CN102121000B - Method for extracting mitochondrial DNA of cotton - Google Patents
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Abstract
The invention discloses a method for extracting mitochondrial deoxyribonucleic acid (DNA) of cotton. The method comprises the following steps of: (1) homogenizing cotton, and centrifuging and collecting precipitate; (2) removing nuclear DNA, adding ethylene diamine tetraacetic acid (EDTA) solution, and centrifuging to obtain the precipitate; (3) adding buffer solution, and centrifuging to obtain mitochondria; (4) adding lysis solution and acetate solution, centrifuging, collecting supernatant, and extracting by using chloroform-isoamylol; (5) adding acetate solution and absolute ethanol, centrifuging to obtain the precipitate, and washing by using ethanol; (6) dissolving in triisopropylphenylsulfonyl (Tris)-EDTA (TE); (7) removing ribonucleic acid (RNA) and protein, extracting by using Tris saturated phenol-chloroform-isoamylol, and centrifuging and collecting the supernatant; and (8) adding the acetate solution and the ethanol, and centrifuging to obtain the precipitate; and washing by using the ethanol to obtain the mitochondrial DNA. Aiming at defects of the conventional extraction of the mitochondrial DNA, the conventional cetyltrimethylammonium bromide (CTAB) method is improved, and the extraction conditions are optimized by adjusting the time and times of centrifugation, changing reagents, increasing extraction and washing times, and the like. On the basis of ensuring that the mechanical damage degree of the mitochondria is minimum, the nuclear DNA, phenols and polysaccharides outside the mitochondria are effectively removed, pollution rate is reduced, the purity and the quality of the mitochondrial DNA are improved, and the mitochondrial DNA can meet the requirement of subsequent experiments of the gene engineering of the cotton mitochondria.
Description
Technical field
The present invention relates to a kind of method of extracting the Mitochondrial DNA of cotton.
Background technology
Plastosome (mitochondria) is the most important organoid of higher organism, is one of plasma inheritance system.Outside the relatively independent nucleus of Mitochondrial Genome Overview DNA (mtDNA), can self-replacation, controlling numerous inherited character.Its configuration mainly contains open loop, closed loop, superhelix, wire and polymer.With the higher animal ratio, the Mitochondrial Genome Overview of higher plant is big and complicated.
The plant cytoplasmic male sterility is the more ubiquitous phenomenon of nature, and research plant cytoplasmic male sterilty is an important topic of Heredity theory research.Area researches such as a large amount of genetics, molecular biology show that cause that the gene of plant cytoplasmic male sterilty mainly is distributed in the mitochondrial genome, promptly Mitochondrial Genome Overview directly influences the fertility of plant cytoplasmic male sterilty.1976; Levings and Pring have delivered mtDNA and plant cytoplasmic male sterility (cytoplasmic male sterility on " Science "; CMS) result of study; Think that mtDNA and CMS have confidential relation, this proposition has caused the extensive interest of investigators to mtDNA research, so the research of plant mtDNA is called one of focus of molecular biology of plants research in modern age.The deep molecular level research of carrying out to the plant mitochondria genome structure is the important channel of this spontaneous phenomenon of announcement CMS.Want to carry out the molecular level research of mtDNA 26S Proteasome Structure and Function, at first must from vegetable material, extract highly purified mtDNA, therefore extracting high-quality mtDNA is the basis of carrying out research work.
At present, being used for method that higher plant mtDNA extracts has the CsC1/EB density gradient centrifugation of the CTAB of Leving, Dolferus, differential centrifugation etc., and the method that these mtDNA extract is widely used.But these method operating process more complicated, cost height and the mtDNA purity of extracting are not high, influence subsequent experimental.In the leaching process of different plant mtDNA, various influence factors are also unusual complicated, be difficult to hold, and also also increasingly high to the requirement of the quality of extracting mtDNA, purity, integrity in the genetically engineered follow-up test.Because cotton contains than Polyphenols and polysaccharose substance, oxidation easily in separation and purifying mtDNA process causes certain difficulty for purifying mtDNA.
Summary of the invention
The purpose of this invention is to provide a kind of method of extracting the Mitochondrial DNA of cotton.
