CN105713900A - Nucleic acid extraction method based on magnetic graphene nano-composites - Google Patents
Nucleic acid extraction method based on magnetic graphene nano-composites Download PDFInfo
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- 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 nucleic acid extraction method based on magnetic graphene nano-composites and belongs to the technical field of novel nucleic acid extraction. According to the nucleic acid extraction method, carboxy groups on the surface of graphene oxide are taken as connecting loci and are connected with amino magnetic beads, so that synthesis of the magnetic graphene nano-composites is realized; cell lysis and nucleic acid release are realized with a method of matching a lysis solution with proteinase K; after cell debris is removed through centrifugation, the magnetic graphene nano-composites are added to a supernatant for capturing the nucleic acid; finally, a nucleic acid sample is obtained through magnetic separation, cleaning and purification. The method has the characteristics of simple extraction process, high extraction efficiency and RNA protection function, is easy to operate and convenient to store and has great significance in development of molecular biology and related disciplines.
Description
Technical field
The invention belongs to novel nucleic acids extractive technique field, particularly to a kind of method for extracting nucleic acid based on magnetic graphene nano-complex.
Background technology
Nucleic acid, as the carrier of hereditary information, is biological information molecule important in organism, is also the main object of molecular biology research.Explore and physiological significance correlational study to realize nucleic acid function, it usually needs nucleic acid is easily separated purification.Therefore, nucleic acid extraction is one of operation most important, most basic in molecular biology experiment technology, is widely used in.At present, extracting DNA genome and mainly adopt " paramagnetic particle method ", " adsorption column method ", " CTAB method ", " SDS method " etc. to go to realize the extraction of DNA in biological sample, there is the shortcoming such as operating process complexity, extracting cycle length in these methods.The extraction of RNA is then frequently with classical TRIzol isolation kit method, TRIzol reagent is mainly composed of guanidinium isothiocyanate and phenol, wherein guanidinium isothiocyanate cleavable cell, promote ribosomal dissociating, make RNA and Separation of Proteins, and RNA is discharged in solution.When adding chloroform, it can the acid phenol of extracting, and acid phenol can promote RNA to enter aqueous phase, can form aqueous layer and organic layer after centrifugal, and such RNA just separates with the protein remained in organic facies and DNA.Aqueous layer is mainly RNA, and organic layer is mainly DNA and protein.But, the method existence processes that sample size is limited, easily pollute RNase, the shortcoming such as degradable.It should be noted that guanidinium isothiocyanate is a kind of poisonous, chemical reagent that can be carcinogenic, in extraction process, bring huge injury to experimenter.Therefore, development of new, quickly, the method for extracting nucleic acid of high-fidelity, bio-compatibility significant for the development of molecular biology and related discipline.
In recent years, along with the fast development of nanotechnology, nano material applies to life science widely.Wherein, graphene oxide becomes " star " nano material especially, and is widely used in the subjects such as electronics, materialogy, diagnostics.It is known that graphene oxide can pass through " π-π is stacking " effect realizes the purpose of absorption nucleic acid, this effect inspires the author to utilize the oxidation specific seizure nucleic acid of graphene oxide, thus realizing the purpose of nucleic acid extraction.In addition; I discloses a kind of RNA guard method based on graphene oxide composite material at seminar's early stage patent (Chinese patent: 201310178816.9); the method discloses graphene oxide and has the RNA protective capability of excellence, and has the feature of protective efficacy height, protection timeliness length.Therefore; integrate graphene oxide RNA protection talent, Graphene fenestral fabric and absorption nucleic acid performance; we have developed a kind of method for extracting nucleic acid based on magnetic graphene nano-complex, the method is simple to operate, consuming time short and can be effectively protected for RNA offer.
Summary of the invention
In order to the shortcoming of existing nucleic acid extraction technology is with not enough, it is an object of the invention to provide a kind of method for extracting nucleic acid based on magnetic graphene nano-complex.The present invention adopts magnetic graphene as nano-carrier, utilizes the absorption nucleic acid ability of Graphene rigid surface, provides wide absorption cross section for nucleic acid, thus reaches to catch the purpose of nucleic acid.
