CN107475248A - A kind of plant genome DNA rapid extracting method - Google Patents
A kind of plant genome DNA rapid extracting method Download PDFInfo
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- CN107475248A CN107475248A CN201710795010.2A CN201710795010A CN107475248A CN 107475248 A CN107475248 A CN 107475248A CN 201710795010 A CN201710795010 A CN 201710795010A CN 107475248 A CN107475248 A CN 107475248A
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Abstract
The invention discloses a kind of plant genome DNA rapid extracting method, it is characterised in that comprises the following steps：(1) Plant tissue samples are taken, add liquid nitrogen by Plant tissue samples grind into powder, and be collected into the medium step of centrifuge tube.SDS energy cell lysis films in the BufferA of the present invention make protein denaturation, BufferB can act on SDS, effectively remove the impurity such as isolating protein and polysaccharide, and the PVP40 in BufferA can effectively remove the polysaccharide polyphenol material in plant tissue, improve DNA purity, make DNA integralities more preferable, can be directly used for PCR and various endonuclease reactions；The present invention need not use the organic solvents such as phenol chloroform in whole extraction process simultaneously, therefore the steps such as ethanol precipitation need not be carried out, the extracting method of the present invention compares the extraction DNA that traditional extracting method can more rapidly, easy, the operation of its single sample can generally be completed in 1 hour, greatly shorten extraction time.
Description
Technical field
The present invention relates to biological technical field, in particular to a kind of plant genome DNA rapid extracting method.
Background technology
The extraction of plant genome DNA is that all kinds of plants are carried out with the basis of molecular biology further investigation, and is carried
The quality for taking DNA mass is to influence the principal element of downstream molecular biology test success or failure.
The Method of Plant DNA Extraction of document report is many at present, wherein by most widely used Plant Genome
DNA extraction method is traditional SDS method and CTAB methods.
CTAB is a kind of cationic detergent, and it can dissolve cell membrane, and form a kind of compound with nucleic acid.It is this
Compound is solvable in hypersaline environment, is extracted using organic solvent (phenol, chloroform), can effectively remove phenols, polysaccharide, egg
The impurity such as white matter, nucleic acid can be separated by adding ethanol or isopropanol precipitating.
SDS is a kind of anionic detergent.Under the conditions of high temperature (60~70 DEG C), SDS energy cell lysis films, make protein
Denaturation, nucleic acid and protein dissociation, extracted using organic solvent (phenol, chloroform), can effectively remove phenols, polysaccharide, egg
The impurity such as white matter, nucleic acid can be separated by adding ethanol or isopropanol precipitating.
But traditional SDS methods and CTAB methods exists in DNA extraction process, and cumbersome, time-consuming, using toxic reagent,
Extract the shortcomings of DNA mass is unstable.
The content of the invention
It is an object of the invention to overcome the drawbacks described above present in traditional plant genome DNA extracting method, there is provided
A kind of plant genome DNA rapid extracting method.
The purpose of the present invention is achieved through the following technical solutions：A kind of plant genome DNA rapid extracting method, including with
Lower step：
(1) Plant tissue samples are taken, add liquid nitrogen by Plant tissue samples grind into powder, and be collected into centrifuge tube；
(2) BufferA is added into centrifuge tube, water-filling of going forward side by side bath, Plant tissue samples is mixed with BufferA；
(3) BufferB is added into centrifuge tube to mix, and carry out centrifugal treating；
(4) supernatant is collected, and adds into supernatant and is fully mixed with reference to liquid LN, obtains mixed liquor；
(5) equilibrium liquid LB is added into adsorption column, and the filtrate in collecting pipe is outwelled after centrifugal treating；
(6) mixed liquor in step (4) is added in adsorption column, waste liquid is outwelled after centrifugal treating；
(7) rinsing liquid PW is added into adsorption column, waste liquid is outwelled after centrifugation；Repeat step (7) performs step (8) afterwards；
(8) adsorption column is centrifuged, removes remaining liq, and adsorption column is placed under room temperature environment；
(9) adsorption column is put into clean centrifuge tube, elution buffer is added dropwise to the center of the adsorbed film on adsorption column
EB, centrifugal treating is carried out after placing at room temperature, collect DNA sample.
