CN108676791B - Kit for extracting DNA by paramagnetic particle method and extraction method - Google Patents
Kit for extracting DNA by paramagnetic particle method and extraction method Download PDFInfo
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- CN108676791B CN108676791B CN201810375852.7A CN201810375852A CN108676791B CN 108676791 B CN108676791 B CN 108676791B CN 201810375852 A CN201810375852 A CN 201810375852A CN 108676791 B CN108676791 B CN 108676791B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
Abstract
The invention discloses a magnetic bead DNA extraction kit and a method for rapidly and briefly extracting polysaccharide fungus DNA. The kit comprises a solution I, a solution II, a solution III, a solution IV, a washing solution, an eluent and a magnetic bead suspension. The polysaccharide substance of the sclerotium-producing fungi is removed by the innovative use solution II, so that the interference of extracellular polysaccharide is avoided in the DNA extraction process. The magnetic beads used by the kit are also innovatively pretreated in the test process, DNA is used as a molecular target, the adsorption capacity of the magnetic beads on RNA and protein is reduced, and the operation has the outstanding characteristic of high specificity. The kit is suitable for DNA extraction of polysaccharide fungi, is simple and convenient to detect, and reduces interference of exopolysaccharide to DNA extraction.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a sclerotium fungi DNA extraction kit by a paramagnetic particle method, and a method for rapidly extracting sclerotium fungi DNA with strong specificity.
Background
The sclerotium is a dormant body formed by closely connecting and interweaving hyphae, and has the function of mainly resisting adverse environment. Common sclerotium-producing fungi are sclerotium and rhizoctonia fungi, which contain large amounts of exopolysaccharides. The fungus exopolysaccharide has the characteristics of high adsorption and high viscosity, and is one of the difficulties which troubles the separation and extraction of high-purity DNA from the fungus producing sclerotium. Molecular biology means is one of the indispensable techniques for identifying fungi, and DNA separation and purification are the basis for molecular biology research. At present, the conventional CTAB method is a common method for extracting fungal DNA, and the main principle is that CTAB is a cationic detergent, can form a complex with protein, precipitates protein substances, achieves the aim of separating and purifying nucleic acid, and realizes the extraction work of DNA after purification centrifugation, washing, elution and other steps of phenol, chloroform and other substances. However, the DNA extraction of the fungus producing sclerotium having a high polysaccharide content cannot achieve an ideal effect. Moreover, the product is long in service life and the solvent is harmful to human body.
Later, the development of the column-type fungal DNA extraction kit greatly improved the convenience of DNA extraction. The main principle is that the DNA is effectively adsorbed in a filtering membrane in the extraction process by utilizing the adsorption effect of a purification column embedded with a purification filler on DNA molecules, and the DNA is extracted by the steps of centrifuging, washing, eluting and the like, so that the operation speed is accelerated, and the concentration of the DNA is increased. At present, the column type method for recovering DNA is widely applied, but has the defects of the column type method, and fungi with higher polysaccharide substance content often cause the blockage of a filter screen in the filter screen, so that a large amount of DNA fragments cannot be adsorbed on the filter screen, and finally, the DNA extraction concentration is low or the DNA extraction fails.
In recent years, magnetic bead method has attracted more and more attention in the extraction application of DNA from fungi due to the particularity of nano-magnetic iron oxide. The principle is that the surface of the magnetic bead is modified with special chemical groups or depends on huge surface energy, specific adsorption and desorption effects can be formed on DNA molecules under different conditions, the DNA molecules adsorbed in the magnetic bead can be separated from the magnetic bead under the action of an external magnetic field, and the DNA extraction work can be simply and conveniently completed. Although most fungi DNA extraction by the magnetic bead method is simple and convenient, the method has the defect that the influence of polysaccharide substances on DNA adsorption cannot be eliminated.
In addition, although there are many classical methods for DNA extraction from organisms, these conventional methods are not increasingly satisfactory for tests that require very pure DNA and are not expected to contain any other impurities, such as proteins, peptide chains, RNA, polysaccharides or other non-DNA substances, which interfere with the test. Sometimes, although the sample has DNA, the target DNA cannot be detected, which may be caused by interference of impurities contained in the sample with the test system, and although the content of the impurities is small, the impurities still interfere with the detection result, and sometimes even cause the detection to fail. There is a need for improvement of the conventional method, and it is desired to obtain DNA of high purity without containing impurities.
Disclosure of Invention
The invention aims to provide a DNA extraction kit for producing sclerotinia sclerotiorum by a paramagnetic particle method. The kit can effectively remove impurities in a sample solution and specifically adsorb DNA, so that the purity of the obtained DNA reaches more than 99.5 percent. In particular, the magnetic beads are treated with a specific reagent, which allows the beads to specifically adsorb only DNA, but not other impurities, such as protein fragments, polypeptides, RNA, or other impurities. The specificity referred to here is that 99.9% of DNA can be adsorbed, while no or little other non-DNA substances, such as RNA, proteins or polypeptides, or polysaccharides, are adsorbed.
