the formation of flocculent protein substances floating on the surface of a solution is avoided, the centrifugation time is greatly shortened, the buffer solution not only can be used for extraction of high-copy and low-copy plasmids, but also is suitable for extraction of escherichia coli plasmids cultured in different culture mediums, the time of experimental researchers is greatly saved, and the experimental efficiency is improved.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a rapid precipitation buffer solution applied to plasmid DNA extraction, a plasmid DNA purification method, a plasmid DNA preparation method, application of the buffer solution in plasmid DNA preparation and purification and a kit.
Background
The extraction and purification of plasmid DNA is one of the basic steps in molecular biology, and relates to the fields of gene cloning, gene sequence analysis, nucleic acid vaccine, gene therapy, various gene recombinations, etc. Plasmids generally have three conformations, supercoiled, linear and open circular (open circular means that plasmid DNA in the form of a double-stranded circle is partially melted and thus electrophoretically slowest). The sequence of the three conformations of plasmids before and after agarose electrophoresis was supercoiled > linear > open loop, linear plasmid in the middle and open loop plasmid in the end. One index for determining the quality of a plasmid is the amount of supercoiled plasmid. Because the supercoiled plasmid is most efficient when transfecting eukaryotic cells with the plasmid, the supercoiled plasmid content in the plasmid is required to be more than 90%, which is also a requirement for recombinant plasmids as nucleic acid vaccines. The classical steps for the extraction and purification of plasmid DNA from prokaryotes are: and (3) cracking thalli, separating and purifying plasmids. Currently, the most common methods for the lysis of bacteria are: one-step plasmid extraction method and alkaline cracking method. The one-step plasmid extraction method was invented by Eppendorf, Germany. It is an extraction method based on lysozyme lytic bacteria. The method uses lysis solution containing lysozyme and detergent to lyse cells, the lysate is clear and non-sticky, after lysis, the lysate can be directly combined with a filter membrane without centrifugation, and then the supercoiled plasmid DNA is obtained by cleaning and elution through rapid centrifugation. The extraction method is convenient, rapid and good in repeatability, but is not suitable for extracting low-copy plasmids and extracting escherichia coli plasmids cultured by rich culture medium. The alkaline lysis method plasmid DNA purification technology was invented by Birnboim & Doly in 1979. The method is still one of the most common methods in molecular cloning research, and the operation comprises three solution treatments, and the extraction of the plasmid can be completed within about 20 minutes generally. The extraction method has wide application range and good repeatability, but the experimental period is relatively long, the content of supercoiled plasmid is low, and the experimental process of researchers is influenced.
The existing methods for extracting and purifying plasmid DNA still need to be improved.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art.
The present invention is based on the discovery and recognition by the inventors of the following facts and problems:
based on an alkaline lysis method, the whole plasmid extraction process can be optimized by adopting an innovative rapid precipitation buffer solution applied to plasmid DNA extraction. The effect of the chaotropic salt and the surfactant is integrated by the rapid precipitation buffer solution, so that the denatured protein in the plasmid extraction process can be promoted to form a tighter lump, and the formation of flocculent protein substances floating on the surface of the solution is avoided. Greatly shortened centrifugal time, can make whole plasmid extraction shorten to about 6 minutes from traditional 20 minutes, supercoiled plasmid content is more than 90% simultaneously, can not only be used for the extraction of high copy and low copy plasmid, also is applicable to the extraction that different culture mediums cultivateed the escherichia coli plasmid, has saved experimental researcher's time greatly, has improved experimental efficiency.
According to a first aspect of the present invention, the present invention provides a rapid precipitation buffer for plasmid DNA extraction, comprising: sodium iodide; sarcosyl; sodium deoxycholate; 3- [ (3-cholesterylaminopropyl) dimethylamino ] -1-propanesulfonic acid; potassium acetate; acetic acid; and deionized water.