The purpose of the present invention is achieved through the following technical solutions:
Based on the method for extracting nucleic acid of magnetic graphene nano-complex, comprise the steps:
First, utilize surface of graphene oxide carboxylic group as connection site so that it is to connect with amino magnetic bead, thus realizing the synthesis of magnetic graphene nano-complex；Subsequently, adopt the method that lysate coordinates E.C. 3.4.21.64, it is achieved the cracking of cell, discharge nucleic acid；After centrifugal segregation cell debris, supernatant adds magnetic graphene nano-complex and catches nucleic acid；Finally, after Magneto separate cleans and purifies, nucleic acid samples is namely obtained.
Described cell can derive from zooblast, gram positive bacteria, gram negative bacteria, animal tissue, animal blood or plant tissue；
Described cell needs to carry out pre-treatment before cracking；
Described nucleic acid samples includes DNA and RNA；
Described nucleic acid samples adds DNA enzymatic, obtains RNA sample；
Described nucleic acid samples adds RNase, obtains DNA sample；
The described method for extracting nucleic acid based on magnetic graphene nano-complex, including step in detail below:
1) sample pre-treatments
1. zooblast sample treatment
Zooblast cell membrane is relatively thin, rigidity is weak, easily crack, and sample pre-treatments comprises the steps of
A. the centrifugal mode collected is adopted for free cultured cells, make cell aggregation bottom centrifuge tube, then clean the cell culture fluid of twice removal residual with PBS and can obtain cell precipitation.
B. Digestive system digestion or cell scraper is adopted to make the de-wall of cell and dissociate to culture fluid for the cell of adhere-wall culture, mode by centrifugal collection, make cell aggregation bottom centrifuge tube, then with PBS clean twice removal residual cell culture fluid can obtain cell precipitation.
C. adding Sampleprotector in the cell precipitation collected in step a or b, and soak 30min, the RNase in degenerating cell, thus avoiding cell to occur degraded in pretreatment process.
D. removing Sampleprotector, centrifugal collecting cell precipitates, and is frozen in-80 DEG C of refrigerators standby.
2. gram positive bacteria sample pre-treatments
Gram-positive bacteria cell wall is thicker, rigidity by force, more difficult cracking, sample pre-treatments can be passed through two kinds of methods and realize:
A. lysozyme dissolved cell wall
A) the centrifugal gram positive bacteria collecting liquid cultivation, after removing culture fluid, cleans 3 times with PBS.Obtain bacterial sediment.
B) adding Sampleprotector in the cell precipitation collected in step a), and soak 30min, the RNase in degeneration thalline, thus avoiding thalline to occur degraded in pretreatment process.
C) centrifugal bacterial sediment of collecting, removal Sampleprotector, be frozen in-80 refrigerators by gained thalline standby.
D) add the lysozyme 200 μ L of 20mg/mL, hatch half an hour for 37 DEG C.
B. liquid nitrogen grinding removes cell wall
A) the centrifugal gram positive bacteria collecting liquid cultivation, after removing culture fluid, cleans 3 times with PBS.Obtain bacterial sediment.
B) adding Sampleprotector in the cell precipitation collected in step a), and soak 30min, the RNase in degeneration thalline, thus avoiding thalline to occur degraded in pretreatment process.
C) thalline of gained in step b) is moved in the middle of mortar, topple over liquid nitrogen, and after acutely pulverizing, collect powder and cold preservation is standby in-80 DEG C of refrigerators.
3. gram negative bacteria sample pre-treatments
Gram-negative bacteria cell wall is relatively thin, it is easy to broken, only needs with reference to step 2. a；
4. animal tissue's sample pre-treatments
Preferably " now cutting existing use ", to prevent nucleolysis, the particularly RNA of easily degraded, the pretreatment process of animal tissue's sample mainly comprises the steps that animal tissue's sample
A. take fresh animal tissue, cut into fritter (about 3 millimeters) with operating scissors and add Sampleprotector, and soak 1 hour, the RNase in denatured tissues.
B. centrifugal segregation Sampleprotector, gained tissue piece can show use, or cold preservation is standby in-80 DEG C of refrigerators.