The reagent for processing the magnetic beads comprises a N- (phosphonomethyl) iminodiacetic acid solution and a guanidinium isothiocyanate solution, and then the DNA in a sample is adsorbed by the processed magnetic beads, and the purity of the DNA can be obviously improved. PMIDA belongs to a commonly used pesticide, the surface energy of magnetic beads can be increased by treating PMIDA, the adsorption capacity is enhanced, guanidine isothiocyanate is a commonly used protein denaturation reagent, the two reagents are used for treating the magnetic beads, the purity of DNA can be obviously improved, and the purity of the DNA is obviously improved compared with the purity of the conventional DNA extracted conventionally.
In some embodiments, the present invention provides a DNA extraction kit for a sclerotinia sclerotiorum by a magnetic bead method, comprising: solution I, solution II, solution III, solution IV, washing liquid, eluent and magnetic bead suspension;
the magnetic bead suspension is: the magnetic beads are made of nano magnetic iron oxide and have the diameter of 1.2-2 mm; the solution I consists of glucose, Tris-HCl and EDTA, and the pH value is 8.0;
the solution II is absolute ethyl alcohol and potassium acetate solution;
the solution III is SDS solution (diluted by sodium hydroxide solution);
the solution IV comprises guanidine hydrochloride and potassium acetate, and the pH value is 3.5-4.5;
the washing solution is an ethanol solution;
the eluent is deionized water;
wherein the magnetic bead suspension is processed by the following processing steps: soaking the magnetic beads in a N- (phosphonomethyl) iminodiacetic acid solution for 5-10 hours, washing with PBS (pH 7.0) for 5-8 times, soaking the taken magnetic beads in guanidine isothiocyanate solution for 5-6 hours, and washing with PBS for 5-8 times for later use.
Preferably, the concentration of the N- (phosphonomethyl) iminodiacetic acid solution is 5mol/L, and the concentration of the guanidinium isothiocyanate solution is 8 mol/L.
On the other hand, the method for extracting the DNA of the sclerotinia sclerotiorum by the magnetic bead method is characterized by comprising the following steps:
the method comprises the following steps: clamping sclerotium of the cultured fungus into a 1.5ml centrifuge tube under an aseptic condition, wherein the mass of the sclerotium is not less than 300mg, and freezing and grinding by using liquid nitrogen; adding 250 mu L of the solution I, adding 50 mu L of RNase A, and fully and uniformly mixing;
step two: adding 50 μ L protease K, mixing, and water bathing at 60 deg.C for 20 min. Centrifuging at 12000rpm for 5min, and collecting supernatant to a new tube;
step three: adding 150 mu L of solution II, and slightly turning over the solution for 5-6 times; centrifuging at 12000rpm for 5min, and collecting supernatant to a new tube;
step four: adding 350 mu L of solution III, and slightly turning over the solution up and down to ensure that the bacterial solution is fully cracked into a transparent solution. Centrifuging at 12000rpm for 5min, and collecting supernatant to a new tube;
step five: adding 350 μ L of solution IV, slightly turning over until white floccule is formed, and standing at room temperature for 2 min;
step six: adding 50 μ L of magnetic bead suspension, slightly turning over, and standing for 2 min;
step seven: placing the centrifuge tube on a magnetic frame, standing for 1min to make the solution transparent, and discarding the solution;
wherein the magnetic bead suspension is processed by the following processing steps: soaking the magnetic beads in a N- (phosphonomethyl) iminodiacetic acid solution for 5-10 hours, washing the magnetic beads with PBS (pH 7.0) for 5-8 times, soaking the taken magnetic beads in guanidinium isothiocyanate solution for 5-6 hours, and washing the magnetic beads with the PBS for 5-8 times for later use;
the concentration of glucose in the solution I is 50mmol/L, the concentration of Tris-HCl is 25mmol/L, and the concentration of EDTA is 10 mmol/L;
the concentration of the potassium acetate solution in the solution II is 8 mol/L;
the concentration of NaOH solution in the solution III is 0.2mol/L, and the concentration of SDS is 1-1.5%;
the concentration of guanidine hydrochloride in the solution IV is 3.0-4.5M, and the concentration of KAC is 0.75M.
Preferably, the concentration of the N- (phosphonomethyl) iminodiacetic acid solution is 5mol/L, and the concentration of the guanidinium isothiocyanate solution is 8 mol/L.
Preferably, the volume ratio of the N- (phosphonomethyl) iminodiacetic acid solution to the guanidinium isothiocyanate solution is 1: 1.
Preferably, the method further comprises the following steps of washing and eluting the magnetic beads after the step seven: adding 500 mu L of absolute ethyl alcohol into a centrifugal tube containing the adsorbed DNA magnetic beads to wash DNA, fully shaking the solution, discarding the solution, repeatedly washing the solution for 3 times, and washing the organic solution adhered to the surfaces of the magnetic beads as much as possible; and opening a centrifugal tube cover, standing at room temperature for 3-5min to volatilize the absolute ethyl alcohol on the surface of the magnetic beads as much as possible, adding 50 mu L of deionized water, standing for 2min, and then using a pipettor to put the solution containing the DNA into a new tube.
