CN102382821A - Method for extracting DNA from genome - Google Patents
Method for extracting DNA from genome Download PDFInfo
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- CN102382821A CN102382821A CN201110368801XA CN201110368801A CN102382821A CN 102382821 A CN102382821 A CN 102382821A CN 201110368801X A CN201110368801X A CN 201110368801XA CN 201110368801 A CN201110368801 A CN 201110368801A CN 102382821 A CN102382821 A CN 102382821A
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Abstract
The invention relates to a method for extracting DNA from a genome. The method comprises the following steps of separating DNA, separating out DNA, eluting and drying DNA, and dissolving and storing DNA; DNA aqueous solution obtained in the DNA separating step is averagely subpackaged into 1.5-5 ml small centrifugal tubes; and the quality of DNA in the small centrifugal tubes is predicted before the DNA dissolving and storing step. Multiple parts of a little DNA are obtained by subpackaging, so that the total contact area of DNA and the solution is increased during the dissolution and storage of DNA, and the extracted DNA can be quickly and fully dissolved on the premise that the length of DNA is not affected; and the quality of DNA evenly subpacked in the small centrifugal tubes is predicted before the DNA dissolving and storing step, so that the concentration of DNA can be controlled to meet sequent experimental requirements through the quality prediction.
Description
Technical field
The present invention relates to a kind of genome DNA extracting method, relate in particular to a kind of disposable process for extracting that obtains more DNA.
Background technology
Extract genomic dna and be used for PCR isolated genes, Southern hybridization, genomic library construction etc.Because it is long to extract the genomic dna spended time, therefore, can use manpower and material resources sparingly, and for DNA demand bigger experiment such as genomic library construction the disposable just suitable necessity of more DNA of obtaining usually through the more DNA of disposable extraction.
The method of extracting genomic dna is a lot, and like dense salt method, anionic detergent method, phenol chloroform extraction method, water extraction process etc., but total method steps and principle are:
DNA separates: the reagent that provides according to dense salt method, anionic detergent method, phenol chloroform extraction method or the water extraction process genomic dna in the solution of smudge cells separates with protein, lipid, carbohydrate, RNA etc., obtains aqueous dna;
DNA separates out: the Virahol of 0.8 times of volume of adding or the absolute ethyl alcohol of 2 times of volumes are separated out DNA in aqueous dna;
It is dry that DNA washs: behind the precipitated dna, obtain the DNA deposition, will precipitate again with 70% washing with alcohol 2-3 time through the centrifugal supernatant of abandoning, and in drying at room temperature;
DNA dissolves preservation: add entry or TE solution (10mmol/L Tris-Cl, 1mmol/L EDTA disodium, pH 8.0) and dissolve exsiccant DNA, put-20 ℃ and deposit.
For obtaining the primary method that adopts of more DNA is the amount that increases the material that is used to extract DNA, adapts with it, selects the bigger container of volume when extracting DNA, generally adopts 5-50ml centrifuge tube when DNA (extract a small amount of the general 1.5ml of use centrifuge tube).Certainly, also can suitably increase the amount of DNA extraction through the improvement of working specification in the experimentation and certain operations method.
In the existing document; Second piece of extraction of testing nine plant genome DNAs by " molecular biology experiment guidance " (the 2nd edition) that Higher Education Publishing House published in 2007, Wei Qun edits; Adopt the phenol chloroform extraction method to extract genomic dna, adopting the 50ml centrifuge tube is that container is with the more experiment material of disposable extraction; By press of Wuhan University 2009 publish, the extraction and the enzyme of experiment one plant genome DNA of Yan Haiyan chief editor " genetically engineered and molecular biology experiment study course " cut; Adopt the phenol chloroform extraction method to extract genomic dna, adopting the 7ml centrifuge tube is that container is with the more experiment material of disposable extraction.But because the DNA of disposable extraction amount is more, when in the end adding entry or TE solution dissolving DNA, DNA is agglomerating in a large number can't dissolve.Though can adopt high temperature bath heating or inhale the method for beating solution with the rifle head and impel the DNA dissolving, general careful employing is because high temperature bath heating required time length, can cause dna degradation and dissolve still incomplete; The suction of rifle head is beaten solution and can be caused the DNA mechanicalness to shear, and makes DNA length not reach the subsequent experimental requirement.DNA can not abundant fast dissolving directly cause its concentration low, and then the virtual mass of DNA is low, can't satisfy the requirement of subsequent experimental to the DNA quality, especially makes up genomic library.And; Extract in the genomic dna method existing; DNA is not done prediction of quality; DNA is not done the proposition of the concrete grammar of concentration prediction yet, the volume of water that when dissolving DNA, adds or TE solution only or by rule of thumb with fortune, can't control, very easily cause concentration not reach the concentration range that subsequent experimental requires and the DNA extraction experiment fallen short of success for lack of final effort.Therefore, necessary searching is a kind of under the situation that does not influence DNA length, makes DNA fully dissolving fast, and guarantees that DNA concentration reaches the process for extracting that subsequent experimental requires.
