CN114395553A - Magnetic bead method plasmid extraction kit, preparation method and plasmid extraction method - Google Patents
Magnetic bead method plasmid extraction kit, preparation method and plasmid extraction method Download PDFInfo
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Abstract
The invention discloses a magnetic bead method plasmid extraction kit, which comprises a solution SI, a solution I of magnetic beads, a solution II of magnetic beads and an eluent; the SI solution is used for cracking cells and releasing nucleic acid; the SII solution is used for precipitating impurities such as protein and the like; the magnetic bead I solution contains a magnetic bead I and a polycarboxyl compound, the magnetic bead I comprises a first magnetic core and a plurality of carboxyl groups modified on the surface of the first magnetic core, and the magnetic bead I can adsorb protein and other impurity precipitates so as to remove the protein and other impurity precipitates; the solution of the magnetic bead II contains a magnetic bead II which is used for combining plasmids. The invention also provides a preparation method of the plasmid extraction kit by the paramagnetic particle method. The invention also provides a method for extracting plasmids by using the kit.
Description
Technical Field
The invention relates to the technical field of nucleic acid extraction, and particularly relates to a plasmid extraction kit by a paramagnetic particle method, a preparation method and a plasmid extraction method.
Background
In molecular biotechnology, the extraction of nucleic acids is also of critical importance. Through long-term development, there are many methods for extracting nucleic acid, and the most common methods for extracting nucleic acid at present are centrifugal adsorption column extraction and magnetic bead extraction. The extraction of plasmids by a centrifugal adsorption column needs to be performed by multiple times of centrifugation, is difficult to realize automation, and is limited by a certain limitation of a centrifuge on the quantity processing of samples. The automation and high flux of nucleic acid extraction can be realized by the automatic nucleic acid extractor matched with the nucleic acid extraction kit by the magnetic bead method. However, in the existing method for extracting plasmids by using the magnetic bead method, the automatic extraction is mainly performed after most impurities are removed by cracking and centrifuging the thalli, the step of removing the impurities by centrifuging greatly increases the labor cost for sample treatment, and limits the automation range of plasmid extraction.
Disclosure of Invention
In view of the above, it is necessary to provide a plasmid extraction kit, a preparation method and a plasmid extraction method by magnetic bead method, which can avoid the centrifugation in the plasmid extraction process, and are of decisive significance for realizing the automation of the whole plasmid extraction process.
The invention provides a first purpose of a magnetic bead method plasmid extraction kit, which comprises a solution SI, a solution I of magnetic beads, a solution II of magnetic beads and an eluent;
the solution SI contains sodium hydroxide, lauryl sodium sulfate and ethylene diamine tetraacetic acid or salts thereof;
the STII solution contains sylvite and an acidic pH regulator;
the magnetic bead I solution contains a magnetic bead I and a polycarboxyl compound, the magnetic bead I comprises a first magnetic core and a plurality of carboxyl groups modified on the surface of the first magnetic core, and the polycarboxyl compound at least contains two carboxyl groups;
the magnetic bead II solution contains magnetic beads II, alcohol and sodium acetate, and the magnetic beads II are used for binding nucleic acid.
In one embodiment, the polycarboxyl compound is selected from one or more of citric acid, ethylenediaminetetraacetic acid, tartaric acid, and the corresponding salts of these compounds.
In one embodiment, the carboxyl group modified on the surface of the magnetic bead I is obtained by adding the polycarboxyl compound during the preparation process of the magnetic bead I.
The second purpose of the invention is to provide a preparation method of the plasmid extraction kit by the paramagnetic particle method, which comprises the step of mixing the components respectively according to the proportion.
In one embodiment, the preparation method of the solution of magnetic beads i comprises the following steps:
mixing a ferrous compound, a ferric compound and water, and stirring under an anaerobic condition;
adding ammonia water into the stirred mixture, and reacting at a first temperature of 50-120 ℃;
adding the polycarboxyl compound and the antioxidant into a product obtained after the reaction at the first temperature, and stirring for reaction at a second temperature, wherein the second temperature is 50-120 ℃;
performing magnetic separation and washing on the product obtained after the reaction at the second temperature by using water until the pH value of a washing liquid is 6.5-7.5;
mixing the washed product with a solution containing the polycarboxyl compound.
The third purpose of the invention is to provide a method for extracting plasmids by using the plasmid extraction kit by the magnetic bead method, which comprises the following steps:
mixing a sample containing bacterial liquid with the SI solution to obtain a first mixed solution;
mixing the first mixed solution, the SII solution and the magnetic bead I solution to obtain a second mixed solution;
performing magnetic attraction separation on the second mixed solution, and collecting to obtain a first supernatant;
mixing the first supernatant with the magnetic bead II solution to obtain a third mixed solution;
carrying out magnetic attraction separation on the third mixed solution, and removing supernatant to obtain magnetic beads II with nucleic acid combined on the surface;
washing the magnetic bead II with the surface combined with the nucleic acid to remove impurities on the surface of the magnetic bead II;
and eluting the washed magnetic beads II with the nucleic acid combined on the surface by using the eluent so as to collect the nucleic acid in the eluent.