In some preferred modes, larger particles in the sample are filtered to remove, or some high-content substances, such as polysaccharides and larger protein substances are primarily removed, and then treated magnetic beads are added for adsorption, and the magnetic beads are found to only adsorb DNA in the sample, but not other substances in the sample, such as protein fragments, polypeptides, RNA or other impurities.
Polysaccharide substances are precipitated and DNA is adsorbed, so that the DNA concentration of the sclerotium-producing fungi is improved by more than 80 percent, and the sclerotium-producing fungi can be directly applied to downstream molecular biology experiments such as sequencing, connection, PCR amplification and the like.
The second purpose of the invention is to provide a method for rapidly extracting DNA of the sclerotium-producing fungi by a magnetic bead method. And matching with a magnetic bead suspension consisting of a N- (phosphonomethyl) iminodiacetic acid solution, a guanidine isothiocyanate solution and magnetic beads. The kit can improve the specific adsorption capacity of the magnetic beads to DNA molecules, so that the DNA purity of the sclerotium-producing fungi is improved by over 90 percent.
In order to achieve the purpose, the invention adopts the following technical scheme: a DNA extraction kit for producing sclerotium fungi by a paramagnetic particle method. Comprises a solution I, a solution II, a solution III, a solution IV, a washing solution, an eluent and a magnetic bead suspension;
the magnetic bead suspension is: the magnetic beads are made of nano magnetic iron oxide and have the diameter of 1.2-2 mm;
the solution I consists of glucose, Tris-HCl and EDTA, and the pH value is 8.0;
the solution II consists of absolute ethyl alcohol and potassium acetate;
the solution III is SDS solution (diluted by sodium hydroxide solution);
the solution IV comprises guanidine hydrochloride and potassium acetate, and the pH value is 3.5-4.5;
the washing solution is an absolute ethyl alcohol solution;
the eluent is deionized water;
the concentration of the magnetic bead suspension is 40-50 mg/ml;
the concentration of glucose in the solution I is 50mmol/L, the concentration of Tris-HCl is 25mmol/L, and the concentration of EDTA is 10 mmol/L;
the concentration of the potassium acetate solution in the solution II is 8 mol/L;
the concentration of NaOH solution in the solution III is 0.2mol/L, and the concentration of SDS is 1-1.5%;
the concentration of guanidine hydrochloride in the solution IV is 3.0-4.5M, and the concentration of KAC is 0.75M;
wherein, the flow rate of the water is controlled by the control unit.
The method for rapidly extracting the DNA of the sclerotium-producing fungi by the kit comprises the following steps:
the method comprises the following steps: and clamping the sclerotium of the cultured fungus into a 1.5ml centrifuge tube under the aseptic condition, wherein the mass of the sclerotium is not less than 500mg, and freezing and grinding by using liquid nitrogen. Add 250. mu.L of solution I and then 50. mu.L of RNase A and mix well.
Step two, adding 50 mu L of proteinase K, fully and uniformly mixing, and carrying out constant-temperature water bath at 60 ℃ for 20 min. Centrifuge at 12000rpm for 5min, and take the supernatant to a new tube.
Step three: adding 150 μ L of solution II, and gently turning over 5-6 times. Centrifuge at 12000rpm for 5min, and take the supernatant to a new tube.
Step four: adding 350 mu L of solution III, and slightly turning over the solution up and down to ensure that the bacterial solution is fully cracked into a transparent solution. Centrifuge at 12000rpm for 5min, and take the supernatant to a new tube.
Step five: adding 350 μ L of solution IV, turning over slightly until white floccule is formed, and standing at room temperature for 2 min.
Step six: adding 50 μ L of magnetic bead suspension, slightly turning over, and standing for 2 min.
Step seven: placing the centrifuge tube on a magnetic frame, standing for 1min to make the solution transparent, and discarding the solution.
Step eight: add 500. mu.L of washing solution to wash the DNA, shake well and discard the solution.
Step nine: opening the centrifugal tube cover, standing at room temperature for 3-5min, adding 50 μ L of eluent, standing for 2min, collecting solution, transferring to new tube, and standing at-20 deg.C.
The application creatively provides that polysaccharide substances aiming at the sclerotium-producing fungi are effectively precipitated, so that the DNA concentration of the polysaccharide substances is improved, the influence of extracellular polysaccharide of the sclerotium-producing fungi on the DNA concentration is weakened, and the phenomenon that the downstream test fails due to low concentration in the traditional DNA extraction process can be avoided.
The invention has the beneficial effects that:
(1) the extraction rate of DNA is high, and the concentration can reach more than 200 ng/. mu.L. The method can settle polysaccharide substances based on potassium acetate and absolute ethanol solution, remove extracellular polysaccharide of fungi from DNA in the environment of buffer solution, reduce interference of polysaccharide substances during extraction of DNA molecules, and has high DNA extraction rate and the extracted DNA concentration of more than 200 ng/mu L.
