CN112391384B - Kit for rapidly extracting genomic DNA of gram-negative bacteria and extraction method - Google Patents
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Abstract
A kit for rapidly extracting genomic DNA of gram-negative bacteria comprises a working solution A, a working solution B, a magnetic bead binding solution, a working solution C and a working solution D. The extraction method using the kit is simple and mainly comprises the following four steps: bacteria cracking, magnetic bead adsorption, washing and elution. The invention adopts monodisperse and superparamagnetic silicon oxide nanometer magnetic beads, needs other reagents which are conventional nontoxic reagents, has low cost and improved experimental safety, and the extracted genomic DNA fragment of the gram-negative bacteria has good integrity, high yield and high purity, and is suitable for downstream molecular biology experiments such as PCR detection, nucleic acid hybridization, DNA library construction and the like.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a kit for rapidly extracting genomic DNA of gram-negative bacteria and an extraction method.
Background
Food safety is a public health safety problem which is highly concerned globally and directly related to people, food-borne pathogenic bacteria are one of the main reasons for frequent events of the food safety problem, pathogenic bacteria in food are timely and accurately detected, screening and detection of harmful bacteria are enhanced, food pollution is effectively prevented, and the food safety detection kit is a fundamental measure for controlling pathogenic bacteria pollution and spread of various infectious diseases in food.
The DNA extraction technology is the first step of the food-borne pathogenic bacteria biosensor detection technology and is also the most critical step, and whether high-purity DNA is obtained or not is directly related to the safety and success or failure of downstream experiments. The conventional bacterial genome DNA extraction methods mainly comprise an alkaline lysis method, a phenol-chloroform method and the like, and the methods generally have the problems of impure products, complex operation and the like and cannot get rid of the dependence on a high-speed centrifuge. The biomagnetic beads are favored in nucleic acid extraction due to the advantages of small particles, large specific surface area, large coupling capacity, good suspension stability, being beneficial to the smooth proceeding of coupling reaction and the like.
The conventional bacterial genome DNA extraction method mostly adopts organic reagents such as phenol, chloroform and the like for extraction, and the method has the defects of organic reagent pollution, incapability of getting rid of dependence on a high-speed centrifuge, low extraction efficiency due to impure products and the like. At present, the commercial nucleic acid extraction kit has the advantages of convenience, rapidness, no need of organic solvent phenol-simulated extraction and the like, and replaces the traditional nucleic acid extraction method. Especially, the newly developed extraction method based on magnetic beads greatly simplifies the nucleic acid purification process, is the best method for nucleic acid purification recognized at present, has already been widely applied in European and American countries, and has a huge market. However, the efficiency of nucleic acid extraction by the bead extraction methods is greatly different from that of the conventional bead extraction methods, and thus it is necessary to improve the technique for extracting nucleic acid by the bead method.
The method has the advantages of efficiently extracting the genomic DNA of the gram-negative bacteria, providing an important technical guarantee for timely and accurately detecting the pathogenic bacteria in the food, and having important significance for promoting the food safety in China.
Disclosure of Invention
One of the purposes of the invention is to provide a kit for rapidly extracting genomic DNA of gram-negative bacteria, and the genomic DNA fragment of gram-negative bacteria extracted by the kit has good integrity, high yield and high purity.
The invention also aims to provide an extraction method of the kit for rapidly extracting the genomic DNA of the gram-negative bacteria, which shortens the extraction time on the premise of ensuring the full lysis of the gram-negative bacteria and provides a guarantee for improving the extraction efficiency.
In order to achieve the purpose, the invention adopts the following technical scheme:
a kit for rapidly extracting genomic DNA of gram-negative bacteria comprises a plurality of working solutions, wherein each working solution is prepared by the following steps:
in the working solution A, the mass concentration of CTAB is 0.5-1.0%, the concentration of NaCl is 1.5-3.5mol/L, the mass concentration of Triton-X-100 is 1.0 to 2.0%, the concentration of Tris-HCl is 50-120mmol/L, the pH value is 7.0-8.0, the concentration of EDTA is 8-25mmol/L, the mass concentration of polyvinylpyrrolidone PVP is 0.5 to 1%, and the solvent is sterile deionized water;
the concentration of NaCl in the working solution B is 1.0-2.0mol/L, the concentration of guanidinium isothiocyanate is 3-4.5mol/L, the concentration of ribonuclease A is 0.3-0.45mg/mL, the concentration of sodium citrate is 0.07-0.lmol/L, and the solvent is sterile deionized water;
the working solution C is an ethanol solution, and the volume fraction of the working solution C is 60-75%;
working solution D contains Tris-HCl with concentration of 10-15mmol/L, pH of 7.0-8.0, bacteriolysis deionized water, and pH of 7.5-8.0;
the concentration of NaCl in the magnetic bead binding solution is 0.5-1.5mol/L, the concentration of guanidine hydrochloride is 1.2-1.8mol/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 magnetic beads are suspended in the magnetic bead binding solution, wherein the pH value of the magnetic bead binding solution is 5.0-6.5.