Further, the Buffer A in the step (2) include Tris-HCl, Na2EDTA, NaCl, SDS and
PVP40；And Tris-HCl concentration is 50~100mM, Na in every 1000ml Buffer A2EDTA concentration be 10~50mM,
NaCl concentration is 200~500mM, SDS volume ratio is 0.5%~2%, PVP40 volume ratio is 1%~2%.
BufferB in the step (3) is the liquor kalii acetici that concentration is 3-5M.
The combination liquid LN added in the step (4) volume is 2 times of supernatant volume.
Combination liquid LN in the step (4) includes the volume ratio of isopropanol and combination liquid, the isopropanol and combination liquid
For 1：1；Include Tris-HCl, Na in the combination liquid2EDTA, guanidine hydrochloride and Tween20, and per 1000ml in combination liquid
Tris-HCl concentration is 50-100mM, Na2EDTA concentration is 1-10mM, the concentration of guanidine hydrochloride is 4-6M, Tween 20
Content is 50-100ml.
Equilibrium liquid LB in the step (5) includes Nacl, MOPS, isopropanol and Triton-100, and often
Nacl concentration is 500~800mM in 1000ml equilibrium liquid LB, MOPS concentration is 30~80mM, the content of isopropanol is
100-200ml, Triton-100 content are 1-2ml.
The absolute ethyl alcohol that the rinsing liquid PW used in the step (7) contains 700~800ml per 1000ml.
Elution buffer EB in the step (9) is the Tris-HCl solution that concentration is 10-20mM.
The pH value of the Tris-HCl is 7.5-8.5.
Bath temperature in the step (2) is 70 DEG C, and water bath time is 10~30min.
The present invention compared with the prior art, has advantages below and beneficial effect：SDS energy in the BufferA of the present invention
Cell lysis film makes protein denaturation, and BufferB can act on SDS, effectively remove the impurity such as isolating protein and polysaccharide, and
PVP40 in BufferA can effectively remove the polysaccharide polyphenol material in plant tissue, improve DNA purity, make DNA integralities
More preferably, PCR and various endonuclease reactions be can be directly used for；The present invention need not use phenol chloroform etc. in whole extraction process simultaneously
Organic solvent, therefore need not carry out the steps such as ethanol precipitation, extracting method of the invention compares traditional extracting method can be with
More rapidly, easy extraction DNA, the operation of its single sample can generally be completed in 1 hour, when greatly shortening extraction
Between.
Brief description of the drawings
The Ago-Gel for the DNA that the DNA and traditional extraction process that Fig. 1 is extracted for the extracting method of the present invention are extracted
Electrophoretogram.
Embodiment
The present invention is described in further detail with reference to embodiment, but embodiments of the present invention are not limited to
This.
Embodiment
The present embodiment is with the plant genome DNA rapid extracting method of the present invention and traditional Tiangeng plant genome DNA
Extracting method compares：
The plant genome DNA rapid extracting method of the present invention, comprises the following steps：
(1) take 100mg fresh corns leaf sample to add liquid nitrogen grinding into powder, and be collected into the sterile centrifugations of 1.5ml
Guan Zhong.
(2) 600ul BufferA is added into centrifuge tube, after rapid reverse mixing, centrifuge tube is placed in 70 DEG C of water-baths 10
~30 minutes, overturn centrifuge tube for several times therebetween, Plant tissue samples is mixed with BufferA.Before centrifuge tube is added,
BufferA is first preheated under 70 DEG C of environment.
