CN113388611A - Kit for extracting stable bacterial genome DNA by paramagnetic particle method and extraction method thereof - Google Patents
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Abstract
The invention provides a kit for extracting stable bacterial genome DNA by a paramagnetic particle method and an extraction method thereof. The kit comprises a lysis reagent group for lysing and releasing bacterial genomic DNA and an extraction reagent group for extracting the genomic DNA; the lysis reagent group consists of buffer solution and lysis solution; the extraction reagent group consists of magnetic bead binding liquid, washing liquid and eluent; the magnetic bead binding solution consists of sodium citrate or sodium acetate, reducing agent dithiothreitol, guanidine hydrochloride and magnetic bead suspension. According to the invention, the raw materials are reasonably compounded to obtain the optimal proportion of a magnetic bead binding solution compounding system and combined extraction conditions, and the extraction method is optimized, so that the extracted bacterial genome DNA has high purity, complete fragments and difficult degradation, the stability is far superior to that of a commercial kit, and the advantages in subsequent experiments such as DNA storage, hybridization, PCR and the like are obvious; meanwhile, the reagents are non-toxic and have little pollution to the environment.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a kit for extracting stable bacterial genome DNA by a paramagnetic particle method and an extraction method thereof.
Background
The nucleic acid extraction and purification technology is a key technology of the molecular biology technology in clinical detection application at present, the operation of a conventional commercialized bacterial genome extraction kit is complex, ten steps are needed from collecting bacteria to obtaining genomic DNA, and the adsorption efficiency of the genomic DNA in the extraction process is low. The magnetic bead method for purifying DNA is based on the specific adsorption of polynucleotide and magnetic beads with negative electricity on the surface under the condition of high-concentration salt ions, so that the purpose of DNA purification is achieved; the method has the advantages of high extraction efficiency, simple operation, few steps and easy realization of automation. Therefore, the magnetic bead method DNA purification method has great popularization value in the field of clinical detection.
Bacteria usually have hard cell walls, and the conventional boiling method is difficult to effectively destroy the cell walls of the bacteria, so that the extraction efficiency of genome DNA is extremely low. Although the existing bacterial genome DNA extraction kit on the market generally uses an SDS (sodium dodecyl sulfate) lysis method, a lysozyme lysis method, a CTAB (cetyltrimethyl ammonium bromide) method and the like, the kit is influenced by the effect of lysis solution on reagent extraction efficiency, and the commonly used commercial magnetic bead method bacterial genome DNA extraction kit has poor fragment stability, is easy to degrade and difficult to store or transport for a long time, and cannot be used for subsequent experiments such as amplification, cloning and the like. Therefore, it is necessary to develop a kit which can stably extract DNA fragments and can be stored for a long period of time.
The invention patent with the application number of CN202110030644.5 discloses a kit for rapidly extracting genomic DNA of gram-negative bacteria and an extraction method. The kit comprises working solution A, working solution B, magnetic bead binding solution, working solution C and working solution D. And (3) adopting monodisperse and superparamagnetic silicon oxide nano magnetic beads to extract the genomic DNA of the gram-negative bacteria. The concentration of NaCl in the magnetic bead binding solution is 0.5-1.5 mol/L, the concentration of guanidine hydrochloride is 1.2-1.8 mol/L, the volume ratio of absolute ethyl alcohol is 20-30%, the volume ratio of isopropanol is 30-60%, the mass fraction of polyethylene glycol is 3% -10%, the molecular weight of polyethylene glycol is 6000-8000, and the pH value is 5.0-6.5.
The invention patent with the application number of CN201810061626.1 discloses a kit for extracting bacterial genome DNA by a magnetic bead method and application thereof. The kit comprises the following six components: magnetic bead suspension A, buffer B, buffer C, buffer D, buffer E and eluent. The magnetic beads used in the magnetic bead suspension A are monodisperse superparamagnetic silicon hydroxyl nano magnetic beads, the particle size of the magnetic beads is 300-500 nm, the concentration of the magnetic beads is 100mg/mL, and the magnetic beads are suspended in 20% ethanol.