Compared with the existing centrifugal impurity removal technology, the method has the advantages that:
the invention adopts a double-magnetic bead method to respectively realize the separation of impurities and the extraction of plasmids. The SI solution is used for cracking cells and releasing nucleic acid; the SII solution is used for precipitating impurities such as protein and the like; the magnetic bead I solution contains a magnetic bead I and a polycarboxyl compound, the magnetic bead I comprises a first magnetic core and a plurality of carboxyl groups modified on the surface of the first magnetic core, and the magnetic bead I can adsorb protein and other impurity precipitates so as to remove the protein and other impurity precipitates; the magnetic bead II solution contains magnetic beads II for binding nucleic acid. The inventive magnetic bead I can adsorb impurities such as fragments, proteins and the like after cracking of a sample, avoids a centrifugation step after cracking, and realizes full automation of plasmid extraction.
Drawings
FIG. 1 is a transmission electron microscope image of a magnetic bead I prepared in example 1 of the present invention;
FIG. 2 is a TEM image of a magnetic bead II prepared in example 2 of the present invention;
FIG. 3 is a comparative diagram of the supernatant after the bacteria liquid is cracked to remove impurities in example 3 of the present invention;
FIG. 4 is an agarose electrophoresis picture of the plasmid extracted in example 4 of the present invention;
FIG. 5 is a comparison graph of the supernatant after the bacteria liquid of comparative example 1 of the present invention is cracked to remove impurities;
FIG. 6 is an agarose electrophoresis image of the plasmid extracted in comparative example 2 of the present invention;
FIG. 7 is an agarose electrophoresis image of the plasmid extracted in example 5 of the present invention.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
In the present invention, "first aspect", "second aspect", "third aspect" and the like are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor are they to be construed as implicitly indicating the importance or quantity of the technical feature indicated.
As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. As used herein, the terms "comprising," "including," and "comprising" are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps. In the present invention, the technical features described in the open type include a closed technical solution composed of the listed features, and also include an open technical solution including the listed features.
In the present invention, the technical features described in the open type include a closed technical solution composed of the listed features, and also include an open technical solution including the listed features.
In the present invention, the numerical range is defined to include both end points of the numerical range unless otherwise specified.
The percentage contents referred to in the present invention mean, unless otherwise specified, mass percentages for solid-liquid mixing and solid-solid phase mixing, and volume percentages for liquid-liquid phase mixing.
The percentage concentrations referred to in the present invention refer to the final concentrations unless otherwise specified. The final concentration refers to the ratio of the additive component in the system to which the component is added.
In a first aspect, an embodiment of the present invention provides a plasmid extraction kit by a paramagnetic particle method, including an si solution, an S ii solution, a paramagnetic particle i solution, a paramagnetic particle ii solution, and an eluent;
the solution SI contains sodium hydroxide, lauryl sodium sulfate and ethylene diamine tetraacetic acid or salts thereof;
the STII solution contains sylvite and an acidic pH regulator;
the magnetic bead I solution contains a magnetic bead I and a polycarboxyl compound, the magnetic bead I comprises a first magnetic core and a plurality of carboxyl groups modified on the surface of the first magnetic core, and the polycarboxyl compound at least contains two carboxyl groups;
the magnetic bead II solution contains magnetic beads II, alcohol and sodium acetate, and the magnetic beads II are used for binding nucleic acid.
The SI solution is used for cracking cells and releasing nucleic acid; the SII solution is used for precipitating impurities such as protein and the like; the magnetic bead I solution contains a magnetic bead I and a polycarboxyl compound, the magnetic bead I comprises a first magnetic core and a plurality of carboxyl groups modified on the surface of the first magnetic core, and the magnetic bead I can adsorb protein and other impurity precipitates so as to remove the protein and other impurity precipitates; the magnetic bead II solution contains magnetic beads II used for binding nucleic acid, and the eluent is used for eluting the nucleic acid from the magnetic beads II.
The invention adopts a double-magnetic bead method to respectively realize the separation of impurities and the extraction of plasmids. The inventive magnetic bead I can adsorb impurities such as fragments, proteins and the like after cracking of a sample, avoids a centrifugation step after cracking, and realizes full automation of plasmid extraction. Can realize automatic separation in an automatic nucleic acid extractor, retain nucleic acid in a supernatant solution, and continue to combine the nucleic acid with the magnetic bead II. The purification and separation of nucleic acid can be realized through magnetic attraction separation, and the separation of impurities can be realized without adopting a centrifugal method.
The kit provided by the invention can be used with a full-automatic nucleic acid extractor to realize full-automatic plasmid extraction, does not need manual operation, liberates manpower, and is favorable for industrial extraction of plasmids. When a small amount of plasmids are extracted, the reagent in each step can be packaged into a deep-hole plate according to a program, so that direct sample loading and extraction are convenient. When a large amount of plasmids are extracted, the reagent in each step can be filled into a liquid feeding bottle according to a program, and the plasmid extraction is completed through an automatic sample injection system. In addition, the invention can complete extraction in a closed integrated instrument in the whole plasmid extraction process, thereby avoiding transfer of a lysis sample, avoiding pollution in the manual operation process and ensuring the stability of plasmid extraction.
