CN110592077B - DNA extracting solution of pedunculate herpetospermum seeds, extraction kit, application of DNA extracting solution and extraction method of DNA extracting solution - Google Patents
DNA extracting solution of pedunculate herpetospermum seeds, extraction kit, application of DNA extracting solution and extraction method of DNA extracting solution Download PDFInfo
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- 238000000605 extraction Methods 0.000 title claims abstract description 21
- 241000530044 Herpetospermum Species 0.000 title claims description 22
- 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 abstract description 13
- 241000530047 Herpetospermum pedunculosum Species 0.000 claims abstract description 13
- 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 abstract description 9
- 238000007400 DNA extraction Methods 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 38
- 239000006228 supernatant Substances 0.000 claims description 34
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 31
- 238000002156 mixing Methods 0.000 claims description 29
- 239000011259 mixed solution Substances 0.000 claims description 22
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 19
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 16
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 16
- 239000002244 precipitate Substances 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 14
- 239000000843 powder Substances 0.000 claims description 12
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 238000005406 washing Methods 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 9
- 235000019441 ethanol Nutrition 0.000 claims description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims description 8
- 239000007984 Tris EDTA buffer Substances 0.000 claims description 7
- 238000005119 centrifugation Methods 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 abstract 2
- 239000011543 agarose gel Substances 0.000 description 7
- 230000000052 comparative effect Effects 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- FGUUSXIOTUKUDN-IBGZPJMESA-N C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 Chemical compound C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 FGUUSXIOTUKUDN-IBGZPJMESA-N 0.000 description 5
- 238000000227 grinding Methods 0.000 description 5
- GNFTZDOKVXKIBK-UHFFFAOYSA-N 3-(2-methoxyethoxy)benzohydrazide Chemical compound COCCOC1=CC=CC(C(=O)NN)=C1 GNFTZDOKVXKIBK-UHFFFAOYSA-N 0.000 description 4
- 238000010200 validation analysis Methods 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 229920000459 Nitrile rubber Polymers 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 208000020694 gallbladder disease Diseases 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 208000019423 liver disease Diseases 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
- 239000011780 sodium chloride Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
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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
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Abstract
The invention provides a herpetospermum pedunculosum seed DNA extracting solution, an extraction kit, an application and an extraction method thereof, and relates to the technical field of DNA extraction, wherein the DNA extracting solution comprises 100 mmol/L of Tris-HCl, 24 mmol/L of EDTA and 2.4% of SDS (sodium dodecyl sulfate) by mass-volume ratio, and the pH value of the Tris-HCl is 8.0.
Description
Technical Field
The invention belongs to the technical field of DNA extraction, and particularly relates to a pedunculate herpetospermum seed DNA extracting solution, an extraction kit, application of the extracting kit and an extraction method of the extracting kit.
Background
The Herpetospermum pedunculosum (Herpetospermum) is a Herpetospermum plant of Cueurbitaceae Herpetospermum, grows in alpine mountains with the elevation of 2300-3000 m, is mainly distributed in Tibet, Yunnan and Sichuan of China, and is also distributed in India and Nipol. The species has important medicinal value in treating liver and gallbladder diseases, and is widely used in Tibetan medicine.
When carrying out omics research on the herpetospermum pedunculosum, the extraction of seed genome DNA is one of necessary steps. Many other methods for extracting plant tissue genome DNA are disclosed at present, but the methods are mostly directed at extracting DNA of plant tissue materials with high contents of polyphenol and polysaccharide, are not applicable to extracting DNA of herpetospermum pedunculosum seeds with high content of grease, and have low purity of extracted DNA and no band in DNA electrophoresis.
Disclosure of Invention
In view of the above, the present invention aims to provide a pedunculate herpetospermum seed DNA extract, an extraction kit, and applications and an extraction method thereof, wherein the extracted DNA has high purity, no tailing in electrophoresis bands, and low cost.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a herpetospermum pedunculosum seed DNA extracting solution which comprises 100 mmol/L of Tris-HCl, 24 mmol/L of EDTA and 2.4% of SDS by mass-volume ratio, wherein the pH value of the Tris-HCl is 8.0.
