CN102643800B - Method for extracting plant deoxyribonucleic acid (DNA) and special kit thereof - Google Patents
Method for extracting plant deoxyribonucleic acid (DNA) and special kit thereof Download PDFInfo
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Abstract
The invention aims to provide a method for extracting plant deoxyribonucleic acid (DNA) and a special kit thereof. The special kit comprises a buffer solution A which consists of solute and solvent, wherein the final concentrations of the solute and the solute in the buffer solution A are as follows: 4-6mmol.L<-1> of ethylene diamine tetraacetic acid, 0.2-0.3M of NaCl, 1-3g/100ml of polyvinyl pyrrolidone, 1M of Tris-HCl of which the pH value is 8.0 and 8-12ml/100ml of buffer solution; and the solvent is water. The plant DNA extracted by a mCTAB method has the advantages of being high in yield, better in quality, high in PCR (polymerase chain reaction) amplification success rate, low in cost and good in generality, can satisfy the requirements of extracting DNA in molecular biological studies and is especially suitable for researchers with insufficient cost of scientific research and extraction of the plant DNA on a large scale.
Description
Technical field
The present invention relates to a kind of method and dedicated kit thereof that extracts DNA of plants.
Background technology
DNA is the basic genetic material of plant, is the carrier of plant genetic information.A certain amount of and high-quality DNA sample is the basis of carrying out restriction enzyme digestion, pcr amplification, molecular hybridization, analysis of genetic polymorphisms and the biological study of genomics equimolecular.Therefore, how to obtain a certain amount of and high-quality DNA sample and seem very important.
Vegetable cell has cell walls, contain the more secondary metabolites such as polysaccharide, and in different plants, kind and the content difference of secondary metabolite is very large, sometimes kind and the content of this raw metabolite of kindred plant Different Organs or tissue are also different, and causing obtaining high-quality DNA has certain difficulty.DNA extraction method for certain specialized species optimization is not necessarily applicable to other species.Traditional CT AB method is maximum DNA extraction method of applying at present, but due to differences such as vegetable material chemical composition, weave constructions, the extraction effect of traditional CTAB method is sometimes not good enough, is subject to certain restrictions in the use.Therefore in the urgent need to a kind of easy, efficient, economy and the good Method of Plant DNA Extraction of versatility.
Summary of the invention
An object of the present invention is to provide a kind of method and dedicated kit thereof that extracts DNA of plants.
Provided by the present invention for extracting the test kit of DNA of plants, comprise buffer A; Described buffer A is by solute and solvent composition; Described solute and the final concentration in described buffer A thereof are as follows: disodium ethylene diamine tetraacetate 4-6mmolL
-1, NaCl 0.2-0.3M, Polyvinylpyrolidone (PVP) 1-3g/100ml; Concentration is the Tris-HCl damping fluid 8-12ml/100ml that 1M and pH are 8.0; Described solvent is water.
In mentioned reagent box, also can comprise buffer B; Described buffer B is by solute and solvent composition: described solute and the final concentration in described buffer B thereof are as follows: concentration is the Tris-HCl damping fluid 8-12ml/100ml that 1M and pH are 8.0, disodium ethylene diamine tetraacetate 20-30mmolL
-1, NaCl 1.2-1.6M, CTAB2.5-3.5g/100ml, sodium metabisulfite 0.5-1.5g/100ml, sodium ascorbate 0.5-1.5g/100ml, Polyvinylpyrolidone (PVP) 1-3g/100ml, beta-mercaptoethanol 80-120ul/100ml; Described solvent is water.
In above-mentioned arbitrary described test kit, described buffer A specifically can be as follows: described solute and the final concentration in described buffer A thereof are as follows: disodium ethylene diamine tetraacetate 5mmolL
-1, NaCl 0.25M, Polyvinylpyrolidone (PVP) 2g/100ml; Concentration is the Tris-HCl damping fluid 10ml/100ml that 1M and pH are 8.0;
In above-mentioned arbitrary described test kit, described buffer B specifically can be as follows: described solute and the final concentration in described buffer B thereof are as follows: concentration is the Tris-HCl damping fluid 10ml/100ml that 1M and pH are 8.0, disodium ethylene diamine tetraacetate 25mmolL
-1, NaCl 1.4M, CTAB 3g/100ml, sodium metabisulfite 1g/100ml, sodium ascorbate 1g/100ml, Polyvinylpyrolidone (PVP) 2g/100ml, beta-mercaptoethanol 100ul/100ml.