The following detailed description of the automated plasmid extraction principle of the kit of the invention corresponds to the specific components:
in the solution SI, the sodium hydroxide solution can promote cell lysis and release nucleic acid. Sodium Dodecyl Sulfate (SDS) is capable of binding to proteins, denaturing the proteins. EDTA can be combined with metal ions such as Mg, Ca, Mn, Fe and the like, so that the activity of nuclease is inhibited, and the integrity of nucleic acid is ensured.
The STII solution contains potassium ions, and impurities such as SDS and proteins bound to the SDS can form precipitates insoluble in water. Preferably, the sii solution may include a weakly basic potassium salt, such as potassium acetate, or an acidic pH adjusting agent may be added to make the solution acidic to neutralize the alkalinity of the sii solution and make the impurities more easily settle.
In some embodiments, the potassium salt is selected from one or more of potassium acetate, potassium sulfate, potassium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and potassium chloride.
Preferably, the solution of the sI does not contain potassium ions, so that precipitation caused by reaction of SDS and potassium ions is avoided.
The potassium salt precipitate of SDS can be adsorbed by carboxyl groups modified on the surface of magnetic beads I. The polycarboxyl compound is used for removing impurities from magnetic beads, but the more or less carboxyl groups, the better, for example, polyacrylic acid cannot achieve the impurity removing effect. In some embodiments, the polycarboxyl compound may be selected from one or more of citric acid, ethylenediaminetetraacetic acid, tartaric acid, and the corresponding salts of these compounds. Preferably, a polycarboxyl compound is additionally added into the solution of the magnetic beads I, so that the magnetic beads I can form a stable suspension and cannot settle quickly. Preferably, a polycarboxyl compound is added to the solution of the SI to ensure the carboxyl group. Preferably, the solution of the magnetic bead I contains an antioxidant for protecting the magnetic bead I from being oxidized, so that the long-term storage stability of the magnetic bead I is guaranteed. The choice of antioxidant is not particularly critical, but does not necessarily affect the overall reaction.
After magnetic attraction separation, the magnetic beads I and the protein and other impurities bound on the surfaces of the magnetic beads I are removed, and the nucleic acid is left in the supernatant.
Then, the extraction and separation of the nucleic acid from which impurities are removed are performed by nucleic acid extraction magnetic beads commonly used in the art.
In some embodiments, the sii solution comprises the following mass ratios: the proportion of the sodium hydroxide, the lauryl sodium sulfate, the ethylene diamine tetraacetic acid or the salt thereof and the sodium citrate is (0.2-5%) (0.15-10%) (10 mM-100 mM): 2 mM-50 mM).
In some embodiments, the SII solution comprises potassium acetate, potassium chloride, and glacial acetic acid, wherein the molar ratio of potassium acetate to potassium chloride is (0.05-3) M (10-100) mM.
In some embodiments, the solution of the bead I comprises the bead I, sodium citrate and an antioxidant, and the mass of the bead I and the sodium citrate is (10-100) mg/mL (2-50) mM.
In some embodiments, the solution of the magnetic bead II comprises the magnetic bead II, absolute ethyl alcohol, isopropanol and sodium acetate, and the mass of the magnetic bead II and the mass of the sodium acetate are (1-50) mg/mL (0.5-3) M.
In some embodiments, the magnetic bead ii comprises a second magnetic core and a silica layer coated on the surface of the second magnetic core.
The alcohol is one or more of methanol, ethanol, isopropanol, isoamylol, glycerol and polyethylene glycol.
In some embodiments, the concentration of the liquid, by mass concentration,
the solution of the SII comprises 0.2-5% (e.g., 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%) of sodium hydroxide, 0.15-10% (e.g., 0.15%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%) of sodium lauryl sulfate, 10-100 mM (e.g., 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM) of ethylenediaminetetraacetic acid or a salt thereof, 2-50 mM (e.g., 2mM, 5mM, 10mM, 20mM, 30mM, 40mM, 50mM) of sodium citrate;
the STII solution contains 0.05M to 3M (e.g., 0.05M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2M, 2.1M, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M, 2.7M, 2.8M, 2.9M, 3M) of potassium acetate, 10mM to 100mM (e.g., 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM) of potassium chloride and glacial acetic acid, and the pH of the STII solution is 3.5.8, 4.8, 4.6, 4.8, 4.3.4.4, 3.8, 3.4, 3.4.8, 3.8M);
the solution of the magnetic bead I comprises 10mg/mL to 100mg/mL (such as 10mg/mL, 20mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, 100mg/mL) of the magnetic bead I, 2mM to 50mM (such as 2mM, 5mM, 10mM, 20mM, 30mM, 40mM, 50mM) of sodium citrate and 2mM to 50mM (such as 2mM, 5mM, 10mM, 20mM, 30mM, 40mM, 50mM) of antioxidant;
the solution of the magnetic bead II comprises 1mg/mL to 50mg/mL (e.g., 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg/mL, 10mg/mL20mg/mL, 30mg/mL, 40mg/mL, 50mg/mL) of the magnetic bead II, ethanol, isopropanol, 0.5M to 3M (e.g., 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 2M, 3M) of sodium acetate, and the total volume fraction of the ethanol and the isopropanol in the solution of the magnetic bead II is 40% to 75% (e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%).