Summary of the invention
Technical problem to be solved by this invention provides a kind of genome DNA extracting method, under the prerequisite that does not influence DNA length, can make quick, the fully dissolving of DNA, and guarantee that DNA concentration reaches the subsequent experimental requirement.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is:
A kind of genome DNA extracting method comprises that DNA separates, DNA separates out, DNA washs drying and the DNA dissolving is preserved, and said DNA separates the aqueous dna average mark that obtains and installs in the little centrifuge tube of a plurality of 1.5-5ml; Before said DNA dissolving is preserved DNA in the said little centrifuge tube is carried out prediction of quality.
Aqueous dna carries out that DNA separates out after through packing more respectively, DNA washing drying and DNA dissolving are preserved; Because the amount of DNA is few in each little centrifuge tube; Adding less water or TE solution can dissolve exsiccant DNA fast when then the DNA dissolving was preserved; This method utilization increases the principle that solute and solvent contact area improve dissolution rate and dissolution degree, even need not blow and beat, need not heat and just can make DNA fully dissolving fast, thereby solution DNA dissolves the problem of difficulty; Do not blow and beat and do not heat the probability that has yet reduced dna break, guaranteed the length of DNA.Through average packing, make the DNA prediction of quality become possibility, so that control DNA concentration reaches the subsequent experimental requirement.
Preferably, said aqueous dna average mark installs in the little centrifuge tube of a plurality of 1.5ml.1.5ml little centrifuge tube bottom taper helps the DNA deposition and is affixed on the pipe end when centrifugal, and adds few solvent to 5 μ l volumes and just can cover and manage the end, covering DNA, even naked eyes are difficult for seeing and can guarantee that also it is covered fully and dissolves after the DNA drying.
Said DNA prediction of quality detects DNA concentration for wash in dried arbitrary the little centrifuge tube water that adds 5-50 μ l or TE solution to said DNA after, and obtains the quality of DNA according to this concentration and volume.Because average packing; The amount of DNA is roughly the same in each little centrifuge tube; Add suitable water or TE solution according to predicted quality to all the other little centrifuge tubes and can guarantee that concentration reaches the requirement of subsequent experimental, be unlikely to add blindly or by rule of thumb entry or TE solution dissolving DNA and cause concentration not reach the subsequent experimental requirement.The may command of DNA concentration has been practiced thrift experimental period and experimental cost, has improved the success ratio of experiment greatly.
Said DNA prediction of quality can also detect DNA concentration behind the water that respectively adds equal volume in the only little centrifuge tube of dried any 2-4 or the TE solution and averages for washing to said DNA, and obtains the average quality of DNA according to this concentration and volume.Average quality to obtain the control that makes DNA concentration more accurate.
Said detection DNA concentration can be through measuring light absorption value OD
260/ OD
280Perhaps obtain through agarose gel electrophoresis.
The present invention has increased total contact area of DNA and solvent through the packing aqueous dna when the DNA dissolving is preserved, under the prerequisite that does not influence DNA length, can make the DNA fully dissolving fast of extraction; Through packing, also make the DNA prediction of quality become possibility, the concentration of the DNA that then can guarantee to extract reaches the subsequent experimental requirement.Compared with prior art, its dissolution rate improved greatly when the DNA dissolving was preserved, and dissolution degree becomes dissolving fully by the agglomerating difficult dissolving of prior art especially; DNA quality measurable makes its concentration may command when the DNA dissolving is preserved, and solved prior art and added dissolution with solvents DNA blindly or by rule of thumb and cause concentration not reach requiring and the problem of failure.
Description of drawings
Fig. 1 extracts the schema of DNA for embodiment of the present invention;
Fig. 2 by the embodiment of the invention the electrophoretogram of extraction DNA.
Embodiment
The invention is not restricted to following embodiment or embodiment, all modification and distortion of being made without prejudice to the present invention's spirit all should be included within the scope of the invention.