The invention also provides a kit for extracting the DNA of the seeds of the pedunculate herpetospermum seeds, which comprises the DNA extracting solution.
The invention also provides application of the DNA extracting solution or the DNA extracting kit in extracting the DNA of the seeds of the pedunculate herpetospermum seeds.
The invention also provides a method for extracting the DNA of the seeds of the pedunculate herpetospermum seeds, which comprises the following steps:
(1) mixing the ground powder of the pedunculate herpetospermum seeds by using liquid nitrogen with the preheated DNA extracting solution at 65 ℃, and then carrying out water bath at 65 ℃ for 35min to obtain homogenate;
(2) mixing the homogenate with the mixed solution in equal volume, standing for 8min, and centrifuging at 14000rpm at 4 ℃ for 10 min; the mixed solution comprises the following components in a volume ratio of 24: 1 chloroform and isoamyl alcohol;
(3) mixing the centrifuged supernatant in the step (2) with the mixed solution, standing for 8min, and centrifuging at 14000rpm at 4 ℃ for 10 min;
(4) mixing the centrifuged supernatant in the step (3) with absolute ethyl alcohol, standing for 15min, centrifuging at 8000rpm at 4 ℃ for 10min, and removing the supernatant;
(5) mixing and washing the precipitate obtained after the centrifugation in the step (4) with ethanol with the volume ratio of 70%, centrifuging at 12000rpm at 4 ℃ for 4min, and removing the supernatant;
(6) and (3) drying the precipitate obtained after centrifugation in the step (5), mixing with 8mM NaOH solution, dissolving for 8-12 h, and adjusting the pH to 7-8 by using TE buffer solution.
Preferably, the mass-to-volume ratio of the powder and the DNA extraction solution in the step (1) is 0.04 g: 700. mu. L.
Preferably, during the water bath in the step (1), the mixture is inverted and mixed every 10 min.
Preferably, the washing in step (5) is performed 2 times.
The invention provides a herpetospermum pedunculosum seed DNA extracting solution, which improves the concentration ratio of the extracting solution, so that the herpetospermum pedunculosum DNA extracting efficiency is higher, and the herpetospermum pedunculosum seed DNA extracting solution can be prepared into a herpetospermum pedunculosum DNA extracting kit.
The invention also provides a method for extracting DNA by using the DNA extracting solution or the DNA extracting kit, and in the method, in addition to the application of the DNA extracting solution, the method also comprises the steps of dissolving DNA precipitate by using an NAOH solution, and adjusting the pH value by using a TE solution, so that the extracted DNA has fewer impurities, high purity, clear bands and no dragging.
Drawings
FIG. 1 is a drawing of agarose gel of DNA extracted in example 1 and comparative examples 2 to 4;
FIG. 2 is an agarose gel image of DNA extracted in comparative example 1.
Detailed Description
The invention provides a herpetospermum pedunculosum seed DNA extracting solution which comprises 100 mmol/L Tris-HCl, 24 mmol/L EDTA and 2.4% SDS by mass-volume ratio, wherein the pH value of the Tris-HCl is 8.0.
The invention also provides a kit for extracting the DNA of the seeds of the pedunculate herpetospermum seeds, which comprises the DNA extracting solution.
The invention also provides application of the DNA extracting solution or the DNA extracting kit in extracting the DNA of the seeds of the pedunculate herpetospermum seeds.
The invention also provides a method for extracting the DNA of the seeds of the pedunculate herpetospermum seeds, which comprises the following steps:
(1) mixing the ground powder of the pedunculate herpetospermum seeds by using liquid nitrogen with the preheated DNA extracting solution at 65 ℃, and then carrying out water bath at 65 ℃ for 35min to obtain homogenate;
(2) mixing the homogenate with the mixed solution in equal volume, standing for 8min, and centrifuging at 14000rpm at 4 ℃ for 10 min; the mixed solution comprises the following components in a volume ratio of 24: 1 chloroform and isoamyl alcohol;
(3) mixing the centrifuged supernatant in the step (2) with the mixed solution, standing for 8min, and centrifuging at 14000rpm at 4 ℃ for 10 min;
(4) mixing the centrifuged supernatant in the step (3) with absolute ethyl alcohol, standing for 15min, centrifuging at 8000rpm at 4 ℃ for 10min, and removing the supernatant;
(5) mixing and washing the precipitate obtained after the centrifugation in the step (4) with ethanol with the volume ratio of 70%, centrifuging at 12000rpm at 4 ℃ for 4min, and removing the supernatant;
(6) and (3) drying the precipitate obtained after centrifugation in the step (5), mixing with 8mM NaOH solution, dissolving for 8-12 h, and adjusting the pH to 7-8 by using TE buffer solution.