Further, the Buffer A include Tris-HCl, Na2EDTA, NaCl, SDS and PVP40；And per 1000ml
Tris-HCl concentration is 50~100mM, Na in Buffer A2EDTA concentration is 10~50mM, NaCl concentration be 200~
500mM, SDS volume ratio are 0.5%~2%, PVP40 volume ratio is 1%~2%.Specifically, the concentration of the Tris-HCl
Can be 50mM or 100mM, the present embodiment is arranged to optimal 80mM；The Na2EDTA concentration can be 10mM or 50mM, make
To be preferred, the present embodiment is arranged to 30mM；The concentration of the NaCl can be 200mM or 500mM, and the present embodiment is arranged to optimal
350mM；SDS volume ratio is 0.5% or 2% in per 1000ml Buffer A, and the present embodiment is arranged to 1.5%；Per 1000ml
PVP40 volume ratio 1% or 2%, the present embodiment are arranged to 1.5% in Buffer A.The pH value of the Tris-HCl be 7.5~
8.5, specifically, the pH value of the Tris-HCl can be 7.5 or 8.5, the present embodiment is arranged to 8.0.
(3) BufferB that 150ul is added into centrifuge tube is mixed, and 3min is centrifuged under the conditions of 12,000rpm.Specifically
, the BufferB is the liquor kalii acetici that concentration is 3-5M.
(4) supernatant is collected using a new centrifuge tube, and adds into supernatant and fully mixed with reference to liquid LN, obtained
Mixed liquor；Wherein, the combination liquid LN of addition volume is 2 times of supernatant volume.
Include isopropanol specifically, this combines liquid LN and combine liquid, the volume ratio of the isopropanol and combination liquid is 1：1.Its
In, combine in liquid and include Tris-HCl, Na2EDTA, guanidine hydrochloride and Tween 20, and Tris- in liquid is combined per 1000ml
HCl concentration is 50~100mM, Na2EDTA concentration is 1~10mM, the concentration of guanidine hydrochloride is 4~6M, Tween 20 contains
Measure as 50~100ml；Specifically, the concentration of the Tris-HCl is 50mM or 100mM, the present embodiment is arranged to 80mM；Na2EDTA
Concentration be 1mM or 10mM, the present embodiment sets 6mM；The concentration of guanidine hydrochloride is 4M or 6M, and the present embodiment is arranged to 5M；Often
Tween 20 content is 50ml or 100ml in 1000ml combination liquid, and the present embodiment is arranged to optimal 80ml.The Tris-
HCl pH value is 7.5~8.5, specifically, the pH value of the Tris-HCl is 7.5 or 8.5, the present embodiment is arranged to 8.0.
(5) 500ul equilibrium liquid LB is added into adsorption column, and adsorption column is centrifuged 1min under the conditions of 12,000rpm,
Outwell the filtrate in collecting pipe；By equilibrium liquid LB come equilibrium adsorption post, enable the more preferable adsorption of DNA of adsorption column.
Equilibrium liquid LB is Nacl, MOPS, isopropanol and Triton-100 mixed solution, wherein, Nacl concentration
Concentration for 500~800mM, MOPS is 30~80mM, per 1000ml equilibrium liquids LB in the isopropanol containing 100~200ml and
1-2ml Triton-100.Specifically, the concentration of the Nacl can be 500mM or 800mM, the present embodiment is arranged to optimal
650mM；The concentration of the MOPS can be 30mM or 80mM, and the present embodiment is arranged to optimal 50mM；Per 1000ml equilibrium liquids LB
The volume of middle isopropanol can be 100ml or 200ml, and the present embodiment is arranged to optimal 150ml；Per in 1000ml equilibrium liquids LB
Triton-100 volume can be 1ml or 2ml, and the present embodiment is arranged to optimal 1.5ml.
(6) mixed liquor in step (4) is added in adsorption column, and centrifuged 30 seconds under the conditions of 12,000rpm, make DNA
Absorption outwells waste liquid on the adsorbed film of adsorption column.