However, none of the above-mentioned kits uses a biological reagent capable of stabilizing nucleic acids, and the stability of the extracted genomic DNA is not investigated, and thus it is impossible to provide effective preliminary assurance for subsequent biological experiments such as amplification and genetic information analysis.
In view of the above, there is a need to design an improved kit for extracting stable bacterial genomic DNA by the magnetic bead method and an improved method for extracting stable bacterial genomic DNA by the magnetic bead method, so as to solve the above problems.
Disclosure of Invention
The invention aims to provide a kit for extracting stable bacterial genome DNA by a paramagnetic particle method and an extraction method thereof.
In order to achieve the above purpose, the present invention provides a kit for extracting stable bacterial genomic DNA by a magnetic bead method, which comprises a lysis reagent group for lysis and release of bacterial genomic DNA and an extraction reagent group for extraction of genomic DNA;
the lysis reagent group consists of buffer solution and lysis solution; the extraction reagent group consists of magnetic bead binding liquid, washing liquid and eluent; the magnetic bead binding solution consists of sodium citrate or sodium acetate, a reducing agent dithiothreitol, guanidine hydrochloride and a magnetic bead suspension;
in the magnetic bead binding solution, the concentration of sodium salt is 0.05-0.15M, the content of reducing agent dithiothreitol is 0.5-1.5 wt%, and the concentration of guanidine hydrochloride is 2-5M.
As a further improvement of the invention, in the magnetic bead suspension, the particle size of the magnetic beads is 0.5-5 μm.
As a further improvement of the invention, the surface of the magnetic bead is modified with hydroxyl or carboxyl.
As a further improvement of the invention, the magnetic bead suspension is prepared by mixing magnetic beads and isopropanol according to a mass ratio of 1: (30-50), and the pH value of the magnetic bead binding solution is 7.5-8.5.
As a further improvement of the invention, in the magnetic bead binding solution, the concentration of sodium salt is 0.1M, the content of dithiothreitol is 1 wt%, and the concentration of guanidine hydrochloride is 5M.
As a further improvement of the invention, the washing solution is an isopropanol water solution or an ethanol water solution with the volume fraction of 75-85%; the eluent is one of distilled water, deionized water and ultrapure water.
As a further improvement of the invention, the buffer solution is a mixed water solution containing 0.5-1.5 mass percent of triton, 1-10 mM of disodium ethylene diamine tetraacetate, 15-25 mM of tris hydrochloride and 0.1-0.5 mass percent of lysozyme, and the pH value of the buffer solution is 7.0-8.0.
As a further improvement of the invention, the lysis solution is a mixed aqueous solution containing 0.2-0.4M of sodium chloride and 1-3% of hexadecyl trimethyl ammonium bromide by mass fraction, and the pH value of the lysis solution is 7.0-8.0.
In order to achieve the above object, the present invention further provides an extraction method for extracting stable bacterial genomic DNA by using the above kit, comprising the following steps:
s1, taking 1-4 mL of fresh overnight-cultured bacteria culture solution into a centrifuge tube, centrifuging for 1-5 minutes at 10000-12000 rpm, discarding the supernatant, adding 150-300 mu L of buffer solution, blowing and beating for 10-15 seconds by using a pipette, uniformly mixing the bacteria solution, and then placing the mixture in a 37 ℃ water bath pot for incubation for 30-60 minutes;
s2, adding 300-800 mu L of lysis solution into the centrifugal tube in the step S1, blowing and beating the lysis solution for 10-15S by using a pipette, uniformly mixing the lysis solution, and then placing the lysis solution in a 65 ℃ water bath kettle for incubation for 10-30 minutes;
s3, adding 10-20 mu L of magnetic bead binding solution into the centrifuge tube in the step S2, blowing and beating for 10-15S by using a pipette, standing for 1-3 minutes, then carrying out magnetic separation by using a magnetic rack, and removing the liquid;
s4, adding 500-1000 mu L of washing liquid into the centrifugal tube in the step S3, blowing and beating the washing liquid for 10-15 seconds by using a pipette, standing the washing liquid for 10-30 seconds, then carrying out magnetic separation by using a magnetic frame, sucking away the liquid, and standing the washing liquid for 5-10 minutes at room temperature after uncovering;
s5, adding 50-100 mu L of eluent into the centrifuge tube in the step S5, blowing and beating the mixture for 10-15S by using a pipette, standing the mixture for 1-3 minutes, then carrying out magnetic separation by using a magnetic frame, and absorbing supernatant liquid into a new centrifuge tube to obtain the stable bacterial genome DNA.
