CN107904229A - Genome DNA extracting method and extracts kit - Google Patents
Genome DNA extracting method and extracts kit Download PDFInfo
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- CN107904229A CN107904229A CN201710613331.6A CN201710613331A CN107904229A CN 107904229 A CN107904229 A CN 107904229A CN 201710613331 A CN201710613331 A CN 201710613331A CN 107904229 A CN107904229 A CN 107904229A
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- C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
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Abstract
The invention discloses a kind of genome DNA extracting method and extracts kit, it is characterised in that：Sample to be extracted is by cell lysis successively and removes removing protein, then is purified through absorption and drying dissolve to obtain the Genomic DNA solution in sample to be extracted；Wherein, removing protein is removed by the way that albumen precipitation is realized；The sample to be extracted is selected from animal blood or animal tissue cell.Genome DNA extracting method provided by the invention and extracts kit are safe and non-toxic using simply having operated, and it is low that component lacks use cost, is applicable to all types of animal tissues' extractions, DNA yield is high, and be widely used prospect.
Description
Technical field
The present invention relates to a kind of genome DNA extracting method and extracts kit, belongs to biologic medical field.
Background technology
Nucleic acid is the carrier of hereditary information, is most important biological information molecule, is the main right of molecular biology research
As therefore, the extraction of nucleic acid is operation most important, most basic in molecular biology experiment technology.Genetic test is modern point
Sub- biological study and the precisely main means of medical treatment.The DNA of high quality is extracted from human body or the blood or tissue of animal is
Genetic test, precisely medical treatment and other molecular biology researches and the premise of clinically relevant detection, follow-up experiment such as PCR,
QPCR etc. is required on the basis of nucleic acid extraction preferably carrying out.
At present, common DNA extraction method includes phenol chloroform extraction method, centrifugal column method and paramagnetic particle method etc., these methods
Respectively there are advantage and disadvantage.Phenol chloroform extraction method needs frequently to use toxic reagent, easily brings safety problem, and extraction process is numerous
It is trivial, extraction efficiency is low.Column centrifugal process using silicon matrix material under certain high salt buffer system efficiently, exclusively adsorption of DNA,
It is also required to use such as phenol, chloroform toxic reagent, and centrifugal column method extraction process takes more, have impact on overall point
Analyse the speed of detection.Paramagnetic particle method be by nanometer new material adsorption of DNA Novel extraction method, have reagent safety dosage it is few,
The advantages that DNA molecular structural integrity, but the DNA mass and the quality of magnetic bead extracted are closely related, and the cost of magnetic bead in itself is higher,
So that the extraction cost of magnetic bead extraction method is higher than traditional extraction process.
Due to the basic of DNA extractions and routinely so that research of the scientific research personnel of biological field in DNA extractions field
Never stopped.For example, Chinese patent application CN 106868000A disclose a kind of body fluid suspension cell DNA extraction kit,
Including re-suspension liquid, Proteinase K Solution, RNase A solution, lysate, precipitating reagent, cleaning solution A, cleaning solution B and DNA lysate, make
With the method for agent box extraction body fluid suspension cell DNA, comprise the following steps：Centrifugation, precipitation are resuspended, addition lysate, albumen
Enzyme K solution cracks, addition precipitating reagent centrifugation, adds cleaning solution A, and cleaning solution B centrifuge washings, and it is molten to add DNA after drying at room temperature
Solve liquid.This method is suitable only for collection and extraction work, the narrow scope of application of body fluid suspension cell DNA, and is washed in this method
Wash liquid A and B need to shift to an earlier date it is cumbersome in -20 degree precooling treatments 30 minutes, operation.Chinese patent application CN 106906207A are disclosed
A kind of Rapid nucleic acid extraction kit, the mixed enzyme working solution, cracking adsorption liquid, washing individually prepared are included in kit
Liquid, rinsing liquid and eluent, mixed enzyme working solution include Proteinase K and lysozyme, and cracking adsorption liquid includes Gu-Hcl, Tris-
Hcl, EDTA, isopropanol, SDS, Triton-100, beta -mercaptoethanol and sodium citrate, cleaning solution include Licl, Nacl, MOPS
And ethanol, rinsing liquid are ethanol, eluent is EDTA and Tris-Hcl.This method not only needs to use poisonous and harmful reagent, and
And need to use magnetic Nano microsphere and Magneto separate frame in experimentation, add use cost.Chinese patent application CN
106867992A discloses a kind of whole blood DNA extracts kit, parcel DNA is adsorbed using nanometer magnetic bead, to reach separating-purifying
Purpose, which is made of lysate, nanometer magnetic bead, cleaning solution and eluent, wherein lysate include guanidinium isothiocyanate,
Lithium iodide, trisodium citrate, Tween 20, the nanometer magnetic bead are enclosed with the Fe of silica3O4, cleaning solution includes different
Guanidine thiocyanate, Tris-HCl, NaCl and absolute ethyl alcohol, eluent include Tris-HCl and EDETATE SODIUM.The kit need to include
At least ten kinds of components, not only reagent is poisonous, and required equipment is more, and step is complicated, and the cycle of extraction is also longer.