In some embodiments, the kit further comprises a wai solution comprising ethanol, isopropanol, and 0.1M to 2M (e.g., 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 2M) sodium acetate, wherein the total volume fraction of ethanol and isopropanol in the wai solution is 50% to 75% (e.g., 50%, 55%, 60%, 65%, 70%, 75%).
In some embodiments, the kit further comprises a wai solution comprising 50% to 80% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%) by volume of an ethanol solution.
In some embodiments, the eluent is a TE solution or water.
The first magnetic core or the second magnetic core can be selected from the following magnetic microspheres: fe. Co, Ni, Fe2O3、Fe3O4、Co2O3、Co3O4One or a mixture of two or more of them. Preferably from Fe3O4。
The magnetic microsphere has superparamagnetism, can display magnetism in a magnetic field, eliminates the magnetism after being separated from the magnetic field, and is dispersed again.
The surface of the magnetic bead II is provided with a coating layer. Compared with an organic polymer shell material, the silicon dioxide layer has good chemical stability, protects an internal iron oxide core structure from being oxidized easily, and prevents the core-shell structure from being damaged due to corrosion under an acidic condition. The surface of the silicon dioxide has good biocompatibility, and a large number of silicon hydroxyl groups are convenient for modifying surface functional groups.
The method for coating silicon dioxide on the surface of the magnetic core generally comprises a Stober method (sol-gel method), a microemulsion method and the like, wherein the Stober (sol-gel) method is mainly based on the principle that a silicon precursor is hydrolyzed and polymerized on the surface of magnetic beads to form a silicon-oxygen network structure layer with uniform thickness. The method is widely applied because the reaction time is adjustable, the yield is high, and the thickness of the shell structure is uniform.
In a second aspect, the embodiment of the present invention provides a method for preparing a plasmid extraction kit by a magnetic bead method according to any one of the above embodiments, which includes mixing the components separately according to a certain ratio.
In some embodiments, the method for preparing the solution of magnetic beads i comprises:
mixing a ferrous compound, a ferric compound and water, and stirring under an anaerobic condition;
adding ammonia water into the stirred mixture, and reacting at a first temperature of 50-120 ℃;
adding the polycarboxyl compound and the antioxidant into a product obtained after the reaction at the first temperature, and stirring for reaction at a second temperature, wherein the second temperature is 50-120 ℃;
performing magnetic separation and washing on the product obtained after the reaction at the second temperature until the pH value of a washing liquid is 6.5-7.5;
mixing the washed product with a solution containing the polycarboxyl compound.
Preferably, the ratio of the amount of the ferric ion to the ferrous ion is n (Fe)3+)/n(Fe2+)＝1.5～2。
Preferably, the ferrous compound is ferrous sulfate, and the ferric compound is ferric chloride.
Preferably, N (NH)3·H2O)/n(Fe3+And Fe2+) Not less than 1.5, and n is mole number.
Preferably, n (polycarboxy compound and antioxidant)/n (Fe)3+And Fe2+) 0.2 to 10 percent, and n is mole number. The molar ratio is, for example, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%.
In some embodiments, the method for preparing the solution of magnetic beads ii comprises:
mixing a ferrous compound, a ferric compound and water, and stirring under an anaerobic condition;
adding ammonia water into the stirred mixture, and reacting at a third temperature of 50-120 ℃;
performing magnetic separation and washing on the product obtained after the reaction at the third temperature until the pH value of a washing liquid is 6.5-7.5;
dispersing the product obtained after washing in alcohol, adding tetraethoxysilane and ammonia water, and carrying out stirring reaction at a fourth temperature of 20-35 ℃;
and mixing the product obtained after the reaction at the third temperature with an alcohol solution.
Preferably, the ratio of the amount of the ferric ion to the ferrous ion is n (Fe)3+)/n(Fe2+)＝1.5～2
Preferably, the ferrous compound is ferrous sulfate, and the ferric compound is ferric chloride.
Preferably, N (NH)3·H2O)/n(Fe3+And Fe2+) Not less than 1.5, and n is mole number.
In a third aspect, the embodiment of the present invention provides a method for extracting plasmids by using a plasmid extraction kit by a magnetic bead method according to any one of the above embodiments, including the following steps:
mixing a sample containing bacterial liquid with the SI solution to obtain a first mixed solution;
mixing the first mixed solution, the SII solution and the magnetic bead I solution to obtain a second mixed solution;
performing magnetic attraction separation on the second mixed solution, and collecting to obtain a first supernatant;
mixing the first supernatant with the magnetic bead II solution to obtain a third mixed solution;
carrying out magnetic attraction separation on the third mixed solution, and removing supernatant to obtain magnetic beads II with nucleic acid combined on the surface;
washing the magnetic bead II with the surface combined with the nucleic acid to remove impurities on the surface of the magnetic bead II;
and eluting the washed magnetic beads II with the nucleic acid combined on the surface by using the eluent so as to collect the nucleic acid in the eluent.
