CN102643800A - 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 more secondary metabolites such as polysaccharide; And the kind and the content difference of secondary metabolite is very big in the different plants; Sometimes this kind and content of giving birth to metabolite of kindred plant Different Organs or tissue is also different, and causing obtaining high-quality DNA has certain degree of difficulty.DNA extraction method to certain specialized species optimization not necessarily is applicable to other species.Traditional CT AB method is maximum DNA extraction method of using at present, but because differences such as vegetable material chemical ingredients, weave construction, the extraction effect of traditional CTAB method is not good enough sometimes, receives certain restriction in the use.Therefore press for a kind of easy, efficient, economy and versatility DNA of plants process for extracting preferably.
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.
The test kit that is used to extract DNA of plants provided by the present invention comprises buffer A; Said buffer A is by solute and solvent composition; Said solute and the final concentration in said buffer A thereof are following: EDTA Disodium 4-6mmolL
-1, NaCl 0.2-0.3M, PVP K120 1-3g/100ml; Concentration is that 1M and pH are 8.0 Tris-HCl damping fluid 8-12ml/100ml; Said solvent is a water.
Also can comprise buffer B in the mentioned reagent box; Said buffer B is by solute and solvent composition: said solute and the final concentration in said buffer B thereof are following: concentration is that 1M and pH are 8.0 Tris-HCl damping fluid 8-12ml/100ml, EDTA Disodium 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, PVP K120 1-3g/100ml, beta-mercaptoethanol 80-120ul/100ml; Said solvent is a water.
In above-mentioned arbitrary said test kit, said buffer A specifically can be as follows: said solute and the final concentration in said buffer A thereof are following: EDTA Disodium 5mmolL
-1, NaCl 0.25M, PVP K120 2g/100ml; Concentration is that 1M and pH are 8.0 Tris-HCl damping fluid 10ml/100ml;
In above-mentioned arbitrary said test kit, said buffer B specifically can be as follows: said solute and the final concentration in said buffer B thereof are following: concentration is that 1M and pH are 8.0 Tris-HCl damping fluid 10ml/100ml, EDTA Disodium 25mmolL
-1, NaCl 1.4M, CTAB 3g/100ml, sodium metabisulfite 1g/100ml, sodium ascorbate 1g/100ml, PVP K120 2g/100ml, beta-mercaptoethanol 100ul/100ml.
The method of extraction DNA of plants provided by the present invention comprises the steps:
(1) with buffer A and plant tissue powder mixing, ice bath, centrifugal, abandon supernatant, collecting precipitation;
(2) with buffer B and step (1) gained deposition mixing, place, centrifugal, collect supernatant;
(3) from step (2), extract DNA in the gained supernatant with chloroform isoamyl alcohol solution, go the step of RNA and deposit D NA again, obtain target DNA;
Said buffer A and said buffer B are buffer A and the buffer B in above-mentioned arbitrary said test kit.
In the aforesaid method, in the said step (1), the time of ice bath is 10min-20min.
In the aforesaid method, in the said step (2), the method for said placement is 60 ℃ of-70 ℃ of water-bath 90-120min, is specially 65 ℃ of water-bath 100min.
In the aforesaid method, in said step (1) afterwards, said step (2) also comprises following repeating step before: will go up step gained deposition and said buffer A mixing, and ice bath, centrifugal, abandon supernatant, collecting precipitation; Up to supernatant thickness not.
In the aforesaid method, in the said step (1), the proportioning of said buffer A and plant tissue powder is the 1mL buffer A: 20mg plant tissue powder; Put upside down mixing 2-3 time in the said ice bath process; Said centrifugal be the centrifugal 10min of 7000 * g; The time of said ice bath is 15min;
In the aforesaid method, in the said step (2), the proportioning of said buffer B and said plant tissue powder is the 0.7mL buffer B: 20mg plant tissue powder; Put upside down mixing in the said water-bath process for several times; Said centrifugal be the centrifugal 10min of 10000 * g.