The method of the Mitochondrial DNA of extraction provided by the invention cotton comprises two steps of extraction of mitochondrial extraction and Mitochondrial DNA;
Said mitochondrial extraction comprises the steps (1) to step (3):
(1) the cotton sample is carried out homogenate, centrifugal collecting precipitation in buffer A; The preparation method of said buffer A is following: 0.05mol Tris-HCl, 1.2mol NaCl, 2mmol EDTA, 6-10g Vinylpyrrolidone polymer (PVP), 2-5mmol β-thin basic ethanol and 0.1-0.8g BSA water are settled to 1 liter, and adjust pH is to 7.5-8.5;
(2) deposition to step (1) adds buffer B and DNase I, to remove nuclear DNA, adds the EDTA aqueous solution (or EDTA-Na then
2The aqueous solution), the centrifugal deposition that obtains; The preparation method of said buffer B is following: with 0.05mol Tris-HCl, 0.1-0.5mol sucrose, 0.01-0.05mol MgCl
2Be settled to 1 liter with 1-5g Vinylpyrrolidone polymer (PVP) water, adjust pH is to 7.5-8.5;
(3) deposition to step (2) adds damping fluid C and damping fluid D, the centrifugal plastosome that obtains; The preparation method of said damping fluid C is following: with 0.05mol Tris-HCl, 0.02mol EDTA-Na
2Be settled to 1 liter, adjust pH to 7.5 with 0.1-0.5mol sucrose water; The preparation method of said damping fluid D is following: with 0.05molTris-HCl, 0.02mol EDTA-Na
2Be settled to 1 liter with 0.2-1.0mol sucrose water, adjust pH is to 7.5-8.5;
The extraction of said Mitochondrial DNA comprises the steps (4) to step (8):
(4) the plastosome adding lysate to step (3) carries out cracking, adds acetate aqueous solution then, and centrifugal collection supernatant carries out extracting with isopyknic solution first, the centrifugal deposition that obtains; Said solution first is made up of chloroform and primary isoamyl alcohol, and the volume ratio of chloroform and primary isoamyl alcohol is 24: 1;
(5) deposition to step (4) adds acetate aqueous solution, adds absolute ethyl alcohol again, and the centrifugal deposition that obtains is with 70% (volumn concentration) aqueous ethanolic solution washing deposition;
(6) with the deposition of TE solution or T50E10 solution dissolving step (5);
(7) add RNaes then, to remove RNA; Add Proteinase K then, to remove albumen; Carry out extracting with isopyknic solution second then, centrifugal collection supernatant; Said solution second is made up of the saturated phenol of Tris, chloroform and primary isoamyl alcohol, and the volume ratio of the saturated phenol of Tris, chloroform and primary isoamyl alcohol is 25: 24: 1;
(8) supernatant to step (7) adds acetate aqueous solution, adds absolute ethyl alcohol again, the centrifugal deposition that obtains; With 70% (volumn concentration) aqueous ethanolic solution washing deposition, obtain the Mitochondrial DNA of cotton.
In the step (4), said acetate can be at least a in potassium acetate, amine acetate and the sodium acetate.
In the step (5), said acetate can be at least a in potassium acetate, amine acetate and the sodium acetate.
In the step (8), said acetate can be at least a in potassium acetate, amine acetate and the sodium acetate.
In the step (4): said lysate can be the CTAB lysate; Said lysate is preferably the solution that following method prepares: with 0.01mol Tris-HCl, 0.02mol EDTA-Na
2, 2g CTAB, 2g Vinylpyrrolidone polymer (PVP) and 1.4mol NaCl water be settled to 1 liter.
In the step (6), said TE solution specifically can be the solution that following method prepares: with the Tris-Cl aqueous solution of 1 milliliter of 1mol/LpH8.0 and the EDTA-Na of 200 microlitre 0.5mol/L pH 8.0
2The aqueous solution joins in 98.8 ml distilled waters.In the step (6), said T50E10 solution specifically can be the solution that following method prepares: with the EDTA-Na of 5 milliliters, the Tris-Cl aqueous solution of 1mol/L, pH8.0 and 2 milliliters, 0.5mol/L, pH 8.0
2The aqueous solution joins in 93 ml distilled waters.