(2) The DNA purity is higher, and the RNA and protein content is less. The method is based on the N- (phosphonomethyl) iminodiacetic acid solution and the guanidinium isothiocyanate solution to crack RNA and dissolve protein, the specific adsorption capacity of magnetic beads on DNA is enhanced in the buffer solution environment, the RNA and the protein cannot be adsorbed on the magnetic beads, the DNA purity is improved, and the OD260/280 value is 1.80-1.90.
(3) Convenient operation, time saving and safety. The method is based on a magnetic bead method and matched with the use of a kit solution I, a solution II, a solution III, a solution IV, a washing solution and an eluent, so that the working efficiency can be greatly improved, the operation method is brief and efficient, and only about 10-30 minutes is needed for completing one-time DNA lifting.
Detailed Description
Example 1: magnetic bead method sclerotium fungi DNA extraction kit
1.1. The kit for extracting the DNA of the sclerotinia sclerotiorum by the paramagnetic particle method comprises (A7-A8 samples):
the method comprises the following steps: clamping sclerotium of cultured fungus (yeast) into a 1.5ml centrifuge tube under aseptic condition, wherein the sclerotium mass is not less than 500mg, and freezing and grinding with liquid nitrogen. Add 250. mu.L of solution I and then 50. mu.L of RNase A and mix well.
Step two: adding 50 μ L protease K, mixing, and water bathing at 60 deg.C for 20 min. Centrifuge at 12000rpm for 5min, and take the supernatant to a new tube.
Step three: adding 150 μ L of solution II, and gently turning over 5-6 times. Centrifuge at 12000rpm for 5min, and take the supernatant to a new tube.
Step four: adding 350 mu L of solution III, and slightly turning over the solution up and down to ensure that the bacterial solution is fully cracked into a transparent solution. Centrifuge at 12000rpm for 5min, and take the supernatant to a new tube.
Step five: adding 350 μ L of solution IV, turning over slightly until white floccule is formed, and standing at room temperature for 2 min.
Step six: adding 50 μ L of untreated magnetic bead suspension (purchased from Shanghai Carboxyphenanthrene biomedical science and technology Co., Ltd., nanometer hydroxyl magnetic bead specially used for extracting DNA, lot number: FE10001, average particle diameter of 500 nm), gently turning over, and standing for 2 min.
Step seven: placing the centrifuge tube on a magnetic frame, standing for 1min to make the solution transparent, and discarding the solution.
Step eight: adding 500 mu L of absolute ethyl alcohol into a centrifugal tube containing the adsorbed DNA magnetic beads to wash DNA, fully shaking the solution, discarding the solution, repeatedly washing the solution for 3 times, and washing the organic solution adhered to the surfaces of the magnetic beads as much as possible; and opening a centrifugal tube cover, standing at room temperature for 3-5min to volatilize the absolute ethyl alcohol on the surface of the magnetic beads as much as possible, adding 50 mu L of deionized water, standing for 2min, and then using a pipettor to put the solution containing the DNA into a new tube.
The concentration of glucose in the solution I is 50mmol/L, the concentration of Tris-HCl is 25mmol/L, and the concentration of EDTA is 10 mmol/L;
the concentration of the potassium acetate solution in the solution II is 8 mol/L;
the concentration of NaOH solution in the solution III is 0.2mol/L, and the concentration of SDS is 1-1.5%;
the concentration of guanidine hydrochloride in the solution IV is 3.0-4.5M, and the concentration of KAC is 0.75M.
1.2 traditional CTAB extraction (A1-A3 samples):
the method comprises the following steps: clamping sclerotium of cultured fungus (yeast) into a 1.5ml centrifuge tube under aseptic condition, wherein the sclerotium mass is not less than 500mg, and freezing and grinding with liquid nitrogen. Add 500. mu.L of preheated CTAB solution and mix well, then water bath at 65 ℃ for 50 min.
Step two: after cooling to room temperature, 500. mu.L of phenol, chloroform and isoamyl alcohol was added thereto, and after thoroughly mixing, the mixture was centrifuged at 12000rpm for 15min, and the supernatant was taken out to a new tube.
Step three: then 500. mu.L phenol, chloroform and isoamyl alcohol were added, and after thorough mixing, centrifugation was carried out at 12000rpm for 15min, and the supernatant was taken out to a new tube.
Step four: adding 500 mu L of isoamyl alcohol and 50 mu L of NaAC, slightly rotating the centrifugal tube to fully mix the liquid in the tube, and freezing the tube in a refrigerator at the temperature of 20 ℃ below zero for 2 hours.
Step five: centrifuge at 12000rpm for 15min, and discard the supernatant.
Step six: air dried and 50. mu.L of deionized water was added to dissolve the DNA.
1.3Axygen column extraction (A4-A6 samples):
the method comprises the following steps: clamping sclerotium of cultured fungus (yeast) into a 1.5ml centrifuge tube under aseptic condition, wherein the sclerotium mass is not less than 500mg, and freezing and grinding with liquid nitrogen. Add 500. mu.L of preheated CTAB solution and mix well, then water bath at 65 ℃ for 50 min.