C. the tissue piece of gained in step b is moved in the middle of mortar, topple over liquid nitrogen, and after acutely pulverizing, collect powder and cold preservation is standby in-80 DEG C of refrigerators.
5. animal blood sample pre-treatment
Animal blood sample needs to accomplish in " existing enchashment is used " blood containing abundant enzyme, and the extraction for nucleic acid exists huge hidden danger.Therefore, the extraction of nucleic acids in blood is huge difficult problem all the time, and corresponding sample pre-treatments is accomplished by different because of actual demand:
A. Objective extraction nucleic acid is in hemocyte
A) as target nucleic acid is present in hemocyte, then need first to collect hemocyte, make blood cell sedimentation by high speed centrifugation, remove supernatant, collect cell.
B) cell precipitation collected in step a) adds Sampleprotector, and soak 30min, the RNase in degeneration hemocyte.
C) centrifugal bacterial sediment of collecting, removal Sampleprotector, be frozen in-80 refrigerators by gained hemocyte standby.
B. Objective extraction nucleic acid is in serum
The serum obtained after washed corpuscles, can directly freeze pure (-80 DEG C), goes out Deproteinization then through going through lysis process.
6. Plant tissue samples pre-treatment
A. take fresh plant tissue, cut into fritter (about 2 millimeters) with operating scissors and add Sampleprotector, and soak 1 hour, the RNase in denatured tissues.
B. centrifugal segregation Sampleprotector, gained tissue piece can show use, or cold preservation is standby in-80 DEG C of refrigerators.
C. the tissue piece of gained in step b is moved in the middle of mortar, topple over liquid nitrogen, and after acutely pulverizing, collect powder and cold preservation is standby in-80 DEG C of refrigerators.
2) lysis, nucleic acid capture and cleaning
1. lysis
The present invention, for Listeria monocytogenes (Listeriamonocytogenes), specifically teaches the lysis process after living through sample pre-treatments:
A. cell pyrolysis liquid is configured
Configuration SDS lysis buffer, between each composition, concentration is as follows: Tris-HCl (pH value 8.0, final concentration 50mM)；EDTA (pH value 8.0, final concentration 20mM)；NaCl (0.4M)；SDS (2%).Fully after mixing, sterilizing is standby.
B. lysis
Add the SDS cell pyrolysis liquid of gained in step a.Under normal circumstances, 107Individual cell adds 1.5mL, after fully concussion mixes, and add E.C. 3.4.21.64 heating in water bath to 70 DEG C 15 minutes.Until mixed liquor clarification.
C. the clear liquor of step b) gained is cooled to room temperature, and is centrifuged off a small amount of precipitation.Preservation supernatant is standby.
The present invention, for Listeria monocytogenes, demonstrates the feasibility of this cleavage method, shown in the atomic force phenogram of experimental result such as Fig. 2 B and C, and complete Listeria monocytogenes, cracking fully after experience sample pre-treatments.Thus, the feasibility of this cleavage method obtains sufficient checking.
2. nucleic acid capture and cleaning
A. in step 1. gained supernatant, add magnetic graphene nano-complex 50 μ L (1mg/mL) fully shake mixing.
B. incubated at room ten minutes, make nucleic acid be adsorbed on the surface of magnetic graphene nano-complex fully.
C. Magneto separate, magnetic graphene nano-complex is made to be gathered in bottom, suck supernatant, it is sequentially added into ethanol, the PBS washing and precipitating of 70%, and fully shake, making magnetic graphene nano-complex spread out, recirculation above step can obtain being adsorbed with magnetic graphene nano-complex for two to three times, and is dried to no liquid residual.
D. in step c gained be adsorbed with magnetic graphene nano-complex, add TE buffer 100 μ L, and heat to 70 DEG C, make nucleic acid separate with magnetic graphene.Magneto separate removes magnetic graphene nano-complex, collects supernatant and can obtain nucleic acid extractive.
The present invention, relative to prior art, has such advantages as and effect:
(1) present invention extracts process simply, easily operates.
(2) extraction efficiency of the present invention is high.