The method of extraction DNA of plants provided by the present invention, comprises the steps:
(1) buffer A and plant tissue powder are mixed, ice bath, centrifugal, abandon supernatant, collecting precipitation;
(2) buffer B and step (1) gained precipitation are mixed, place, centrifugal, collect supernatant liquor;
(3) with chloroform isoamyl alcohol solution, from step (2), in gained supernatant liquor, extract DNA, then go the step of RNA and precipitation DNA, obtain target DNA;
Described buffer A and described buffer B are buffer A and the buffer B in above-mentioned arbitrary described test kit.
In aforesaid method, in described step (1), the time of ice bath is 10min-20min.
In aforesaid method, in described step (2), the method for described placement is 60 ℃ of-70 ℃ of water-bath 90-120min, is specially 65 ℃ of water-bath 100min.
In aforesaid method, in described step (1) afterwards, described step (2) before, also comprises following repeating step: previous step gained precipitation is mixed with described buffer A, and ice bath, centrifugal, abandon supernatant, collecting precipitation; Until supernatant thickness not.
In aforesaid method, in described step (1), the proportioning of described buffer A and plant tissue powder is 1mL buffer A: 20mg plant tissue powder; In described ice bath process, put upside down and mix 2-3 time; Described centrifugal be the centrifugal 10min of 7000 * g; The time of described ice bath is 15min;
In aforesaid method, in described step (2), the proportioning of described buffer B and described plant tissue powder is 0.7mL buffer B: 20mg plant tissue powder; In described water-bath process, put upside down and mix for several times; Described centrifugal be the centrifugal 10min of 10000 * g.
In aforesaid method, in described step (3), the described method of extracting DNA with chloroform isoamyl alcohol solution from step (2) in gained supernatant liquor is as follows: in step (2) gained supernatant liquor, add 0.7mL chloroform isoamyl alcohol solution, put upside down and mix 10min, 10000 * g, centrifugal 10min, collects supernatant; Chloroform isoamyl alcohol Chlorine in Solution is imitative: the volume ratio of primary isoamyl alcohol is 24: 1; Repeat above-mentioned chloroform isoamyl alcohol step, until do not precipitate between centrifugal latter two liquid level; Then add 0.5mL Virahol, mix, place 20min for-20 ℃, 10000 * g, centrifugal 10min, abandons supernatant;
In aforesaid method, in described step (3), described in go the method for RNA to comprise the steps: that adding the concentration of 0.1mL is 100mgL
-1rNase solution, place 30-60min for 37 ℃; Add 0.1mL deionized water, 0.1mL5MNaCl solution, 0.8mL 95% dehydrated alcohol, mixes, and the centrifugal 10min of 10000 * g, abandons supernatant;
In aforesaid method, in described step (3), the method for described precipitation DNA is as follows: add 0.5mL 75% aqueous ethanolic solution, and the precipitation of upspringing, 10000 * g, centrifugal 2min, abandons supernatant.
In aforesaid method, described plant tissue is plant leaf.
In aforesaid method, described plant is as lower any: short sharp leaf wall moss, Matteuccia strthiopteris, Chinese pine, two Qiao Yulan, grape, Flos Rosae Chinensis, Amaranthus retroflexus, beautiful Sunflower Receptacle, indocalamus, Tibet Cymbidium hookerianum.
MCTAB method of the present invention is extracted DNA of plants and is had that productive rate is high, quality better, pcr amplification success ratio is high, cost is low, the large advantage of versatility five, the needs of general molecular biology research DNA extraction be can meet, investigator and the extraction of DNA of plants in enormous quantities that research funding is not abundant are particularly suitable for.
Accompanying drawing explanation
Fig. 1 is the DNA agarose gel electrophoresis detected result by 10 kind of plant of Lung biopsy extraction.The concentration of λ DNA is from left to right respectively 10ng, 25ng and 50ng.Numbering 1-10 is sample number into spectrum (with table 1).I, II, III and IV represent respectively the DNA extraction test kit of Si Ge company.