In some embodiments, the volume ratio of the solution of SI, the solution of beads I and the solution of beads II added is (200-300): (200-300): (80-120): (400-1000). In some embodiments, the volume ratio of the solution of si, the solution of beads i, the solution of beads ii, the solution of wi, and the solution of wi is (200-300): (200-300): (80-120): (400-1000): (450-550): (450-550). Preferably, the volume ratio of the solution SI to the solution II to the solution I to the magnetic beads II is (200-300): (200-300): (80-120): (450-550). More preferably, the volume ratio of the solution SI, the solution of magnetic beads I and the magnetic beads II is 250: 250: 100: 500.
the nucleic acids of the invention are particularly directed to plasmids.
The sample is in particular a sample to be tested, for example a bacterial suspension, which contains plasmids.
The sample to be detected may be obtained from any source, including but not limited to prokaryotes (e.g., bacteria), eukaryotes (e.g., protozoa, parasites, fungi, yeast, plants, animals including mammals and humans).
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. It will be appreciated by those skilled in the art that various other changes, modifications, substitutions, combinations, and omissions may be made in the form and detail of the invention without departing from the spirit and scope of the invention.
Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
1 part of the SI solution of each of the following examples is 250. mu.L; 1 part of the STII solution means 250. mu.L; 1 part of the solution of the magnetic beads I is 100 mu L; 1 part of the solution of the magnetic bead II is 500. mu.L, and 1 part of the solution of the W I is 500. mu.L; 1 part of the WII solution is 500. mu.L.
EXAMPLE 1 preparation of solution of magnetic beads I
S1, respectively weighing 27g of ferric chloride hexahydrate and 15.88g of ferrous sulfate heptahydrate, dissolving in 100mL of deionized water, adding into a reaction kettle, and stirring at high speed under an oxygen-free condition;
s2, adding 50mL of ammonia water, reacting at 50 ℃ for 30min, adding 1g of sodium citrate and 0.2g of ascorbic acid, heating to 80 ℃, and continuing to react for 30 min;
s3, after the reaction is finished, performing magnetic separation and washing on the product in the S2 by using pure water until the washing liquid is neutral;
and S4, storing the washed magnetic beads I in a solution of 0.5% of sodium citrate and 0.1% of ascorbic acid to form a magnetic bead I solution.
FIG. 1 shows a TEM image of the magnetic beads I prepared in example 1.
EXAMPLE 2 preparation of solution of magnetic beads II
S1, respectively weighing 27g of ferric chloride hexahydrate and 15.88g of ferrous sulfate heptahydrate, dissolving in 100mL of deionized water, adding into a reaction kettle, and stirring at high speed under an oxygen-free condition;
s2, adding 50mL of ammonia water, reacting at 50 ℃ for 30min, heating to 80 ℃ and continuing to react for 30 min;
s3, after the reaction is finished, performing magnetic separation and washing on the product by using a 50% ethanol solution until the washing solution is neutral;
s4, dispersing 10g of the product obtained in the step S3 in absolute ethyl alcohol, transferring the product into a reaction kettle, adding 0.5mL of ethyl orthosilicate and 0.2mL of ammonia water, and stirring the mixture at room temperature for 3 hours;
s5: the product in S4 was magnetically separated and washed three times with pure water, and stored in 70% isopropyl alcohol solution to constitute a magnetic bead ii solution.
FIG. 2 is a transmission electron micrograph of the magnetic bead II prepared in example 2.
Example 3 method of Using a plasmid extraction kit based on the paramagnetic particle method
S1: transferring 2 parts of the bacterial liquid into a deep hole plate, and recording the serial numbers of the first step and the second step;
s2: adding a part of S I solution into the bacterial liquid, uniformly mixing for a period of time, then adding 1 part of S II solution and 1 part of magnetic bead I solution, and continuously uniformly mixing for a period of time;
s3: meanwhile, adding one part of the solution SI into the bacterial liquid, uniformly mixing for a period of time, then adding 1 part of the solution SI and 2 parts of the solution of the magnetic beads I, and continuously mixing for a period of time;
s4: magnetic attraction separation is carried out, and supernate I and supernate II are collected;
s5: simultaneously adding 1 part of bacterial liquid into a centrifugal tube, adding one part of the SI solution, uniformly mixing for a period of time, adding the SI solution, and continuously mixing for a period of time;
s6: centrifuging the centrifugal tube, and collecting supernatant liquid;
s7: supernatants collected at S4 and S6 were compared.
The solution SI comprised 0.8% sodium hydroxide, 1% sodium lauryl sulfate, 10mM disodium EDTA, 10mM sodium citrate.
The SII solution comprised 3M potassium acetate, 10mM potassium chloride, and was adjusted to pH 3.5 with glacial acetic acid.
A solution of magnetic beads I was prepared as described in example 1.
FIG. 3 is a comparative graph of the supernatant obtained after the lysis of the bacterial liquid to remove impurities. The result shows that the clarity of the supernatant liquid is the highest. The magnetic bead I prepared in the embodiment 1 of the invention has stronger binding capacity with protein and better impurity removal effect when used for removing impurities.