Compared with the existing buffer solution, the rapid precipitation buffer solution applied to plasmid DNA extraction is added with sodium iodide, sodium dodecyl sarcosinate, sodium deoxycholate and 3- [ (3-cholesteryl aminopropyl) dimethylamino ] -1-propanesulfonic acid, wherein the sodium iodide is chaotropic salt and can play a role in cracking cells and denatured proteins, improving the salt concentration in a solution system and adsorbing the released proteins and nucleic acid on a silica gel adsorption membrane. Sodium dodecyl sarcosinate, sodium deoxycholate and 3- [ (3-cholesteryl aminopropyl) dimethylamino ] -1-propanesulfonic acid all belong to chemical reagents of surfactant class, have the function of assisted lysis to cells, and under the condition that chaotropic salt exists, denatured protein formed in the process of plasmid extraction can form more compact lumps, avoid forming flocculent protein substances floating on the surface of the solution, and greatly shorten the time of centrifugation. Meanwhile, the buffer solution is in an acid environment by potassium acetate and acetic acid, so that the buffer solution plays a role in neutralization, and plasmids obtained by alkaline cleavage play a role in renaturation. Therefore, after alkaline lysis, the rapid precipitation buffer solution applied to plasmid DNA extraction is added, so that most of protein, genome DNA, non-supercoiled plasmid DNA and RNA exist in an insoluble state, all insoluble substances can be precipitated to the bottom of a tube after centrifugation for 1min, and the supernatant can be subjected to the next operation. Greatly shortened centrifugal time, can make whole plasmid extraction shorten to about 6 minutes from traditional 20 minutes, supercoiled plasmid content is more than 90% simultaneously, can not only be used for the extraction of high copy and low copy plasmid, also is applicable to the extraction that different culture mediums cultivateed the escherichia coli plasmid, has saved experimental researcher's time greatly, has improved experimental efficiency.
According to some embodiments of the invention, the rapid precipitation buffer applied to plasmid DNA extraction comprises, based on 100ml of the buffer: 60-75 g of sodium iodide; 0.25-0.5 g of sodium dodecyl sarcosinate; 0.25-0.5 g of sodium deoxycholate; 3- [ (3-Cholesterol aminopropyl) dimethylamino ] -1-propanesulfonic acid 0.5-1.5 g; 2-6 g of potassium acetate; 1-5 ml of acetic acid; and the balance deionized water. By utilizing the rapid precipitation buffer solution applied to plasmid DNA extraction, the whole optimized plasmid extraction process can be completed within 6 minutes, and meanwhile, the content of supercoiled plasmid is more than 90%, so that the time of an experimental researcher is greatly saved, and the experimental efficiency is further improved.
According to a second aspect of the present invention, there is provided a method of purifying plasmid DNA comprising:
(1) mixing a sample containing plasmid DNA with the rapid precipitation buffer applied to plasmid DNA extraction;
(2) centrifuging the mixed solution obtained in the step (1), and collecting supernatant; and
(3) subjecting the supernatant obtained in step (2) to affinity chromatography to obtain purified plasmid DNA. By using the method, the plasmid extraction process can be completed within 6 minutes, and meanwhile, the content of the supercoiled plasmid is more than 90%, so that the time of an experimental researcher is greatly saved, and the experimental efficiency is improved. The method is efficient, rapid and simple, and the plasmid DNA purified by the method can be suitable for various conventional operations including enzyme digestion, PCR, sequencing, connection, transformation, library screening, in vitro translation, transfection of some conventional passage cells and the like.
According to some embodiments of the invention, the sample containing plasmid DNA is obtained by alkaline lysis of a microorganism containing plasmid DNA.
According to some embodiments of the invention, the alkaline lysis comprises:
(a-1) adding the suspension to the plasmid DNA-containing microorganism so as to obtain a first solution;
(a-2) adding an alkaline lysis solution to the first solution so as to obtain the plasmid DNA-containing sample. This can further improve the experimental efficiency.