It is usually enzymic digestion, strong acid and strong base precipitation and centrifugation point to the impurity removing method in sample in existing technology
From etc., remove in sample such as RNA, the impurity of protein one kind, but these purification modes are not thorough or excessively there is removal of impurities
Removal of impurities influences the problem of DNA structure.Protein especially in people's blood and histocyte, dedoping step are pure to the DNA after purification
Have an impact on degree and extracting concentration.Chinese patent application CN104560959A discloses a kind of saturated sodium-chloride method rapid batch
The kit and method of blood DNA are extracted, which includes erythrocyte cracked liquid, protein liquid removal, protein precipitant, adjusting knot
Liquid, eluent and Proteinase K Solution are closed, wherein 70% ethanol of protein liquid removal position, deproteinized precipitated liquid is saturated sodium-chloride
Solution.This method can realize the extraction to DNA in blood, but since body fluid components are complex, suspend carefully in the body fluid of part
Born of the same parents' amount is few, therefore extraction of this method for DNA in body fluid is difficult to meet the requirement to purity, yield and integrality, wherein
Protein precipitation needs low temperature, and rear to need to dissolve by heating again to carry out subsequent reactions, reaction condition is cumbersome, big using difficulty.In the party
In method, sodium chloride solution can precipitate albumen in sample as albumen precipitation liquid, but sodium chloride solution is soluble easily in water, be slightly soluble in second
Alcohol, causes to be not easy to remove salt ion totally during cleaning below, has an impact to the quality of DNA, also to be follow-up
Experimental implementation brings hidden danger.
To sum up, it is a kind of easy to operate, low, nontoxic to extraction instrument and personnel requirement there is an urgent need to have, and can be with
The effective method for extracting nucleic acid and extracts kit for removing impurity, not influencing DNA structure.
The content of the invention
In view of the above-mentioned problems existing in the prior art, the object of the present invention is to provide it is a kind of it is easy to operate, security is good
Genome DNA extracting method and extracts kit.
First goal of the invention of the present invention is to provide a kind of genome DNA extracting method, sample to be extracted in this method
By cell lysis successively and removing protein is removed, then is purified through absorption and drying dissolves to obtain the genome in sample to be extracted
DNA solution；Wherein, removing protein is removed by the way that albumen precipitation is realized；The sample to be extracted is selected from animal blood or animal tissue
Cell.Since the cell composition in blood is different from histocyte, the genome DNA extracting method in the present invention is extraction
Genomic DNA in leucocyte or histocyte.As long as the animal tissue containing DNA can carry as the DNA in the present invention
Take object.As a preferred embodiment, described be organized as buccal swab.
Preferably, the DNA extraction method in the blood sample includes the following steps：
A) red blood cell is removed：Erythrocyte cracked liquid is added into sample to be extracted, cracks the red blood cell in sample to be extracted,
3000rpm centrifuges 5min after mixing, is cracked completely to red blood cell, extracting waste precipitation；Wherein blood sample and erythrocyte cracked liquid
Volume ratio be 1：4.