Example 4 method of Using a plasmid extraction kit based on the paramagnetic particle method
S1: transferring 2 parts of the bacterial liquid into a deep hole plate, and recording the serial numbers of the first step and the second step;
s2: adding a part of S I solution into the bacterial liquid, uniformly mixing for a period of time, then adding 1 part of S II solution and 1 part of magnetic bead I solution, and continuously uniformly mixing for a period of time;
s3: meanwhile, adding one part of the solution SI into the bacterial liquid, uniformly mixing for a period of time, then adding 1 part of the solution SI and 2 parts of the solution of the magnetic beads I, and continuously mixing for a period of time;
s4: magnetic attraction separation, removing magnetic substances, and retaining supernatant;
s5: adding a magnetic bead II solution into the supernatant of S4, uniformly mixing for a period of time, and removing the supernatant through a magnetic separation device;
s6: adding 1 part of the W I solution, uniformly mixing for a period of time, and removing supernatant through a magnetic separation device;
s7: adding 1 part of WII solution, uniformly mixing for a period of time, and removing supernatant through a magnetic separation device;
s8: and (2) drying the magnetic beads in the S6 at room temperature for 5min, adding 1 part of TE eluent, uniformly mixing for a period of time, and collecting supernate through a magnetic separation device to obtain purified plasmids (i) and (ii).
S9: meanwhile, the same state and volume of bacteria liquid is purified by an adsorption column plasmid extraction kit (Tiangen Biochemical, DP103-03) to obtain purified plasmid III.
The composition of the solution of SI was the same as in example 3.
The composition of the SII solution was the same as in example 3.
The solution of beads I was the same as in example 3.
The W I solution comprises absolute ethyl alcohol, isopropanol and 0.8M sodium acetate, and the proportion of alcohol solvent is 70%.
The WII solution is 80% ethanol solution.
A solution of magnetic beads II was prepared as in example 2.
Example 4 agarose electrophoresis of extracted plasmids is shown in FIG. 4. The result shows that when the kit is used for extracting nucleic acid, the impurity removal is strong, the nucleic acid extraction effect is good, and the method is even superior to the traditional adsorption column centrifugation method.
Comparative example 1 preparation of solution of magnetic beads I
Based on the preparation conditions for preparing the magnetic bead I, proper experimental conditions are not adopted (sodium citrate is not added in the preparation process), and the process for preparing the magnetic bead I is completed according to the following steps:
s1, respectively weighing 27g of ferric chloride hexahydrate and 15.88g of ferrous sulfate heptahydrate, dissolving in 100mL of deionized water, adding into a reaction kettle, and stirring at high speed under an oxygen-free condition;
s2, adding 50mL of ammonia water, reacting at 50 ℃ for 30min, adding 0.2g of ascorbic acid, heating to 80 ℃, and continuing to react for 30 min;
s3, after the reaction is finished, performing magnetic separation and washing on the product in the S2 by using pure water until the washing liquid is neutral;
s4, storing the washed magnetic beads I in a 0.1% ascorbic acid solution to form a magnetic bead I solution.
Comparative example 2 the method of using the plasmid extraction kit based on the paramagnetic particle method was as follows:
s1: transferring 2 parts of the bacterial liquid into a deep hole plate, and recording the numbers (fourth) and (fifth);
s2: adding one part of the solution SI into the bacterial liquid, uniformly mixing for a period of time, then adding 1 part of the solution SI and 1 part of the solution of the magnetic beads I, and continuously uniformly mixing for a period of time;
s3: meanwhile, adding one part of the SI solution into the bacteria liquid, uniformly mixing for a period of time, then adding 1 part of the SI solution and 2 parts of the magnetic bead I solution, and continuously mixing for a period of time;
s4: magnetic attraction separation, collecting supernatant liquid;
s5: simultaneously adding 1 part of bacterial liquid into a centrifugal tube, adding one part of the SI solution, uniformly mixing for a period of time, adding the SI solution, and continuously mixing for a period of time;
s6: centrifuging the tube, and collecting the supernatant;
s7: supernatants collected at S4 and S6 were compared.
The composition of the solution of SI was the same as in example 3.
The composition of the SII solution was the same as in example 3.
A solution of beads I was prepared as in comparative example 1.
Comparative example 2 comparative graph of supernatant after removing impurities by lysis of bacterial liquid is shown in fig. 5. The results showed that the clarity of the supernatants iv and v was poor. It is shown that when the magnetic beads I prepared in comparative example 1 of the present invention are used for nucleic acid extraction, the protein binding capacity is poor, resulting in poor impurity removal effect.
Comparative example 3
Extracting plasmids from the supernatant liquid collected in the comparative example 1 by a magnetic bead method, and extracting plasmids from the supernatant liquid by an adsorption column method plasmid extraction kit by the following steps:
s1: adding 1 part of the magnetic bead II solution in the example 2 into the supernate IV and the supernate IV collected in the comparative example 1, uniformly mixing for a period of time, and removing the supernate through a magnetic separation device;
s2: adding 1 part of the W I solution, uniformly mixing for a period of time, and removing supernatant through a magnetic separation device;
s3: adding 1 part of WII solution, uniformly mixing for a period of time, and removing supernatant through a magnetic separation device;
s4: drying the magnetic beads in S6 at room temperature for 5min, adding 1 part of TE eluent, uniformly mixing for a period of time, and collecting supernatant through a magnetic separation device to obtain purified plasmids (iv) and (v);
s5: and purifying the supernatant by using an adsorption column method plasmid extraction kit to obtain the plasmid.