(7) 600ul rinsing liquid PW is added into adsorption column, and is outwelled after being centrifuged 30 seconds under the conditions of 12,000rpm useless
Liquid；Repeat step (7), step (8) is performed after being rinsed again.Wherein, anhydrous second is contained in every 1000ml rinsing liquids PW
Alcohol 700-800ml.
(8) adsorption column is centrifuged into 1min under the conditions of 12,000rpm, to ensure thoroughly to remove the raffinate on adsorption column
Body, and adsorption column is placed on 5~10min under room temperature environment, 10min is placed in the present embodiment, is remained with removing on adsorption column
Ethanol.
(9) adsorption column is put into clean centrifuge tube, washing for 30-80ul is added dropwise to the center of the adsorbed film on adsorption column
De- buffer solution EB, and 2min is centrifuged under the conditions of 12,000rpm, collects DNA sample after placement 2min at room temperature.
Specifically, elution buffer EB dripping quantity can be 30ul or 80ul, preferably, the present embodiment elution buffer
Liquid EB dripping quantity is 50ul.Elution buffer EB is the Tris-HCl solution that concentration is 10-20mM；Specifically, the Tris-
HCl concentration can be 10mM or 20mM, and Tris-HCl concentration is optimal 15mM in the present embodiment.In addition, the Tris-
HCl pH value is 7.5-8.5, and specific Tris-HCl pH value can be 7.5 or 8.5, preferably, in this implementation
Tris-HCl pH value is arranged to 8.0.
Traditional Tiangeng plant genome DNA extracting method step is as follows：
First, maize leaf sample 100mg is taken, liquid nitrogen is added and is fully milled into powder.
2nd, ground sample powder is quickly transferred to be pre-loaded with 700 μ l and CTAB solution through 65 DEG C of preheatings
In centrifuge tube (mercaptoethanol is added in the CTAB solution of preheating before experiment, makes its final concentration of 0.1%), rapid reverse mixing
Afterwards, centrifuge tube is placed on 65 DEG C of water-bath 20min.
3rd, 700 μ l chloroforms are added into centrifuge tube, are fully mixed, and under the conditions of 12,000rpm (~13,400 × g)
Centrifuge 5min.
4th, upper strata aqueous phase is transferred in a new centrifuge tube, and adds 700 μ l CTAB solution, fully mixed, obtain
Mixed liquor.
5th, the mixed liquor of step 4 is transferred in adsorption column CB3, and under the conditions of 12,000rpm (~13,400 × g) from
Heart 30sec, discards waste liquid.
6th, 500 μ l guanidine hydrochloride solution is added into adsorption column CB3, and in 12,000rpm (~13,400 × g) condition
Lower centrifugation 30sec, outwells waste liquid, and adsorption column CB3 is put into collecting pipe.
7th, added into adsorption column CB3 600 μ l concentration be 80% absolute ethyl alcohol, 12,000rpm (~13,400 ×
G) 30sec is centrifuged under the conditions of, outwells waste liquid；Step 8 is performed after repeat step seven.
8th, adsorption column CB3 is centrifuged into 2min under the conditions of 12,000rpm (~13,400 × g), removes remaining liq.Will
Adsorption column CB3 is placed in room temperature and placed several minutes, thoroughly to dry rinsing liquid remaining in sorbing material.
9th, adsorption column CB3 is transferred in a clean centrifuge tube, 50 μ l is vacantly added dropwise to the middle part of adsorbed film and wash
De- buffer solution TE, room temperature place 2-5min, 12,000rpm (~13,400 × g) centrifugation 2min, collect DNA sample.