The present invention extracts DNA step such as Fig. 1, and concrete steps are following:
DNA separates: the reagent that provides according to dense salt method, anionic detergent method, phenol chloroform extraction method or the water extraction process genomic dna in the solution of smudge cells separates with protein, lipid, carbohydrate, RNA etc., obtains aqueous dna;
The aqueous dna packing: the aqueous dna average mark that separation is obtained installs in the little centrifuge tube of 1.5-5ml.Aqueous dna is through packing; The amount of DNA is few in each little centrifuge tube; The total contact area of DNA and water or TE solution increased when then the DNA dissolving was preserved; Improved dissolution rate and the dissolution degree of DNA, even need not blow and beat, need not heat and just can make fast fully dissolving of DNA, thereby also solve the difficult problem of DNA dissolving; Do not blow and beat and do not heat the probability that has yet reduced dna break, guaranteed the length of DNA.Especially 1.5ml centrifuge tube, its bottom taper more helps at the bottom of the DNA deposited tube, and adds few solvent to 5 μ l volumes and just can cover the pipe end, covering DNA, helps DNA and dissolves.
DNA separates out: in packing add 0.8 times of volume in the little centrifuge tube of aqueous dna Virahol or 2 times of volume of ethanol DNA is separated out;
It is dry that DNA washs: behind the precipitated dna, centrifugal and topple over supernatant then DNA be deposited on the pipe end, use 70% washing with alcohol DNA2-3 time again, and in drying at room temperature;
DNA prediction of quality: for the DNA that average packing obtains, after the washing drying, in arbitrary little centrifuge tube, add water or the TE solution of 5-50 μ l, get part through measuring light absorption value OD
260/ OD
280Perhaps obtaining DNA concentration through agarose gel electrophoresis, also can be to add entry or TE measured in solution DNA concentration in centrifugal and average to any 2-4 is only little, obtains the quality of DNA according to the concentration that obtains and volume.
DNA dissolves preservation: according to the concentration requirement of predicted quality and subsequent experimental, in all the other little centrifuge tubes, add the water or the TE solution of suitable volumes, put-20 ℃ and deposit.Through to the DNA prediction of quality, can control the concentration of DNA, be unlikely to add blindly or by rule of thumb entry or TE solution dissolving DNA and cause concentration not reach the subsequent experimental requirement.Practice thrift experimental period and experimental cost, improved the success ratio of experiment greatly.
Compared with prior art, its dissolution rate improved greatly when the DNA dissolving was preserved, and dissolution degree becomes dissolving fully by the agglomerating difficult dissolving of prior art especially; DNA quality measurable makes its concentration may command when the DNA dissolving is preserved, and solved prior art and added dissolution with solvents DNA blindly or by rule of thumb and cause concentration not reach requiring and the problem of failure.
Embodiment:
Combine the inventive method to extract the locust genomic dna with the phenol chloroform extraction method, concrete steps are following:
101.DNA separate: get fresh locust leg muscle rapid grinding powder in liquid nitrogen; In a 50ml centrifuge tube, add the said powder of the about 1.5ml of volume, add 15mlDNA lysate (0.1mol/L NaCl, 0.01mol/L EDTA disodium, 0.1mol/L Tris, 2%SDS, pH 8.0) again, piping and druming is dissolved powder fully gently; Adding the RNA enzyme, to make final concentration be 20 μ g/mL, and adding Proteinase K, to make final concentration be 100 μ g/mL, 50 ℃ of incubations 3 hours, lysing cell, digestible protein; Add isopyknic phenol, chloroform, iso pentane alcohol mixture (volume ratio 25: 24: 1) again, mixing after the layering, is centrifugal 10 minutes of 6500rpm with the rotating speed gently, draws the upper strata in another 50ml centrifuge tube; In the upper solution that obtains, add isopyknic chloroform, iso pentane alcohol mixture (volume ratio 24: 1), mixing gently, after the layering, centrifugal 10 minutes of rotating speed 6500rpm, the upper strata is aqueous dna (about 10ml).
102.DNA aqueous solution packing: the 101 aqueous dna average marks that obtain are installed in 25 1.5ml centrifuge tubes the about 400 μ l of every pipe solution.With prior art supernatant is packed into entirely and to compare in the 50ml centrifuge tube, few through the amount of the DNA in each centrifuge tube of packing, the abundant dissolving of DNA in TE solution and dissolving fast after helping; Through average packing; The amount of the contained DNA of solution of each centrifuge tube is identical; Then after DNA washing and drying, the DNA in wherein 1 or several the little centrifuge tubes to be carried out concentration detect, calculate DNA weight, the concentration that can control DNA in all the other little centrifuge tubes reaches the subsequent experimental requirement.