As a further improvement of the invention, the stable bacterial genome DNA extracted by the extraction method has high purity, complete fragments and good stability, and is not degraded after being stored for 2 days at 4 ℃.
The invention has the beneficial effects that:
1. according to the kit for extracting the stable bacterial genome DNA by the magnetic bead method, sodium citrate or sodium acetate, dithiothreitol and guanidine hydrochloride are mixed with the magnetic bead suspension, the obtained magnetic bead binding solution has excellent binding force and binding stability on the bacterial genome DNA, the bacterial genome DNA extracted by the magnetic bead binding solution has high purity, complete fragments and good stability, is not easy to degrade, has stability far superior to that of a commercially available kit, and has remarkable advantages in storage of DNA, hybridization, PCR and other subsequent experiments; meanwhile, the reagents are non-toxic and have little pollution to the environment, and can be used for replacing most similar products on the market.
2. In the kit for extracting the stable bacterial genome DNA by the paramagnetic particle method, sodium salt sodium citrate or sodium acetate acts as follows in a magnetic bead binding liquid system with the pH value of 7.5-8.5: the inorganic salt cation can neutralize the negative charge on DNA molecules, reduce the repulsive force among electronegative DNA molecules and promote the aggregation and precipitation of DNA. Meanwhile, the citrate or acetate is adsorbed on the surface of the magnetic bead, so that the activity of silicon hydroxyl or carboxyl groups on the surface of the magnetic bead can be effectively protected, and the firm adsorption of the bacterial genome DNA on the surface of the magnetic bead is facilitated.
The effect of the reducing agent dithiothreitol is as follows: can prevent the magnetic beads from being oxidized to a certain extent to reduce the magnetism, and can also effectively prevent the nucleic acid from being oxidized to be denatured. The effect of the nuclease inhibitor guanidine hydrochloride is as follows: can not only denature protein, but also inhibit the degradation of nucleic acid by nuclease. The magnetic beads with the particle size of 0.5-5 mu m and the modified hydroxyl or carboxyl on the surfaces have the following functions: hydroxyl or carboxyl magnetic beads can form a salt bridge structure of 'anion-cation-anion' by utilizing the electronegativity of the surface and electronegative nucleic acid in the presence of salt ions, and the nucleic acid is specifically extracted; meanwhile, the magnetic separation of nucleic acid can be effectively ensured by the proper magnetic bead particle size of 0.5-5 mu m, so that the purpose of separation and purification can be achieved.
According to the invention, through reasonable compounding of the raw material components, the optimal proportion and the combined extraction condition of the magnetic bead binding solution compound system are obtained, so that the raw materials can exert a synergistic effect, and the magnetic bead binding solution system has excellent binding force and binding stability to bacterial genome DNA. The mechanism of this synergy is as follows: when the bacteria are cracked by the cracking liquid, components such as protein, nuclease, nucleic acid and the like in the bacteria are released, and the protein and the nucleic acid are adsorbed together due to the electrostatic interaction between the protein and the nucleic acid, so that certain loss is caused to the nucleic acid; nucleases can constantly degrade nucleic acids, which themselves can begin to undergo oxidative denaturation. When sodium citrate or sodium acetate with proper concentration, reducing agent dithiothreitol, guanidine hydrochloride and hydroxyl or carboxyl magnetic beads are added, sodium ions can effectively protect the activity of silicon hydroxyl groups on the surfaces of the magnetic beads and enhance the binding capacity of the magnetic beads to nucleic acids while increasing the adsorption capacity of the magnetic beads to the nucleic acids; guanidine hydrochloride denatures protein, can further enhance the binding capacity of magnetic beads and DNA, and simultaneously inhibits degradation of nuclease to nucleic acid so as to improve the stability of the nucleic acid to a certain extent; dithiothreitol can prevent the magnetic bead from oxidation and magnetism from weakening, ensure effective combination of the magnetic bead and nucleic acid, and can further prevent the nucleic acid from oxidation and denaturation.