The DNA mass extracted using spectrophotometer method detection above two method：
1ulDNA samples are respectively taken, DNA concentration and quality such as following table are put forward using ultramicrospectrophotometer detection：
Table 1 is the DNA testing results extracted using traditional Tiangeng plant genome DNA extracting method：
| Detection project | First time detected value | Second of detected value | Average value |
| A260/A280 | 1.98 | 1.95 | 1.965 |
| Concentration (ng/ul) | 131.3 | 143.5 | 137.4 |
The DNA testing results that table 2 is extracted for the plant genome DNA rapid extracting method of the use present invention：
It can be seen from Tables 1 and 2, the DNA mass of two methods extraction is all higher, does not have RNA and protein contamination substantially,
But the DNA A260/A280 ratios that are extracted of the plant genome DNA rapid extracting method of the present invention are closer to 1.8, i.e.,
The quality for the DNA that the plant genome DNA rapid extracting method of invention is extracted is extracted higher than traditional Tiangeng plant genome DNA
The DNA of method extraction quality；Meanwhile the DNA's that is extracted of plant genome DNA rapid extracting method of the invention is average dense
Spend for 163.75ng/ul, and the DNA of traditional Tiangeng plant genome DNA extracting method extraction mean concentration is 137.4ng/
Ul, i.e., the mean concentration for the DNA that plant genome DNA rapid extracting method of the invention is extracted is than traditional Tiangeng plant gene
The DNA of group DNA extraction method extraction mean concentration；Therefore, plant genome DNA rapid extracting method of the invention is extracted
DNA better quality, purity it is higher, and extraction time is shorter.
The DNA extracted using agarose gel electrophoresis detection above two method quality：
The DNA that above two method is extracted respectively takes 5ul to carry out electrophoresis inspection on the Ago-Gel that concentration is 1.2%
Survey, as shown in figure 1, the DNA bands that are extracted of above two method are clear, DNA quality meets the requirements testing result.
As described above, the present invention can be realized well.
Claims (10)
1. a kind of plant genome DNA rapid extracting method, it is characterised in that comprise the following steps：
(1) Plant tissue samples are taken, add liquid nitrogen by Plant tissue samples grind into powder, and be collected into centrifuge tube；
(2) BufferA is added into centrifuge tube, water-filling of going forward side by side bath, Plant tissue samples is mixed with BufferA；
(3) BufferB is added into centrifuge tube to mix, and carry out centrifugal treating；
(4) supernatant is collected, and adds into supernatant and is fully mixed with reference to liquid LN, obtains mixed liquor；
(5) equilibrium liquid LB is added into adsorption column, and the filtrate in collecting pipe is outwelled after centrifugal treating；
(6) mixed liquor in step (4) is added in adsorption column, waste liquid is outwelled after centrifugal treating；
(7) rinsing liquid PW is added into adsorption column, waste liquid is outwelled after centrifugation；Repeat step (7) performs step (8) afterwards；
(8) adsorption column is centrifuged, removes remaining liq, and adsorption column is placed under room temperature environment；
(9) adsorption column is put into clean centrifuge tube, elution buffer EB is added dropwise to the center of the adsorbed film on adsorption column,
Centrifugal treating is carried out after placing at room temperature, collects DNA sample.
A kind of 2. plant genome DNA rapid extracting method according to claim 1, it is characterised in that the step (2)
In Buffer A include Tris-HCl, Na2EDTA, NaCl, SDS and PVP40；And Tris- in every 1000ml Buffer A
HCl concentration is 50~100mM, Na2EDTA concentration is 10~50mM, NaCl concentration is 200~500mM, SDS volume
Than being 1%~2% for 0.5%~2%, PVP40 volume ratio.
A kind of 3. plant genome DNA rapid extracting method according to claim 1, it is characterised in that the step (3)
In BufferB be concentration be 3-5M liquor kalii acetici.
A kind of 4. plant genome DNA rapid extracting method according to claim 1, it is characterised in that the step (4)
The combination liquid LN of middle addition volume is 2 times of supernatant volume.