Fig. 2 is the situation by the conventional fragment of four kinds of DNA barcodes of DNA cloning of 10 kind of plant of Lung biopsy extraction.Primer situation: A.rbcLaf/r; B.trnH/psbA; C.matK472F/1248R; D.ITS1/ITS4.Numbering 1-10 is sample number into spectrum (with table 1); MD is mCTAB; I, II, III and IV represent respectively the DNA extraction test kit of Si Ge company.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
The raw material using in following each embodiment and comparative example is as follows: choose 10 kinds of frequently seen plants (table 1), respectively as the representative of each large branches (APG III, 2009) such as bryophyte, pteridophyte, gymnosperm, Basal angiosperms, monocotyledons, rose class plant, spirea.This 10 kind of plant has comprised the different habits such as arbor, shrub, liana, perennial herb, annual herb simultaneously, representative.Plant sample all picks up from Institute of Botany, Chinese Academy of Sciences Botanical gardens, and digital image information and voucher specimen deposit in Institute of Botany, Chinese Academy of Sciences's sample shop (PE).Gather the fresh blade of this 10 plant material, after 40 ℃ of oven dry, use preservation in silica standby.
Table 1. is for the material of comparative study.
As follows to the detection method of the productive rate of DNA and quality in following embodiment and comparative example:
(1) electrophoresis detection
By 1% agarose gel electrophoresis, detect clip size, degraded situation and the concentration of the DNA of five kinds of DNA extraction methods extractions.Get 1 μ L DNA stoste, add 9 μ L electrophoretic buffers, click and enter 1% sepharose after mixing, 5V/cm electrophoresis 30min, observes and takes pictures on ultraviolet gel imaging instrument.With the λ DNA of 10ng, 25ng and tri-kinds of concentration of 50ng, be standard, Quantity One (BIO-RAD) calculates the amount of DNA, thereby calculates DNA productive rate.
(2) spectrophotometer detects
Get 1 μ L DNA stoste ，Yong U.S. NanoDrop 2000 micro-spectrophotometers and measure OD value and the A260/280 ratio of the DNA of five kinds of DNA extraction methods extractions.According to the OD value of the DNA of spectrophotometric determination, calculate the productive rate of DNA.
(3) PCR detects
The DNA stoste that five kinds of DNA extraction methods are extracted is diluted to 50ng/ μ L, then uses DNA of plants barcode candidate segment (Kress et al., 2005; Plant Working Group 2009; Li et al.2011) carry out pcr amplification, the primer is: rbcLaf/r (Kress et al., 2007), trnH/psbA (Kress et al., 2005), matK472F/1248R (Yu et al., 2011) and ITS1/ITS4 (Henrion et al., 1992).
Pcr amplification adopts 10 μ L reaction systems, comprising 10 * PCR Buffer, (contains MgCl
2) 1 μ L, 2mmolL
-1dNTPs 1 μ L, 5 μ molL
-1primer F/R 0.5 μ L, 0.5U Taq DNA polymerase0.1Ml, Template DNA 1 μ L, ddH
2o 5.9 μ L.
Pcr amplification reaction completes on BIO-RAD C1000TM Thermal Cycler PCR instrument.Pcr amplification program is 94 ℃ of denaturation 4min; 94 ℃ of sex change 30s; 52 ℃ of annealing 40s; 72 ℃ are extended 1min; 35 circulations; Last 72 ℃ are extended 10min.PCR product detects DNA cloning success situation with 1% agarose gel electrophoresis, and ultraviolet gel imaging instrument is observed and takes pictures.
The method of embodiment 1, extraction DNA of plants of the present invention (hereinafter to be referred as mCTAB method)
1, take 20mg plant dry substance, add quartz sand, grind into powder, by powder transfer in the centrifuge tube of 2.0mL.
2, the buffer A that adds 1mL precooling, mixes rear ice bath 15min; In ice bath process, put upside down and mix 2-3 time.7000 * g is centrifugal, and 10min abandons supernatant.
3, repeating step 2, until supernatant thickness not.
4, add 0.7mL buffer B (table 3), mix rear 65 ℃ of water-bath 90-120min, in water-bath process, put upside down and mix for several times.10000 * g, centrifugal 10min, sucts clearly in new 2.0mL centrifuge tube.
