CN116855490A - Method for rapidly extracting genomic DNA of hibiscus maritima and application thereof - Google Patents
Method for rapidly extracting genomic DNA of hibiscus maritima and application thereof Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 34
- 235000005206 Hibiscus Nutrition 0.000 title description 9
- 235000007185 Hibiscus lunariifolius Nutrition 0.000 title description 9
- 241001075721 Hibiscus trionum Species 0.000 title 1
- 239000006166 lysate Substances 0.000 claims abstract description 20
- 241000196324 Embryophyta Species 0.000 claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 17
- 239000002244 precipitate Substances 0.000 claims abstract description 16
- 238000001179 sorption measurement Methods 0.000 claims abstract description 15
- 239000006228 supernatant Substances 0.000 claims abstract description 15
- 244000130592 Hibiscus syriacus Species 0.000 claims abstract description 12
- 235000018081 Hibiscus syriacus Nutrition 0.000 claims abstract description 12
- 239000011324 bead Substances 0.000 claims abstract description 11
- 238000000605 extraction Methods 0.000 claims abstract description 10
- 238000000227 grinding Methods 0.000 claims abstract description 10
- 239000000843 powder Substances 0.000 claims abstract description 9
- 238000002156 mixing Methods 0.000 claims abstract description 7
- 238000010438 heat treatment Methods 0.000 claims abstract description 6
- 238000001556 precipitation Methods 0.000 claims abstract description 6
- 239000000725 suspension Substances 0.000 claims abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 30
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- 239000003480 eluent Substances 0.000 claims description 10
- 239000004570 mortar (masonry) Substances 0.000 claims description 8
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 4
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 claims description 4
- 239000007983 Tris buffer Substances 0.000 claims description 4
- 230000002934 lysing effect Effects 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 235000011056 potassium acetate Nutrition 0.000 claims description 4
- 239000013049 sediment Substances 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 3
- 230000003993 interaction Effects 0.000 abstract description 2
- 210000001519 tissue Anatomy 0.000 description 15
- 241000218033 Hibiscus Species 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 6
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- 240000007124 Brassica oleracea Species 0.000 description 3
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 description 3
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 description 3
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 description 3
- 238000007400 DNA extraction Methods 0.000 description 3
- 108010052285 Membrane Proteins Proteins 0.000 description 3
- 102000018697 Membrane Proteins Human genes 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- GLXHFRGBLGCWIK-UHFFFAOYSA-N 2-[carbamimidoyl(methyl)amino]tetradecanoic acid Chemical compound C(CCCCCCCCCCC)C(C(=O)O)N(C)C(N)=N GLXHFRGBLGCWIK-UHFFFAOYSA-N 0.000 description 1
- 235000010149 Brassica rapa subsp chinensis Nutrition 0.000 description 1
- 235000000536 Brassica rapa subsp pekinensis Nutrition 0.000 description 1
- 241000499436 Brassica rapa subsp. pekinensis Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 102000007999 Nuclear Proteins Human genes 0.000 description 1
- 108010089610 Nuclear Proteins Proteins 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000633 nuclear envelope Anatomy 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of biological extraction. The invention provides a method for rapidly extracting genomic DNA of hibiscus syriacus, which comprises the steps of grinding plant tissues into powder, mixing with a lysate, and heating in water bath; adding the precipitate, and collecting supernatant; adding the adsorption liquid and the suspension magnetic beads to obtain a precipitate for later use; rinsing and eluting to obtain genome DNA solution, and storing. The method can rapidly, efficiently and stably extract the genomic DNA of the hibiscus syriacus through the interaction of three reagents, namely unique lysate, precipitation solution and adsorption solution.
Description
Technical Field
The invention relates to the technical field of biological extraction, in particular to a method for rapidly extracting genomic DNA of hibiscus maritima.