B) leucocyte and digestible protein are cracked：200 μ L of cell pyrolysis liquid and albumen are added into white precipitate obtained by step a
Enzyme K20 μ L, piping and druming mix, 56 DEG C of water-bath 10min incubation reaction systems；In order to further obtain good digestion effect, step b
Further include piping and druming when being incubated to 5min and mix reaction system；
C) removing protein is removed：200 μ L of protein precipitant are added into the system of step b, the protein in precipitation reaction system,
Promote the precipitation of DNA, 4 DEG C of standing 10min, 12000rpm centrifugation 1min, retain supernatant；
D) DNA absorption purification：DNA precipitating reagents are added into supernatant obtained by step c, add cation after mixing by several times
In DNA chromatographic columns, waste liquid, 70% ethanol, 700 μ L washing chromatographic columns, 12000rpm centrifugation 1min, repeated washing chromatography are abandoned in centrifugation
Column at least 2 times；Wherein：DNA precipitating reagents are isopropanol or absolute ethyl alcohol；
E) DNA solution is collected：The chromatographic column of washed centrifugation in step d is centrifuged again, chromatographic column after centrifugation is moved into
New centrifuge tube, centrifuge tube are uncapped air-dried 5min, are added eluent and are stood, and DNA solution is collected after 12000rpm centrifugations 2min ,-
20 DEG C save backup.
Preferably, the DNA extraction method in the tissue sample includes the following steps：
A) cell lysis and digestible protein：Preserved to the histocyte gathered and cell pyrolysis liquid and protease are added in liquid
K, cracks cell in this step reaction system and digests the albumen in this step reaction system, incubation reaction system；
B) removing protein is removed：Add protein precipitant into the system obtained by step a, the protein in precipitation reaction system,
Promote the precipitation of DNA, centrifuged after standing, retain supernatant；
C) DNA absorption purification：DNA precipitating reagents are added into the supernatant obtained by step b, add cation after mixing by several times
In DNA chromatographic columns, waste liquid is abandoned in centrifugation, is centrifuged after washing chromatographic column；
D) DNA solution is collected：Chromatographic column obtained by the washed centrifugations of step c is centrifuged, moves into chromatographic column newly after centrifugation
Centrifuge tube, air-dry centrifuge tube, add eluent and stand, collected after centrifugation DNA solution again.
The DNA extraction method of above-mentioned tissue sample use with the DNA extraction method of blood sample using identical reagent and
Operating method, the step for differing only in not comprising red blood cell is removed.
Preferably, the erythrocyte cracked liquid is 5mol/L NaCl 0.2mL, 1M Tris-Hcl 2mL and 0.5M EDTA
The mixed solution of 0.02mL.Wherein, remove red blood cell to crack completely to red blood cell, as once-through operation is not thorough, can repeatedly weigh
Multiple operating procedure a is cracked completely to red blood cell.
Preferably, it is described per 100ml cell pyrolysis liquids SDS containing 1g, 1M Tris-HCl 1mL, NaCl 0.585g and
0.5M EDTA 1mL, solution ph 8.0.
Preferably, Proteinase K is 20mg/mL proteinases.
Preferably, protein precipitant is ammonium acetate solution, and concentration is not less than 4mol/L.As a kind of preferable embodiment party
Case, the concentration of protein precipitant select ammonium acetate solution as big as possible, such as the ammonium acetate solution of 10mol/L.Ammonium acetate is readily soluble
Yu Shui, is soluble in ethanol, and solution is in neutrality, and ammonium acetate is as protein precipitant, NH therein4+One side and the SDS in reagent
Generation microsolubility material is attached on protein, promotes the genomic DNA that the protein in system and protein connected
Sedimentation；One side DNA is in negativity, adds cation NH4+Electric charge is neutralized, DNA can be promoted to be precipitated in absolute ethyl alcohol.
Preferable DNA precipitating reagents are isopropanol or absolute ethyl alcohol in the present invention.