Drawings
FIG. 1 is an agarose electrophoresis of E.coli genomic DNAs extracted in example 1 and comparative examples 1 to 2 (wherein lanes M are Marker bands; lanes 1 and 2 are bacterial genomic DNA bands extracted with the kit of example 1 of the present invention; lanes 3 and 4 are bacterial genomic DNA bands extracted with the commercially available Omega kit of comparative example 1; lanes 5 and 6 are bacterial genomic DNA bands extracted with the commercially available Tiangen kit of comparative example 2).
FIG. 2 is an agarose electrophoresis of E.coli genomic DNAs extracted in example 1 and comparative examples 1 to 2 after storage at 4 ℃ for 2 days (wherein lanes M are Marker bands; lanes 1 and 2 are bacterial genomic DNA bands extracted with the kit of example 1 of the present invention; lanes 3 and 4 are bacterial genomic DNA bands extracted with the commercially available Omega kit of comparative example 1; lanes 5 and 6 are bacterial genomic DNA bands extracted with the commercially available Tiangen kit of comparative example 2).
FIG. 3 is an agarose electrophoresis of bacterial genomic DNAs extracted in example 1 and comparative examples 3 to 5 of the present invention after storage at 4 ℃ for 2 days (wherein lane M is a Marker band; lane 1 is a bacterial genomic DNA band of example 1; lane 2 is a bacterial genomic DNA band of comparative example 3; lane 3 is a bacterial genomic DNA band of comparative example 4; and lane 4 is a bacterial genomic DNA band of comparative example 5).
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in detail with reference to the accompanying drawings and specific embodiments.
It should be noted that, in order to avoid obscuring the present invention with unnecessary details, only the structures and/or processing steps closely related to the aspects of the present invention are shown in the drawings, and other details not closely related to the present invention are omitted.
In addition, it is also to be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
The invention also provides an extraction method of the stable bacterial genome DNA, which comprises the following steps:
s1, taking 1-4 mL of fresh overnight-cultured bacteria culture solution into a centrifuge tube, centrifuging for 1-5 minutes at 10000-12000 rpm, discarding the supernatant, adding 150-300 mu L of buffer solution, blowing and beating for 10-15 seconds by using a pipette, uniformly mixing the bacteria solution, and then placing the mixture in a 37 ℃ water bath pot for incubation for 30-60 minutes;
s2, adding 300-800 mu L of lysis solution into the centrifugal tube in the step S1, blowing and beating the lysis solution for 10-15S by using a pipette, uniformly mixing the lysis solution, and then placing the lysis solution in a 65 ℃ water bath kettle for incubation for 10-30 minutes;
s3, adding 10-20 mu L of magnetic bead binding solution into the centrifuge tube in the step S2, blowing and beating for 10-15S by using a pipette, standing for 1-3 minutes, then carrying out magnetic separation by using a magnetic rack, and removing the liquid;
s4, adding 500-1000 mu L of washing liquid into the centrifugal tube in the step S3, blowing and beating the washing liquid for 10-15 seconds by using a pipette, standing the washing liquid for 10-30 seconds, then carrying out magnetic separation by using a magnetic frame, sucking away the liquid, and standing the washing liquid for 5-10 minutes at room temperature after uncovering;
s5, adding 50-100 mu L of eluent into the centrifuge tube in the step S5, blowing and beating the mixture for 10-15S by using a pipette, standing the mixture for 1-3 minutes, then carrying out magnetic separation by using a magnetic frame, and absorbing supernatant liquid into a new centrifuge tube to obtain the stable bacterial genome DNA.