The magnetic beads are monodisperse superparamagnetic silicon oxide nano magnetic beads, the particle size of the magnetic beads is 200-300nm, and the concentration of the magnetic beads is 10 mg/mL.
An extraction method of a kit for rapidly extracting genomic DNA of gram-negative bacteria mainly comprises the steps of bacteria lysis, magnetic bead adsorption, washing and elution, and specifically comprises the following steps:
step one, adding 500 mu L of gram-negative bacteria culture solution cultured for 30 hours into a centrifuge tube, then adding 300 mu L of working solution A, uniformly mixing, and cracking at 70 ℃ for 10 minutes to clarify the solution;
step two, adding 300 mu L of working solution B into the centrifuge tube, turning upside down, mixing uniformly, standing at room temperature for 5min, and centrifuging at 12000r/min for 5min;
step three, sucking 200 mu L of centrifuged water phase supernatant into another clean centrifugal tube, then adding 10 mu L of magnetic bead binding solution, uniformly mixing, standing at room temperature for 3-5min, placing the centrifugal tube on a magnetic frame for 5min, carrying out magnetic separation until the centrifugal tube is completely clear, sucking the supernatant, and taking out the centrifugal tube from the magnetic frame;
step four, adding 500 mu L of working solution C, uniformly mixing, placing the centrifuge tube on a magnetic frame for 3min, carrying out magnetic separation until the centrifuge tube is completely clarified, absorbing the working solution C as much as possible, and then taking out the centrifuge tube from the magnetic frame;
step five, repeating the step four 1 time;
sixthly, drying the centrifugal tube in a thermostat with the temperature of 55 ℃ for 5-10min until no liquid remains in the centrifugal tube;
step seven, adding 100 mu L of working solution D, mixing uniformly, suspending magnetic beads, and placing the centrifugal tube in a water bath kettle at the temperature of 60-70 ℃ for 8-10min;
and step eight, taking out the centrifuge tube from the water bath, placing the centrifuge tube on a magnetic frame for 2min, and carefully sucking the supernatant into a new centrifuge tube after the magnetic beads are completely sucked onto the wall of the centrifuge tube to obtain the DNA product.
Compared with the prior art, the invention has the following advantages:
although many studies have conducted lysis and binding of a biological sample in the same step, this method can theoretically shorten the nucleic acid extraction time, but in the mixed solution environment of a lysis solution and a binding solution, nucleic acid dissociation is greatly affected by impurities, which results in increased difficulty and reduced efficiency of nucleic acid extraction. The reasonable compound system of the working solution A can ensure that a biological sample is fully cracked, the cracking reaction temperature is controlled to be 60-70 ℃, the cracking time is preferably 10min, the extraction time is shortened on the premise of ensuring the full cracking of gram-negative bacteria, and the improvement of the extraction efficiency is guaranteed. Meanwhile, the high-salt environment provided by the working solution B can destroy the secondary structure of the protein, so that the protein in the nucleic acid-protein complex is quickly denatured and separated from DNA, and the aim of removing the protein and releasing free nucleic acid is fulfilled. And finally, the high-efficiency adsorption of the magnetic beads on the DNA is realized, the influence of impurities on the nucleic acid extraction is effectively reduced, and the nucleic acid extraction efficiency and the nucleic acid purity are further improved. The gram-negative bacterium genome DNA fragment extracted by the kit has good integrity, high yield and high purity, and is more suitable for downstream molecular biology experiments such as PCR detection, nucleic acid hybridization and DNA library construction. The specific description is as follows:
compared with the prior art, the working solution A is specific to the characteristics of gram-negative bacteria, a reasonable compound system of the surfactant, PVP and EDTA in the system can rapidly and effectively crack the bacteria, has excellent effects on the sedimentation and dissociation of impurities such as protein, lipid, carbohydrate, salt and the like in a sample, and can effectively improve the nucleic acid extraction efficiency and the nucleic acid extraction purity particularly for biological samples containing lipopolysaccharide on cell walls such as gram-negative bacteria and the like. Meanwhile, the system provides a buffer environment with better biocompatibility and prevents the genomic DNA from being damaged.