Background
The principle of the extraction method of plant genome DNA commonly used at present is as follows: and grinding different tissues of the plant under the condition of low-temperature liquid nitrogen, so as to ensure the stability of the DNA of the sample. The ion type surfactant such as sodium dodecyl creatine, cetyltrimethylammonium bromide and sodium dodecyl sulfate is added into the extraction buffer solution to dissolve cell membrane and nuclear membrane protein, depolymerize the nuclear protein and free DNA. The addition of an antioxidant or reducing agent beta-mercaptoethanol, dithiothreitol, reduces the activity of enzymes. Adding organic solvents such as phenol and chloroform to denature proteins, centrifuging for several times, extracting with organic solvents, precipitating DNA with absolute ethanol, air drying, and dissolving to obtain plant genome DNA. The traditional method has high DNA concentration, but can not ensure the quality of genome DNA, and greatly reduces the reaction efficiency of DNA. Meanwhile, the traditional method has complicated experimental steps and involves multiple uses of dangerous chemicals such as chloroform, so that the health of experimenters is affected, the environment is polluted, and the experimental cost is high.
Disclosure of Invention
Accordingly, the present invention is directed to a method for rapidly extracting genomic DNA of hibiscus maritima, which solves the above-mentioned problems.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for rapidly extracting genomic DNA of hibiscus syriacus comprises the following steps:
1) Sample grinding: placing fresh plant tissues into a mortar, adding liquid nitrogen into the mortar, and fully grinding into powder;
2) Lysing the cells: mixing plant tissue powder with the lysate, and heating in water bath;
3) Removing sediment: adding the precipitate into the mixture in the step 2), centrifuging, and taking the supernatant for later use;
4) Adsorption: adding adsorption liquid and suspension magnetic beads into the supernatant, and uniformly mixing; standing at room temperature for 8-10min, centrifuging, placing on a magnetic rack, removing supernatant, and standing precipitate;
5) Rinsing: adding rinsing liquid into the precipitate, standing on a magnetic rack for 55-65s, removing supernatant, and repeating the rinsing step for one time to obtain precipitate for later use;
6) Eluting DNA: adding eluent into the precipitate obtained in the step 5), uniformly mixing, standing for 5-6 minutes, placing on a magnetic rack for 1-2 minutes, transferring the eluted genomic DNA solution to a new container after the solution becomes clear, and preserving.
Preferably, preheating is required at 45-55deg.C before adding the lysate or eluent.
Preferably, the water bath heating temperature in the step 2) is 45-55 ℃, and the heating time is 25-35min.
Preferably, the adsorption liquid comprises 10-30% PEG8000 and 15-20% NaCl.
Preferably, the lysate comprises 0.4-0.6M NaCl,100-200mM Tris,50-100mM EDTA,2-5% SDS, pH5-6.
Preferably, the lysate further comprises 10mg/ml RNaseA.
Preferably, the precipitation solution comprises 5-6M potassium acetate solution; the rinsing liquid comprises 70% -80% absolute ethyl alcohol; the eluent comprises 1mM-3mM Tris-HCl, pH7.5-8.5.
Preferably, the magnetic beads have a size of 10-100 nm.
It is another object of the present invention to apply the above method to one or more 20mg or 100mg fresh plant sample DNA purifications.
Compared with the prior art, the invention has the following beneficial effects:
each reagent in the kit can exert the unique specificity. The invention can extract genome DNA rapidly, efficiently and stably through the interaction of three reagents, namely the unique lysate, the impurity removing liquid and the DNA extraction aid. The components in the lysate synergistically act to destroy cell walls, rapidly infiltrate cells, lyse cells, release genomic DNA and maintain their stability. On one hand, the DNA extraction aid can effectively remove polyphenol, polysaccharide, terpenoid, membrane protein and the like released by plant cells after the plant cells are cracked. Meanwhile, the DNA extraction aid can protect genomic DNA and prevent DNA dimerization caused by excessively long reaction time. Finally, the impurity removing liquid quickly dissolves the membrane protein and damages the structure of the protein.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is an electrophoretogram of genomic DNA obtained by the method of the present invention under 1% agarose gel; in FIG. 1, 1-12 are maritime shrubalthea, 13-16 are other species (cabbage) controls, M is Marker, hindIII cleaves lambda DNA.