A kind of 5. plant genome DNA rapid extracting method according to any one of Claims 1 to 4, it is characterised in that institute
State the combination liquid LN in step (4) and include isopropanol and combination liquid, the isopropanol and the volume ratio for combining liquid are 1：1；It is described
Include Tris-HCl, Na in combination liquid2EDTA, guanidine hydrochloride and Tween 20, and Tris-HCl in liquid is combined per 1000ml
Concentration be 50-100mM, Na2The content that EDTA concentration is 1-10mM, the concentration of guanidine hydrochloride is 4-6M, Tween 20 is 50-
100ml。
A kind of 6. plant genome DNA rapid extracting method according to claim 1, it is characterised in that the step (5)
In equilibrium liquid LB include Nacl, MOPS, isopropanol and Triton-100, and Nacl in the equilibrium liquid LB per 1000ml
Concentration is 500~800mM, MOPS concentration is 30~80mM, the content of isopropanol is containing for 100-200ml, Triton-100
Measure as 1-2ml.
A kind of 7. plant genome DNA rapid extracting method according to claim 1, it is characterised in that the step (7)
The absolute ethyl alcohol that the middle rinsing liquid PW used contains 700~800ml per 1000ml.
A kind of 8. plant genome DNA rapid extracting method according to claim 1, it is characterised in that the step (9)
In elution buffer EB be concentration be 10-20mM Tris-HCl solution.
A kind of 9. plant genome DNA rapid extracting method according to claim 8, it is characterised in that the Tris-
HCl pH value is 7.5-8.5.
A kind of 10. plant genome DNA rapid extracting method according to claim 1, it is characterised in that the step
(2) bath temperature in is 70 DEG C, and water bath time is 10~30min.
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Cited By (6)
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| CN112029762A (en) * | 2020-07-24 | 2020-12-04 | 汉远化生医国际科技(北京)有限公司 | Plant tissue DNA rapid extraction method, extraction kit and extraction device |
| CN112048503A (en) * | 2020-09-09 | 2020-12-08 | 中国农业科学院作物科学研究所 | Kit for extracting plant genome DNA by high-throughput rapid magnetic bead method and extraction method |
| CN112326395A (en) * | 2020-09-24 | 2021-02-05 | 汉远化生医国际科技(北京)有限公司 | Sample processing method for rapidly extracting biological DNA |
| CN112779246A (en) * | 2020-12-30 | 2021-05-11 | 浙江大学 | Plant genome DNA extracting solution and plant genome DNA extracting method |
| CN113151255A (en) * | 2021-05-26 | 2021-07-23 | 中国科学院植物研究所 | Method for rapidly extracting plant leaf genome DNA |
| CN114426967A (en) * | 2020-10-29 | 2022-05-03 | 西南大学 | Non-toxic plant universal high-purity genome DNA rapid extraction method and kit |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112029762A (en) * | 2020-07-24 | 2020-12-04 | 汉远化生医国际科技(北京)有限公司 | Plant tissue DNA rapid extraction method, extraction kit and extraction device |
| CN112048503A (en) * | 2020-09-09 | 2020-12-08 | 中国农业科学院作物科学研究所 | Kit for extracting plant genome DNA by high-throughput rapid magnetic bead method and extraction method |
| CN112326395A (en) * | 2020-09-24 | 2021-02-05 | 汉远化生医国际科技(北京)有限公司 | Sample processing method for rapidly extracting biological DNA |
| CN114426967A (en) * | 2020-10-29 | 2022-05-03 | 西南大学 | Non-toxic plant universal high-purity genome DNA rapid extraction method and kit |
| CN112779246A (en) * | 2020-12-30 | 2021-05-11 | 浙江大学 | Plant genome DNA extracting solution and plant genome DNA extracting method |
| CN113151255A (en) * | 2021-05-26 | 2021-07-23 | 中国科学院植物研究所 | Method for rapidly extracting plant leaf genome DNA |
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