CN104450681A - Method for extracting and purifying radix tetrastigme genome DNA - Google Patents
Method for extracting and purifying radix tetrastigme genome DNA Download PDFInfo
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- CN104450681A CN104450681A CN201410763362.6A CN201410763362A CN104450681A CN 104450681 A CN104450681 A CN 104450681A CN 201410763362 A CN201410763362 A CN 201410763362A CN 104450681 A CN104450681 A CN 104450681A
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- lespedezae buergeri
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Abstract
The invention discloses a method for extracting and purifying radix tetrastigme genome DNA. The method comprises the following steps: purifying radix tetrastigme leaves and then adding a cell nucleus lysis buffer; extracting with an extract liquid after a water bath at 65 DEG C, taking supernate, and adding isometric isopropanol sediments, so as to obtain a DNA sediment; sequentially washing the DNA sediment with 70% ethanol and 75% ethanol, drying, and adding TE buffer; and adding RNA digestive enzyme after dissolving the DNA sediment, and digesting in a water bath at 37 DEG C, so as to obtain the radix tetrastigme genome DNA. The radix tetrastigme genome DNA prepared by the method is good in quality, low in chlorophyll DNA and mitochondria DNA, and few in impurities such as polysaccharide, polyphenol, pigments and protein; the OD260/OD280 of the purified DNA reaches 1.954; the concentration is 866ng/muL; and the method can be applied to researches in genetic map building, genomic sequencing and the like.
Description
Technical field
The invention belongs to DNA extraction technical field of purification, particularly a kind of method of Radix Apioris Fortunei (Radix Lespedezae Buergeri) extracting genome DNA purifying.
Background technology
Radix Apioris Fortunei (Radix Lespedezae Buergeri) has another name called gold thread hoist, silk thread hangs Jin Zhong, the flat rattan of three leaves, stone mouse etc., be Vitaceae Tetrastigma plant Radix Apioris Fortunei (Radix Lespedezae Buergeri) Tetrastigma hemsleyanum Diels et Gilg., with block root or all herbal medicine, have clearing heat and detoxicating, dispelling pathogenic wind and eliminating phlegm, the effects such as promoting blood circulation and stopping pain, cure mainly children with high fever induced convulsions, Whooping cough, furunculosis ulcer, tuberculous lymphadenitis, dysentery, bronchitis, pneumonia, pharyngolaryngitis, hepatitis and viral meningitis, venomous snake bite is controlled in external application, tonsillitis, phlegmon, wound, mainly be distributed in Zhejiang, Jiangxi, Fujian, Hubei, Hunan, Guangdong, the areas such as Sichuan.
The extraction preparation of Radix Apioris Fortunei (Radix Lespedezae Buergeri) all has good therapeutic action to the multiple primary carcinoma such as esophagus cancer, cancer of the stomach, lung cancer, liver cancer, kidney, carcinoma of the pancreas, carcinoma of gallbladder, mammary cancer, cervical cancer, leukemia, lymphatic cancer, ovarian cancer, bladder cancer, prostate cancer, metastatic carcinoma etc.To all untoward reactions that chemotherapy of tumors brings, as low in appetite, vomiting, feel sick, epilation and oligoleukocythemia etc. all improve significantly, have alleviating pain effect to patients with terminal, effectively can also improve the immunologic function of human body.Before 30 years, China Radix Apioris Fortunei (Radix Lespedezae Buergeri) resource very abundant, wild Radix Apioris Fortunei (Radix Lespedezae Buergeri) except do medicinal except, also often cook dish eat.Along with the development of society, the deterioration of physical environment and people's unauthorized and excessive mining without limit, cause wild Radix Apioris Fortunei (Radix Lespedezae Buergeri) wild resource exhausted, endangered.Radix Apioris Fortunei (Radix Lespedezae Buergeri) is perennial plant, and secondary metabolite is many, and DNA extraction purifying is difficult, how to effectively utilize Radix Apioris Fortunei (Radix Lespedezae Buergeri) resource, its DNA of extraction purification, becomes the difficult problem that everybody pays close attention to.
Summary of the invention
The object of the invention is to overcome the shortcoming that exists in prior art with not enough, a kind of method of Radix Apioris Fortunei (Radix Lespedezae Buergeri) extracting genome DNA purifying is provided.