(3) present invention stores conveniently.
(4) present invention has RNA defencive function.
Accompanying drawing explanation
Fig. 1 is based on the method for extracting nucleic acid schematic diagram of magnetic graphene nano-complex.
Fig. 2 is the nucleic acid extraction result in embodiment 2 for Listeria monocytogenes: wherein, and Fig. 2 A. extracts process substep figure；Fig. 2 B. Listeria monocytogenes atomic force microscope characterizes；Fig. 2 C. lysis debris atoms force microscope characterizes；Fig. 2 D. extracts product electrophoretogram.
Fig. 3 is for the RNA of Listeria monocytogenes or DNA extraction result in embodiment 2: wherein, Fig. 3 A.RNA, DNA genome extract electrophoretogram；Fig. 3 B. gray analysis figure
The RNA that Fig. 4 is zooblast, tissue, blood sample and Plant tissue samples extracts result；Wherein, Fig. 4 A. zooblast and tissue RNA extract electrophoretogram；Fig. 4 B. animal blood and Plant tissue samples RNA extract electrophoretogram.
Fig. 5 is the comparing result of the RNA extraction efficiency of the present invention and the extraction efficiency of TRIzol test kit and fluorescence degradation rate；Fig. 5 A. magnetic graphene nano-complex method for extracting nucleic acid and the contrast of TRIzol method electrophoresis；Fig. 5 B. magnetic graphene nano-complex method for extracting nucleic acid and TRIzol method fluorescence results；Fig. 5 C. magnetic graphene nano-complex method for extracting nucleic acid and TRIzol method fluorescence intensity comparison diagram；Fig. 5 D. magnetic graphene nano-complex method for extracting nucleic acid and TRIzol method fluorescence degradation rate comparison diagram.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
The structure of embodiment 1 magnetic graphene nano-complex and sample pre-treatments
1) structure of magnetic graphene nano-complex
The present invention adopts graphene oxide as synthetic material, utilizes the carboxylic group of surface of graphene oxide as connection site, and is coated magnetic bead by amido link with amino and is connected, and finally realizes the synthesis of magnetic graphene nano-complex.The synthesis of magnetic graphene nano-complex mainly comprises the steps that
1. the graphene oxide (particle diameter is 1 micron) taking 10mg is dissolved in 30mL distilled water, and ultrasonic one and a half hours, make graphene oxide fully dissolve, obtain the graphene oxide solution of 0.33mg/mL, and it is standby to be stored in 4 DEG C of refrigerators.
2. graphene oxide activated carboxylic
4mgN-N-Hydroxysuccinimide (NHS) is added in step 1. middle gained graphene oxide solution, and after fully mixing, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) of 5mg, fully after mixing, stirring reaction 1 hour at 37 DEG C, and ultrasonic half an hour make the graphene oxide of activated carboxylic be again dispersed into monolayer without assemble solution.
3. amido link connects
In the solution of step 2. gained, add 10mg amino magnetic bead (particle diameter is 200nm), and fully mix.After sealing, 37 DEG C of stirring reactions cross liquid, obtain the thick product of magnetic graphene nano-complex.
4. purification
Step 3. in the thick product of gained, be not connected with the Graphene of magnetic bead containing small part, it is necessary to be purified to remove in solution and do not have magnetic graphene oxide.The present invention adopts the method for Magneto separate, the magnetic graphene oxide of tool is made to converge at one end, magnetic field, removal does not have magnetic supernatant solution, add isopyknic PBS (concentration is 1 ×), after removing magnetic field, mixing is shaken, and then carries out Magneto separate operation again and can obtain purer magnetic graphene nano-complex (MGO) for 2 times.
2) sample pre-treatments
1. zooblast sample treatment
Zooblast cell membrane is relatively thin, rigidity is weak, easily crack, and sample pre-treatments comprises the steps of
A. the centrifugal mode collected is adopted for free cultured cells, make cell aggregation bottom centrifuge tube, then clean the cell culture fluid of twice removal residual with PBS and can obtain cell precipitation.