Guanidine isothiocyanate and high-salt environment in the working solution B system can destroy the secondary structure of protein, so that the protein in the nucleic acid-protein complex is quickly denatured and separated from DNA, and the aim of removing the protein and releasing free nucleic acid is fulfilled. Meanwhile, the ribonuclease A in the working solution B can rapidly and effectively degrade RNA in a system, remove RNA pollution and further increase the adsorption amount of magnetic beads on DNA.
The efficient adsorption of magnetic beads on DNA is the key technology of the invention. Ethanol precipitation is the most common method for recovering nucleic acid in a sample, but the recovery rate of nucleic acid in the sample is lower by ethanol precipitation, the precipitation efficiency of DNA by isopropanol is higher than that of ethanol, but the isopropanol precipitation has the defect that a large amount of protein and salt are precipitated, and the purity of the product is influenced. According to the invention, through repeated experiments, the optimal proportion and reaction conditions of the optimal complex system of the magnetic bead binding solution are obtained, the system creates a hydrophobic environment with high salt and low pH, and the magnetic bead binding solution enhances the hydrophobic effect of nucleic acid and the surface of the magnetic bead, promotes the adsorption effect of the magnetic bead on the nucleic acid, and thus enhances the nucleic acid extraction efficiency. Because the DNA molecules are negatively charged, na + in the system can neutralize the negative charge on the DNA molecules, so that the DNA is easy to aggregate and precipitate, and the adsorption quantity of magnetic beads to the DNA is increased. Meanwhile, the high salt concentration in the system can destroy a hydrophilic membrane on the surface of the nucleic acid to form a hydrophobic environment, so that the nucleic acid is favorably and firmly adsorbed on the surface of the magnetic bead, and impurities such as residual protein, lipid, carbohydrate, salt and the like in a sample can be effectively removed.
The extraction method provided by the invention has simple steps, can quickly complete the whole extraction process, saves the cost, and is suitable for high-throughput extraction experiments.
Other reagents required in the invention are conventional non-toxic reagents, so that the cost is low, and the experimental safety is improved.
Drawings
FIG. 1 shows Shigella flexneri and Escherichia coli hemorrhagic 157 extracted by the method of the present invention: agarose gel electrophoresis of H7 genomic DNA: lane 1,2 is extracted shigella flexneri O157: h7 genomic DNA; lane 3,4 is genomic DNA extracted from Escherichia coli hemorrhagic bacterium, yongdao M is DL5000 DNA Marker of TAKARA company;
FIG. 2 is an agarose gel electrophoresis of genomic DNA of Escherichia coli (O157: H7) extracted by three different methods, and lane 1 shows the DNA of Escherichia coli hemorrhagic bacterium extracted by phenol-chloroform extraction O157: h7 genomic DNA, lane 2, commercially available kit extracted escherichia coli O157: h7 genomic DNA, lane 3, enterobacter hemorrhagic bacteria O157: h7 genomic DNA, lane M is DL5000 DNA Marker.
Detailed Description
The invention will be further illustrated by the following examples.
Working solution A, working solution B, working solution C and working solution D are named for the convenience of distinguishing different buffers used for expressing different steps, and letters A, B, C and D have no meanings.
(1) Working solution A: CTAB with a mass concentration of 0.8%, 2.5mol/L NaCl, triton-X-100 with a mass concentration of 2%, 80mmol/L Tris-HCl (pH 7.0-8.0), 15mmol/L EDTA, 1% PVP with a mass concentration, and sterile deionized water as a solvent.
(2) Working solution B:2.0mol/L NaCl, 4.0mol/L guanidine isothiocyanate, 0.45mg/mL ribonuclease A and 0.07mol/L sodium citrate, and the solvent is sterile deionized water.
(3) The working solution C is an ethanol solution, and the volume fraction of the working solution C is 70%.
(4) Working solution D:15mmol/L Tris-HCl with pH7.0-8.0; the solvent was sterile deionized water, pH8.0.
(5) Magnetic bead binding solution: 1.5mol/L NaCl, 1.2mol/L guanidine hydrochloride, 20 percent of absolute ethyl alcohol by volume ratio, 30 percent of isopropanol by volume ratio and 7 percent of polyethylene glycol 8000 by mass fraction, and suspending 10 mg/mL of magnetic beads in the solution, wherein the used magnetic beads are monodisperse and superparamagnetic silicon oxide nanometer magnetic beads, and the particle size of the magnetic beads is 200-300nm, so as to obtain the magnetic bead binding solution with the pH value of 5.0.