Object of the present invention is achieved through the following technical solutions: a kind of method of Radix Apioris Fortunei (Radix Lespedezae Buergeri) extracting genome DNA purifying, comprise the steps: to add Nuclei lysis buffer by after Radix Apioris Fortunei (Radix Lespedezae Buergeri) blade purifying, after 65 DEG C of water-bath 40min, adopt extraction liquid to extract, get supernatant liquor, add isopyknic isopropanol precipitating, obtain DNA throw out; Use 70% ethanol, 75% washing with alcohol DNA throw out successively, drying, adds TE damping fluid (Tris-EDTA damping fluid), adds RNA digestive ferment, in 37 DEG C of water-bath digestion 30min after DNA precipitate dissolves, obtain Radix Apioris Fortunei (Radix Lespedezae Buergeri) genomic dna, save backup in-20 DEG C.
Described Radix Apioris Fortunei (Radix Lespedezae Buergeri) blade preferably grinds to form granular Radix Apioris Fortunei (Radix Lespedezae Buergeri) blade;
Described Radix Apioris Fortunei (Radix Lespedezae Buergeri) blade is preferably adopted and is ground to form granular Radix Apioris Fortunei (Radix Lespedezae Buergeri) blade with the following method: clip Radix Apioris Fortunei (Radix Lespedezae Buergeri) is raw fresh blade then, washed with de-ionized water three times, mortar is put into after drying, add pvp (polyvinylpyrrolidone), in liquid nitrogen, be ground to granular Radix Apioris Fortunei (Radix Lespedezae Buergeri) blade.
Described purifying is preferably adopted and is carried out with the following method: Radix Apioris Fortunei (Radix Lespedezae Buergeri) blade is placed in the centrifuge tube filling nucleus refined solution and carries out purifying, centrifugal, gets precipitation, obtains the Radix Apioris Fortunei (Radix Lespedezae Buergeri) blade after purifying;
Described nucleus refined solution is become to be grouped into by following: 100mM Tris-HCl pH 8.0,20mM EDTA, 0.25M NaCl and 2wt% beta-mercaptoethanol.
Described Nuclei lysis buffer is become to be grouped into by following: 2wt%CTAB, 2wt%PVP, 100mM Tris-HCl pH8.0,25mM EDTA and 2.0M NaCl.
The chloroform-isoamyl alcohol of described extraction liquid to be volume ratio be 24:1.
The number of times of described extraction is preferably twice.
Described precipitation is adopted and is carried out with the following method: 30min placed by-20 DEG C of refrigerators.
The present invention has following advantage and effect relative to prior art: the present invention, by carrying out cracking by adding Nuclei lysis buffer after Radix Apioris Fortunei (Radix Lespedezae Buergeri) blade purifying, then by TE buffer solution and the digestion of RNA digestive ferment, obtains Radix Apioris Fortunei (Radix Lespedezae Buergeri) genomic dna.The quality of this Radix Apioris Fortunei (Radix Lespedezae Buergeri) genomic dna is good, its chlorophyll D NA and mitochondrial DNA content low, the impurity such as contained polysaccharide, polyphenol, pigment, protein are few, after purifying, the OD260/OD280 of DNA reaches 1.954, concentration is 866ng/ μ L, may be used for building the research of the aspect such as genetic map and gene order-checking.
Accompanying drawing explanation
Fig. 1 is the schematic flow sheet of the Radix Apioris Fortunei (Radix Lespedezae Buergeri) extracting genome DNA purifying of embodiment 1.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1
As shown in Figure 1, the invention provides a kind of method of Radix Apioris Fortunei (Radix Lespedezae Buergeri) extracting genome DNA purifying, comprise the steps:
Clip Radix Apioris Fortunei (Radix Lespedezae Buergeri) is raw fresh blade then, and washed with de-ionized water three times, puts into mortar after drying, add pvp, be ground to powder in liquid nitrogen, powder is proceeded in the centrifuge tube filling nucleus refined solution and carry out purifying (nucleus refined solution is become to be grouped into by following: 100mMTris-HCl pH 8.0, 20mM EDTA, 0.25M NaCl and 2wt% beta-mercaptoethanol), by centrifugal for the nucleus refined solution obtained, get precipitation, add preheated Nuclei lysis buffer, cracking digestion 60min is carried out in 65 DEG C of water-baths, and (Nuclei lysis buffer is become to be grouped into by following: 2wt%CTAB, 2wt%PVP, 100mM Tris-HCl (pH8.0), 25mM EDTA, 2.0M NaCl), get supernatant, add isopyknic chloroform-isoamyl alcohol (V:V=24:1) to extract, centrifugal, get supernatant, re-extract 1 time, merge supernatant, add isopyknic Virahol, light and slowly put upside down mixing, 30min placed by-20 DEG C of refrigerators, centrifugal, get precipitation, use 70% and 75% ethanol rinse successively each 1 time, room temperature is micro-dry, add Tris-EDTA buffer solution, add RNA digestive ferment again, 37 DEG C of water-bath digestion RNA 30min, obtain Radix Apioris Fortunei (Radix Lespedezae Buergeri) genomic dna, save backup in-20 DEG C.