B. Digestive system digestion or cell scraper is adopted to make the de-wall of cell and dissociate to culture fluid for the cell of adhere-wall culture, mode by centrifugal collection, make cell aggregation bottom centrifuge tube, then with PBS clean twice removal residual cell culture fluid can obtain cell precipitation.
C. adding Sampleprotector (Takara) in the cell precipitation collected in step a or b, and soak 30min, the RNase in degenerating cell, thus avoiding cell to occur degraded in pretreatment process.
D. removing Sampleprotector, centrifugal collecting cell precipitates, and is frozen in-80 DEG C of refrigerators standby.
2. gram positive bacteria sample pre-treatments
Gram-positive bacteria cell wall is thicker, rigidity by force, more difficult cracking, sample pre-treatments can be passed through two kinds of methods and realize:
A. lysozyme dissolved cell wall
A) the centrifugal gram positive bacteria collecting liquid cultivation, after removing culture fluid, cleans 3 times with PBS.Obtain bacterial sediment.
B) adding Sampleprotector in the cell precipitation collected in step a), and soak 30min, the RNase in degeneration thalline, thus avoiding thalline to occur degraded in pretreatment process.
C) centrifugal bacterial sediment of collecting, removal Sampleprotector, be frozen in-80 refrigerators by gained thalline standby.
D) add the lysozyme 200 μ L of 20mg/mL, hatch half an hour for 37 DEG C.
B. liquid nitrogen grinding removes cell wall
A) the centrifugal gram positive bacteria collecting liquid cultivation, after removing culture fluid, cleans 3 times with PBS.Obtain bacterial sediment.
B) adding Sampleprotector in the cell precipitation collected in step a), and soak 30min, the RNase in degeneration thalline, thus avoiding thalline to occur degraded in pretreatment process.
C) thalline of gained in step b) is moved in the middle of mortar, topple over liquid nitrogen, and after acutely pulverizing, collect powder and cold preservation is standby in-80 DEG C of refrigerators.
3. gram negative bacteria sample pre-treatments
Gram-negative bacteria cell wall is relatively thin, it is easy to broken, only needs with reference to step 2. a；
4. animal tissue's sample pre-treatments
Preferably " now cutting existing use ", to prevent nucleolysis, the particularly RNA of easily degraded, the pretreatment process of animal tissue's sample mainly comprises the steps that animal tissue's sample
A. take fresh animal tissue, cut into fritter (about 3 millimeters) with operating scissors and add Sampleprotector, and soak 1 hour, the RNase in denatured tissues.
B. centrifugal segregation Sampleprotector, gained tissue piece can show use, or cold preservation is standby in-80 DEG C of refrigerators.
C. the tissue piece of gained in step b is moved in the middle of mortar, topple over liquid nitrogen, and after acutely pulverizing, collect powder and cold preservation is standby in-80 DEG C of refrigerators.
5. animal blood sample pre-treatment
Animal blood sample needs to accomplish in " existing enchashment is used " blood containing abundant enzyme, and the extraction for nucleic acid exists huge hidden danger.Therefore, the extraction of nucleic acids in blood is huge difficult problem all the time, and corresponding sample pre-treatments is accomplished by different because of actual demand:
A. Objective extraction nucleic acid is in hemocyte
A) as target nucleic acid is present in hemocyte, then need first to collect hemocyte, make blood cell sedimentation by high speed centrifugation, remove supernatant, collect cell.
B) cell precipitation collected in step a) adds Sampleprotector, and soak 30min, the RNase in degeneration hemocyte.
C) centrifugal bacterial sediment of collecting, removal Sampleprotector, be frozen in-80 refrigerators by gained hemocyte standby.
B. Objective extraction nucleic acid is in serum
The serum obtained after washed corpuscles, can directly freeze pure (-80 DEG C), goes out Deproteinization then through going through lysis process.
6. Plant tissue samples pre-treatment
A. take fresh plant tissue, cut into fritter (about 2 millimeters) with operating scissors and add Sampleprotector, and soak 1 hour, the RNase in denatured tissues.
B. centrifugal segregation Sampleprotector, gained tissue piece can show use, or cold preservation is standby in-80 DEG C of refrigerators.