103.DNA separate out: respectively add 800 μ l absolute ethyl alcohols to the 1.5ml of said packing centrifuge tube, DNA was separated out in 8-12 hour in-20 ℃ of placements.
104.DNA washing is dry: the 1.5ml centrifuge tube that will place 8-12 hour is in centrifugal 10 minutes of 4 ℃, 10000rpm, the supernatant that inclines, and the DNA deposition is assembled also to be affixed on and is managed at the end; With a spot of 70% ethanol rinsing deposition 2 times, centrifugal 10 minutes of 4 ℃, 6500rpm, deposition is affixed on the pipe end, topples over supernatant, and in the air-dry deposition of room temperature.
105.DNA quality examination: add 20 μ lTE dissolving to 1.5ml centrifuge tube wherein, and get 1-2 μ l and detect light absorption value OD260/OD280 value, obtain concentration and be about 300mg/ml, calculate that DNA weight is 6mg in this 1.5ml centrifuge tube.Compared with prior art, 1.5ml centrifuge tube pipe vertex (vertices) is thin, and deposition is affixed on the pipe end, and adding fewly is enough to cover deposition to 5 μ lTE solution, even the air-dry back of DNA naked eyes are difficult for seeing it is covered fully and dissolve; Obtain the volume of suitable TE solution through the amount of measuring wherein DNA in the 1.5ml centrifuge tube, can make DNA concentration in all the other 1.5ml centrifuge tubes reach the requirement of subsequent experimental, be unlikely to because the reason of concentration causes the failure of an experiment.
106.DNA dissolving is preserved: according to the weight of DNA in the 105 little centrifuge tubes that obtain; And subsequent experimental is to the DNA concentration requirement; If it is 500-1000mg/ml that subsequent experimental requires DNA concentration, then can add 10 μ lTE solution to all the other little centrifuge tubes, concentration is that 600mg/ml reaches requirement; Can dna solution be incorporated in 1 centrifuge tube ,-20 ℃ of preservations are for use.
Obtain dna solution that concentration is about 600mg/ml totally 240 μ l with aforesaid method; The OD260/OD280 value is 1.86, and purity is high; Observe DNA electrophorogram such as Fig. 2 (M is Low Range PFG DNAMarker, the 1 DNA sample for extraction), the DNA length of extraction is at 80-200kb.The DNA purity that this method obtains is high, length is long, can be applicable to PCR isolated genes, Southern hybridization, genomic library construction etc. preferably.
Embodiment that the present invention lifts or embodiment have carried out further detailed description to the object of the invention, technical scheme and advantage; Institute is understood that; Abovely lift embodiment or embodiment is merely preferred implementation of the present invention; Not in order to restriction the present invention, all within the present invention spirit and principle to any modification that the present invention did, be equal to replacement, improvement etc., all should be included within the protection domain of the present invention.
Claims (6)
1. a genome DNA extracting method comprises that DNA separates, DNA separates out, DNA washs drying and the DNA dissolving is preserved, and it is characterized in that said DNA separates the aqueous dna average mark that obtains and installs in the little centrifuge tube of 1.5-5ml; Before said DNA dissolving is preserved DNA in the said little centrifuge tube is carried out prediction of quality.
2. the method for claim 1 is characterized in that, said little centrifuge tube is 1.5ml.
3. the method for claim 1 is characterized in that, said qualitative forecasting method detects DNA concentration for wash in dried arbitrary the little centrifuge tube water that adds 5-50 μ l or TE solution to said DNA after, obtains the quality of DNA according to DNA concentration and volume.
4. the method for claim 1; It is characterized in that; Said qualitative forecasting method detects DNA concentration behind the water that respectively adds equal volume in the only little centrifuge tube of dried any 2-4 or the TE solution and averages for washing to said DNA, obtains the quality of DNA according to DNA concentration and volume.
5. like claim 3 or 4 described methods, it is characterized in that said DNA concentration is through measuring light absorption value OD
260/ OD
280Obtain.
6. like claim 3 or 4 described methods, it is characterized in that said DNA concentration obtains through agarose gel electrophoresis.
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Effective date of registration: 20171024 Address after: 266011 Shandong province Qingdao City, Nanchang Road No. 118 building -1-1 Patentee after: Shandong Bai Mao Biotechnology Co., Ltd. Address before: 400065 Chongqing Nan'an District huangjuezhen pass Chongwen Road No. 2 Patentee before: Chongqing University of Posts and Telecommunications |
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