The mass-to-volume ratio of the powder to the DNA extract in step (1) of the present invention is preferably 0.04 g: 700. mu. L.
In the water bath process in the step (1), the mixture is preferably inverted and mixed once every 10 min.
The washing in step (5) of the present invention is preferably carried out 2 times.
The following examples are provided to describe the extraction solution and the extraction kit for the DNA of the pedunculate herpetospermum seeds, and the application and the extraction method thereof in detail, but they should not be construed as limiting the scope of the present invention.
Example 1
a) Grinding 0.04g of the seed material of the pedunculate herpetospermum seeds with the seed coat removed by liquid nitrogen, and transferring the powder to a centrifugal tube of 1.5m L;
b) adding extract preheated at 700 mu L65 ℃ into the centrifugal tube in the step a), carrying out water bath at 65 ℃ for 35min, and reversing the centrifugal tube every 10min for uniformly mixing, wherein the extract comprises 100 mmol/L Tris-HCl (pH8.0), 24 mmol/L EDTA and 2.4% SDS;
c) adding 700 mu L mixed solution into the homogenate liquid in the step b), lightly shaking, standing for 8min, and centrifuging for 10min at 14000rpm at 4 ℃, wherein the mixed solution comprises chloroform and isoamylol in a volume ratio of 24: 1;
d) transferring the supernatant obtained in the step c) to a new 1.5m L centrifuge tube, adding 600 mu L of mixed solution, slightly reversing and uniformly mixing, standing for 8min, and centrifuging for 10min at 14000rpm at 4 ℃;
e) transferring the supernatant obtained in the step d) to a new 1.5m L centrifuge tube, adding 500 mu L of absolute ethyl alcohol, standing for 15min, centrifuging at 4 ℃ and 8000rpm for 10min, and removing the supernatant;
f) washing the precipitate obtained in step e) with 500. mu. L70% ethanol, centrifuging at 12000rpm at 4 deg.C for 4min, discarding the supernatant
g) Repeating step f) once;
h) the precipitate obtained in step g) was air dried, dissolved overnight in 50. mu. L8 mM NaOH and adjusted to pH between 7 and 8 with 50. mu. L TE buffer.
The DNA obtained was subjected to agarose gel assay, specifically bands shown by reference numerals 31 to 40 in FIG. 1.
Comparative example 1
a) Grinding 0.04g of the seed material of the pedunculate herpetospermum seeds with the seed coats removed by liquid nitrogen, and transferring the powder to a 2m L centrifugal tube;
b) adding an extracting solution preheated at 1000 mu L65 ℃ and 3 mu Lβ -mercaptoethanol into the powder obtained in the step a), shaking for 60min in a water bath kettle at 65 ℃, and reversing and uniformly mixing a centrifugal tube every 10min, wherein the extracting solution comprises 100 mol/L Tris-HCl (pH8.0), 1.4 mol/L NaCl, 20 mol/L EDTA and 2% CTAB;
c) adding the homogenate obtained in the step b) into the mixed liquid with the same volume, slowly turning over the centrifugal tube for 10min, fully and uniformly mixing, centrifuging at 4 ℃ and 10000rpm for 10min, taking the supernatant, and transferring the supernatant to a new centrifugal tube of 1.5m L, wherein the mixed liquid 1 comprises chloroform and isoamylol with the volume ratio of 24: 1;
d) repeating step c);
e) adding 2/3 volumes of-20 deg.C pre-cooled isopropanol into the supernatant obtained in d), and gently shaking to precipitate at-20 deg.C for 40 min;
f) centrifuging the mixed solution obtained in the step e) at 4 ℃ and 10000rpm for 6min, removing the supernatant, adding 500 mu L75% ethanol, centrifuging at 4 ℃ and 10000rpm for 4min, and removing the supernatant;
g) repeating the step f), and airing the precipitate;
h) dissolving the precipitate obtained in g) in 50 μ L8 mM NaOH overnight, adjusting pH to 7-8 with 50 μ L TE, centrifuging at 12000rpm at 4 deg.C for 10min, and collecting supernatant to new 1.5m L centrifuge tube.