In the said method, the said EDTA aqueous solution (or the EDTA-Na of said cotton sample, said buffer A, said buffer B, said DNase I, step (2)
2The aqueous solution), the proportioning of the said acetate aqueous solution of the said acetate aqueous solution of the said acetate aqueous solution of said damping fluid C, said damping fluid D, said lysate, step (4), step (5), said TE solution, said RNase, said Proteinase K and step (8) can be: 30-100g cotton sample: 200-800 milliliter buffer A: 5-40 milliliter buffer B: the EDTA aqueous solution (or the EDTA-Na of 10-40UDNase I:100-400 μ L 0.5mol/L pH7.5-8.5
2The aqueous solution): 5-40 milliliter damping fluid C:5-40 milliliter damping fluid D:1-10 milliliter lysate: 200-450 microlitre 2mol/L acetate aqueous solution: 400-900 microlitre 1mol/L acetate aqueous solution: 100-600 microlitre TE solution: 1-10URNaes:1-10U Proteinase K: 20-60 microlitre 2-5mol/L acetate aqueous solution.
Step (2) specifically can be: after the deposition of step (1) added said buffer B and DNase I, 4 ℃ were reacted 30-60 minute, and added the said EDTA aqueous solution then.In the step (2), said centrifugal parameter specifically can be: 10000-17000g, 5-40 minute.
In the step (3), said centrifugal parameter specifically can be: 10000-17000g, 5-20 minute.
Step (4) specifically can be: said plastosome to step (3) adds said lysate, 65 ℃ cracking 15-60 minute, add said acetate aqueous solution then.
Step (7) specifically can be: in the solution of step (6), add said RNaes, 37 ℃ water-bath 30-60 minute; Add said Proteinase K then, 37 ℃ water-bath 30-60 minute.
Said cotton sample specifically can be the cotton etiolated seedling.
The kind of said cotton specifically can be Xu cotton 244.
The invention provides the method for separating the cotton Mitochondrial DNA, is one of molecular biological basic step, belongs to the plant gene engineering technology field.The present invention is directed to the deficiency that exists in the present extraction Mitochondrial DNA process; On traditional CTAB method, improve targetedly; Adjust the centrifugal time, suitably increase centrifugal number of times, change reagent, increase extracting washing times etc., optimize its extraction conditions.The present invention guarantees in leaching process on the basis to plastosome physical abuse degree minimum; Effectively remove nuclear DNA and phenols, polysaccharose substance beyond the plastosome; Reduce pollution rate, improve the purity and the quality of Mitochondrial DNA, can satisfy cotton chondriogen engineering subsequent experimental.
Description of drawings
Fig. 1 is the agarose gel electrophoresis figure of the Mitochondrial DNA of embodiment 1 extraction; 1-4 is for extracting the Mitochondrial DNA that obtains.
Fig. 2 is the agarose gel electrophoresis figure of the Mitochondrial DNA of embodiment 2 extractions; 1-4 is for extracting the Mitochondrial DNA that obtains.
Fig. 3 is the agarose gel electrophoresis figure of the Mitochondrial DNA of embodiment 3 extractions; 1-4 is for extracting the Mitochondrial DNA that obtains.
The Mitochondrial DNA enzyme that Fig. 4 extracts for embodiment 1 to 3 is cut the agarose gel electrophoresis figure of product; 1:D15000plus DNA ladder; 2,3: the enzyme that embodiment 1 extracts the Mitochondrial DNA that obtains is cut product; 4,5: the enzyme that embodiment 2 extracts the Mitochondrial DNA that obtains is cut product; 6,7: the enzyme that embodiment 3 extracts the Mitochondrial DNA that obtains is cut product.
The Mitochondrial DNA enzyme that Fig. 5 extracts for the CTAB method is cut the agarose gel electrophoresis figure of product; The enzyme of the Mitochondrial DNA that the 1-5:CTAB method is extracted is cut product; 6:D2000DNA ladder.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.
Xu cotton 244 (having another name called Soviet Union cotton No. 20): available from Jiangsu Xu Nong kind industry Science and Technology Ltd.; Cultivate by Xuzhou Academy of Agricultural Sciences, be numbered Soviet Union and examine cotton 200202 through Jiangsu Province's crop varietal approval committee in 2002.