(1) Working solution A: CTAB at a mass concentration of 0.5%, 3.5mol/L NaCl, triton-X-100 at a mass concentration of 1%, 50mmol/L Tris-HCl (pH 7.0-8.0), 25mmol/L EDTA, 1% PVP at a mass concentration, and sterile deionized water as a solvent.
(2) Working solution B:1.5mol/L NaCl, 3.0mol/L guanidine isothiocyanate, 0.40mg/mL ribonuclease A and 0.1mol/L sodium citrate, and the solvent is sterile deionized water.
(3) The working solution C is an ethanol solution, and the volume fraction of the working solution C is 60%.
(4) Working solution D:15mmol/L Tris-HCl with pH7.0-8.0; the solvent was sterile deionized water, pH8.0.
(5) Magnetic bead binding solution: 0.5mol/L NaCl, 1.8mol/L guanidine hydrochloride, 30% absolute ethyl alcohol by volume ratio, 60% isopropyl alcohol by volume ratio and 3% polyethylene glycol 7000 by mass fraction, and suspending 10 mg/mL magnetic beads in the sodium alginate nano magnetic beads, wherein the used magnetic beads are monodisperse and superparamagnetic silicon oxide nano magnetic beads, the particle size of the magnetic beads is 200-300nm, and the pH value is 5.0.
(1) Working solution A: CTAB with a mass concentration of 1.0%, 1.5mol/L NaCl, triton-X-100 with a mass concentration of 1%, 120mmol/L Tris-HCl (pH 7.0-8.0), 8mmol/L EDTA, PVP with a mass concentration of 0.5%, and sterile deionized water as a solvent.
(2) Working solution B:1.0mol/L NaCl, 4.5mol/L guanidine isothiocyanate, 0.3mg/mL ribonuclease A and 0.08mol/L sodium citrate, and the solvent is sterile deionized water.
(3) The working solution C is an ethanol solution, and the volume fraction of the ethanol solution is 75%.
(4) Working solution D:10mmol/L Tris-HCl, solvent sterile deionized water, pH8.0.
(5) Magnetic bead binding solution: 1.0mol/L NaCl, 1.5mol/L guanidine hydrochloride, 20 percent of absolute ethyl alcohol by volume ratio, 45 percent of isopropanol by volume ratio and 10 percent of polyethylene glycol 6000 by mass fraction, and suspending 10 mg/mL of magnetic beads in the solution, wherein the used magnetic beads are monodisperse and superparamagnetic silicon oxide nanometer magnetic beads, the particle size of the magnetic beads is 200-300nm, and the pH value is 5.0.
The monodisperse superparamagnetic silica nano magnetic beads described in the above embodiments may be made of the following materials: ZL 2015 10488579.5, named: magnetic beads disclosed in the section of "silica-coated ferroferric oxide nanoclusters as magnetic labeling materials and preparation method thereof".
Example 4, the method for extracting the shigella flexneri genomic DNA using the kit for extracting gram-negative bacterial genomic DNA prepared in the above example 1 mainly includes the steps of bacterial lysis, magnetic bead adsorption, washing and elution, and specifically includes the following steps:
(1) Adding 500 mu L of Shigella flexneri culture solution cultured for 30h into a centrifuge tube, adding 300 mu L of working solution A, mixing uniformly, and cracking at 70 ℃ for 10 minutes to clarify the solution.
(2) Adding 300 mu L of working solution B into the centrifuge tube, turning upside down, mixing uniformly, standing at room temperature for 5min, and centrifuging at 12000r/min for 5min.
(3) Sucking 200 μ L of centrifuged supernatant of the water phase into another clean centrifuge tube, adding 10 μ L of magnetic bead binding solution, mixing, standing at room temperature for 3-5min, placing the centrifuge tube on a magnetic frame for 5min, magnetically separating to completely clarify, sucking off the supernatant, and taking out the centrifuge tube from the magnetic frame.
(4) Adding 500 μ L of working solution C, mixing, placing the centrifuge tube on a magnetic frame for 3min, performing magnetic separation until the solution is completely clear, absorbing the working solution C as much as possible, and taking out the centrifuge tube from the magnetic frame.
(5) Repeat step four 1 times.