Step two: after cooling to room temperature, 500. mu.L of phenol, chloroform and isoamyl alcohol was added thereto, and after thoroughly mixing, the mixture was centrifuged at 12000rpm for 15min, and the supernatant was taken out to a new tube.
Step three: then 500. mu.L phenol, chloroform and isoamyl alcohol were added, and after thorough mixing, centrifugation was carried out at 12000rpm for 15min, and the supernatant was taken out to a new tube.
Step four: add 500. mu.L of isoamyl alcohol and 50. mu.L of NaAC, and gently rotate the centrifuge tube to mix the liquid in the tube well.
Step five: pouring the mixed solution into a filter column containing a DNA adsorption membrane, centrifuging at 5000rpm for 2min, and washing with anhydrous ethanol for 3 times.
Step six: air-drying, adding 50 μ L deionized water to dissolve DNA in the adsorption membrane, and centrifuging the dissolved DNA product at 5000rpm for 2min to a new tube.
And (3) detecting an OD value: OD measurements were performed on 9 DNA product samples using a 721 UV spectrophotometer (see Table 1). The detection result shows that the DNA product extracted from the sclerotinia sclerotiorum by using the kit has higher concentration and good purity.
Table 1: the results of measuring the OD value and concentration of the DNA product extracted from 100mg of the mycogenic fungi in example 1
(A1-A3: conventional CTAB method; A4-A6: Axygen column method; A7-A9: the kit)
As can be seen from the results of extracting DNA of the fungus causing the sclerotium in the table 1 by adopting untreated magnetic beads, the concentration of the DNA extracted by adopting the kit disclosed by the invention is the highest and can reach more than 200 ng/mu L, while the concentration of the DNA extracted by the traditional CTAB method and the Axygen column method is obviously lower, and the results show that the kit disclosed by the invention can effectively remove polysaccharide substances of the fungus causing the sclerotium and avoid the interference of the polysaccharide substances on the DNA extraction. However, the OD values measured by the DNA sample solution extracted by the three methods are all low, and the DNA purity in the sample solution is not effectively improved under the condition of interference of impurities such as protein, RNA and the like. Thus, the present invention also provides for the handling of DNA-adsorbed magnetic beads, since high purity DNA, generally having OD readings between 1.8 and 2.0, mostly contains RNA below 1.8 and mostly contains protein above 2.0. From the above experimental results, it can be seen that the extraction of DNA using magnetic beads purchased directly can increase the concentration, but the concentration of the extracted DNA contains impurities, particularly RNA, and the purity of DNA is not satisfactory, and sometimes the extracted DNA cannot be used for DNA detection for specific applications or other high requirements.
Example 2: DNA extraction of samples using treated magnetic beads
The extraction process described in example 1 was followed, with the following differences: the purchased magnetic beads were processed before the extraction of DNA using the magnetic beads, and the DNA was extracted using the processed magnetic beads, and other methods and reagents were used in the same manner as in the magnetic bead extraction method of example 1.
And (3) processing the magnetic beads, wherein the processing method comprises the following steps:
magnetic beads purchased from Shanghai Carbonfaphy biomedical science and technology Co., Ltd. (same lot as that of example 1) and used exclusively for extracting DNA were treated and soaked with N- (phosphonomethyl) iminodiacetic acid (5mol/L) and guanidine isothiocyanate (8 mol/L). The treatment method comprises the following steps: soaking 2g of magnetic beads in 10 ml of N- (phosphonomethyl) iminodiacetic acid solution for 5-10 hours, washing with PBS (pH 7.0) for 5-8 times, soaking the magnetic beads in 10 ml of guanidine isothiocyanate solution for 5-6 hours, and washing with PBS for 5-8 times for later use. And adsorbing the DNA by using the treated magnetic beads, and finally eluting the magnetic beads by using deionized water to obtain the target DNA.
Of course, the amount or weight of the magnetic beads depends on the concentration of the solution of PMIDA and guanidinium isothiocyanate needed, and this can be arbitrarily adjusted according to the spirit of the present invention, for example, 2g of the magnetic beads of the present invention can be treated with 10 ml of PMIDA and 10 ml of guanidinium isothiocyanate, which can be adjusted according to the diameter of the magnetic beads for limited experiments. The treated 2g magnetic beads can adsorb about 200-250 ng/. mu.L DNA, the magnetic beads are generally placed in excess, when the concentration of the DNA in the solution is very low, the excess magnetic beads can be placed in, the excess magnetic beads can adsorb DAN, and the enrichment can be performed, so that high-concentration DNA is obtained, and meanwhile, the enrichment is specific and selective, only the DNA is enriched, but the RAN or the protein is not enriched, so that high-purity DNA is obtained.
Untreated magnetic beads were used as a control (same as the magnetic bead extraction method in example 1).
The detection result shows that the magnetic beads in the kit have higher concentration and good purity of the DNA product extracted from the sclerotinia sclerotiorum, and the content of the extracted DNA can be improved by more than 80 percent.