In the aforesaid method, in the said step (3), said from step (2), to extract the method for DNA in the gained supernatant with chloroform isoamyl alcohol solution following: in step (2) gained supernatant, add 0.7mL chloroform isoamyl alcohol solution; Put upside down mixing 10min; 10000 * g, centrifugal 10min collects supernatant; Chloroform in the chloroform isoamyl alcohol solution: the volume ratio of primary isoamyl alcohol is 24: 1; Repeat above-mentioned chloroform isoamyl alcohol step, between centrifugal latter two liquid level, do not precipitate; Add the 0.5mL Virahol then, mixing is placed 20min for-20 ℃, 10000 * g, and centrifugal 10min abandons supernatant;
In the aforesaid method, in the said step (3), the concentration that the method for the said RNA of going comprises the steps: to add 0.1mL is 100mgL
-1RNase solution, place 30-60min for 37 ℃; Add 0.1mL deionized water, 0.1mL5MNaCl solution, 0.8mL 95% absolute ethyl alcohol, mixing, the centrifugal 10min of 10000 * g abandons supernatant;
In the aforesaid method, in the said step (3), the method for said deposit D NA is following: add 0.5mL 75% aqueous ethanolic solution, and the deposition of upspringing, 10000 * g, centrifugal 2min abandons supernatant.
In the aforesaid method, said plant tissue is a plant leaf.
In the aforesaid method, said plant is any in following: 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 extraction DNA of plants of the present invention has the productive rate height, quality is better, the pcr amplification success ratio is high, low, the versatility five big advantages of cost; Can satisfy the needs of general molecular biology research DNA extraction, be particularly suitable for the extraction of not abundant investigator of research funding and DNA of plants in enormous quantities.
Description of drawings
Fig. 1 is the DNA agarose gel electrophoresis detected result with 10 kind of plant of five kinds of methods extractions.The concentration of λ DNA from left to right is respectively 10ng, 25ng and 50ng.Numbering 1-10 is sample number into spectrum (with table 1).I, II, III and IV represent the DNA extraction test kit of four companies respectively.
Fig. 2 is four kinds of DNA barcodes of the DNA cloning segmental situation commonly used with 10 kind of plant of five kinds of methods extractions.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 the DNA extraction test kit of four companies respectively.
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
The raw material that uses in following each embodiment and the Comparative Examples is following: choose 10 kinds of frequently seen plants (table 1); Respectively as the representative of each big branches (APG III, 2009) such as bryophyte, pteridophyte, gymnosperm, base portion angiosperm, monocotyledons, rose class plant, spirea.This 10 kind of plant has comprised different habits such as arbor, shrub, liana, perennial herb, annual herb simultaneously, and is representative.Plant sample all picks up from the 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 sample shop (PE).Gather the fresh blade of this 10 plant material, preserve subsequent use with silica gel after 40 ℃ of oven dry.
Table 1. is used for the material of comparative study.
Detection method to the productive rate of DNA and quality in following embodiment and the Comparative Examples is following:
(1) electrophoresis detection
Detect clip size, degraded situation and the concentration of the DNA of five kinds of DNA extraction methods extractions through 1% agarose gel electrophoresis.Get 1 μ L DNA stoste, add 9 μ L electrophoretic buffers, click and enter 1% sepharose behind the mixing, 5V/cm electrophoresis 30min observes on the ultraviolet gel imaging appearance and takes pictures.Use the λ DNA of 10ng, 25ng and three kinds of concentration of 50ng to be standard, Quantity One (BIO-RAD) calculates the amount of DNA, thereby calculates the DNA productive rate.
(2) spectrophotometer detects
Get 1 μ L DNA stoste, measure OD value and the A260/280 ratio of the DNA of five kinds of DNA extraction methods extractions with U.S. NanoDrop 2000 micro-spectrophotometers.Calculate the productive rate of DNA according to the OD value of the DNA of spectrophotometric determination.
(3) PCR detects
The DNA stoste that five kinds of DNA extraction methods are extracted is diluted to 50ng/ μ L, uses DNA of plants barcode candidate segment (Kress et al., 2005 then; Plant Working Group 2009; Li et al.2011) carries 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, (contains MgCl comprising 10 * PCR Buffer
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 is accomplished on BIO-RAD C1000TM Thermal Cycler PCR appearance.The pcr amplification program is 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 30s; 52 ℃ of annealing 40s; 72 ℃ are extended 1min; 35 circulations; Last 72 ℃ are extended 10min.The PCR product detects DNA cloning success situation with 1% agarose gel electrophoresis, and ultraviolet gel imaging appearance is observed and taken pictures.
The method of embodiment 1, extraction DNA of plants of the present invention (hereinafter to be referred as the mCTAB method)
1, take by weighing 20mg plant dry substance, add silica sand, grind into powder, with powder transfer in the centrifuge tube of 2.0mL.
2, the buffer A that adds the 1mL precooling, ice bath 15min behind the mixing; Put upside down mixing 2-3 time in the ice bath process.7000 * g is centrifugal, and 10min abandons supernatant.