The bead II solution was the same as in example 4.
The WI solution was the same as in example 4.
The waii solution was the same as in example 4.
Comparative example 3 agarose electrophoresis of the extracted plasmid is shown in FIG. 6. The result shows that when the method of the comparative example is used for extracting nucleic acid, the subsequent nucleic acid extraction effect is poor due to poor impurity-containing ability of the magnetic bead I combined with protein and poor impurity removal effect, and the nucleic acid cannot be extracted.
Example 5
S1: transferring 3 parts of the bacterial liquid into a deep hole plate, and recording numbers (c), (b) and (c);
s2: adding 250 mu L S I solution into the seventh, the eighth and the ninth components, uniformly mixing for a period of time, then adding 250 mu LS II solution, and continuously uniformly mixing for a period of time;
s3: adding 100 mu L, 50 mu L and 10 mu L of magnetic bead I solution into the air of the samples and uniformly mixing for a period of time;
s4: magnetically attracting and separating to collect supernatant fluid in the seventh step, the eighth step and the ninth step respectively;
s5: adding 1 part of the magnetic bead II solution obtained in the example 2 into each of the three parts of the supernatant, uniformly mixing for a period of time, and removing the supernatant through a magnetic separation device;
s6: respectively adding 1 part of the W I solution, uniformly mixing for a period of time, and removing supernatant through a magnetic separation device;
s7: respectively adding 1 part of WII solution, uniformly mixing for a period of time, and removing supernatant through a magnetic separation device;
s8: drying the magnetic beads in the S7 at room temperature for 5min, adding 1 part of TE eluent respectively, mixing uniformly for a period of time, and collecting supernate through a magnetic separation device to obtain purified plasmids (c), (b) and (c);
the composition of the solution of SI was the same as in example 3.
The composition of the SII solution was the same as in example 3.
The solution of beads I was the same as in example 3.
The bead II solution was the same as in example 4.
The WI solution was the same as in example 4.
The waii solution was the same as in example 4.
Example 5 agarose electrophoresis of the extracted plasmid is shown in FIG. 7. The result shows that after the impurity removal is finished by adopting the magnetic bead I solution with different dosage, the final extraction effect is obviously different when the plasmid is extracted, and the extraction effect after adding 100 mu L of the magnetic bead I solution to remove the impurity is obviously better than the extraction effect after adding 50 mu L and 10 mu L of the magnetic bead I solution to remove the impurity. The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, so as to understand the technical solutions of the present invention specifically and in detail, but not to be understood as the limitation of the patent protection scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the patent of the invention is subject to the appended claims, and the description can be used for explaining the contents of the claims.
Claims (10)
1. A magnetic bead method plasmid extraction kit is characterized by comprising a solution SI, a solution of magnetic beads I, a solution of magnetic beads II and an eluent;
the solution SI contains sodium hydroxide, lauryl sodium sulfate and ethylene diamine tetraacetic acid or salts thereof;
the STII solution contains sylvite and an acidic pH regulator;
the magnetic bead I solution contains a magnetic bead I and a polycarboxyl compound, the magnetic bead I comprises a first magnetic core and a plurality of carboxyl groups modified on the surface of the first magnetic core, and the polycarboxyl compound at least contains two carboxyl groups;
the magnetic bead II solution contains magnetic beads II, alcohol and sodium acetate, and the magnetic beads II are used for binding nucleic acid.
2. The magnetic bead method plasmid extraction kit of claim 1, wherein the polycarboxyl compound is selected from one or more of citric acid, ethylenediaminetetraacetic acid, tartaric acid, and the corresponding salts of these compounds;
preferably, the carboxyl group modified on the surface of the magnetic bead I is obtained by adding the polycarboxyl compound in the preparation process of the magnetic bead I.
3. The kit for extracting plasmids by the magnetic bead method according to claim 1, wherein the magnetic bead II comprises a second magnetic core and a silica layer coated on the surface of the second magnetic core.
4. The kit for extracting plasmids by magnetic bead method according to claim 1, wherein the potassium salt is selected from one or more of potassium acetate, potassium sulfate, potassium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and potassium chloride.