Table 2: OD value measurement results in DNA products extracted from treated and untreated magnetic beads in example 2
As can be seen from the results of detecting the OD values of the DNA products extracted from the treated and untreated magnetic beads in Table 2, the OD values of the DNA products extracted from the magnetic beads of the present invention are both between 1.80 and 1.90, and the DNA products extracted from the untreated magnetic beads have higher purity, while the OD values of the DNA products extracted from the untreated magnetic beads are significantly lower than 1.80, wherein impurities are mixed and the purity is lower, and the purity of the DNA extracted from the magnetic beads of the present invention is significantly higher (P <0.01) than that of the magnetic beads not treated through analysis of variance. Therefore, the purity of DNA can be obviously improved by adopting the magnetic bead.
Example 3: determination of DNA adsorption Capacity Using treated magnetic beads
An ultrasonic cell disruptor is used for cell wall disruption of bacteria (staphylococcus aureus) to be detected, and the specific disruption method is as follows:
the bacteria were scraped off and made into a suspension with distilled water (10)7And each/mL), then placing the beaker containing the suspension into a crusher, placing a probe of the crusher into the beaker, inserting the beaker into the spore suspension, setting the crushing time to be 10min under an ice bath environment, and taking out a sample after cell crushing for carrying out magnetic bead adsorption DNA detection.
A predetermined weight of magnetic beads (2 to 5 g, however) (treated magnetic beads in example 2) of the present invention was added to the disrupted sample liquid, the beaker was placed on a magnetic holder to adsorb DNA magnetic beads, the magnetic beads were taken out after 10min, the magnetic beads were washed 3 times with a washing solution, 50. mu.L of an eluent was added to elute DNA on the magnetic beads, the resulting solution containing DNA was transferred to a new tube by a pipette after standing for 2min to obtain a DNA sample, and the OD value and concentration thereof were measured, and untreated magnetic beads were used as controls.
The method for eluting the magnetic beads is as follows:
adding 500 mu L of absolute ethyl alcohol into a centrifugal tube containing the adsorbed DNA magnetic beads to wash DNA, fully shaking the solution, discarding the solution, repeatedly washing the solution for 3 times, and washing the organic solution adhered to the surfaces of the magnetic beads as much as possible; and opening a centrifugal tube cover, standing at room temperature for 3-5min to volatilize the absolute ethyl alcohol on the surface of the magnetic beads as much as possible, adding 50 mu L of deionized water, standing for 2min, and then using a pipettor to put the solution containing the DNA into a new tube.
The detection result shows that the magnetic bead of the invention has higher adsorption capacity to DNA in cells, higher product concentration and good purity, less interference of RNA and protein and capability of leading the concentration of extracted DNA to reach more than 200 ng/muL.
Table 3: results of detecting OD value and concentration in DNA product extracted from treated and untreated magnetic beads in example 3
(A1-A3: magnetic beads of the present invention; A4-A6: untreated magnetic beads)
As can be seen from the results of detecting the OD values of the DNA products extracted by the treated and untreated magnetic beads, the OD values of the DNA sample solution extracted by the magnetic beads are all between 1.80 and 1.90, the DNA sample solution has higher purity, the adsorption of impurities such as RNA, protein and the like can be well eliminated, the concentration of the impurities can reach more than 100 ng/muL, and the purity and the concentration of the DNA in the sample solution extracted by the untreated magnetic beads are lower. The result shows that the magnetic bead of the invention has remarkable DNA adsorption property.
Example 4: determination of DNA adsorption Capacity Using treated magnetic beads (extraction of plasmid DNA)
1.1 the conventional method is as follows:
the extracted plasmid DNA can be directly used for enzyme digestion, PCR amplification and silver staining sequence analysis. The method comprises the following steps:
1: 1% plasmid containing Escherichia coli cells in 2ml LB medium.
2: the cells were cultured overnight at 37 ℃ with shaking.
3: 1.5ml of the cells were put in an Ep tube (centrifuge tube), centrifuged at 4000rpm for 3min, and the supernatant was discarded.
4: add 0.lml solution I (1% glucose, 50mM/LEDTApH8.0, 25mM/LTris-HClpH8.0) and mix well.
5: 0.2ml of solution II (0.2mM/LNaOH, 1% SDS) was added, mixed by gentle tumbling and placed in an ice bath for 5min.
6: 0.15m1 precooled solution III (5mol/LKAc, pH4.8) was added, mixed by gentle tumbling and placed in an ice bath for 5min.
7: centrifuging at 10,000 rpm for 20min, and collecting supernatant in another new Ep tube
8: adding isovaleric alcohol with the same volume, uniformly mixing, and standing for 10min.
9: centrifuge at 10,000 rpm for 20min and discard the supernatant.
10: washed once with 0.5ml of 70% ethanol and all the liquid was drained.
11: after the precipitate was dried, it was dissolved in 50ul of TE buffer (or 60 ℃ in deionized water).