5, add 0.7mL chloroform isoamyl alcohol solution (chloroform: primary isoamyl alcohol=24: 1), put upside down and mix 10min.10000 * g, centrifugal 10min, sucts clearly in new 1.5mL centrifuge tube.
6, repeating step 5, until do not precipitate between centrifugal latter two liquid level.
7, add the pre-cold isopropanol of 0.5mL, mix gently, place 20min for-20 ℃.10000 * g, centrifugal 10min, abandons supernatant.
8, the RNase (100mgL that adds 0.1mL
-1), place 30-60min for 37 ℃.Add 0.1mL deionized water, 0.1mL5M NaCl, 0.8mL precooling 95% dehydrated alcohol, mixes gently.The centrifugal 10min of 10000 * g, abandons supernatant.
9, add 0.5mL 75% ethanol, the precipitation of upspringing gently, 10000 * g, centrifugal 2min, abandons supernatant.
10, repeating step 9.
11, air-dry ethanol, adds 0.1mLTE dissolving DNA.
Buffer A is by following solute and solvent composition:
Solute and the final concentration in buffer A thereof are as follows: disodium ethylene diamine tetraacetate 5mmolL
-1, NaCl 0.25M, Polyvinylpyrolidone (PVP) 2g/100ml; The Tris-HCl damping fluid 10ml/100ml of 1M and pH8.0; Solvent is water.
Buffer B is by following solute and solvent composition:
Solute and the final concentration in buffer B thereof are as follows: the Tris-HCl damping fluid 10ml/100ml of 1M and pH=8.0, disodium ethylene diamine tetraacetate 25mmolL
-1, NaCl 1.4M, CTAB 3g/100ml, sodium metabisulfite 1g/100ml, sodium ascorbate 1g/100ml, Polyvinylpyrolidone (PVP) 2g/100ml, beta-mercaptoethanol 100ul/100ml.Solvent is water.
Method is substantially in the same manner as in Example 1, and difference is as follows:
Buffer A is by solute and solvent composition; Solute and the final concentration in buffer A thereof are as follows: disodium ethylene diamine tetraacetate 4mmolL
-1, NaCl 0.2M, Polyvinylpyrolidone (PVP) 1g/100ml; The Tris-HCl damping fluid 8ml/100ml of 1M and pH8.0; Described solvent is water.
Buffer B is by solute and solvent composition: solute and the final concentration in buffer B thereof are as follows: the Tris-HCl damping fluid 8ml/100ml of 1M and pH=8.0, disodium ethylene diamine tetraacetate 20mmolL
-1, NaCl 1.2M, CTAB2.5g/100ml, sodium metabisulfite 0.5g/100ml, sodium ascorbate 0.5g/100ml, Polyvinylpyrolidone (PVP) 1g/100ml, beta-mercaptoethanol 80ul/100ml; Described solvent is water.
The time of ice bath is 10min; The condition of water-bath is 60 ℃ of water-bath 90min.
Method is substantially in the same manner as in Example 1, and difference is as follows:
Buffer A is by solute and solvent composition; Solute and the final concentration in buffer A thereof are as follows: disodium ethylene diamine tetraacetate 6mmolL
-1, NaCl 0.3M, Polyvinylpyrolidone (PVP) 3g/100ml; The Tris-HCl damping fluid 12ml/100ml of 1M and pH8.0; Described solvent is water.
Buffer B is by solute and solvent composition: solute and the final concentration in buffer B thereof are as follows: the Tris-HCl damping fluid 12ml/100ml of 1M and pH=8.0, disodium ethylene diamine tetraacetate 30mmolL
-1, NaCl 1.6M, CTAB3.5g/100ml, sodium metabisulfite 1.5g/100ml, sodium ascorbate 1.5g/100ml, Polyvinylpyrolidone (PVP) 3g/100ml, beta-mercaptoethanol 120ul/100ml; Described solvent is water.
The time of ice bath is 20min; The condition of water-bath is 70 ℃ of water-bath 120min.
Comparative example 1,
The DNA of plants of selling with the I of company is on the market extracted test kit, is extracted to specifications.Hereinafter to be referred as the I of company.