The DNA obtained was subjected to agarose gel validation as shown in FIG. 2.
Comparative example 2
a) Grinding 0.04g of the seed material of the pedunculate herpetospermum seeds with the seed coat removed by liquid nitrogen, and transferring the powder to a centrifugal tube of 1.5m L;
b) adding extract preheated at 700 mu L65 ℃ into the centrifugal tube in the step a), carrying out water bath at 65 ℃ for 35min, and reversing the centrifugal tube every 10min for uniformly mixing, wherein the extract comprises 100 mmol/L Tris-HCl (pH8.0), 24 mmol/L EDTA and 2.4% SDS;
c) adding 700 mu L mixed solution into the homogenate liquid in the step b), lightly shaking, standing for 8min, and centrifuging for 10min at 14000rpm at 4 ℃, wherein the mixed solution comprises chloroform and isoamylol in a volume ratio of 24: 1;
d) transferring the supernatant obtained in the step c) to a new 1.5m L centrifuge tube, adding 600 mu L of mixed solution, slightly reversing and uniformly mixing, standing for 8min, and centrifuging for 10min at 14000rpm at 4 ℃;
e) transferring the supernatant obtained in the step d) to a new 1.5m L centrifuge tube, adding 500 mu L of absolute ethyl alcohol, standing for 15min, centrifuging at 4 ℃ and 8000rpm for 10min, and removing the supernatant;
f) washing the precipitate obtained in step e) with 500. mu. L70% ethanol, centrifuging at 12000rpm at 4 deg.C for 4min, discarding the supernatant
g) Repeating step f) once;
h) the pellet obtained in step g) was air dried and dissolved in 50. mu. L TE buffer.
The obtained DNA was subjected to agarose gel validation, and the bands shown by the reference numerals 1 to 10 in FIG. 1 were shown.
Comparative example 3
a) Grinding 0.04g of the seed material of the pedunculate herpetospermum seeds with the seed coat removed by liquid nitrogen, and transferring the powder to a centrifugal tube of 1.5m L;
b) adding an extracting solution preheated at 700 mu L65 ℃ into the centrifugal tube in the step a), carrying out water bath at 65 ℃ for 35min, and reversing the centrifugal tube every 10min for uniformly mixing, wherein the extracting solution comprises 10 mmol/L Tris-HCl (pH7.8), 5 mmol/L EDTA and 0.5% SDS;
c) adding 700 mu L mixed solution into the homogenate liquid in the step b), lightly shaking, standing for 8min, and centrifuging for 10min at 14000rpm at 4 ℃, wherein the mixed solution comprises chloroform and isoamylol in a volume ratio of 24: 1;
d) transferring the supernatant obtained in the step c) to a new 1.5m L centrifuge tube, adding 600 mu L of mixed solution, slightly reversing and uniformly mixing, standing for 8min, and centrifuging for 10min at 14000rpm at 4 ℃;
e) transferring the supernatant obtained in the step d) to a new 1.5m L centrifuge tube, adding 500 mu L of absolute ethyl alcohol, standing for 15min, centrifuging at 4 ℃ and 8000rpm for 10min, and removing the supernatant;
f) washing the precipitate obtained in step e) with 500. mu. L70% ethanol, centrifuging at 12000rpm at 4 deg.C for 4min, discarding the supernatant
g) Repeating step f) once;
h) the precipitate obtained in step g) was air dried, dissolved overnight in 50. mu. L8 mM NaOH and adjusted to pH between 7 and 8 with 50. mu. L TE buffer.
The obtained DNA was subjected to agarose gel validation, and the bands shown by the reference numerals 11 to 20 in FIG. 1 were shown.