(6) Drying the centrifugal tube in a thermostat at 55 ℃ for 5-10min until no liquid remains in the centrifugal tube.
(7) Adding 100 μ L of working solution D, mixing, suspending magnetic beads, and placing the centrifuge tube in 60-70 deg.C water bath for 8-10min.
(8) And (3) taking out the centrifuge tube from the water bath, placing the centrifuge tube on a magnetic frame for 2min, and carefully sucking the supernatant into a new centrifuge tube after the magnetic beads are completely sucked to the wall of the centrifuge tube, thereby obtaining the DNA product.
Example 5, escherichia coli O157 (blood) was extracted using the genomic DNA extraction kit for gram-negative bacteria prepared in example 1: the extraction method of the H7 genome DNA mainly comprises the steps of bacterial lysis, magnetic bead adsorption, washing and elution, and specifically comprises the following steps:
(1) 500. Mu.L of 30-hour cultured Escherichia coli O157: adding the H7 culture solution into a centrifuge tube, then adding 300 mu L of the working solution A, uniformly mixing, and cracking at 70 ℃ for 10 minutes to clarify the solution.
(2) Adding 300 mu L of working solution B into the centrifuge tube, turning upside down, mixing uniformly, standing at room temperature for 5min, and centrifuging at 12000r/min for 5min.
(3) Sucking 200 μ L of centrifuged supernatant of the water phase into another clean centrifuge tube, adding 10 μ L of magnetic bead binding solution, mixing, standing at room temperature for 3-5min, placing the centrifuge tube on a magnetic frame for 5min, magnetically separating to completely clarify, sucking off the supernatant, and taking out the centrifuge tube from the magnetic frame.
(4) Adding 500 μ L of working solution C, mixing, placing the centrifuge tube on a magnetic frame for 3min, performing magnetic separation until the solution is completely clarified, absorbing the working solution C as much as possible, and taking out the centrifuge tube from the magnetic frame.
(5) Repeat step four 1 times.
(6) Drying the centrifuge tube in 55 deg.C thermostat for 5-10min until there is no residual liquid in the centrifuge tube.
(7) Adding 100 μ L of working solution D, mixing, suspending magnetic beads, and placing the centrifuge tube in 60-70 deg.C water bath for 8-10min.
(8) And (4) taking out the centrifuge tube from the water bath, placing the centrifuge tube on a magnetic frame for 2min, and carefully sucking the supernatant into a new centrifuge tube after the magnetic beads are completely sucked onto the wall of the centrifuge tube to obtain a DNA product.
(9) Agarose gel electrophoresis was used to detect genomic DNA of two gram-negative bacteria extracted in examples 4 and 5 of the present invention, lanes 1 and 2 are extracted genomic DNA of Shigella flexneri; lane 3,4 is the extracted hemorrhagic escherichia coli O157: h7 genome DNA, yongdao M is DL5000 DNA Marker of TAKARA company. The results show that the obtained genomic DNA product has good parallelism, clear bands and no tailing and impurity bands, and indicate that the extracted genomic DNA has good integrity and no other DNA pollution (shown in figure 1).
(10) Detecting OD value of the extracted 4 parts of genome DNA recovery products by using an ultraviolet-visible spectrophotometer, wherein the purity is OD 260nm /OD 280nm The ratio is measured, and the detection result shows that the genome DNA product extracted by the method has high content, good purity and high concentration, and is shown in table 1.
TABLE 1 genomic DNA extraction results of gram-negative bacteria of the kit of the invention
Comparative example
Extracting Escherichia coli O157 hemorrhagic by phenol-chloroform extraction and commercial kit extraction respectively: h7 genomic DNA, the procedure was performed according to the respective instructions, and the extracted genomic DNA was analyzed by agarose gel electrophoresis. FIG. 2 is an agarose gel electrophoresis of genomic DNA extracted by three different methods, and lane 1 shows the DNA of Escherichia coli hemorrhagic bacterium O157 extracted by phenol-chloroform extraction: h7 genomic DNA, lane 2 is a commercially available kit-extracted hemorrhagic Escherichia coli O157: h7 genomic DNA, lane 3 is the hemorrhagic Escherichia coli O157: h7 genomic DNA, lane M is DL5000 DNA Marker. As can be seen from FIG. 2, the genomic DNA product extracted by the kit of the invention has clear bands, no tailing and miscellaneous bands, good integrity of the extracted genomic DNA and high purity compared with the other two methods.