Comparative example 2,
The DNA of plants of selling with the II of company is on the market extracted test kit, is extracted to specifications.Hereinafter to be referred as the II of company.
Comparative example 3,
The DNA of plants of selling with the III of company is on the market extracted test kit, is extracted to specifications.Hereinafter to be referred as the III of company.
Comparative example 4,
The DNA of plants of selling with the IV of company is on the market extracted test kit, is extracted to specifications.Hereinafter to be referred as the IV of company.
Result is as follows:
1, the productive rate of DNA
Electrophoresis result: different DNA extraction methods is caught the different of degree because of the difference of lysis method from DNA in subsequent operations, and its DNA productive rate is different, can judge the height of DNA productive rate from the brightness of DNA electrophoretic band.In above-mentioned Lung biopsy, its electrophoretic band of DNA that the mCTAB method of embodiment 1 is extracted is obviously than the DNA electrophoretic band of additive method bright (Fig. 1), illustrate that the productive rate of DNA of mCTAB extraction of embodiment 1 is high.
Spectrophotometric determination result (table 2): in Lung biopsy, the DNA productive rate that the mCTAB method of embodiment 1 is extracted is apparently higher than other four kinds of kit methods, and 10 kind of plant mean values exceed more than one times than each kit method mean value.Especially for No. 5 sample grapes, the mCTAB method of embodiment 1 has been extracted more DNA, the DNA extraction test kit of the I of company has only extracted micro-DNA, and the IV of III, company of II, company of company there is no acquisition DNA, explanation is for the higher material of difficulty, the mCTAB method extraction effect of embodiment 1 is fine, and the test kit effect of the I of company is all undesirable.
DNA productive rate (every milligram of dry substance nanogram DNA, the ng.mg of 10 kind of plant that table 2. is estimated with spectrophotometric
-1)
2, the quality of DNA
In prior art, it is generally acknowledged, by UV spectrophotometer measuring, the A260/280 ratio of high purity DNA should be between 1.8～2.0.When A260/280 is less than 1.8, the pollution that has protein in DNA sample is described, when A260/280 is greater than 2.0, illustrate that in DNA sample, rna content is higher.As can be seen from Table 3, the DNA purity that different methods extracts has different.The DNA that the mCTAB method of embodiment 1 is extracted has 7 sample A260/280 ratios between 1.8～1.89.The DNA that the I of company test kit extracts has 9 sample A260/280 ratios to be greater than 2.0, illustrates that in DNA sample, rna content is higher.The A260/280 ratio of the most samples of DNA that the test kit of the IV of IIIHe company of II, company of company extracts is all less than 1.8, illustrates in DNA sample and has the organic pollutions such as protein.As can be seen here, the DNA purity that the mCTAB method of embodiment 1 is extracted is the highest.
Table 3. extracts the DNA purity (A260/280) of 10 kind of plant with Lung biopsy.
| Sample | mCTAB | I | | III | IV | |
| 1 | 1.81 | 3.06 | 1.71 | 1.70 | 1.73 | |
| 2 | 1.89 | 2.25 | 1.74 | 1.52 | 1.77 | |
| 3 | 1.80 | 2.11 | 1.80 | 1.67 | 1.77 | |
| 4 | 1.88 | 2.09 | 1.81 | 1.79 | 1.73 | |
| 5 | 1.66 | 5.69 | 1.66 | 1.82 | 1.60 | |
| 6 | 1.85 | 4.51 | 1.61 | 1.25 | 1.79 | |
| 7 | 2.16 | 2.14 | 1.71 | 1.75 | 1.79 | |
| 8 | 1.68 | 1.98 | 1.25 | 1.29 | 1.47 | |
| 9 | 1.83 | 2.21 | 1.78 | 1.80 | 1.06 | |
| 10 | 1.88 | 2.12 | 1.83 | 1.84 | 1.84 |
Pcr amplification success ratio is one of important evidence of passing judgment on DNA quality.Overall pcr amplification success ratio by Fig. 2 can find out, the mCTAB of embodiment 1 is identical with the I of company, amplifies band maximum; IVPCR is identical for IIHe company of company, amplifies band slightly few; III is the poorest in company.With regard to concrete gene fragment, matK and the ITS of the mCTAB of embodiment 1 and the I of company are obviously better than additive method.