1.2 after step 7 (omitting the conventional steps of 8 to 11), adding the treated magnetic beads of the present invention for DNA adsorption and adding the non-treated magnetic beads for DNA adsorption, and then performing the elution method of the present invention,
the method for eluting the magnetic beads is as follows:
adding 500 mu L of absolute ethyl alcohol into a centrifugal tube containing the adsorbed DNA magnetic beads to wash DNA, fully shaking the solution, discarding the solution, repeatedly washing the solution for 3 times, and washing the organic solution adhered to the surfaces of the magnetic beads as much as possible; and opening a centrifugal tube cover, standing at room temperature for 3-5min to volatilize the absolute ethyl alcohol on the surface of the magnetic beads as much as possible, adding 50 mu L of deionized water, standing for 2min, and then using a pipettor to put the solution containing the DNA into a new tube.
Table 4: results of detecting OD value and concentration in DNA product extracted from treated and untreated magnetic beads in example 4
(A1-A3: magnetic beads of the present invention; A4-A6: untreated magnetic beads; A7-A9 are conventional methods)
As can be seen from the results of detecting the OD values in the plasmid DNA products extracted by the treated and untreated magnetic beads, the OD values in the DNA sample solution extracted by the magnetic beads are all between 1.80 and 1.90, the DNA sample solution has higher purity, the adsorption of impurities such as RNA, protein and the like can be well eliminated, the concentration of the impurities can reach more than 100 ng/mu L, and the purity and the concentration of the DNA in the sample solution extracted by the untreated magnetic beads are lower. The result shows that the magnetic bead of the invention has remarkable DNA adsorption property.
The terms and expressions which have been employed herein as terms of description and not of limitation are not intended to be exclusive and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or their equivalents, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it is believed that while the invention has been set forth herein in a variety of specific embodiments and with any features described in detail, it is believed that alterations to the design disclosed herein will be resorted to those skilled in the art, and that such alterations will be consistent with the appended claims. The contents of the articles, patents, patent applications, and all other documents, and the useful electronic information mentioned and cited herein are incorporated by reference in their entirety, to the extent any portion of this disclosure is specifically identified. The applicants have the right to incorporate into this application any and all information and material from such articles, patents, patent applications, or other documents as part of the disclosure of this patent specification.
Claims (5)
1. A magnetic bead method produces sclerotinia sclerotiorum DNA extraction kit, its characterized in that includes:
solution I, solution II, solution III, solution IV, washing liquid, eluent and magnetic bead suspension;
the magnetic bead suspension is: the magnetic beads are made of nano magnetic iron oxide and have the diameter of 1.2-2 mm;
the solution I consists of glucose, Tris-HCl and EDTA, and the pH value is 8.0;
the solution II is absolute ethyl alcohol and potassium acetate solution;
the solution III is SDS solution diluted by sodium hydroxide;
the solution IV comprises guanidine hydrochloride and potassium acetate, and the pH value is 3.5-4.5;
the washing solution is an ethanol solution;
the eluent is deionized water;
wherein the magnetic bead suspension is processed by the following processing steps: soaking the magnetic beads in a N- (phosphonomethyl) iminodiacetic acid solution for 5-10 hours, washing the magnetic beads with PBS (phosphate buffer solution) with the pH value of 7.0 for 5-8 times, soaking the taken magnetic beads in guanidinium isothiocyanate solution for 5-6 hours, and washing the magnetic beads with the PBS for 5-8 times for later use;
the concentration of the PMIDA solution is 5mol/L, and the concentration of the guanidinium isothiocyanate solution is 8 mol/L.
2. A method for extracting DNA of sclerotium fungi generated by a magnetic bead method is characterized by comprising the following steps:
the method comprises the following steps: clamping sclerotium of the cultured fungus into a 1.5ml centrifuge tube under an aseptic condition, wherein the mass of the sclerotium is not less than 300mg, and freezing and grinding by using liquid nitrogen; adding 250 mu L of the solution I, adding 50 mu L of RNase A, and fully and uniformly mixing;
step two: adding 50 μ L protease K, mixing, soaking in 60 deg.C constant temperature water for 20min, centrifuging at 12000rpm for 5min, and collecting supernatant;
step three: adding 150 mu L of solution II, and slightly turning over the solution for 5-6 times; centrifuging at 12000rpm for 5min, and collecting supernatant to a new tube;
step four: adding 350 μ L of solution III, slightly turning over to fully crack the bacteria liquid into transparent solution, centrifuging at 12000rpm for 5min, and taking the supernatant to a new tube;
step five: adding 350 μ L of solution IV, slightly turning over until white floccule is formed, and standing at room temperature for 2 min;
step six: adding 50 μ L of magnetic bead suspension, slightly turning over, and standing for 2 min;
step seven: placing the centrifuge tube on a magnetic frame, standing for 1min to make the solution transparent, and discarding the solution;
wherein the magnetic bead suspension is processed by the following processing steps: soaking the magnetic beads in a N- (phosphonomethyl) iminodiacetic acid solution for 5-10 hours, washing the magnetic beads with PBS (phosphate buffer solution) with the pH value of 7.0 for 5-8 times, soaking the taken magnetic beads in guanidinium isothiocyanate solution for 5-6 hours, and washing the magnetic beads with the PBS for 5-8 times for later use;
the concentration of glucose in the solution I is 50mmol/L, the concentration of Tris-HCl is 25mmol/L, and the concentration of EDTA is 10 mmol/L;
the concentration of the potassium acetate solution in the solution II is 8 mol/L;
the concentration of NaOH solution in the solution III is 0.2mol/L, and the concentration of SDS is 1-1.5%;
the concentration of guanidine hydrochloride in the solution IV is 3.0-4.5M, and the concentration of KAC is 0.75M;
the concentration of the PMIDA solution is 5mol/L, and the concentration of the guanidinium isothiocyanate solution is 8 mol/L.