3, repeating step 2, up to supernatant thickness not.
4, add 0.7mL buffer B (table 3), 65 ℃ of water-bath 90-120min behind the mixing put upside down mixing for several times in the water-bath process.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 mixing 10min.10000 * g, centrifugal 10min sucts clearly in new 1.5mL centrifuge tube.
6, repeating step 5, between centrifugal latter two liquid level, do not precipitate.
7, add 0.5mL precooling Virahol, mixing is placed 20min for-20 ℃ gently.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% absolute ethyl alcohol, mixing gently.The centrifugal 10min of 10000 * g abandons supernatant.
9, add 0.5mL 75% ethanol, the deposition of upspringing gently, 10000 * g, centrifugal 2min abandons supernatant.
10, repeating step 9.
11, air-dry ethanol adds the 0.1mLTE dissolving DNA.
Buffer A is by following solute and solvent composition:
Solute and the final concentration in buffer A thereof are following: EDTA Disodium 5mmolL
-1, NaCl 0.25M, PVP K120 2g/100ml; The Tris-HCl damping fluid 10ml/100ml of 1M and pH8.0; Solvent is a water.
Buffer B is by following solute and solvent composition:
Solute and the final concentration in buffer B thereof are following: the Tris-HCl damping fluid 10ml/100ml of 1M and pH=8.0, EDTA Disodium 25mmolL
-1, NaCl 1.4M, CTAB 3g/100ml, sodium metabisulfite 1g/100ml, sodium ascorbate 1g/100ml, PVP K120 2g/100ml, beta-mercaptoethanol 100ul/100ml.Solvent is a water.
Method basically with embodiment 1 in identical, difference is following:
Buffer A is by solute and solvent composition; Solute and the final concentration in buffer A thereof are following: EDTA Disodium 4mmolL
-1, NaCl 0.2M, PVP K120 1g/100ml; The Tris-HCl damping fluid 8ml/100ml of 1M and pH8.0; Said solvent is a water.
Buffer B is by solute and solvent composition: solute and the final concentration in buffer B thereof are following: the Tris-HCl damping fluid 8ml/100ml of 1M and pH=8.0, EDTA Disodium 20mmolL
-1, NaCl 1.2M, CTAB2.5g/100ml, sodium metabisulfite 0.5g/100ml, sodium ascorbate 0.5g/100ml, PVP K120 1g/100ml, beta-mercaptoethanol 80ul/100ml; Said solvent is a water.
The time of ice bath is 10min; The condition of water-bath is 60 ℃ of water-bath 90min.
Method basically with embodiment 1 in identical, difference is following:
Buffer A is by solute and solvent composition; Solute and the final concentration in buffer A thereof are following: EDTA Disodium 6mmolL
-1, NaCl 0.3M, PVP K120 3g/100ml; The Tris-HCl damping fluid 12ml/100ml of 1M and pH8.0; Said solvent is a water.
Buffer B is by solute and solvent composition: solute and the final concentration in buffer B thereof are following: the Tris-HCl damping fluid 12ml/100ml of 1M and pH=8.0, EDTA Disodium 30mmolL
-1, NaCl 1.6M, CTAB3.5g/100ml, sodium metabisulfite 1.5g/100ml, sodium ascorbate 1.5g/100ml, PVP K120 3g/100ml, beta-mercaptoethanol 120ul/100ml; Said solvent is a water.
The time of ice bath is 20min; The condition of water-bath is 70 ℃ of water-bath 120min.
Comparative Examples 1,
The DNA of plants of using the I of company on the market to sell extracts test kit, extracts to specifications.Hereinafter to be referred as the I of company.
Comparative Examples 2,
The DNA of plants of using the II of company on the market to sell extracts test kit, extracts to specifications.Hereinafter to be referred as the II of company.
Comparative Examples 3,
The DNA of plants of using the III of company on the market to sell extracts test kit, extracts to specifications.Hereinafter to be referred as the III of company.
Comparative Examples 4,
The DNA of plants of using the IV of company on the market to sell extracts test kit, extracts to specifications.Hereinafter to be referred as the IV of company.
The result is following:
1, the productive rate of DNA
Electrophoresis result: different DNA extraction methods is caught the different of degree because of DNA in the difference of lysis method and the 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 five kinds of methods, 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), and the productive rate of the DNA that the mCTAB of illustrative embodiment 1 extracts is high.