The extraction of embodiment 1, cotton Mitochondrial DNA
One, solution preparation
The preparation method of buffer A is following: 0.05mol Tris-HCl, 1.2mol NaCl, 2mmol EDTA, 8g Vinylpyrrolidone polymer (PVP), 4mmol β-thin basic ethanol and 0.5g bovine serum albumin (BSA) water are settled to 1 liter, adjust pH to 8.0.
The preparation method of buffer B is following: with 0.05mol Tris-HCl, 0.3mol sucrose, 0.03mol MgCl
2Be settled to 1 liter, adjust pH to 8.0 with 3g Vinylpyrrolidone polymer (PVP) water.
The preparation method of damping fluid C is following: with 0.05mol Tris-HCl, 0.02mol EDTA-Na
2Be settled to 1 liter with 0.3mol sucrose water, adjust pH is to 7.5.
The preparation method of damping fluid D is following: with 0.05mol Tris-HCl, 0.02mol EDTA-Na
2Be settled to 1 liter, adjust pH to 8.0 with 0.6mol sucrose water.
The preparation method of lysate is following: with 0.0lmol Tris-HCl, 0.02mol EDTA-Na
2, 2g CTAB, 2g Vinylpyrrolidone polymer (PVP) and 1.4mol NaCl water be settled to 1 liter.
The preparation method of TE solution is following: with the EDTA-Na of 1 milliliter, the Tris-Cl aqueous solution of 1mol/L, pH8.0 and 200 microlitres, 0.5mol/L, pH 8.0
2The aqueous solution joins in 98.8 ml distilled waters.
The preparation method of T50E10 solution is following: with the EDTA-Na of 5 milliliters, the Tris-Cl aqueous solution of 1mol/L, pH8.0 and 2 milliliters, 0.5mol/L, pH 8.0
2The aqueous solution joins in 93 ml distilled waters.
The preparation method of solution first is following: be made up of chloroform and primary isoamyl alcohol, the volume ratio of chloroform and primary isoamyl alcohol is 24: 1.
The preparation method of solution second is following: the saturated phenol of Tris, chloroform and primary isoamyl alcohol are formed, and the volume ratio of the saturated phenol of Tris, chloroform and primary isoamyl alcohol is 25: 24: 1.
Two, the extraction of Mitochondrial DNA
(1) 50g cotton 244 etiolated seedlings of Xu and 400 milliliters of buffer A mixings are carried out homogenate, centrifugal collecting precipitation.
(2) deposition to step (1) adds 20 milliliters of buffer B and 4 ℃ of reactions of 20U DNase I 30min successively, adds the EDTA-Na of 200 microlitres, 0.5mol/L, pH7.5 then
2The aqueous solution, the centrifugal 5min of 10000 * g obtains deposition.
(3) deposition to step (2) adds 20 milliliters of damping fluid C, injects 20 milliliters of damping fluid D with syringe gently to the bottom then, ice bath 30min, and the centrifugal 5min of 10000 * g gets pure complete plastosome.
(4) plastosome to step (3) adds 5 milliliters of 65 ℃ of cracking 15min of lysate (cracking is complete); The potassium acetate solution ice bath 30min that adds 300 microlitres, 2mol/L then; Centrifugal collection supernatant carries out extracting with isopyknic solution first, the centrifugal deposition that obtains.
(5) add the aqueous sodium acetate solution of 450 microlitres, 1mol/L to the deposition of step (4), add absolute ethyl alcohol again, the centrifugal mitochondrial pellet that obtains is through 70% (volumn concentration) aqueous ethanolic solution washing mitochondrial pellet.
(6) mitochondrial pellet of drying step (5) adds the fully dissolving of spending the night of 400 microlitre T50E10 solution.
(7) in the lysate of step (6), add 5U RNase, 30min in 37 ℃ of water-baths; Add the 5U Proteinase K then, 30min in 37 ℃ of water-baths afterwards, carries out extracting with isopyknic solution second, centrifugal collection supernatant.
(8) in the supernatant of step (7), add the aqueous sodium acetate solution of 40 microlitres, 3mol/L and the absolute ethyl alcohol that diploid amasss, centrifugal collecting precipitation; With 70% (volumn concentration) aqueous ethanolic solution washing deposition, obtain Mitochondrial DNA.Drying precipitated, add TE and fully dissolve.