FIG. 2 shows the measurement results of genomic DNA obtained by the method of example 2 at Nanodrop 2000; in the figure, 1-12 are different seashore shrubalthea. M1 is lamdaDNA, M2 is 1 KBDNAMaroker.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
A method for rapidly extracting genomic DNA of hibiscus syriacus comprises the following steps: 1) Sample grinding: taking plant tissue of 100mg of fresh tissue or 20mg of dry weight tissue, putting the plant tissue into a mortar, adding liquid nitrogen into the mortar, and fully grinding the plant tissue into powder; 2) Lysing the cells: 600ul of lysate (preheated at 50 ℃) was added to a 2ml EP tube and 50-100mg of tissue powder was immediately mixed. The EP tube was water-bath at 50℃for 30 minutes; 3) Removing sediment: 200ul of the precipitate is added and mixed upside down. The EP tube was centrifuged at 5000g for 10min. Transfer supernatant about 400ul to a new 22ml ep tube; 4) Adsorption: 400ul of adsorption solution and 20ul of magnetic beads (room temperature) were added and the mixture was poured into a tube 20 times. Place at room temperature for 10min, centrifuge gently for 5s at 5000g, place on a magnet rack for about 3min (until the solution becomes clear). Removing the supernatant without touching the precipitate; 5) Rinsing: to the EP tube, 1ml of 70% rinse solution was added and the EP tube was placed on a magnet rack for about 60s. The supernatant was removed without touching the pellet. And repeated once more. Uncapping, and allowing the magnetic beads to air dry for 1min; 6) Eluting DNA: 80ul of eluent (preheated at 50 ℃) was added to the EP tube and the light EP tube was removed from the suspension. After the EP tube was gently centrifuged, it was placed on a magnetic rack for about 2min (until the solution cleared). Transfer 50-100ul of the eluted genomic DNA solution to a new 1.5ml EP tube, -20 preserve.
Further, the method comprises the steps of: lysate, precipitation solution, adsorption solution, rinse solution, eluent, magnetic beads, magnetic rack, collection pipe, etc.; the adsorption liquid comprises 10-30% PEG8000 and 15-20% NaCl.
Further, the lysate comprises 0.4-0.6M NaCl,100-200mM Tris,50-100mM EDTA,2-5% SDS, pH5-6.
Further, the lysate also included 10mg/ml RNaseA.
Further, the precipitation solution comprises 5-6M potassium acetate solution.
Further, the rinsing liquid comprises 70% -80% absolute ethyl alcohol.
Further, the eluent comprises 1mM-3mM Tris-HCl, pH7.5-8.5.
Further, the magnetic beads have the size of 10-100 nanometers, and the magnetic rack is matched with the collecting pipe, and can be 1.5ml or 300 ul; 1.5ml was applied to a single tube and 300ul to a 96 well plate.
Example 1
A method for rapidly extracting genomic DNA of hibiscus syriacus comprises the following steps:
1) Sample grinding: taking plant tissue of 100mg of fresh tissue or 20mg of dry weight tissue, putting the plant tissue into a mortar, adding liquid nitrogen into the mortar, and fully grinding the plant tissue into powder;
2) Lysing the cells: 600ul of lysate (preheated at 50 ℃) was added to a 2ml EP tube and 50-100mg of tissue powder was immediately mixed. The EP tube was water-bath at 50℃for 30 minutes;
3) Removing sediment: 200ul of the precipitate is added and mixed upside down. The EP tube was centrifuged at 5000g for 10min. Transfer supernatant about 400ul to a new 22ml ep tube;
4) Adsorption: 400ul of adsorption solution and 20ul of magnetic beads (room temperature) were added and the mixture was poured into a tube 20 times. Place at room temperature for 10min, centrifuge gently for 5s at 5000g, place on a magnet rack for about 3min (until the solution becomes clear). Removing the supernatant without touching the precipitate;
5) Rinsing: to the EP tube, 1ml of 70% rinse solution was added and the EP tube was placed on a magnet rack for about 60s. The supernatant was removed without touching the pellet. And repeated once more. Uncapping, and allowing the magnetic beads to air dry for 1min;
6) Eluting DNA: 80ul of eluent (preheated at 50 ℃) was added to the EP tube and the light EP tube was removed from the suspension. After the EP tube was gently centrifuged, it was placed on a magnetic rack for about 2min (until the solution cleared). Transfer 50-100ul of the eluted genomic DNA solution to a new 1.5ml EP tube, -20 preserve.