3. The method of claim 2, wherein the volume ratio of the solution of N- (phosphonomethyl) iminodiacetic acid to the solution of guanidinium isothiocyanate is 1: 1.
4. The method of claim 2, further comprising washing and eluting the magnetic beads after step seven: adding 500 mu L of absolute ethyl alcohol into a centrifugal tube containing the adsorbed DNA magnetic beads to wash DNA, fully shaking the solution, discarding the solution, repeatedly washing the solution for 3 times, and washing the organic solution adhered to the surfaces of the magnetic beads as much as possible; and opening a centrifugal tube cover, standing at room temperature for 3-5min to volatilize the absolute ethyl alcohol on the surface of the magnetic beads as much as possible, adding 50 mu L of deionized water, standing for 2min, and transferring the solution containing the DNA to a new tube by using a pipettor.
5. A method for extracting plasmid DNA from Escherichia coli comprises the following steps:
1) inoculating 1% of escherichia coli cells containing plasmids into 2ml of LB culture medium;
2) shaking and culturing at 37 ℃ overnight;
3) taking 1.5ml of thalli in an Ep tube, centrifuging at 4000rpm for 3min, and removing supernatant;
4) 0.lml solution I of 1% glucose, 50mmol/L EDTA and 25mmol/L Tris-HCl solution at pH 8.0;
5) adding 0.2ml of solution II, slightly turning and uniformly mixing, and placing in an ice bath for 5min, wherein the solution II is 0.2mmol/L NaOH and 1% SDS solution;
6) adding 0.15m1 precooled solution III, slightly turning and mixing uniformly, and placing in an ice bath for 5min, wherein the solution III is 5mol/L KAc solution, and the pH value is 4.8;
7) centrifuging at 10000rpm for 20min, and taking the supernatant in another new Ep tube;
8) adding excessive magnetic beads to the Ep tube in the step 7 and separating the magnetic beads, wherein the magnetic beads are processed by the following method: soaking the magnetic beads in a N- (phosphonomethyl) iminodiacetic acid solution for 5-10 hours, washing the magnetic beads with PBS (phosphate buffer solution) with the pH value of 7.0 for 5-8 times, soaking the taken magnetic beads in the guanidinium isothiocyanate solution for 5-6 hours, washing the magnetic beads with the PBS buffer solution for 5-8 times for later use, wherein the concentration of the N- (phosphonomethyl) iminodiacetic acid solution is 5mol/L, and the concentration of the guanidinium isothiocyanate solution is 8 mol/L;
9) adding 500 mu L of absolute ethyl alcohol into the centrifuge tube containing the adsorbed DNA magnetic beads to wash DNA, fully shaking the centrifuge tube, discarding the solution, repeatedly washing the centrifuge tube for 3 times, and washing the organic solution adhered to the surfaces of the magnetic beads as much as possible; and opening a centrifugal tube cover, standing at room temperature for 3-5min to volatilize the absolute ethyl alcohol on the surface of the magnetic beads as much as possible, adding 50 mu L of deionized water, standing for 2min, and transferring the solution containing the DNA to a new tube by using a pipettor.
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| CN104212793A (en) * | 2014-08-08 | 2014-12-17 | 中国人民解放军疾病预防控制所 | Kit for magnetic bead method for bacterial genome DNA extraction and extraction method thereof |
| CN104673783A (en) * | 2015-01-19 | 2015-06-03 | 宝瑞源生物技术(北京)有限公司 | Kit for extracting DNA/RNA of virus through magnetic bead method and using method |
| CN104805073A (en) * | 2015-04-21 | 2015-07-29 | 益百尚(北京)生物技术有限责任公司 | Kit for extracting viral genome nucleic acid and use method thereof |
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| CN104212793A (en) * | 2014-08-08 | 2014-12-17 | 中国人民解放军疾病预防控制所 | Kit for magnetic bead method for bacterial genome DNA extraction and extraction method thereof |
| CN104673783A (en) * | 2015-01-19 | 2015-06-03 | 宝瑞源生物技术(北京)有限公司 | Kit for extracting DNA/RNA of virus through magnetic bead method and using method |
| CN104805073A (en) * | 2015-04-21 | 2015-07-29 | 益百尚(北京)生物技术有限责任公司 | Kit for extracting viral genome nucleic acid and use method thereof |
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