Mitochondrial DNA is carried out agarose gel electrophoresis, see Fig. 1.
The extraction of embodiment 2, cotton Mitochondrial DNA
One, solution preparation
The preparation method of buffer A is following: 0.05mol Tris-HCl, 1.2mol NaCl, 2mmol EDTA, 6g Vinylpyrrolidone polymer (PVP), 2mmol β-thin basic ethanol and 0.1g BSA water are settled to 1 liter, adjust pH to 7.5.
The preparation method of buffer B is following: with 0.05mol Tris-HCl, 0.1mol sucrose, 0.01mol MgCl
2Be settled to 1 liter, adjust pH to 7.5 with 1g Vinylpyrrolidone polymer (PVP) water.
The preparation method of damping fluid C is following: with 0.05mol Tris-HCl, 0.02mol EDTA-Na
2Be settled to 1 liter with 0.1mol sucrose water, adjust pH is to 7.5.
The preparation method of damping fluid D is following: with 0.05mol Tris-HCl, 0.02mol EDTA-Na
2Be settled to 1 liter, adjust pH to 7.5 with 0.2mol sucrose water.
The preparation method of lysate is following: with 0.01mol Tris-HCl, 0.02mol EDTA-Na
2, 2g CTAB, 2g Vinylpyrrolidone polymer (PVP) and 1.4mol NaCl water be settled to 1 liter.
The preparation method of TE solution is following: with the EDTA-Na of 1 milliliter, the Tris-Cl aqueous solution of 1mol/L, pH8.0 and 200 microlitres, 0.5mol/L, pH 8.0
2The aqueous solution joins in 98.8 ml distilled waters.
The preparation method of T50E10 solution is following: with the EDTA-Na of 5 milliliters, the Tris-Cl aqueous solution of 1mol/L, pH8.0 and 2 milliliters, 0.5mol/L, pH 8.0
2The aqueous solution joins in 93 ml distilled waters.
The preparation method of solution first is following: be made up of chloroform and primary isoamyl alcohol, the volume ratio of chloroform and primary isoamyl alcohol is 24: 1.
The preparation method of solution second is following: the saturated phenol of Tris, chloroform and primary isoamyl alcohol are formed, and the volume ratio of the saturated phenol of Tris, chloroform and primary isoamyl alcohol is 25: 24: 1.
Two, the extraction of Mitochondrial DNA
(1) 30g cotton 244 etiolated seedlings of Xu and 200 milliliters of buffer A mixings are carried out homogenate, centrifugal collecting precipitation.
(2) deposition to step (1) adds 5 milliliters of buffer B and 4 ℃ of reactions of 10U DNase I 45min successively, adds the EDTA aqueous solution of 100 microlitres, 0.5mol/L, pH8.0 then, and the centrifugal 20min of 15000 * g obtains deposition.
(3) deposition to step (2) adds 5 milliliters of damping fluid C and 5 milliliters of damping fluid D successively, and the centrifugal 10min of 15000 * g gets pure complete plastosome.
(4) plastosome to step (3) adds 1 milliliter of 65 ℃ of cracking 40min of lysate (cracking is complete), adds the amine acetate aqueous solution of 200 microlitres, 2mol/L then, and centrifugal collection supernatant carries out extracting with isopyknic solution first, the centrifugal deposition that obtains.
(5) add the aqueous sodium acetate solution of 400 microlitres, 1mol/L to the deposition of step (4), add absolute ethyl alcohol again, the centrifugal mitochondrial pellet that obtains is through 70% (volumn concentration) aqueous ethanolic solution washing mitochondrial pellet.
(6) mitochondrial pellet of drying step (5) adds the fully dissolving of spending the night of 100 microlitre T50E10 solution.
(7) in the lysate of step (6), add 1U RNase, 45min in 37 ℃ of water-baths; Add the 1U Proteinase K then, 45min in 37 ℃ of water-baths afterwards, carries out extracting with isopyknic solution second, centrifugal collection supernatant.