The invention is further illustrated below with respect to specific embodiments.
Example 1
The embodiment 1 of the invention provides a kit for extracting stable bacterial genomic DNA by a paramagnetic particle method, which comprises a lysis reagent group for lysing and releasing bacterial genomic DNA and an extraction reagent group for extracting the genomic DNA; the lysis reagent group consists of buffer solution and lysis solution; the extraction reagent group consists of magnetic bead binding liquid, washing liquid and eluent; the magnetic bead binding solution consists of sodium citrate or sodium acetate, a reducing agent dithiothreitol, guanidine hydrochloride and a magnetic bead suspension; the washing solution is an isopropanol aqueous solution with the volume fraction of 80%; the eluent is distilled water.
The buffer solution is a mixed aqueous solution containing 1% by mass of triton, 5mM of disodium ethylene diamine tetraacetate, 20mM of tris (hydroxymethyl) aminomethane hydrochloride and 0.3% by mass of lysozyme, and the pH value of the buffer solution is 7.5. The lysis solution is a mixed aqueous solution containing 0.3M of sodium chloride and 2% of hexadecyl trimethyl ammonium bromide by mass fraction, and the pH value of the lysis solution is 7.5.
In this embodiment, the magnetic bead binding solution is a mixed magnetic bead solution comprising 0.1M sodium acetate, 1 wt% dithiothreitol, 5M guanidine hydrochloride, and 3mL of a suspension of magnetic beads with 700 nm-sized surface modified with hydroxyl groups, wherein the suspension of magnetic beads is a mixture of magnetic beads and isopropyl alcohol in a mass ratio of 1: 20, and the pH value of the magnetic bead binding solution is 7.5.
The extraction of the E.coli genomic DNA was carried out according to the following procedure:
s1, taking 1mL of fresh overnight-cultured bacteria culture solution into a centrifuge tube, centrifuging for 2 minutes at 10000 rpm, discarding the supernatant, adding 300 mu L of buffer solution, blowing for 10 seconds by using a pipette, mixing the bacteria solution uniformly, and then placing the mixture in a 37 ℃ water bath pot for incubation for 30 minutes;
s2, adding 300 mu L of lysis solution into the centrifuge tube in the step S1, blowing and beating the lysis solution for 15 seconds by using a pipette, uniformly mixing the lysis solution, and then putting the lysis solution in a 65 ℃ water bath pot for incubation for 10 minutes;
s3, adding 10 mu L of magnetic bead solution into the centrifuge tube in the step S2, blowing and beating the solution for 15 seconds by using a pipette, standing the solution for 3 minutes, then carrying out magnetic separation by using a magnetic rack, and absorbing and removing the liquid;
s4, adding 1000 mu L of washing liquid into the centrifugal tube in the step S3, blowing the washing liquid by a pipette for 10 seconds, standing the washing liquid for 10 seconds, then carrying out magnetic separation by a magnetic frame, removing the liquid by suction, and standing the washing liquid for 5 minutes at room temperature after uncovering;
s5, adding 100 mu L of eluent into the centrifuge tube in the step S4, blowing the eluent by a pipette for 10 seconds, standing the mixture for 1 minute, then carrying out magnetic separation by a magnetic frame, absorbing supernatant liquid into a new centrifuge tube, and extracting stable bacterial genome DNA.
Comparative example 1
Comparative example 1 extraction of bacterial genomic DNA was carried out using a commercially available magnetic bead method bacterial DNA extraction kit (M2350) from Omega Bio-Tek.
Comparative example 2
Comparative example 2 extraction of bacterial genomic DNA was carried out using a commercially available magnetic bead method universal genomic DNA extraction kit (DP705) from Tiangen Biochemical technology (Beijing) Ltd.