Preferably, eluent is TE buffer solutions, and Tris-HCl containing 10mM and 1mM EDTA, pH value of solution are in TE buffer solutions
8.0。
The present invention second goal of the invention be to provide a kind of genome DNA extracting reagent kit, the kit includes：
Component is the A liquid of erythrocyte cracked liquid, and component is the B liquid of cell pyrolysis liquid, and component is the C liquid of Proteinase K, and component sinks for albumen
The D liquid of shallow lake agent, component is the E liquid of eluent, and DNA cations chromatographic column is F components.
Preferably, A liquid is the mixing of 5mol/L NaCl 0.2mL, 1M Tris-Hcl 2mL and 0.5M EDTA 0.02mL
Solution；B liquid is per 100ml SDS containing 1g, 1M Tris-HCl 1mL, NaCl 0.585g and 0.5M EDTA 1mL；C liquid is 20mg/
ML proteinases；D liquid is not less than the ammonium acetate solution of 4mol/L for concentration；E liquid is TE buffer solutions.
Experimental data shows：Kit provided by the invention extracts more than 500 example human sample's acquired results, DNA DNA purities
In OD values 1.6-2.1；The genomic DNA yield that 200uL whole blood samples obtain is not less than 1ug.
Mentioned component for the present invention in preferred solution, make any adjustments or replace based on component disclosed above into
Point, as long as corresponding effect can be played, it is considered as falling under the scope of the present invention.
Compared with prior art, genome DNA extracting method provided by the invention and extracts kit have following technology
Effect：
1) composition is simple, and only containing six kinds of compositions, materials safety is reliable, and environmental-friendly；The kit can be removed effectively
Impurity in animal sample to be extracted, especially protein impurities, ensure that the purity and concentration of the DNA extracted；
2) this extracting method is easy to operate, step is succinct, this kit extraction time only needs 1h or so；
3) the kit extraction DNA of this extracting method is utilized, it is only necessary to there is conventional centrifugal equipment can carry out in laboratory,
Step substantially simplifies；
4) DNA concentration of this kit extraction and purity are good, and extraction effect is stablized.
In short, this extracting method operating procedure is simple, composition is few, requires operator relatively low, the simple peace of present invention use
Full genome DNA extracting method can be widely used in hospital, school and research company and be organized for blood, buccal swab etc.
DNA extraction.
Brief description of the drawings
Fig. 1 is the step schematic diagram of genome DNA extracting method provided by the invention.
Fig. 2 is the step flow chart in the embodiment of the present invention 1.
Embodiment
The present invention is made with reference to embodiment further in detail, intactly to illustrate.
The reagent that DNA extraction kit in the present embodiment uses unless otherwise specified, be our company laboratory in oneself
Match somebody with somebody, it is specific as follows：
Erythrocyte cracked liquid (component A)：5mol/L NaCl 0.2mL、1M Tris-Hcl 2mL、0.5M EDTA
0.02mL, high pressure or filter membrane are degerming after mixing；
Cell pyrolysis liquid (component B)：Per 100mL SDS containing 1g, Tris-HCl (1M) (PH=8.0) 1mL,
NaCl0.585g, EDTA (0.5M) (PH=8.0) 1mL, high pressure or filter membrane are degerming after mixing；
Proteinase K (component C)：20mg/mL；Proteinase K powder is purchased from Merck KGaA company, article No.：1245680100
Protein precipitant (components D)：10mol/L ammonium acetates；
TE buffer solutions (component E)：10mM Tris-HCl, pH 8.0,1mM EDTA, pH 8.0, after mixing high pressure or
Filter membrane is degerming.
By taking 12 person-portions as an example, the DNA extraction kit in the present embodiment includes：A liquid 20mL containing component A, containing into
Divide the B liquid 0.25mL of B, the C liquid 3ml containing component C, the D liquid 3mL containing components D, the E liquid 8mL containing component E, further include sun
Ion DNA chromatographic columns (component F), extract the genomic DNA in animal tissue or whole blood with following extracting methods, extraction
Object includes blood, buccal swab etc..Extraction process is as shown in Fig. 1~Fig. 2.