(8) aqueous sodium acetate solution and the absolute ethyl alcohol of adding 20 microlitres, 2mol/L in the supernatant of step (7), centrifugal collecting precipitation; With 70% (volumn concentration) aqueous ethanolic solution washing deposition, obtain Mitochondrial DNA.Drying precipitated, add TE and fully dissolve.
Mitochondrial DNA is carried out agarose gel electrophoresis, see Fig. 2.
The extraction of embodiment 3, cotton Mitochondrial DNA
One, solution preparation
The preparation method of buffer A is following: 0.05mol Tris-HCl, 1.2mol NaCl, 2mmol EDTA, 10g Vinylpyrrolidone polymer (PVP), 5mmol β-thin basic ethanol and 0.8g BSA water are settled to 1 liter, adjust pH to 8.5.
The preparation method of buffer B is following: with 0.05mol Tris-HCl, 0.5mol sucrose, 0.05mol MgCl
2Be settled to 1 liter, adjust pH to 8.5 with 5g Vinylpyrrolidone polymer (PVP) water.
The preparation method of damping fluid C is following: with 0.05mol Tris-HCl, 0.02mol EDTA-Na
2Be settled to 1 liter with 0.5mol sucrose water, adjust pH is to 7.5.
The preparation method of damping fluid D is following: with 0.05mol Tris-HCl, 0.02mol EDTA-Na
2Be settled to 1 liter, adjust pH to 8.5 with 1.0mol sucrose water.
The preparation method of lysate is following: with 0.01mol Tris-HCl, 0.02mol EDTA-Na
2, 2g CTAB, 2g Vinylpyrrolidone polymer (PVP) and 1.4mol NaCl water be settled to 1 liter.
The preparation method of TE solution is following: with the EDTA-Na of 1 milliliter, the Tris-Cl aqueous solution of 1mol/L, pH8.0 and 200 microlitres, 0.5mol/L, pH 8.0
2The aqueous solution joins in 98.8 ml distilled waters.
The preparation method of T50E10 solution is following: with the EDTA-Na of 5 milliliters, the Tris-Cl aqueous solution of 1mol/L, pH8.0 and 2 milliliters, 0.5mol/L, pH 8.0
2The aqueous solution joins in 93 ml distilled waters.
The preparation method of solution first is following: be made up of chloroform and primary isoamyl alcohol, the volume ratio of chloroform and primary isoamyl alcohol is 24: 1.
The preparation method of solution second is following: the saturated phenol of Tris, chloroform and primary isoamyl alcohol are formed, and the volume ratio of the saturated phenol of Tris, chloroform and primary isoamyl alcohol is 25: 24: 1.
Two, the extraction of Mitochondrial DNA
(1) 100g cotton 244 etiolated seedlings of Xu and 800 milliliters of buffer A mixings are carried out homogenate, centrifugal collecting precipitation.
(2) deposition to step (1) adds 40 milliliters of buffer B and 4 ℃ of reactions of 40U DNase I 60min successively, adds the EDTA aqueous solution of 400 microlitres, 0.5mol/L, pH8.5 then, and the centrifugal 40min of 17000 * g obtains deposition.
(3) deposition to step (2) adds 40 milliliters of damping fluid C and 40 milliliters of damping fluid D successively, and the centrifugal 20min of 17000 * g gets pure complete plastosome.
(4) plastosome to step (3) adds 10 milliliters of 65 ℃ of cracking 60min of lysate (cracking is complete), adds the aqueous sodium acetate solution of 450 microlitres, 2mol/L then, and centrifugal collection supernatant carries out extracting with isopyknic solution first, the centrifugal deposition that obtains.
(5) add the amine acetate aqueous solution of 900 microlitres, 1mol/L to the deposition of step (4), add absolute ethyl alcohol again, the centrifugal mitochondrial pellet that obtains is through 70% (volumn concentration) aqueous ethanolic solution washing mitochondrial pellet.
(6) mitochondrial pellet of drying step (5) adds the fully dissolving of spending the night of 600 microlitre TE solution.
(7) in the lysate of step (6), add 10U RNase, 60min in 37 ℃ of water-baths; Add the 10U Proteinase K then, 60min in 37 ℃ of water-baths afterwards, carries out extracting with isopyknic solution second, centrifugal collection supernatant.