The adsorption solution used in example 1 included 10%PEG8000, 15%NaCl. The lysate included 0.4M NaCl,100mM Tris,80mM EDTA,3% SDS, pH5.3. The lysate also included 10mg/ml RNaseA. The precipitation solution included 5M potassium acetate solution. The rinse solution included 75% absolute ethanol. The eluate contained 2mM Tris-HCl, pH8.2.
As shown in FIG. 1, the method of example 1 is used for extracting genomic DNAs of 12 maritime shrubs and 4 cabbages, and the method can be used for extracting genomic DNAs of the maritime shrubes and the cabbages, and has the advantages of obvious main band, good DNA quality and high concentration. The slightly lower DNA concentration of the hibiscus maritima compared with the Chinese cabbage is probably related to the more complex secondary metabolites of the hibiscus maritima leaves and the great extraction difficulty.
Example 2
The genomic DNA of hibiscus maritima was extracted by the method of example 1 (no RNase was added to the lysate) and by the CTAB method, corresponding to samples 1-6,7-12 of FIG. 2, respectively. From the figure, the hibiscus littoralis DNA can be extracted by a CTAB method, but the concentration is low, the impurities are more, no obvious main band exists, and the DNA quality is poor; the hibiscus littoralis DNA extracted by the method has an obvious main band, and has high concentration, less impurities and better quality compared with the DNA extracted by the CTAB method.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant points refer to the description of the method section.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (9)
1. A method for rapidly extracting genomic DNA of hibiscus syriacus is characterized by comprising the following steps:
1) Sample grinding: placing fresh plant tissues into a mortar, adding liquid nitrogen into the mortar, and fully grinding into powder;
2) Lysing the cells: mixing plant tissue powder with the lysate, and heating in water bath;
3) Removing sediment: adding the precipitate into the mixture in the step 2), centrifuging, and taking the supernatant for later use;
4) Adsorption: adding adsorption liquid and suspension magnetic beads into the supernatant, and uniformly mixing; standing at room temperature for 8-10min, centrifuging, placing on a magnetic rack, removing supernatant, and standing precipitate;
5) Rinsing: adding rinsing liquid into the precipitate, standing on a magnetic rack for 55-65s, removing supernatant, and repeating the rinsing step for one time to obtain precipitate for later use;
6) Eluting DNA: adding eluent into the precipitate obtained in the step 5), uniformly mixing, standing, placing on a magnetic rack, transferring the eluted genomic DNA solution to a new container after the solution becomes clear, and preserving.
2. The method for rapid extraction of genomic DNA of hibiscus syriacus according to claim 1 wherein the lysate or eluent is preheated at 45-55deg.C.
3. The method for rapidly extracting genomic DNA from hibiscus syriacus according to claim 1 wherein said step 2) is carried out at a water bath heating temperature of 45-55 ℃ for 25-35min.
4. A method for rapid extraction of genomic DNA of shore a according to any one of claims 1-3, wherein the adsorption solution comprises 10-30% peg8000, 15-20% nacl.
5. A method for rapid extraction of genomic DNA of maritime shrubalthea according to any one of claims 1-3, wherein the lysate comprises 0.4-0.6m naci, 100-200mm tris,50-100mm edta,2-5% sds, ph5-6.
6. The method for rapid extraction of genomic DNA of hibiscus syriacus according to claim 5 wherein the lysate further comprises 10mg/ml RNaseA.
7. A method for rapid extraction of genomic DNA of shore a according to any one of claims 1-3, wherein the precipitation solution comprises 5-6M potassium acetate solution; the rinsing liquid comprises 70% -80% absolute ethyl alcohol; the eluent comprises 1-3mM Tris-HCl, pH7.5-8.5.
8. A method for rapid extraction of genomic DNA of shore a according to any one of claims 1-3, wherein the magnetic beads have a size of 10-100 nm.
9. Use of the method according to any one of claims 1-8, characterized in that: the method is applied to the use of one or more 20mg or 100mg fresh plant sample DNA purification.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202310692173.3A CN116855490A (en) | 2023-06-13 | 2023-06-13 | Method for rapidly extracting genomic DNA of hibiscus maritima and application thereof |
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| CN202310692173.3A CN116855490A (en) | 2023-06-13 | 2023-06-13 | Method for rapidly extracting genomic DNA of hibiscus maritima and application thereof |
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