Embodiment 1
The present embodiment extracts genomic DNA in leucocyte by taking human blood sample sheet as an example, and specific extraction process includes following step
Suddenly：
Experiment starts preceding notice：All experimentss operation carries out at room temperature, and centrifuge tube and chromatographic column are equal in experimentation
Mark need to be carried out, centrifugation and oscillation step are intended to cover tightly lid.
A. red blood cell is removed
The 200 μ L of human blood sample sheet of mixing are taken, add 800 μ LA liquid, overturns and mixes, 3000rpm, centrifuges 5min；
Supernatant is abandoned, adds 800 μ LA liquid, overturns and mixes, 3000rpm, centrifuges 5min.At this time such as the precipitation after centrifuging
It is still red, is centrifuged after repeatable addition A liquid, to white is precipitated as, abandon supernatant, retain white precipitate.
B. leucocyte and digestible protein are cracked
White precipitate in step a is taken, adds 200 μ L of B liquid, mixes, adds 20 μ L of C liquid, is blown and beaten and mixed with pipettor, 56 DEG C of water-baths
It need to be blown and beaten and mixed with pipettor again when 10min, water-bath 5min.
Digestible protein is limpid bright to solution, no residual impurity.As digestion effect is bad, when incubation can be appropriately extended
Between.
C. removing protein is removed
Into the reaction system of step b plus 200 μ L of D liquid, overturn and mix, 4 DEG C or ice bath stand 10min, 12000rpm from
Heart 1min.
D.DNA absorption purifications
500 μ l of E liquid are added, activate chromatographic column F, stand 2min, 12000rpm centrifugation 1min, abandon filtrate, spare.
The supernatant after centrifuging after protein purification is taken into another centrifuge tube, isometric isopropanol is added or adds 2 times
The absolute ethyl alcohol of volume, overturns and mixes.The liquid of mixing is added to chromatographic column F, the 12000rpm centrifugation 1min of activation by several times.
Cleaning：70% ethanol, 700 μ L are added into chromatographic column, 12000rpm centrifugation 1min, abandon waste liquid.Repeated washing is once.
Chromatographic column F is not added with any solution centrifugation, 12000rpm centrifugations 2min.
E. dry dissolving
Chromatographic column is moved into new 1.5mL centrifuge tubes, uncap air-dried 5min.
40 μ l E liquid are added into chromatographic column, stand 5min, 12000rpm centrifugation 2min, the DNA solution after must extracting.-
20 DEG C save backup.
In extraction process, in order to increase the yield of DNA, E liquid can be placed in 56 DEG C of water-baths and preheated.According to different groups
DNA concentration difference in sample is knitted, can suitably increase the dosage of eluent (E liquid).
Embodiment 2
By taking human mouth swab sample as an example, specific extraction process includes the following steps the present embodiment：
Experiment starts preceding notice：All experimentss operation carries out at room temperature, and centrifuge tube and chromatographic column are equal in experimentation
Mark need to be carried out, centrifugation and oscillation step are intended to cover tightly lid.
A. Stomatocyte is collected：
Cell-preservation liquid in centrifuge tube is taken, is put into another new 1.5mL centrifuge tubes, 3000rpm, centrifuges 5min.Abandon
Clear liquid, retains precipitation.
B. cell lysis and digestible protein
Take in step a and precipitate, add 200 μ l of B liquid, mix, add 20 μ L of C liquid, blown and beaten and mixed with pipettor, 56 DEG C of water-baths
60min, need to be blown and beaten with pipettor and mixed again when water-bath is per 20min.
Digestible protein is limpid bright to solution, no residual impurity.As digestion effect is bad, when incubation can be appropriately extended
Between.
C. removing protein is removed
Into the reaction system of step b plus 200 μ L of D liquid, overturn and mix, 4 DEG C or ice bath stand 10min, 12000rpm from
Heart 1min.
D.DNA absorption purifications
500 μ l of E liquid are added, are activated into chromatographic column F, stand 2min, 12000rpm centrifugation 1min, abandon filtrate, spare.