5. The plasmid extraction kit according to any one of claims 1 to 4, wherein the plasmid is extracted from the sample by magnetic bead method,
the SI solution comprises the following substances in mass ratio: the proportion of the sodium hydroxide, the lauryl sodium sulfate, the ethylene diamine tetraacetic acid or the salt thereof and the sodium citrate is (0.2-5%) (0.15-10%) (10 mM-100 mM): 2 mM-50 mM); and/or the presence of a gas in the gas,
the STII solution contains potassium acetate, potassium chloride and glacial acetic acid, and the molar ratio of the potassium acetate to the potassium chloride is (0.05-3) M (10-100) mM; and/or the presence of a gas in the gas,
the solution of the magnetic bead I comprises the magnetic bead I, sodium citrate and an antioxidant, wherein the mass of the magnetic bead I and the mass of the sodium citrate are 10-100 mg/mL (2-50) mM; and/or the presence of a gas in the gas,
the magnetic bead II solution comprises the magnetic bead II, absolute ethyl alcohol, isopropanol and sodium acetate, and the mass of the magnetic bead II and the mass of the sodium acetate are (1-50) mg/mL (0.5-3) M;
preferably, the concentration of the liquid, by mass concentration,
the solution of the S I comprises 0.2 to 5 percent of sodium hydroxide, 0.15 to 10 percent of sodium dodecyl sulfate, 10 to 100mM of ethylenediamine tetraacetic acid or salt thereof and 2 to 50mM of sodium citrate;
the SII solution comprises 0.05-3M of potassium acetate, 10-100 mM of potassium chloride and glacial acetic acid, and the pH value of the SII solution is 3.5-4.8;
the solution of the magnetic bead I comprises 10 mg/mL-100 mg/mL of the magnetic bead I, 2 mM-50 mM of sodium citrate and 2 mM-50 mM of antioxidant;
the magnetic bead II solution contains 1-50 mg/mL of the magnetic bead II, ethanol, isopropanol and 0.5-3M of sodium acetate, and the total volume fraction of the ethanol and the isopropanol in the magnetic bead II solution is 40-75%.
6. The plasmid extraction kit by the magnetic bead method according to any one of claims 1 to 4, wherein the kit further comprises a WI solution, the WI solution contains ethanol, isopropanol and 0.1-2M sodium acetate, and the total volume fraction of the ethanol and the isopropanol in the WI solution is 50-75%;
preferably, the kit further comprises a WII solution, and the WII solution comprises an ethanol solution with the volume fraction of 50-80%.
7. The method for preparing the plasmid extraction kit by the magnetic bead method according to any one of claims 1 to 6, which comprises mixing the components separately according to a certain ratio; preferably, the preparation method of the magnetic bead I solution comprises the following steps:
mixing ferrous compound, ferric compound and water, stirring under oxygen-free condition, preferably, the ratio of the amount of ferric ion to ferrous ion is n (Fe)3+)/n(Fe2+)＝1.5～2；
Adding ammonia water into the stirred mixture, and reacting at a first temperature of 50-120 ℃, preferably, N (NH)3·H2O)/n(Fe3+And Fe2+) More than or equal to 1.5, wherein n is the mole number;
adding the polycarboxyl compound and the antioxidant into a product obtained after the reaction at the first temperature, and stirring for reaction at a second temperature, wherein the second temperature is 50-120 ℃, and preferably, n (polycarboxyl compound and antioxidant)/n (Fe)3+And Fe2+) 0.2 to 10 percent, and n is mole number;
performing magnetic separation and washing on the product obtained after the reaction at the second temperature until the pH value of a washing liquid is 6.5-7.5;
mixing the washed product with a solution containing the polycarboxyl compound;
preferably, the ferrous compound is ferrous sulfate, and the ferric compound is ferric chloride.
8. The method for preparing a plasmid extraction kit by a paramagnetic particle method according to claim 7, wherein the method for preparing the solution of magnetic beads II comprises the following steps:
mixing ferrous compound, ferric compound and water, stirring under oxygen-free condition, preferably, the ratio of the amount of ferric ion to ferrous ion is n (Fe)3+)/n(Fe2+)＝1.5～2；
Adding ammonia water into the stirred mixture, and reacting at a third temperature of 50-120 ℃, preferably, N (NH)3·H2O)/n(Fe3+And Fe2+) More than or equal to 1.5, wherein n is the mole number;
performing magnetic separation and washing on the product obtained after the reaction at the third temperature until the pH value of a washing liquid is 6.5-7.5;
dispersing the product obtained after washing in alcohol, adding tetraethoxysilane and ammonia water, and carrying out stirring reaction at a fourth temperature of 20-35 ℃;
mixing the product obtained after the reaction at the third temperature with an alcohol solution;
preferably, the ferrous compound is ferrous sulfate, and the ferric compound is ferric chloride.
9. The method for extracting plasmids by using the plasmid extraction kit by the magnetic bead method as set forth in any one of claims 1 to 6, which is characterized by comprising the steps of:
mixing a sample containing bacterial liquid with the SI solution to obtain a first mixed solution;
mixing the first mixed solution, the SII solution and the magnetic bead I solution to obtain a second mixed solution;
performing magnetic attraction separation on the second mixed solution, and collecting to obtain a first supernatant;
mixing the first supernatant with the magnetic bead II solution to obtain a third mixed solution;
carrying out magnetic attraction separation on the third mixed solution, and removing supernatant to obtain magnetic beads II with nucleic acid combined on the surface;
washing the magnetic bead II with the surface combined with the nucleic acid to remove impurities on the surface of the magnetic bead II;
and eluting the washed magnetic beads II with the nucleic acid combined on the surface by using the eluent so as to collect the nucleic acid in the eluent.
10. The method for extracting plasmids of claim 9, wherein the volume ratio of the SI solution, the bead I solution and the bead II solution is (200-300): (200-300): (80-120): (450 to 1000), preferably (200 to 300): (200-300): (80-120): (450-550).
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| WO2023125592A1 (en) * | 2021-12-28 | 2023-07-06 | 南京金斯瑞生物科技有限公司 | Magnetic bead, and preparation method therefor and use thereof in nucleic acid extraction |
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