(8) aqueous sodium acetate solution and the absolute ethyl alcohol of adding 60 microlitres, 5mol/L in the supernatant of step (7), centrifugal collecting precipitation; With 70% (volumn concentration) aqueous ethanolic solution washing deposition, obtain Mitochondrial DNA.Drying precipitated, add TE and fully dissolve.
Mitochondrial DNA is carried out agarose gel electrophoresis, see Fig. 3.
Respectively the Mitochondrial DNA that extracts among the embodiment 1 to embodiment 3 was cut 12 hours with Not I enzyme, carried out agarose gel electrophoresis then, with the checking extraction effect.See Fig. 4.
Adopt the CTAB method to extract the Mitochondrial DNA of cotton etiolated seedling, cut 12 hours, carry out agarose gel electrophoresis then, see Fig. 5 with Not I enzyme.
The result shows: conventional CTAB extracts Mitochondrial DNA, and impurity is more, and general restriction endonuclease is cut not opened, and disperse is more serious; And method provided by the invention, the DNA of extraction is purer, and quality is higher, can satisfy the associated molecule test that continues.
Claims (4)
1. method of extracting the Mitochondrial DNA of cotton comprises two steps of extraction of mitochondrial extraction and Mitochondrial DNA;
Said mitochondrial extraction comprises the steps (1) to step (3):
(1) the cotton sample is carried out homogenate, centrifugal collecting precipitation in buffer A; The preparation method of said buffer A is following: 0.05mol Tris-HCl, 1.2mol NaCl, 2mmol EDTA, 6-10g Vinylpyrrolidone polymer, 2-5mmol β-thin basic ethanol and 0.1-0.8g BSA water are settled to 1 liter, and adjust pH is to 7.5-8.5;
(2) deposition to step (1) adds buffer B and DNase I, and 4 ℃ were reacted 30-60 minute, to remove nuclear DNA, adds the EDTA aqueous solution or EDTA-Na then
2The aqueous solution, the centrifugal deposition that obtains; The preparation method of said buffer B is following: with 0.05mol Tris-HCl, 0.1-0.5mol sucrose, 0.01-0.05mol MgCl
2Be settled to 1 liter with 1-5g Vinylpyrrolidone polymer water, adjust pH is to 7.5-8.5;
Said centrifugal parameter is: 10000-17000g, 5-40 minute;
(3) deposition to step (2) adds damping fluid C and damping fluid D successively, the centrifugal plastosome that obtains; The preparation method of said damping fluid C is following: with 0.05mol Tris-HCl, 0.02mol EDTA-Na
2Be settled to 1 liter, adjust pH to 7.5 with 0.1-0.5mol sucrose water; The preparation method of said damping fluid D is following: with 0.05mol Tris-HCl, 0.02mol EDTA-Na
2Be settled to 1 liter with 0.2-1.0mol sucrose water, adjust pH is to 7.5-8.5;
Said centrifugal parameter is: 10000-17000g, 5-20 minute;
The extraction of said Mitochondrial DNA comprises the steps (4) to step (8):
(4) add lysate to the plastosome of step (3) and carry out cracking, 65 ℃ cracking 15-60 minute, add acetate aqueous solution then, centrifugal collection supernatant carries out extracting with isopyknic solution first, centrifugal obtaining precipitated; Said solution first is made up of chloroform and primary isoamyl alcohol, and the volume ratio of chloroform and primary isoamyl alcohol is 24: 1;
Said lysate is the solution that following method prepares: with 0.01mol Tris-HCl, 0.02mol EDTA-Na
2, 2g CTAB, 2g Vinylpyrrolidone polymer and 1.4mol NaCl water be settled to 1 liter;
(5) deposition to step (4) adds acetate aqueous solution, adds absolute ethyl alcohol again, and the centrifugal deposition that obtains is with the aqueous ethanolic solution washing deposition of 70% volumn concentration;
(6) with the deposition of TE solution or T50E10 solution dissolving step (5);
The preparation method of said T50E10 solution is following: with the Tris-Cl aqueous solution of 5 milliliters of 1mol/L pH8.0 and the EDTA-Na of 2 milliliters of 0.5mol/L pH 8.0