According to some embodiments of the invention, the volume ratio of the suspension, the alkaline lysis solution and the rapid precipitation buffer applied to plasmid DNA extraction is 5:5: 14. This can further improve the experimental efficiency.
According to some embodiments of the present invention, after mixing a sample containing plasmid DNA with the rapid precipitation buffer applied to plasmid DNA extraction for 1min, the resulting mixture is centrifuged, and the supernatant is collected. Therefore, the experimental time can be further shortened, and the experimental efficiency can be improved.
According to some embodiments of the invention, the affinity chromatography treatment is performed using a silica membrane adsorption column.
According to a third aspect of the present invention, the present invention provides a method for preparing plasmid DNA, comprising:
(1) culturing 1-3 ml of escherichia coli containing plasmids in a culture medium, and centrifuging at 13000 rpm for 1min to obtain a precipitate;
(2) adding 75 microliters of the suspension to the precipitate, vortexing until the precipitate is completely dissolved, so as to obtain a first solution;
(3) adding 75 microliters of alkaline lysis solution to the first solution, gently flipping up and down until the solution is clear, so as to obtain a second solution;
(4) adding 210 microliters of the rapid precipitation buffer of claim 1 or 2 for plasmid DNA extraction to the second solution to obtain a third solution;
(5) centrifuging the third solution at 13000 rpm for 1min to obtain a supernatant;
(6) and transferring the supernatant into a silicon film adsorption column, centrifuging at 13000 r/min for 1min to remove waste liquid, adding 300 microliters of rinsing liquid, centrifuging at 13000 r/min for 30s to remove waste liquid, adding 50-100 microliters of elution buffer solution, and performing elution centrifugation to obtain a plasmid-containing DNA solution. By using the method, the plasmid extraction process can be completed within 6 minutes, and meanwhile, the content of the supercoiled plasmid is more than 90%, so that the time of an experimental researcher is greatly saved, and the experimental efficiency is improved. The method is efficient, rapid and simple, and the plasmid DNA purified by the method can be suitable for various conventional operations including enzyme digestion, PCR, sequencing, connection, transformation, library screening, in vitro translation, transfection of some conventional passage cells and the like.
According to a fourth aspect of the invention, the invention proposes the use of the aforementioned buffer for the preparation and purification of plasmid DNA.
According to a fifth aspect of the invention, there is provided a kit comprising: the aforementioned rapid precipitation buffer applied to plasmid DNA extraction. By utilizing the kit, the plasmid extraction process can be completed within 6 minutes, and meanwhile, the content of supercoiled plasmid is more than 90 percent, so that the time of an experimental researcher is greatly saved, and the experimental efficiency is improved; the kit is simple and rapid to operate, the content and quality of the prepared plasmid DNA are high, and the purified plasmid DNA can be suitable for various conventional operations including enzyme digestion, PCR, sequencing, connection, transformation, library screening, in vitro translation, transfection of some conventional passage cells and the like.
According to some embodiments of the invention, the kit further comprises: a suspension; an alkaline lysis solution; a rinsing liquid; and (4) eluting the solution. Thus, the experiment time can be further shortened, and the experiment efficiency can be improved.
Drawings
FIG. 1 is a photograph of agarose gel electrophoresis examination of the high copy plasmid pBS purified according to example 1 of the present invention.
FIG. 2 is an agarose gel electrophoresis test of the low copy plasmid pBR322 obtained by purification according to example 2 of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
Examples
Example 1: high copy plasmid pBS was extracted from E.coli cultures.