C. the tissue piece of gained in step b is moved in the middle of mortar, topple over liquid nitrogen, and after acutely pulverizing, collect powder and cold preservation is standby in-80 DEG C of refrigerators.
Embodiment 2 lysis, nucleic acid capture, cleaning and purification
1. lysis
The present invention, for Listeria monocytogenes (Listeriamonocytogenes) (buying in Guangzhou Culture Collection), is shown in Fig. 2；Specifically teach the lysis process after living through sample pre-treatments:
A. cell pyrolysis liquid is configured
Configuration SDS lysis buffer, between each composition, concentration is as follows: Tris-HCl (pH value 8.0, final concentration 50mM)；EDTA (pH value 8.0, final concentration 20mM)；NaCl (0.4M)；SDS (2%).Fully after mixing, sterilizing is standby.
B. lysis
Add the SDS cell pyrolysis liquid of gained in step a.Under normal circumstances, 107Individual cell adds 1.5mL, after fully concussion mixes, and add E.C. 3.4.21.64 heating in water bath to 70 DEG C 15 minutes.Until mixed liquor clarification.
C. the clear liquor of step b gained is cooled to room temperature, and is centrifuged off a small amount of precipitation.Preservation supernatant is standby.
The present invention, for Listeria monocytogenes, demonstrates the feasibility of this cleavage method, shown in the atomic force phenogram of experimental result such as Fig. 2 B and C, and complete Listeria monocytogenes, cracking fully after experience sample pre-treatments.Thus, the feasibility of this cleavage method obtains sufficient checking.
2. nucleic acid capture and cleaning
A. in step 1. gained supernatant, add magnetic graphene nano-complex 50 μ L (1mg/mL) fully shake mixing.
B. incubated at room ten minutes, make nucleic acid be adsorbed on the surface of magnetic graphene nano-complex fully.
C. Magneto separate, magnetic graphene nano-complex is made to be gathered in bottom, suck supernatant, it is sequentially added into ethanol, the PBS washing and precipitating of 70%, and fully shake, making magnetic graphene nano-complex spread out, recirculation above step can obtain being adsorbed with magnetic graphene nano-complex for two to three times, and is dried to no liquid residual.
D. in step c gained be adsorbed with magnetic graphene nano-complex, add TE buffer 100 μ L, and heat to 70 DEG C, make nucleic acid separate with magnetic graphene.Magneto separate removes magnetic graphene nano-complex, collects supernatant and can obtain nucleic acid extractive.
As shown in Figure 2 D, what the present invention can be stable extracts nucleic acid (including DNA and RNA) to nucleic acid extraction experimental result from thalline.Meanwhile, RNA or DNA that addition RNase or DNA enzymatic can be gone out in supernatant respectively in the supernatant of step " 1. lysis " gained, thus obtaining independent DNA genome or rna gene group.Experimental result is as it is shown on figure 3, it follows that the present invention can be used as the extracting method of DNA, it is possible to as the extracting method of RNA, or proposed in the lump by nucleic acid in born of the same parents.
Simultaneously, the present invention is extracted as example with actual sample RNA, describing the present invention and extract process for the RNA of zooblast, tissue, blood sample and Plant tissue samples, experimental result as shown in Figure 4, thus demonstrates this invention for feasibility that the RNA of complex sample extracts.It addition, we compared for the extraction efficiency of traditional RNA extraction method and the present invention, as shown in Fig. 5 A and B, the extraction efficiency of the extraction efficiency of the present invention and TRIzol test kit remains basically stable, and the extraction efficiency thus demonstrating the present invention is better.Comparatively speaking, the RNA defencive function of the present invention is another bright spot of the method, by Fig. 5 C and D it can be seen that by magnetic graphene nano-complex protection RNA can long-acting preservation in atmosphere, compare its degradation rate of TRIzol test kit low.