3, cost keeping
The cost of DNA extraction is one of the factor that must consider in the limited situation of research funding.Through adjusting, the cost of the mCTAB method of embodiment 1 is that the cost of the test kit of the single sample 0.6Yuan， I of company and the II of company is that the cost of single sample 5 III of Yuan， company is that the cost of the single sample 11Yuan， IV of company is that single sample is 30 yuan.Obviously, mCTAB method has extremely significant cost advantage.
Shown in above-described embodiment 2 and embodiment 3, method is also extracted and is obtained target DNA, and result and embodiment 1 are without significant difference.
In sum, mCTAB method of the present invention is extracted DNA of plants and is had that productive rate is high, quality better, pcr amplification success ratio is high, cost is low, the large advantage of versatility five, the needs of general molecular biology research DNA extraction be can meet, investigator and the extraction of DNA of plants in enormous quantities that research funding is not abundant are particularly suitable for.
Claims (7)
1. for extracting a test kit for DNA of plants, it is characterized in that: test kit comprises buffer A;
Described buffer A is by solute and solvent composition; Described solute and the final concentration in described buffer A thereof are as follows: disodium ethylene diamine tetraacetate 5mmolL
-1, NaCl0.25M, Polyvinylpyrolidone (PVP) 2g/100ml; Concentration is the Tris-HCl damping fluid 10ml/100ml that 1M and pH are 8.0; Described solvent is water;
In described test kit, also comprise buffer B; Described buffer B is by solute and solvent composition: described solute and the final concentration in described buffer B thereof are as follows: concentration is the Tris-HCl damping fluid 10ml/100ml that 1M and pH are 8.0, disodium ethylene diamine tetraacetate 25mmolL
-1, NaCl1.4M, CTAB3g/100ml, sodium metabisulfite 1g/100ml, sodium ascorbate 1g/100ml, Polyvinylpyrolidone (PVP) 2g/100ml, beta-mercaptoethanol 100ul/100ml; Described solvent is water.
2. a method of extracting DNA of plants, comprises the steps:
(1) buffer A and plant tissue powder are mixed, ice bath, centrifugal, abandon supernatant, collecting precipitation;
(2) buffer B and step (1) gained precipitation are mixed, place, centrifugal, collect supernatant liquor; The method of described placement is 60 ℃ of-70 ℃ of water-bath 90-120min;
(3) with chloroform isoamyl alcohol solution, from step (2), in gained supernatant liquor, extract DNA, then go the step of RNA and precipitation DNA, obtain target DNA;
Described buffer A and described buffer B are buffer A and the buffer B in test kit described in claim 1.
3. method according to claim 2, is characterized in that: in described step (1), the time of ice bath is 10min-20min.
4. method according to claim 3, is characterized in that: in described step (2), the method for described placement is 65 ℃ of water-bath 100min.
5. method according to claim 4, is characterized in that: in described method, in described step (1) afterwards, described step (2) before, also comprises following repeating step: previous step gained precipitation is mixed to ice bath with described buffer A, centrifugal, abandon supernatant, collecting precipitation; Until supernatant thickness not.
6. method according to claim 5, is characterized in that:
In described step (1), the proportioning of described buffer A and plant tissue powder is 1mL buffer A: 20mg plant tissue powder; In described ice bath process, put upside down and mix 2-3 time; Described centrifugal be the centrifugal 10min of 7000 * g; The time of described ice bath is 15min;
In described step (2), the proportioning of described buffer B and described plant tissue powder is 0.7mL buffer B: 20mg plant tissue powder; In described water-bath process, put upside down and mix for several times; Described centrifugal be the centrifugal 10min of 10000 * g;
In described step (3), the described method of extracting DNA with chloroform isoamyl alcohol solution from step (2) in gained supernatant liquor is as follows: in step (2) gained supernatant liquor, add 0.7mL chloroform isoamyl alcohol solution, put upside down and mix 10min, 10000 * g, centrifugal 10min, collects supernatant; Chloroform isoamyl alcohol Chlorine in Solution is imitative: the volume ratio of primary isoamyl alcohol is 24:1; Repeat the step that above-mentioned chloroform isoamyl alcohol extracts DNA, until do not precipitate between centrifugal latter two liquid level; Then add 0.5mL Virahol, mix, place 20min for-20 ℃, 10000 * g, centrifugal 10min, abandons supernatant;
In described step (3), described in go the method for RNA to comprise the steps: that adding the concentration of 0.1mL is 100mgL
-1rNase solution, place 30-60min for 37 ℃; Add 0.1mL deionized water, 0.1mL5M NaCl solution, 0.8mL95% dehydrated alcohol, mixes, and the centrifugal 10min of 10000 * g, abandons supernatant;
In described step (3), the method for described precipitation DNA is as follows: add 0.5mL75 ﹪ aqueous ethanolic solution, and the precipitation of upspringing, 10000 * g, centrifugal 2min, abandons supernatant.