(1) Carrying out centrifugal separation on 1.5 ml of escherichia coli liquid cultured overnight, wherein the rotating speed of the centrifugal separation is 13000 r/min, the time of the centrifugal separation is 1min, and taking out a precipitate of the centrifugal separation;
(2) adding 75 μ l of suspension (50mM glucose, 25mM tris, 10mM EDTA, pH 8.0) to the precipitate, and vortexing until the precipitate is completely dissolved;
(3) adding 75 microliters of alkaline lysis solution (0.2N sodium hydroxide and 1 percent (mass percentage) of sodium dodecyl sulfate) into the solution, and gently turning the solution up and down for 6-8 times to fully lyse the thalli; and adding 210 microliters of rapid precipitation buffer solution into the solution, immediately and gently turning up and down for 6-8 times, and fully and uniformly mixing. The mixture was then centrifuged at 13000 rpm for 1 minute. Wherein, the buffer solution for rapid precipitation is 65 g of sodium iodide; 0.3 g of sodium dodecyl sarcosinate; 0.3 g of sodium deoxycholate; 1.0 g of 3- [ (3-cholesterylaminopropyl) dimethylamino ] -1-propanesulfonic acid; 4 g of potassium acetate; 4 ml of acetic acid; and deionized water to make up to 100 ml.
(4) Transferring the supernatant obtained in the step (4) into a silicon membrane adsorption column (product of Tiangen Biochemical technology (Beijing) Co., Ltd., catalog number RK132), carrying out adsorption centrifugation, wherein the rotation speed of the centrifugal adsorption is 13000 r/min, the centrifugal adsorption time is 30 seconds, and pouring out waste liquid;
(5) and (3) adding 300 microliters of rinsing liquid (80% ethanol by volume) into the silicon membrane adsorption column in the step (5), desalting and centrifuging at the rotating speed of 13000 rpm for 30 seconds, and pouring out the waste liquid.
(6) And (3) adding 50-100 microliters of elution buffer solution (10mM tris (hydroxymethyl) aminomethane, pH 8.0) into the silicon membrane adsorption column in the step (6), and performing elution centrifugation, wherein the rotation speed of the elution centrifugation is 13000 r/min, and the elution centrifugation time is 30 seconds, so as to obtain the plasmid DNA-containing solution.
(7) The plasmid DNA solution obtained in step (7) was subjected to agarose gel electrophoresis detection, and the detection result is shown in FIG. 1, wherein lane 1: deoxyribonucleic acid molecular weight standards (Marker iv), lane 2: example 1 the obtained plasmid DNA was purified.
Example 2: the low copy plasmid pBR322 was extracted from E.coli cultures.
(1) Carrying out centrifugal separation on 3 ml of escherichia coli liquid cultured overnight, wherein the rotating speed of the centrifugal separation is 13000 r/min, the time of the centrifugal separation is 1min, and taking out a precipitate of the centrifugal separation;
(2) adding 75 μ l of suspension (50mM glucose, 25mM tris, 10mM EDTA, pH 8.0) to the precipitate, and vortexing until the precipitate is completely dissolved;
(3) adding 75 microliters of alkaline lysis solution (0.2N sodium hydroxide and 1 percent (mass percentage) of sodium dodecyl sulfate) into the solution, and gently turning the solution up and down for 6-8 times to fully lyse the thalli;
(4) and adding 210 microliters of rapid precipitation buffer solution into the solution, immediately and gently turning up and down for 6-8 times, and fully and uniformly mixing. The mixture was then centrifuged at 13000 rpm for 1 minute. Wherein, the buffer solution for rapid precipitation is 65 g of sodium iodide; 0.3 g of sodium dodecyl sarcosinate; 0.3 g of sodium deoxycholate; 1.0 g of 3- [ (3-cholesterylaminopropyl) dimethylamino ] -1-propanesulfonic acid; 4 g of potassium acetate; 4 ml of acetic acid; and deionized water to make up to 100 ml.
(5) Transferring the supernatant obtained in the step (4) into a silicon membrane adsorption column (product of Tiangen Biochemical technology (Beijing) Co., Ltd., catalog number RK132), carrying out adsorption centrifugation, wherein the rotation speed of the centrifugal adsorption is 13000 r/min, the centrifugal adsorption time is 30 seconds, and pouring out waste liquid;
(6) and (3) adding 300 microliters of rinsing liquid (80% ethanol by volume) into the silicon membrane adsorption column in the step (5), desalting and centrifuging at the rotating speed of 13000 rpm for 30 seconds, and pouring out the waste liquid. And (3) adding 50-100 microliters of elution buffer solution (10mM tris (hydroxymethyl) aminomethane, pH 8.0) into the silicon membrane adsorption column in the step (6), and performing elution centrifugation, wherein the rotation speed of the elution centrifugation is 13000 r/min, and the elution centrifugation time is 30 seconds, so as to obtain the plasmid DNA-containing solution.