Spectrophotometric determination result (table 2): in five kinds of methods, 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 MVs exceed more than one times than each kit method MV.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 II of company, the III of company, the IV of company do not obtain DNA basically, explain 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.
Table 2. is with DNA productive rate (every milligram of dry substance nanogram DNA, the ng.mg of 10 kind of plant of spectrophotometric estimation
-1)
2, the quality of DNA
In the prior art, it is generally acknowledged, use UV spectrophotometer measuring, the A260/280 ratio of high purity DNA should be between 1.8～2.0.When A260/280 less than 1.8 the time, explain to have proteinic pollution in the DNA sample, when A260/280 greater than 2.0 the time, explain that rna content is higher in the DNA sample.Can find out that from table 3 the DNA purity that different methods extracts has certain difference.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 greater than 2.0, explains that rna content is higher in the DNA sample.The A260/280 ratio of the most samples of the DNA that the test kit of the II of company, the III of company and the IV of company extracts is explained organic pollutions such as there being protein in the DNA sample all less than 1.8.This shows that 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 five kinds of methods.
| 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 |
The pcr amplification success ratio is one of important evidence of passing judgment on the DNA quality.Overall pcr amplification success ratio by Fig. 2 can find out that the mCTAB of embodiment 1 is identical with the I of company, and it is maximum to amplify band; The II of company is identical with the IVPCR of company, and it is few slightly to amplify band; 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 obviously are superior to additive method.
3, cost keeping
The cost of DNA extraction is one of the factor that must consider under the limited situation of research funding.Through adjusting, the cost of the mCTAB method of embodiment 1 is 0.6 yuan of a single sample, and the cost of the test kit of I of company and the II of company is 5 yuan of single samples, and the cost of the III of company is 11 yuan of single samples, and the cost of the IV of company is that single sample is 30 yuan.Obviously, the mCTAB method has extremely significant cost advantage.
The foregoing description 2 also extracts with method shown in the embodiment 3 and obtains target DNA, result and embodiment 1 no significant difference.
In sum; MCTAB method extraction DNA of plants of the present invention has the productive rate height, quality is better, the pcr amplification success ratio is high, low, the versatility five big advantages of cost; Can satisfy the needs of general molecular biology research DNA extraction, be particularly suitable for the extraction of not abundant investigator of research funding and DNA of plants in enormous quantities.
Claims (9)
1. test kit that is used to extract DNA of plants, it is characterized in that: test kit comprises buffer A;
Said buffer A is by solute and solvent composition; Said solute and the final concentration in said buffer A thereof are following: EDTA Disodium 4-6mmolL
-1, NaCl 0.2-0.3M, PVP K120 1-3g/100ml; Concentration is that 1M and pH are 8.0 Tris-HCl damping fluid 8-12ml/100ml; Said solvent is a water.
2. test kit according to claim 1 is characterized in that: also comprise buffer B in the said test kit; Said buffer B is by solute and solvent composition: said solute and the final concentration in said buffer B thereof are following: concentration is that 1M and pH are 8.0 Tris-HCl damping fluid 8-12ml/100ml, EDTA Disodium 20-30mmolL
-1, NaCl 1.2-1.6M, CTAB 2.5-3.5g/100ml, sodium metabisulfite 0.5-1.5g/100ml, sodium ascorbate 0.5-1.5g/100ml, PVP K120 1-3g/100ml, beta-mercaptoethanol 80-120ul/100ml; Said solvent is a water.
3. test kit according to claim 1 and 2 is characterized in that: in the said buffer A, said solute and the final concentration in said buffer A thereof are following: EDTA Disodium 5mmolL
-1, NaCl 0.25M, PVP K120 2g/100ml; Concentration is that 1M and pH are 8.0 Tris-HCl damping fluid 10ml/100ml;
In the said buffer B, said solute and the final concentration in said buffer B thereof are following: concentration is that 1M and pH are 8.0 Tris-HCl damping fluid 10ml/100ml, EDTA Disodium 25mmolL
-1, NaCl 1.4M, CTAB 3g/100ml, sodium metabisulfite 1g/100ml, sodium ascorbate 1g/100ml, PVP K120 2g/100ml, beta-mercaptoethanol 100ul/100ml.
4. a method of extracting DNA of plants comprises the steps:
(1) with buffer A and plant tissue powder mixing, ice bath, centrifugal, abandon supernatant, collecting precipitation;
(2) with buffer B and step (1) gained deposition mixing, place, centrifugal, collect supernatant;
(3) from step (2), extract DNA in the gained supernatant with chloroform isoamyl alcohol solution, go the step of RNA and deposit D NA again, obtain target DNA;
Said buffer A and said buffer B are buffer A and the buffer B in arbitrary said test kit among the claim 1-3.