Comparative example 4
a) Grinding 0.04g of the seed material of the pedunculate herpetospermum seeds with the seed coat removed by liquid nitrogen, and transferring the powder to a centrifugal tube of 1.5m L;
b) adding an extracting solution preheated at 700 mu L65 ℃ into the centrifugal tube in the step a), carrying out water bath at 65 ℃ for 35min, and reversing the centrifugal tube every 10min for uniformly mixing, wherein the extracting solution comprises 10 mmol/L Tris-HCl (pH7.8), 5 mmol/L EDTA and 0.5% SDS;
c) adding 700 mu L mixed solution into the homogenate liquid in the step b), lightly shaking, standing for 8min, and centrifuging for 10min at 14000rpm at 4 ℃, wherein the mixed solution comprises chloroform and isoamylol in a volume ratio of 24: 1;
d) transferring the supernatant obtained in the step c) to a new 1.5m L centrifuge tube, adding 600 mu L of mixed solution, slightly reversing and uniformly mixing, standing for 8min, and centrifuging for 10min at 14000rpm at 4 ℃;
e) transferring the supernatant obtained in the step d) to a new 1.5m L centrifuge tube, adding 500 mu L of absolute ethyl alcohol, standing for 15min, centrifuging at 4 ℃ and 8000rpm for 10min, and removing the supernatant;
f) washing the precipitate obtained in step e) with 500. mu. L70% ethanol, centrifuging at 12000rpm at 4 deg.C for 4min, discarding the supernatant
g) Repeating step f) once;
h) the pellet obtained in step g) was air dried and dissolved in 50. mu. L TE buffer.
The obtained DNA was subjected to agarose gel assay, and the bands are shown by reference numerals 21 to 30 in FIG. 1.
The invention provides a herpetospermum pedunculosum seed DNA extracting solution, an extraction kit, application of the extraction kit and an extraction method of the extraction kit, which can be used for extracting herpetospermum pedunculosum seed DNA with high fat content, and the extracted DNA has the advantages of high purity, uniform strip, no dragging and low cost.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (4)
1. A method for extracting the DNA of the seeds of the pedunculate herpetospermum seeds is characterized by comprising the following steps:
(1) mixing powder of ground herpetospermum pedunculosum seeds by using liquid nitrogen with the DNA extracting solution preheated at 65 ℃, and then carrying out water bath at 65 ℃ for 35min to obtain homogenate, wherein the DNA extracting solution comprises 100 mmol/L Tris-HCl, 24 mmol/L EDTA and SDS with the mass-volume ratio of 2.4 percent, and the pH value of the Tris-HCl is 8.0;
(2) mixing the homogenate with the mixed solution in equal volume, standing for 8min, and centrifuging at 14000rpm at 4 ℃ for 10 min; the mixed solution comprises the following components in a volume ratio of 24: 1 chloroform and isoamyl alcohol;
(3) mixing the centrifuged supernatant in the step (2) with the mixed solution, standing for 8min, and centrifuging at 14000rpm at 4 ℃ for 10 min;
(4) mixing the centrifuged supernatant in the step (3) with absolute ethyl alcohol, standing for 15min, centrifuging at 8000rpm at 4 ℃ for 10min, and removing the supernatant;
(5) mixing and washing the precipitate obtained after the centrifugation in the step (4) with ethanol with the volume ratio of 70%, centrifuging at 12000rpm at 4 ℃ for 4min, and removing the supernatant;
(6) and (3) drying the precipitate obtained after centrifugation in the step (5), mixing with 8mM NaOH solution, dissolving for 8-12 h, and adjusting the pH to 7-8 by using TE buffer solution.
2. The extraction method according to claim 1, wherein the mass-to-volume ratio of the powder to the DNA extraction solution in step (1) is 0.04 g: 700. mu. L.
3. The extraction method according to claim 1 or 2, wherein the water bath in step (1) is performed by mixing the mixture by inversion every 10 min.
4. The extraction method according to claim 1, wherein the washing of step (5) is performed 2 times.
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| CN104017801A (en) * | 2014-06-20 | 2014-09-03 | 湖北省农业科学院经济作物研究所 | Micro-extraction method for extracting DNA (Deoxyribonucleic Acid) from single pepper seed |
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