Above-described embodiment is the present invention preferably embodiment; but embodiments of the present invention are also not restricted to the described embodiments; the change made under other any spirit without departing from the present invention and principle, modification, replacement, combination, simplification; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. based on the method for extracting nucleic acid of magnetic graphene nano-complex, it is characterised in that comprise the steps:
First, utilize surface of graphene oxide carboxylic group as connection site so that it is to connect with amino magnetic bead, thus realizing the synthesis of magnetic graphene nano-complex；Subsequently, adopt the method that lysate coordinates E.C. 3.4.21.64, it is achieved the cracking of cell, discharge nucleic acid；After centrifugal segregation cell debris, supernatant adds magnetic graphene nano-complex and catches nucleic acid；Finally, after Magneto separate cleans and purifies, nucleic acid samples is namely obtained.
2. the method for extracting nucleic acid based on magnetic graphene nano-complex according to claim 1, it is characterised in that: described cell needs to carry out pre-treatment before cracking.
3. the method for extracting nucleic acid based on magnetic graphene nano-complex according to claim 2, it is characterised in that: described cell derived is in zooblast, gram positive bacteria, gram negative bacteria, animal tissue, animal blood or plant tissue.
4. the method for extracting nucleic acid based on magnetic graphene nano-complex according to any one of claims 1 to 3, it is characterised in that: described nucleic acid samples includes DNA and RNA.
5. the method for extracting nucleic acid based on magnetic graphene nano-complex according to any one of claims 1 to 3, it is characterised in that: described nucleic acid samples adds DNA enzymatic, obtains RNA sample.
6. the method for extracting nucleic acid based on magnetic graphene nano-complex according to any one of claims 1 to 3, it is characterised in that: described nucleic acid samples adds RNase, obtains DNA sample.
7. the method for extracting nucleic acid based on magnetic graphene nano-complex according to claim 3, it is characterised in that:
The pre-treatment of described zooblast, comprises the steps:
A. the centrifugal mode collected is adopted for free cultured cells, make cell aggregation bottom centrifuge tube, then clean the cell culture fluid of twice removal residual with PBS and namely obtain cell precipitation；
B. Digestive system digestion or cell scraper is adopted to make the de-wall of cell and dissociate to culture fluid for the cell of adhere-wall culture, mode by centrifugal collection, make cell aggregation bottom centrifuge tube, then with PBS clean twice removal residual cell culture fluid namely obtain cell precipitation；
C. the cell precipitation collected in step a or b adds Sampleprotector, and soaks, the RNase in degenerating cell；
D. removing Sampleprotector, centrifugal collecting cell precipitates, and is frozen in-80 DEG C of refrigerators standby.
8. the method for extracting nucleic acid based on magnetic graphene nano-complex according to claim 3, it is characterised in that: the pre-treatment of described gram positive bacteria, adopt the one in method a and method b, comprise the steps:
A. lysozyme dissolved cell wall
A) the centrifugal gram positive bacteria collecting liquid cultivation, after removing culture fluid, cleans 3 times with PBS, obtains bacterial sediment；
B) cell precipitation collected in step a) adds Sampleprotector, and soak, the RNase in degeneration thalline；
C) centrifugal bacterial sediment of collecting, removal Sampleprotector, be frozen in-80 refrigerators by gained thalline standby；
D) add lysozyme, hatch for 37 DEG C；
B. liquid nitrogen grinding removes cell wall
A) the centrifugal gram positive bacteria collecting liquid cultivation, after removing culture fluid, cleans 3 times with PBS, obtains bacterial sediment；
B) cell precipitation collected in step a) adds Sampleprotector, and soak, the RNase in degeneration thalline；
C) thalline of gained in step b) is moved in the middle of mortar, topple over liquid nitrogen, and after acutely pulverizing, collect powder and cold preservation is standby in-80 DEG C of refrigerators；
The pre-treatment of described gram negative bacteria, with reference to the method a of the pre-treatment of gram positive bacteria.