Claims (2)
1. A kit for rapidly extracting genomic DNA of gram-negative bacteria comprises a plurality of working solutions, and is characterized in that each working solution is specifically prepared as follows:
in the working solution A, the mass concentration of CTAB is 0.5-1.0%, the concentration of NaCl is 1.5-3.5mol/L, the mass concentration of Triton-X-100 is 1.0-2.0%, the concentration of Tris-HCl is 50-120mmol/L, the pH value is 7.0-8.0, the concentration of EDTA is 8-25mmol/L, the mass concentration of polyvinylpyrrolidone PVP is 0.5-1%, and the solvent is sterile deionized water;
the concentration of NaCl in the working solution B is 1.0-2.0mol/L, the concentration of guanidinium isothiocyanate is 3-4.5mol/L, the concentration of ribonuclease A is 0.3-0.45mg/mL, the concentration of sodium citrate is 0.07-0.lmol/L, and the solvent is sterile deionized water;
the working solution C is an ethanol solution, and the volume fraction of the working solution C is 60-75%;
working solution D contains Tris-HCl with concentration of 10-15mmol/L, pH of 7.0-8.0, bacteriolysis deionized water, and pH of 7.5-8.0;
the concentration of NaCl in a magnetic bead binding solution is 0.5-1.5mol/L, the concentration of guanidine hydrochloride is 1.2-1.8mol/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, magnetic beads are suspended in the magnetic bead binding solution, and the pH value of the magnetic bead binding solution is 5.0-6.5;
the magnetic beads are monodisperse superparamagnetic silicon oxide nano magnetic beads, the particle size of the magnetic beads is 200-300nm, and the concentration of the magnetic beads is 10 mg/mL.
2. The extraction method of the kit for rapidly extracting genomic DNA of gram-negative bacteria according to claim 1, which mainly comprises the steps of bacteria lysis, magnetic bead adsorption, washing and elution, and specifically comprises the following steps:
step one, adding 500 mu L of gram-negative bacteria culture solution cultured for 30 hours into a centrifuge tube, then adding 300 mu L of working solution A, uniformly mixing, and cracking at 70 ℃ for 10 minutes to clarify the solution;
step two, adding 300 mu L of working solution B into the centrifuge tube, turning upside down and mixing uniformly, standing at room temperature for 5min, and centrifuging for 5min at 12000r/min;
step three, sucking 200 mu L of centrifuged water phase supernatant into another clean centrifugal tube, then adding 10 mu L of magnetic bead binding solution, uniformly mixing, standing at room temperature for 3-5min, placing the centrifugal tube on a magnetic frame for 5min, carrying out magnetic separation until the centrifugal tube is completely clear, sucking the supernatant, and taking out the centrifugal tube from the magnetic frame;
step four, adding 500 mu L of working solution C, uniformly mixing, placing the centrifuge tube on a magnetic frame for 3min, carrying out magnetic separation until the centrifuge tube is completely clarified, absorbing the working solution C as much as possible, and then taking out the centrifuge tube from the magnetic frame;
step five, repeating the step four 1 time;
sixthly, drying the centrifugal tube in a thermostat with the temperature of 55 ℃ for 5-10min until no liquid remains in the centrifugal tube;
step seven, adding 100 mu L of working solution D, mixing uniformly, suspending magnetic beads, and placing the centrifugal tube in a water bath kettle at the temperature of 60-70 ℃ for 8-10min;
and step eight, taking out the centrifuge tube from the water bath, placing the centrifuge tube on a magnetic frame for 2min, and carefully sucking the supernatant into a new centrifuge tube after the magnetic beads are completely sucked onto the wall of the centrifuge tube, thereby obtaining the DNA product.
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| CN107964545A (en) * | 2017-12-12 | 2018-04-27 | 杭州联川生物技术股份有限公司 | A kind of kit and its method based on magnetic bead technology extraction fungi/bacterial genomes DNA |
| 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 |
| CN109735532A (en) * | 2018-12-30 | 2019-05-10 | 北京优迅医学检验实验室有限公司 | A kind of bead suspension and its application for nucleic acid purification and screening |
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| CN107964545A (en) * | 2017-12-12 | 2018-04-27 | 杭州联川生物技术股份有限公司 | A kind of kit and its method based on magnetic bead technology extraction fungi/bacterial genomes DNA |
| 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 |
| CN109735532A (en) * | 2018-12-30 | 2019-05-10 | 北京优迅医学检验实验室有限公司 | A kind of bead suspension and its application for nucleic acid purification and screening |
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