The quality of the Radix Apioris Fortunei (Radix Lespedezae Buergeri) genomic dna obtained is good, its chlorophyll D NA and mitochondrial DNA content low, the impurity such as contained polysaccharide, polyphenol, pigment, protein are few, after purifying, the OD260/OD280 of DNA reaches 1.954, concentration is 866ng/ μ L, may be used for building the aspect such as genetic map and gene order-checking.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included in protection of the present invention.
Claims (8)
1. the method for a Radix Apioris Fortunei (Radix Lespedezae Buergeri) extracting genome DNA purifying, it is characterized in that, comprise the steps: to add Nuclei lysis buffer by after Radix Apioris Fortunei (Radix Lespedezae Buergeri) blade purifying, after 65 DEG C of water-bath 40min, adopt extraction liquid to extract, get supernatant liquor, add isopyknic isopropanol precipitating, obtain DNA throw out; Use 70% ethanol, 75% washing with alcohol DNA throw out successively, dry, add TE damping fluid, after DNA precipitate dissolves, add RNA digestive ferment, in 37 DEG C of water-bath digestion 30min, obtain Radix Apioris Fortunei (Radix Lespedezae Buergeri) genomic dna.
2. the method for Radix Apioris Fortunei (Radix Lespedezae Buergeri) extracting genome DNA purifying according to claim 1, is characterized in that, described Radix Apioris Fortunei (Radix Lespedezae Buergeri) blade is for grinding to form granular Radix Apioris Fortunei (Radix Lespedezae Buergeri) blade.
3. the method for Radix Apioris Fortunei (Radix Lespedezae Buergeri) extracting genome DNA purifying according to claim 2, it is characterized in that, described Radix Apioris Fortunei (Radix Lespedezae Buergeri) blade is adopted and is ground to form granular Radix Apioris Fortunei (Radix Lespedezae Buergeri) blade with the following method: clip Radix Apioris Fortunei (Radix Lespedezae Buergeri) is raw fresh blade then, washed with de-ionized water three times, mortar is put into after drying, add pvp, in liquid nitrogen, be ground to granular Radix Apioris Fortunei (Radix Lespedezae Buergeri) blade.
4. the method for Radix Apioris Fortunei (Radix Lespedezae Buergeri) extracting genome DNA purifying according to claim 1, it is characterized in that, described purifying is adopted and is carried out with the following method: Radix Apioris Fortunei (Radix Lespedezae Buergeri) blade is placed in the centrifuge tube filling nucleus refined solution and carries out purifying, centrifugal, get precipitation, obtain the Radix Apioris Fortunei (Radix Lespedezae Buergeri) blade after purifying.
5. the method for Radix Apioris Fortunei (Radix Lespedezae Buergeri) extracting genome DNA purifying according to claim 4, it is characterized in that, described nucleus refined solution is become to be grouped into by following: 100mM Tris-HClpH 8.0,20mM EDTA, 0.25MNaCl and 2wt% beta-mercaptoethanol.
6. the method for Radix Apioris Fortunei (Radix Lespedezae Buergeri) extracting genome DNA purifying according to claim 1, it is characterized in that, described Nuclei lysis buffer is become to be grouped into by following: 2wt%CTAB, 2wt%PVP, 100mM Tris-HCl pH8.0,25mM EDTA and 2.0MNaCl.
7. the method for Radix Apioris Fortunei (Radix Lespedezae Buergeri) extracting genome DNA purifying according to claim 1, is characterized in that, the chloroform-isoamyl alcohol of described extraction liquid to be volume ratio be 24:1.
8. the method for Radix Apioris Fortunei (Radix Lespedezae Buergeri) extracting genome DNA purifying according to claim 1, is characterized in that, described precipitation is adopted and carried out with the following method: 30min placed by-20 DEG C of refrigerators.
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| CN105713902A (en) * | 2016-04-14 | 2016-06-29 | 中国科学院寒区旱区环境与工程研究所 | Method for extracting total DNA (deoxyribonucleic acid) from eremophytes |
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| CN105713901A (en) * | 2016-03-31 | 2016-06-29 | 云南省农业科学院花卉研究所 | Improved method for extracting total DNA (deoxyribonucleic acid) from polysaccharide and polyphenol plant Rhododendron lapponicum |
| CN105713902A (en) * | 2016-04-14 | 2016-06-29 | 中国科学院寒区旱区环境与工程研究所 | Method for extracting total DNA (deoxyribonucleic acid) from eremophytes |
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Application publication date: 20150325 |