CN104928283B - Reagent is extracted for the DNA cleaning solution extracted and its application and DNA - Google Patents
Reagent is extracted for the DNA cleaning solution extracted and its application and DNA Download PDFInfo
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Abstract
The present invention provides a kind of cleaning solution extracted for DNA, including guanidine hydrochloride, disodium ethylene diamine tetraacetate, trishydroxymethylaminomethane, hydrochloric acid and 2- fourth oxyethanol, pH value 6.8-7.2.Cleaning solution of the present invention can remove the pollution such as polysaccharide, the polyphenol of different plants during Genomic Purification extensively, improve the purity and concentration of purifying gained DNA, be conducive to downstream application, improve detection sensitivity.
Description
Technical field
The present invention relates to extraction from biological material field more particularly to a kind of cleaning solutions extracted for DNA.
Background technique
Genome be nineteen twenty-four propose for describe biology full gene and genome at concept.1986 by
The genomics that American scientist Thomas Roderick is proposed, which refers to, carries out genomic mapping (including heredity to all genes
Map, physical map, transcript map), nucleotide sequence analysis, the assignment of genes gene mapping and gene function analysis a science.From
Since implementing from the nineteen ninety Human Genome Project, earth-shaking variation is had occurred in genomics, has had developed into one
The forward position of life science and hot fields.And Plant Genome Research and other eucaryotes and human genome research have very greatly
Difference.Firstly, the Genome Size of different plants is very big even if affiliation very close type quality inspection difference；Its
Secondary, many plants are allopolyploids, even there is also more times of relatively broad body cell for some types in diplont
Change phenomenon.
Currently, the research of Plant Genome mainly includes two levels: (1) structural genomics, it is with complete sequence sequencing
Target, construct it is high-resolution by Chromosome recombination exchange based on genetic map and with DNA and nucleotides sequence arranging as base
The physical map of plinth；(2) functional genomics, i.e. " post genome project " are the extensions of Structural genomics research, utilize structure
The hereditary information that genome provides establishes the function map based on transcripting spectrum using EST.
In the Plant Genome Research of different levels, involved by majority be Protocols in Molecular Biology, at present often
Seeing has: Plant Genome sequencing, Plant Genome methylation analysis, Plant Genome in situ hybridization, the analysis of DNA of plants bar code
Deng.
Either the research of Plant Genome different levels or Plant Genome are in various technical applications, head
Wanting premise is the plant genome DNA for obtaining high quality.And evaluate an important indicator of the quality of plant genome DNA just
It is concentration and purity, can this later period application for being directly related to Plant Genome effectively be carried out.And with bacterium, animal tissue,
The biomaterials such as cell are different, the most of plant all polysaccharide containing higher level, polyphenol etc., these substances can severe jamming plant
Object extracting genome DNA is as a result, especially can seriously reduce the purity of plant genome DNA, so that the research in downstream is interfered,
Seriously affect application effect.
In order to which polysaccharide, the polyphenol etc. that are effectively removed in plant genome DNA extraction process pollute, genomic DNA is improved
Purity, different scholars and researcher attempted different methods: Dellaporta etc. thinks that the KAc of high concentration can effectively be gone
Except polysaccharide；Fang etc. thinks that the NaCl cooperation dehydrated alcohol precipitating of 1.0-2.5M can effectively remove the pollution of polysaccharide；Someone utilizes
Polyphenol substance in this high molecular synthetic resin complexing plant genome DNA extraction process of PVP, to reach the mesh of removal polyphenol
's；Somebody effectively removes polyphenol using DTTD.However, these above-mentioned methods can only also remove in general plant
Polysaccharide polyphenol, when plant species higher with these methods processing polysaccharide polyphenol content, the effect is unsatisfactory and it is applied
Differing greatly between species.
Summary of the invention
In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide the cleaning solutions extracted for DNA, including hydrochloric acid
Guanidine, disodium ethylene diamine tetraacetate, trishydroxymethylaminomethane, hydrochloric acid and 2- fourth oxyethanol, pH 6.8-7.2.
Further, the concentration of the guanidine hydrochloride is 1M-5M, the concentration of disodium ethylene diamine tetraacetate is 1mM-50mM, three
The concentration of hydroxymethyl aminomethane is 10mM-100mM, the concentration of hydrochloric acid is 0.1%-1%, and the concentration of 2- fourth oxyethanol is 5%-
20%.
Further, the concentration of guanidine hydrochloride is 3.5M, and disodium ethylene diamine tetraacetate concentration is 20mM, trihydroxy methyl ammonia
Methylmethane concentration is 35mM, and concentration of hydrochloric acid 0.6%, 2- fourth oxyethanol concentration is 10%.
The present invention also provides the cleaning solutions to extract the application on DNA, and the cleaning solution includes guanidine hydrochloride, second two
Amine tetraacethyl disodium, trishydroxymethylaminomethane, hydrochloric acid and 2- fourth oxyethanol, pH 6.8-7.2.
Further, the concentration of the guanidine hydrochloride is 1M-5M, the concentration of disodium ethylene diamine tetraacetate is 1mM-50mM, three
The concentration of hydroxymethyl aminomethane is 10mM-100mM, the concentration of hydrochloric acid is 0.1%-1%, and the concentration of 2- fourth oxyethanol is 5%-
20%.
Further, the concentration of guanidine hydrochloride is 3.5M, and disodium ethylene diamine tetraacetate concentration is 20mM, trihydroxy methyl ammonia
Methylmethane concentration is 35mM, and concentration of hydrochloric acid 0.6%, 2- fourth oxyethanol concentration is 10%.
Preferably, the DNA of extraction is the DNA in plant.