Table 1 shows the results of nucleic acid quality tests of E.coli genomic DNAs extracted with each kit (wherein test Nos. 1 and 2 show the results of bacterial genomic DNAs extracted with the kit of example 1 of the present invention (two sets of parallel examples), 3 and 4 show the results of bacterial genomic DNAs extracted with the commercially available Omega kit of comparative example 1 (two sets of parallel examples), and 5 and 6 show the results of bacterial genomic DNAs extracted with the commercially available Tiangen kit of comparative example 2 (two sets of parallel examples)).
| Experiment number | Concentration (ng/. mu.L) | Purity (A)260/A280) |
| 1 | 482 | 1.92 |
| 2 | 491 | 1.85 |
| 3 | 514 | 2.06 |
| 4 | 489 | 1.82 |
| 5 | 485 | 1.91 |
| 6 | 488 | 1.71 |
As can be seen by combining Table 1 and FIG. 1, the bacterial genomic DNA extracted by the kit provided by the present invention is comparable to the commercially available Omega kit of comparative example 1 in terms of total amount, superior to the commercially available Tiangen kit of comparative example 2, and comparable in terms of fragment integrity and purity to the commercially available competitive products kits provided by comparative example 1 and comparative example 2; however, as can be seen from fig. 2, the bacterial genomic DNA extracted by the kit provided in example 1 of the present invention still had intact DNA fragments after 2 days of storage, and the brightness was not substantially changed, whereas the bacterial genomic DNA extracted by the commercially available Omega and Tiangen kits was significantly degraded after two days of storage, which indicates that the bacterial genomic DNA extracted by the kit was significantly superior in stability to the commercially available competitive product kits provided in comparative examples 1 to 2.
Comparative example 3
The difference from example 1 is that: the sodium salt is conventional sodium chloride. The rest is the same as embodiment 1, and is not described herein again.
Comparative example 4
The difference from example 1 is that: cysteine was used as a conventional reducing agent. The rest is the same as embodiment 1, and is not described herein again.
Comparative example 5
The difference from example 1 is that: the nuclease inhibitor is conventional guanidinium isothiocyanate. The rest is the same as embodiment 1, and is not described herein again.
The stability of the genomic DNA of bacteria extracted in example 1 and comparative examples 3 to 5 was tested, and as shown in FIG. 3, the results showed that: the stability of the bacterial genomic DNA extracted by the kit provided in the embodiment 1 is significantly better than that of the kits provided in the comparative examples 3 to 5, which indicates that in the magnetic bead binding liquid system obtained by mixing and compounding sodium citrate or sodium acetate as a sodium salt, dithiothreitol as a reducing agent, and guanidine hydrochloride as a nuclease inhibitor with a magnetic bead suspension containing magnetic beads modified with hydroxyl or carboxyl groups on the surface and having a particle size of 0.5 to 5 μm, the four components exert a synergistic effect, so that the magnetic bead binding liquid system has excellent binding force and binding stability to the bacterial genomic DNA.
In conclusion, the invention provides a kit for extracting stable bacterial genome DNA by a paramagnetic particle method and an extraction method thereof. The kit comprises a lysis reagent group for lysing and releasing bacterial genomic DNA and an extraction reagent group for extracting the genomic DNA; the lysis reagent group consists of buffer solution and lysis solution; the extraction reagent group consists of magnetic bead binding liquid, washing liquid and eluent; the magnetic bead binding solution consists of sodium citrate or sodium acetate, reducing agent dithiothreitol, guanidine hydrochloride and magnetic bead suspension. According to the invention, the raw materials are reasonably compounded to obtain the optimal proportion of a magnetic bead binding solution compounding system and combined extraction conditions, and the extraction method is optimized, so that the extracted bacterial genome DNA has high purity, complete fragments and difficult degradation, the stability is far superior to that of a commercial kit, and the advantages in subsequent experiments such as DNA storage, hybridization, PCR and the like are obvious; meanwhile, the reagents are non-toxic and have little pollution to the environment.
Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the spirit and scope of the present invention.
Claims (10)
1. The utility model provides a kit of stable form bacterial genome DNA is drawed to paramagnetic particle method which characterized in that: the kit comprises a lysis reagent group for lysis and release of bacterial genomic DNA and an extraction reagent group for extraction of the genomic DNA;
the lysis reagent group consists of buffer solution and lysis solution; the extraction reagent group consists of magnetic bead binding liquid, washing liquid and eluent; the magnetic bead binding solution consists of sodium citrate or sodium acetate, a reducing agent dithiothreitol, a nuclease inhibitor guanidine hydrochloride and a magnetic bead suspension;
in the magnetic bead binding solution, the concentration of sodium salt is 0.05-0.15M, the content of dithiothreitol is 0.5-1.5 wt%, and the concentration of guanidine hydrochloride is 2-5M.
2. The kit for extracting stable bacterial genomic DNA by the magnetic bead method according to claim 1, wherein: in the magnetic bead suspension, the particle size of the magnetic beads is 0.5-5 μm.
3. The kit for extracting stable bacterial genomic DNA by the magnetic bead method according to claim 2, wherein: the surface of the magnetic bead is modified with hydroxyl or carboxyl.
4. The kit for extracting stable bacterial genomic DNA by the magnetic bead method according to claim 2, wherein: the magnetic bead suspension is prepared from magnetic beads and isopropanol according to a mass ratio of 1: (30-50), and the pH value of the magnetic bead binding solution is 7.5-8.5.
5. The kit for extracting stable bacterial genomic DNA by the magnetic bead method according to claim 1, wherein: in the magnetic bead binding solution, the concentration of sodium salt is 0.1M, the content of dithiothreitol is 1 wt%, and the concentration of guanidine hydrochloride is 5M.
6. The kit for extracting stable bacterial genomic DNA by the magnetic bead method according to claim 1, wherein: the washing solution is 75-85% by volume of isopropanol water solution or ethanol water solution; the eluent is one of distilled water, deionized water and ultrapure water.
7. The kit for extracting stable bacterial genomic DNA by the magnetic bead method according to claim 1, wherein: the buffer solution is a mixed water solution containing 0.5-1.5% by mass of triton, 1-10 mM of disodium ethylene diamine tetraacetate, 15-25 mM of tris (hydroxymethyl) aminomethane hydrochloride and 0.1-0.5% by mass of lysozyme, and the pH value of the buffer solution is 7.0-8.0.
8. The kit for extracting stable bacterial genomic DNA by the magnetic bead method according to claim 1, wherein: the lysis solution is a mixed aqueous solution containing 0.2-0.4M of sodium chloride and 1-3% of hexadecyl trimethyl ammonium bromide by mass, and the pH value of the lysis solution is 7.0-8.0.
9. A method for extracting stable bacterial genome DNA is characterized in that: the kit for extracting stable bacterial genomic DNA by using the magnetic bead method according to any one of claims 1 to 8 comprises the following steps:
s1, taking 1-4 mL of fresh overnight-cultured bacteria culture solution into a centrifuge tube, centrifuging for 1-5 minutes at 10000-12000 rpm, discarding the supernatant, adding 150-300 mu L of buffer solution, blowing and beating for 10-15 seconds by using a pipette, uniformly mixing the bacteria solution, and then placing the mixture in a 37 ℃ water bath pot for incubation for 30-60 minutes;
s2, adding 300-800 mu L of lysis solution into the centrifugal tube in the step S1, blowing and beating the lysis solution for 10-15S by using a pipette, uniformly mixing the lysis solution, and then placing the lysis solution in a 65 ℃ water bath kettle for incubation for 10-30 minutes;
s3, adding 10-20 mu L of magnetic bead binding solution into the centrifuge tube in the step S2, blowing and beating for 10-15S by using a pipette, standing for 1-3 minutes, then carrying out magnetic separation by using a magnetic rack, and removing the liquid;
s4, adding 500-1000 mu L of washing liquid into the centrifugal tube in the step S3, blowing and beating the washing liquid for 10-15 seconds by using a pipette, standing the washing liquid for 10-30 seconds, then carrying out magnetic separation by using a magnetic frame, sucking away the liquid, and standing the washing liquid for 5-10 minutes at room temperature after uncovering;
s5, adding 50-100 mu L of eluent into the centrifuge tube in the step S5, blowing and beating the mixture for 10-15S by using a pipette, standing the mixture for 1-3 minutes, then carrying out magnetic separation by using a magnetic frame, and absorbing supernatant liquid into a new centrifuge tube to obtain the stable bacterial genome DNA.