5. method according to claim 4 is characterized in that: in the said step (1), the time of ice bath is 10min-20min.
6. according to claim 4 or 5 described methods, it is characterized in that: in the said step (2), the method for said placement is 60 ℃ of-70 ℃ of water-bath 90-120min, is specially 65 ℃ of water-bath 100min.
7. according to arbitrary described method among the claim 4-6, it is characterized in that: in the said method, afterwards in said step (1); Said step (2) also comprises following repeating step before: will go up step gained deposition and said buffer A mixing, ice bath; Centrifugal, abandon supernatant, collecting precipitation; Up to supernatant thickness not.
8. according to arbitrary described method among the claim 4-7, it is characterized in that:
In the said step (1), the proportioning of said buffer A and plant tissue powder is the 1mL buffer A: 20mg plant tissue powder; Put upside down mixing 2-3 time in the said ice bath process; Said centrifugal be the centrifugal 10min of 7000 * g; The time of said ice bath is 15min;
In the said step (2), the proportioning of said buffer B and said plant tissue powder is the 0.7mL buffer B: 20mg plant tissue powder; Put upside down mixing in the said water-bath process for several times; Said centrifugal be the centrifugal 10min of 10000 * g.
In the said step (3); Said from step (2), to extract the method for DNA in the gained supernatant with chloroform isoamyl alcohol solution following: in step (2) gained supernatant, add 0.7mL chloroform isoamyl alcohol solution, put upside down mixing 10min, 10000 * g; Centrifugal 10min collects supernatant; Chloroform in the chloroform isoamyl alcohol solution: the volume ratio of primary isoamyl alcohol is 24: 1; Repeat above-mentioned chloroform isoamyl alcohol step, between centrifugal latter two liquid level, do not precipitate; Add the 0.5mL Virahol then, mixing is placed 20min for-20 ℃, 10000 * g, and centrifugal 10min abandons supernatant;
In the said step (3), the concentration that the method for the said RNA of going comprises the steps: to add 0.1mL is the RNase solution of 100mgL-1, places 30-60min for 37 ℃; Add 0.1mL deionized water, 0.1mL5M NaCl solution, the 0.8mL95% absolute ethyl alcohol, mixing, the centrifugal 10min of 10000 * g abandons supernatant;
In the said step (3), the method for said deposit D NA is following: add 0.5mL 75% aqueous ethanolic solution, and the deposition of upspringing, 10000 * g, centrifugal 2min abandons supernatant.
9. according to arbitrary described method among the claim 4-8, it is characterized in that: said plant tissue is a plant leaf.
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| 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 |
| KR20220047114A (en) * | 2020-10-08 | 2022-04-15 | 주식회사 인바이러스테크 | Method for extracting nucleic acid from sample rich in polyphenol and/or polysaccharide |
| KR20220047115A (en) * | 2020-10-08 | 2022-04-15 | 주식회사 인바이러스테크 | Method for extracting nucleic acid from sample rich in ribonuclease |
| WO2022234978A1 (en) * | 2021-05-03 | 2022-11-10 | 주식회사 인바이러스테크 | Method for extracting nucleic acids from sample rich in rnase |
| WO2023286980A1 (en) * | 2021-07-12 | 2023-01-19 | 주식회사 인바이러스테크 | Method for extracting nucleic acid from polyphenol-containing sample |
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Cited By (9)
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| CN103966201A (en) * | 2014-05-15 | 2014-08-06 | 中国科学院武汉植物园 | Method for extracting aquatic plant DNA based on high-efficiency sample preservation |
| 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 |
| KR20220047114A (en) * | 2020-10-08 | 2022-04-15 | 주식회사 인바이러스테크 | Method for extracting nucleic acid from sample rich in polyphenol and/or polysaccharide |
| KR20220047115A (en) * | 2020-10-08 | 2022-04-15 | 주식회사 인바이러스테크 | Method for extracting nucleic acid from sample rich in ribonuclease |
| 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 |
| WO2022234978A1 (en) * | 2021-05-03 | 2022-11-10 | 주식회사 인바이러스테크 | Method for extracting nucleic acids from sample rich in rnase |
| WO2023286980A1 (en) * | 2021-07-12 | 2023-01-19 | 주식회사 인바이러스테크 | Method for extracting nucleic acid from polyphenol-containing sample |
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