7. according to arbitrary described method in claim 2-6, it is characterized in that: described plant tissue is plant leaf.
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| CN103966201B (en) * | 2014-05-15 | 2016-02-24 | 中国科学院武汉植物园 | Based on the method for the extraction waterplant DNA of sample efficient fresh-keeping |
| CN104342433A (en) * | 2014-09-19 | 2015-02-11 | 中国农业科学院烟草研究所 | Extracting solution for extracting genome DNA (Deoxyribose Nucleic Acid), application of extracting solution and method for rapidly and efficiently extracting genome DNA of tobaccos by utilizing extracting solution |
| CN109136220A (en) * | 2018-10-17 | 2019-01-04 | 上海应用技术大学 | A kind of Fagaceae sample DNA extracts and purification process |
| KR102415720B1 (en) * | 2020-10-08 | 2022-07-01 | 주식회사 인바이러스테크 | Method for extracting nucleic acid from sample rich in polyphenol and/or polysaccharide |
| KR102421648B1 (en) * | 2020-10-08 | 2022-07-18 | 주식회사 인바이러스테크 | Method for extracting nucleic acid from sample rich in ribonuclease |
| KR20220149944A (en) * | 2021-05-03 | 2022-11-10 | 주식회사 인바이러스테크 | METHOD FOR EXTRACTING NUCLEIC ACID FROM SAMPLE RICH IN RNAase |
| KR20230010395A (en) * | 2021-07-12 | 2023-01-19 | 주식회사 인바이러스테크 | Method for extracting nucleic acid from sample with polyphenol |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1377417A (en) * | 2000-08-01 | 2002-10-30 | 分子农业生物学院 | Isolation and characterization of fiber-specific actin promoter from cotton |
| CN1635895A (en) * | 2001-10-10 | 2005-07-06 | 雀巢产品有限公司 | Coffee plant with reduced alpha-D-galactosidase activity |
-
2012
- 2012-04-25 CN CN201210124356.7A patent/CN102643800B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1377417A (en) * | 2000-08-01 | 2002-10-30 | 分子农业生物学院 | Isolation and characterization of fiber-specific actin promoter from cotton |
| CN1635895A (en) * | 2001-10-10 | 2005-07-06 | 雀巢产品有限公司 | Coffee plant with reduced alpha-D-galactosidase activity |
Non-Patent Citations (4)
| Title |
|---|
| Little DP et al.MAJOR EVOLUTIONARY EVENTS IN THE ORIGIN AND DIVERSIFICATION OF THE FERN GENUS POLYSTICHUM (DRYOPTERIDACEAE).《American Journal of Botany》.2003,第90卷(第3期),第508-514页. |
| MAJOR EVOLUTIONARY EVENTS IN THE ORIGIN AND DIVERSIFICATION OF THE FERN GENUS POLYSTICHUM (DRYOPTERIDACEAE);Little DP et al;《American Journal of Botany》;20030331;第90卷(第3期);第509页最后1段 * |
| 李喜凤 等.蒲公英总DNA的提取及其RAPD-PCR反应条件的优化.《时珍国医国药》.2010,第21卷(第6期),第1501-1503页. |
| 蒲公英总DNA的提取及其RAPD-PCR反应条件的优化;李喜凤 等;《时珍国医国药》;20100620;第21卷(第6期);正文"1材料与仪器"，"2方法" * |
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