10. The method for extracting stable bacterial genomic DNA according to claim 9, wherein: the stable bacterial genome DNA extracted by the extraction method is not degraded after being stored for 2 days at 4 ℃.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114480369A (en) * | 2021-12-24 | 2022-05-13 | 广东润鹏生物技术有限公司 | Magnetic bead preservation solution, preparation method and magnetic bead product |
| CN114717228A (en) * | 2022-05-17 | 2022-07-08 | 广州迪澳基因科技有限公司 | Method for rapidly extracting genome of sputum sample by using paramagnetic particle method |
| CN115058491A (en) * | 2022-06-30 | 2022-09-16 | 南京瑞贝西生物科技有限公司 | Method for rapid desalination and purification of primer based on paramagnetic particle method |
| CN117143963A (en) * | 2023-10-30 | 2023-12-01 | 北京百力格生物科技有限公司 | Method for recovering nucleic acid in PCR product by ethanol-sodium acetate method |
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| CN108220284A (en) * | 2018-01-23 | 2018-06-29 | 南昌大学 | A kind of paramagnetic particle method bacterial genomes DNA extraction kit and application |
| CN110452909A (en) * | 2018-05-07 | 2019-11-15 | 林翠芝 | The really Method and kit for biologically active basic fibroblast growth factor is expressed in bacillus subtilis |
| CN112391384A (en) * | 2021-01-11 | 2021-02-23 | 甘肃省科学院传感技术研究所 | Kit for rapidly extracting genomic DNA of gram-negative bacteria and extraction method |
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| CN108220284A (en) * | 2018-01-23 | 2018-06-29 | 南昌大学 | A kind of paramagnetic particle method bacterial genomes DNA extraction kit and application |
| CN110452909A (en) * | 2018-05-07 | 2019-11-15 | 林翠芝 | The really Method and kit for biologically active basic fibroblast growth factor is expressed in bacillus subtilis |
| CN112391384A (en) * | 2021-01-11 | 2021-02-23 | 甘肃省科学院传感技术研究所 | Kit for rapidly extracting genomic DNA of gram-negative bacteria and extraction method |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114480369A (en) * | 2021-12-24 | 2022-05-13 | 广东润鹏生物技术有限公司 | Magnetic bead preservation solution, preparation method and magnetic bead product |
| CN114480369B (en) * | 2021-12-24 | 2022-12-27 | 广东润鹏生物技术有限公司 | Magnetic bead preservation solution, preparation method and magnetic bead product |
| CN114717228A (en) * | 2022-05-17 | 2022-07-08 | 广州迪澳基因科技有限公司 | Method for rapidly extracting genome of sputum sample by using paramagnetic particle method |
| CN115058491A (en) * | 2022-06-30 | 2022-09-16 | 南京瑞贝西生物科技有限公司 | Method for rapid desalination and purification of primer based on paramagnetic particle method |
| CN117143963A (en) * | 2023-10-30 | 2023-12-01 | 北京百力格生物科技有限公司 | Method for recovering nucleic acid in PCR product by ethanol-sodium acetate method |
| CN117143963B (en) * | 2023-10-30 | 2024-02-27 | 北京百力格生物科技有限公司 | Method for recovering nucleic acid in PCR product by ethanol-sodium acetate method |
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