(7) The plasmid DNA solution obtained in step (6) was subjected to agarose gel electrophoresis, and the detection result is shown in FIG. 2, wherein lane 1: deoxyribonucleic acid molecular weight standards (Marker iv), lane 2: example 2 the obtained plasmid DNA was purified.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (11)
1. A rapid precipitation buffer for use in plasmid DNA extraction, said rapid precipitation buffer comprising, based on 100ml of said rapid precipitation buffer:
60-75 g of sodium iodide;
0.25-0.5 g of sodium dodecyl sarcosinate;
0.25-0.5 g of sodium deoxycholate;
0.5-1.5 g of 3- [ (3-cholesterylaminopropyl) dimethylamino ] -1-propanesulfonic acid;
2-6 g of potassium acetate;
1-5 ml of acetic acid; and
the balance being deionized water.
2. A method of purifying plasmid DNA, comprising:
(1) mixing a sample containing plasmid DNA with the rapid precipitation buffer of claim 1 applied to plasmid DNA extraction;
(2) centrifuging the mixed solution obtained in the step (1), and collecting supernatant; and
(3) subjecting the supernatant obtained in step (2) to affinity chromatography to obtain purified plasmid DNA.
3. The method according to claim 2, wherein the sample containing plasmid DNA is obtained by alkaline lysis of a microorganism containing plasmid DNA.
4. The method of claim 3, wherein the alkaline lysis comprises:
(a-1) adding the suspension to the plasmid DNA-containing microorganism so as to obtain a first solution;
(a-2) adding an alkaline lysis solution to the first solution so as to obtain the plasmid DNA-containing sample.
5. The method of claim 4, wherein the volume ratio of the suspension, the alkaline lysis buffer and the rapid precipitation buffer is 5:5: 14.
6. The method according to claim 2, wherein the sample containing plasmid DNA is mixed with the rapid precipitation buffer for 1min, the resulting mixture is centrifuged, and the supernatant is collected.
7. The method of claim 2, wherein the affinity chromatography is performed using a silica membrane adsorption column.
8. A method for preparing plasmid DNA, comprising:
(1) culturing 1-3 ml of escherichia coli containing plasmids in a culture medium, and centrifuging at 13000 rpm for 1min to obtain a precipitate;
(2) adding 75 microliters of the suspension to the precipitate, vortexing until the precipitate is completely dissolved, so as to obtain a first solution;
(3) adding 75 microliters of alkaline lysis solution to the first solution, gently flipping up and down until the solution is clear, so as to obtain a second solution;
(4) adding 210 microliters of the rapid precipitation buffer of claim 1 for application in plasmid DNA extraction to the second solution to obtain a third solution;
(5) centrifuging the third solution at 13000 rpm for 1min to obtain a supernatant;
(6) and transferring the supernatant into a silicon film adsorption column, centrifuging at 13000 r/min for 1min to remove waste liquid, adding 300 microliters of rinsing liquid, centrifuging at 13000 r/min for 30s to remove waste liquid, adding 50-100 microliters of elution buffer solution, and performing elution centrifugation to obtain a plasmid-containing DNA solution.
9. The use of the rapid precipitation buffer of claim 1 for plasmid DNA extraction in the preparation and purification of plasmid DNA.
10. A kit, comprising: the rapid precipitation buffer of claim 1 applied to plasmid DNA extraction.
11. The kit of claim 10, further comprising:
a suspension;
an alkaline lysis solution;
a rinsing liquid; and
and (4) eluting the solution.
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