2The aqueous solution joins in 93 ml waters;
(7) add RNase then, 37 ℃ water-bath 30-60 minute, to remove RNA; Add Proteinase K then, 37 ℃ water-bath 30-60 minute, to remove albumen; Carry out extracting with isopyknic solution second then, centrifugal collection supernatant; Said solution second is made up of the saturated phenol of Tris, chloroform and primary isoamyl alcohol, and the volume ratio of the saturated phenol of Tris, chloroform and primary isoamyl alcohol is 25: 24: 1;
(8) supernatant to step (7) adds acetate aqueous solution, adds absolute ethyl alcohol again, the centrifugal deposition that obtains; With the aqueous ethanolic solution washing deposition of 70% volumn concentration, obtain the Mitochondrial DNA of cotton;
In the said method, the said EDTA aqueous solution or the EDTA-Na of said cotton sample, said buffer A, said buffer B, said DNase I, step (2)
2The proportioning of the said acetate aqueous solution of the said acetate aqueous solution of the aqueous solution, said damping fluid C, said damping fluid D, said lysate, step (4), the said acetate aqueous solution of step (5), said TE solution, said RNase, said Proteinase K and step (8) is: 30-100g cotton sample: 200-800 milliliter buffer A: 5-40 milliliter buffer B: the EDTA aqueous solution or the EDTA-Na of 10-40U DNase I:100-400 μ L0.5mol/L pH7.5-8.5
2The aqueous solution: 5-40 milliliter damping fluid C:5-40 milliliter damping fluid D:1-10 milliliter lysate: 200-450 microlitre 2mol/L acetate aqueous solution: 400-900 microlitre 1mol/L acetate aqueous solution: 100-600 microlitre TE solution: 1-10U RNase:1-10U Proteinase K: 20-60 microlitre 2-5mol/L acetate aqueous solution.
2. the method for claim 1 is characterized in that: in the step (4), said acetate is at least a in potassium acetate, ammonium acetate and the sodium acetate; In the step (5), said acetate is at least a in potassium acetate, ammonium acetate and the sodium acetate; In the step (8), said acetate is at least a in potassium acetate, ammonium acetate and the sodium acetate.
3. according to claim 1 or claim 2 method, it is characterized in that: in the step (6), said TE solution is the solution that following method prepares: with the Tris-Cl aqueous solution of 1 milliliter of 1mol/L pH8.0 and the EDTA-Na of 200 microlitre 0.5mol/LpH 8.0
2The aqueous solution joins in 98.8 ml waters.
4. method as claimed in claim 3 is characterized in that: said cotton sample is the cotton etiolated seedling; The kind of said cotton is Xu cotton 244.
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| CN107266521B (en) * | 2016-04-08 | 2020-07-14 | 中国农业大学 | Method for extracting cotton mitochondria and protein thereof |
| CN113136384B (en) * | 2021-06-10 | 2023-03-21 | 海南师范大学 | Method for extracting nucleic acid of endangered semi-mangrove plant mallotus japonicus |
| CN113584018A (en) * | 2021-08-24 | 2021-11-02 | 海南师范大学 | Improved CTAB method for extracting nucleic acid from mallotus japonicus |
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| CN101250522A (en) * | 2008-04-14 | 2008-08-27 | 金政策 | Modified high-salt method for extracting mitochondria DNA and uses thereof |
| CN101338310A (en) * | 2008-08-13 | 2009-01-07 | 中国人民解放军第三军医大学第二附属医院 | Process for extracting cytoplasm DNA |
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| CN101250522A (en) * | 2008-04-14 | 2008-08-27 | 金政策 | Modified high-salt method for extracting mitochondria DNA and uses thereof |
| CN101338310A (en) * | 2008-08-13 | 2009-01-07 | 中国人民解放军第三军医大学第二附属医院 | Process for extracting cytoplasm DNA |
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| 孙志栋等.棉花DNA提取方法的探讨.《浙江农业学报》.2004,(第4期),全文. * |
| 宋国立邓.改良CTAB法快速提取棉花DNA.《棉花学报》.1998,(第5期),全文. * |
| 曲延英等.4种快速提取棉花总DNA方法的比较.《新疆农业大学学报》.1999,(第4期),全文. * |
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