9. the method for extracting nucleic acid based on magnetic graphene nano-complex according to claim 3, it is characterised in that:
The pre-treatment of described animal tissue, comprises the steps:
A. taking fresh animal tissue, be cut into small pieces addition Sampleprotector, and soaks, the RNase in denatured tissues；
B. centrifugal segregation Sampleprotector, the existing use of gained tissue piece, or cold preservation is standby in-80 DEG C of refrigerators；
C. the tissue piece of gained in step b is moved in the middle of mortar, topple over liquid nitrogen, and after acutely pulverizing, collect powder and cold preservation is standby in-80 DEG C of refrigerators；
The pre-treatment of described animal blood, comprises the steps:
A. Objective extraction nucleic acid is in hemocyte
A) as target nucleic acid is present in hemocyte, then need first to collect hemocyte, make blood cell sedimentation by high speed centrifugation, remove supernatant, collect cell；
B) cell precipitation collected in step a) adds Sampleprotector, and soak, the RNase in degeneration hemocyte；
C) centrifugal bacterial sediment of collecting, removal Sampleprotector, be frozen in-80 refrigerators by gained hemocyte standby；
B. Objective extraction nucleic acid is in serum
The serum obtained after washed corpuscles, directly freezes pure, goes out Deproteinization then through going through lysis process.
10. the method for extracting nucleic acid based on magnetic graphene nano-complex according to claim 3, it is characterised in that:
The pre-treatment of described plant tissue, comprises the steps:
A. taking fresh plant tissue, be cut into small pieces addition Sampleprotector, and soaks, the RNase in denatured tissues；
B. centrifugal segregation Sampleprotector, the existing use of gained tissue piece, or cold preservation is standby in-80 DEG C of refrigerators；
C. the tissue piece of gained in step b is moved in the middle of mortar, topple over liquid nitrogen, and after acutely pulverizing, collect powder and cold preservation is standby in-80 DEG C of refrigerators.
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Cited By (9)
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| CN111549023A (en) * | 2020-04-22 | 2020-08-18 | 杭州医学院 | Magnetic nano material and application thereof in extraction of human whole blood genome DNA |
| CN112501161A (en) * | 2020-12-23 | 2021-03-16 | 华南师范大学 | Double-magnetic-particle-intervention DNA extraction and purification method |
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| CN106191036A (en) * | 2016-08-08 | 2016-12-07 | 吴江近岸蛋白质科技有限公司 | Rna binding protein is at the application extracted in nucleic acid and extracting method |
| CN106478832A (en) * | 2016-11-07 | 2017-03-08 | 安徽利民生物科技股份有限公司 | A kind of anti-fog haze protects the preparation method of lung ganoderan |
| CN106478832B (en) * | 2016-11-07 | 2019-01-25 | 安徽利民生物科技股份有限公司 | A kind of preparation method of anti-fog haze shield lung ganoderma lucidum polysaccharide |
| CN106755584A (en) * | 2017-01-12 | 2017-05-31 | 广州华弘生物科技有限公司 | A kind of ebb virus's detection kit and detection method |
| CN106928315B (en) * | 2017-04-07 | 2020-09-25 | 上海交通大学 | Nucleic acid binding protein extraction method based on graphene oxide |
| CN106928315A (en) * | 2017-04-07 | 2017-07-07 | 上海交通大学 | Nucleic acid binding protein extracting method based on graphene oxide |
| CN107014658A (en) * | 2017-05-27 | 2017-08-04 | 四川省肿瘤医院 | Extract the method and kit of free cell |
| CN107014658B (en) * | 2017-05-27 | 2023-08-25 | 四川省肿瘤医院 | Method and kit for extracting free cells |
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| CN107236730A (en) * | 2017-07-14 | 2017-10-10 | 博奥生物集团有限公司 | A kind of SPE material and its application in the enrichment and detection of nucleic acid |
| CN111549023A (en) * | 2020-04-22 | 2020-08-18 | 杭州医学院 | Magnetic nano material and application thereof in extraction of human whole blood genome DNA |
| CN112501161A (en) * | 2020-12-23 | 2021-03-16 | 华南师范大学 | Double-magnetic-particle-intervention DNA extraction and purification method |
| CN117046441A (en) * | 2023-08-14 | 2023-11-14 | 中国科学院生态环境研究中心 | Magnetic graphene oxide particles, preparation method and application thereof |
| CN117046441B (en) * | 2023-08-14 | 2024-04-02 | 中国科学院生态环境研究中心 | Magnetic graphene oxide particles, preparation method and application thereof |
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