The supernatant after centrifuging after protein purification is taken into another centrifuge tube, isometric isopropanol is added or adds 2 times
The absolute ethyl alcohol of volume, overturns and mixes.The liquid of mixing is added to chromatographic column F, the 12000rpm centrifugation 1min of activation by several times.
Cleaning：70% ethanol, 700 μ l are added into chromatographic column, 12000rpm centrifugation 1min, abandon waste liquid.Repeated washing is once.
Chromatographic column F is not added with any solution centrifugation, 12000rpm centrifugations 2min.
E. dry dissolving
Chromatographic column is moved into new 1.5mL centrifuge tubes, uncap air-dried 5min.
40 μ l E liquid are added into chromatographic column, stand 5min, 12000rpm centrifugation 2min, the DNA solution after must extracting.-
20 DEG C save backup.
In extraction process, in order to increase the yield of DNA, E liquid can be placed in 56 DEG C of water-baths and preheated.According to different groups
DNA concentration difference in sample is knitted, can suitably increase the dosage of eluent (E liquid).
Comparative example uses the embodiment 2~4 in Chinese patent application CN 106868000A, wherein embodiment 2 and 3
Blood sample, embodiment 4 is saliva sample.According to the embodiment disclosed in this application, solution is configured, according to behaviour
Make step, using the sample in embodiment in the application 1 and embodiment 2, carry out comparative example experiment extraction DNA.Extract result such as
Under：
1 anticoagulated blood sample routine DNA extraction methods of comparative example are contrasted with documents extraction method
2 buccal swab sample routine DNA extraction methods of comparative example are contrasted with documents extraction method
As seen from the above table, the DNA concentration of the DNA extraction method extraction in the present invention is identical compared to what other methods extracted
The DNA concentration of sample, the extraction DNA concentration in the present embodiment is far above the DNA concentration in documents, and DNA purity maintains
In a higher level, illustrate that the extracts kit in the present embodiment and extracting method are applicable to the genome of all types of samples
DNA is extracted, and the DNA stable qualities that method in the present embodiment and kit extract, and extraction efficiency is high.
The result extracted according to more than 500 example blood of human body DNA and buccal swab counts, DNA DNA purities OD values 1.6~
2.1；And the genomic DNA yield that 200ul whole blood samples obtain is not less than 1ug.
Finally be necessary described herein be：Above example is served only for making technical scheme further detailed
Ground explanation, it is impossible to be interpreted as limiting the scope of the invention, those skilled in the art's the above according to the present invention
Some the nonessential modifications and adaptations made belong to protection scope of the present invention.
Claims (10)
- A kind of 1. genome DNA extracting method, it is characterised in that：Sample to be extracted is by cell lysis successively and removes removing protein, Purified again through absorption and drying dissolve to obtain the Genomic DNA solution in sample to be extracted；Wherein, removing protein is removed by by egg White precipitation is realized；The sample to be extracted is selected from animal blood or animal tissue cell.
- 2. genome DNA extracting method according to claim 1, it is characterised in that the DNA extractions in the blood sample Method includes the following steps：A) red blood cell is removed：Erythrocyte cracked liquid is added into sample to be extracted, cracks the red blood cell in sample to be extracted, is centrifuged Separation, cracks completely to red blood cell, extracting waste precipitation；B) leucocyte and digestible protein are cracked：Cell pyrolysis liquid and Proteinase K are added into the white precipitate obtained by step a, is split Solve leucocyte in this step reaction system and digest the albumen in this step reaction system, incubation reaction system；C) removing protein is removed：Add protein precipitant into the system obtained by step b, the protein in precipitation reaction system, promotes The precipitation of DNA, centrifuges after standing, retains supernatant；D) DNA absorption purification：DNA precipitating reagents are added into the supernatant obtained by step c, add cation DNA after mixing by several times In chromatographic column, waste liquid is abandoned in centrifugation, is centrifuged after washing chromatographic column；E) DNA solution is collected：Chromatographic column obtained by the washed centrifugations of step d is centrifuged, after centrifugation by chromatographic column move into it is new from Heart pipe, air-dries centrifuge tube, adds eluent and stands, again collected after centrifugation DNA solution.
- 3. genome DNA extracting method according to claim 1, it is characterised in that the DNA extractions in the tissue sample Method includes the following steps：A) cell lysis and digestible protein：Preserved to the histocyte gathered and cell pyrolysis liquid and Proteinase K are added in liquid, split Solve cell in this step reaction system and digest the albumen in this step reaction system, incubation reaction system；B) removing protein is removed：Add protein precipitant into the system obtained by step a, the protein in precipitation reaction system, promotes The precipitation of DNA, centrifuges after standing, retains supernatant；C) DNA absorption purification：DNA precipitating reagents are added into the supernatant obtained by step b, add cation DNA after mixing by several times In chromatographic column, waste liquid is abandoned in centrifugation, is centrifuged after washing chromatographic column；D) DNA solution is collected：Chromatographic column obtained by the washed centrifugations of step c is centrifuged, after centrifugation by chromatographic column move into it is new from Heart pipe, air-dries centrifuge tube, adds eluent and stands, again collected after centrifugation DNA solution.
- 4. genome DNA extracting method according to claim 2, it is characterised in that：Protein precipitant is ammonium acetate solution, And concentration is not less than 4mol/L.
- 5. genome DNA extracting method according to claim 2, it is characterised in that：Albumen precipitation agent concentration is 10mol/ L。
- 6. genome DNA extracting method according to claim 2, it is characterised in that the concrete operations of the step d are： DNA precipitating reagents are added into supernatant obtained by step c, are added by several times in cation DNA chromatographic column after mixing, waste liquid is abandoned in centrifugation, 70% ethanol, 700 μ l wash chromatographic column, 12000rpm centrifugation 1min, repeated washing chromatographic column at least 2 times；Wherein：DNA precipitating reagents For isopropanol or absolute ethyl alcohol.
- 7. genome DNA extracting method according to claim 2, it is characterised in that the tool of step e in the extracting method Gymnastics conduct：The chromatographic column of washed centrifugation in step d is centrifuged again, chromatographic column after centrifugation is moved into new centrifuge tube, from Heart pipe is uncapped air-dried 5min, is added eluent and is stood, and collects DNA solution after 12000rpm centrifugations 2min, -20 DEG C save backup.
- 8. genome DNA extracting method according to claim 7, it is characterised in that：Eluent is TE buffer solutions, and TE is buffered Tris-HCl containing 10mM and 1mM EDTA, pH value of solution 8.0 in liquid.
- 9. a kind of genome DNA extracting reagent kit, it is characterised in that the kit includes：Component is the A of erythrocyte cracked liquid Liquid, component are the B liquid of cell pyrolysis liquid, and component is the C liquid of Proteinase K, and component is the D liquid of protein precipitant, and component is elution The E liquid of liquid, and DNA cations chromatographic column are F components.
- 10. genome DNA extracting reagent kit according to claim 9, it is characterised in that A liquid is 5mol/L NaCl The mixed solution of 0.2mL, 1M Tris-Hcl 2mL and 0.5M EDTA 0.02mL；B liquid is per 100mL SDS containing 1g, 1M Tris- HCl 1mL, NaCl 0.585g and 0.5M EDTA 1mL；C liquid is 20mg/mL proteinases；D liquid is not less than for concentration The ammonium acetate solution of 4mol/L；E liquid is TE buffer solutions.
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| CN109385419A (en) * | 2018-09-20 | 2019-02-26 | 苏州市爱生生物技术有限公司 | Portable DNA extraction kit and its extracting method |
| CN109609493A (en) * | 2018-12-24 | 2019-04-12 | 北京优迅医学检验实验室有限公司 | Extract the method and kit of genomic DNA in whole blood |
| CN109852607A (en) * | 2018-12-30 | 2019-06-07 | 上海星耀医学科技发展有限公司 | It the reagent of erythroplastid and is applied in DNA extraction in a kind of removal biological sample |
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