It is furthermore preferred that the DNA extracted is the DNA in Caesalpiniaceae plant.
The present invention also provides a kind of for removing the reagent of polysaccharide and polyphenol substance in Plant Genome purification process,
Including guanidine hydrochloride, disodium ethylene diamine tetraacetate, trishydroxymethylaminomethane, hydrochloric acid and 2- fourth oxyethanol, pH 6.8-7.2.
Further, the concentration of the guanidine hydrochloride is 1M-5M, the concentration of disodium ethylene diamine tetraacetate is 1mM-50mM, three
The concentration of hydroxymethyl aminomethane is 10mM-100mM, the concentration of hydrochloric acid is 0.1%-1%, and the concentration of 2- fourth oxyethanol is 5%-
20%.
Further, the concentration of guanidine hydrochloride is 3.5M, and disodium ethylene diamine tetraacetate concentration is 20mM, trihydroxy methyl ammonia
Methylmethane concentration is 35mM, and concentration of hydrochloric acid 0.6%, 2- fourth oxyethanol concentration is 10%.
Further, the Plant Genome is Caesalpiniaceae Plant Genome.
The present invention provides a kind of DNA to extract reagent, including cell suspending liquid, cell pyrolysis liquid, cleaning solution and eluent,
The cleaning solution includes guanidine hydrochloride, disodium ethylene diamine tetraacetate, trishydroxymethylaminomethane, hydrochloric acid and 2- fourth oxyethanol, pH value
For 6.8-7.2.
Further, the concentration of the guanidine hydrochloride is 1M-5M, the concentration of disodium ethylene diamine tetraacetate is 1mM-50mM, three
The concentration that the concentration of hydroxymethyl aminomethane is 10mM-100mM, the concentration of hydrochloric acid is 0.1%-1%, 2- fourth oxyethanol is 5%-
20%
The beneficial effects of the present invention are: cleaning solution of the present invention can remove different plants in Genomic Purification extensively
The pollution such as polysaccharide polyphenol in the process improves the purity and concentration of purifying gained DNA, is conducive to downstream application, improves detection spirit
Sensitivity.
Detailed description of the invention
Fig. 1 tamarind tree plant extracting genome DNA result.
Fig. 2 difference Caesalpiniaceae plant genome DNA purification result.
The contrast and experiment of Fig. 3 cleaning solution of the present invention and other cleaning solutions.
Fig. 4 tamarind tree different parts tissue gene group DNA purification result.
Fig. 5 Different Extraction Method is equipped with the effect of cleaning solution of the present invention.
Fig. 6 cleaning solution of the present invention is applied to the result for extracting not equal platymiscium genome.
Specific embodiment
Below with reference to specific embodiment, the present invention will be described in detail.
Include following components for the DNA cleaning solution extracted:
Guanidine hydrochloride: 1M-5M
Disodium ethylene diamine tetraacetate: 1mM-50mM
Trishydroxymethylaminomethane: 10mM-100mM
Hydrochloric acid: 0.1%-1%
2- fourth oxyethanol: 5%-20%
PH:6.8-7.2
The cleaning solution can be separately as the examination of polysaccharide and/or polyphenol substance in removal Plant Genome purification process
Agent.
A kind of DNA extraction reagent, including cell suspending liquid, cell pyrolysis liquid, cleaning solution and eluent, the cleaning solution packet
Include guanidine hydrochloride, disodium ethylene diamine tetraacetate, trishydroxymethylaminomethane, hydrochloric acid and 2- fourth oxyethanol, pH value 6.8-7.2.?
In one concrete scheme, cleaning solution includes the guanidine hydrochloride of 1M-5M, the disodium ethylene diamine tetraacetate of 1mM-50mM, 10mM-100mM
Trishydroxymethylaminomethane, the hydrochloric acid of 0.1%-1% and the 2- fourth oxyethanol of 5%-20%.
Application of the cleaning solution of the present invention in the DNA extraction method based on silicagel column, comprising the following steps:
1. weighing a certain amount of wood sample, liquid nitrogen is milled.
2. lysate is added and Proteinase K is cracked.
3. column combination liquid was added in centrifuging and taking supernatant.
4. DNA is prepared pipe to be placed in new 2ml centrifuge tube, takes the solution in step 3 and be transferred to and prepare in pipe, 12,
000 × g is centrifuged 1min.
5. abandon filtrate, pipe will be prepared and put back into original 2ml centrifuge tube, be added 700 μ l cleaning solutions, 12,000 × g from
Heart 1min.
6. abandoning filtrate, pipe will be prepared and put back into original 2ml centrifuge tube, 700 μ l are added and remove saline solution, 12,000 × g from
Heart 1min.
7. repeating step 6.
8. abandoning filtrate, pipe will be prepared and put back into original 2ml centrifuge tube, 12,000 × g is centrifuged 1min.
9. DNA is prepared pipe to be placed in the 1.5ml centrifuge tube of another cleaning, add 100 μ l deionizations preparing periosteum center
Water is stored at room temperature 10min, and 12,000 × g is centrifuged 1min eluted dna, obtains genomic DNA.
Application of the cleaning solution of the present invention in the DNA extraction method based on nanometer magnetic microsphere, comprising the following steps:
1. weighing a certain amount of wood sample, liquid nitrogen is milled.
2. lysate is added and Proteinase K is cracked.
3. centrifuging and taking supernatant is added magnetic bead and combines liquid.
4. centrifuge tube is placed on magnetic frame, is inhaled after supernatant clarification and abandon supernatant.
5. adding 700 μ l cleaning solutions, be vortexed washing 1min.
6. 2ml centrifuge tube is placed on magnetic frame, is inhaled after supernatant clarification and abandon supernatant.
7. 700 μ l is added to remove saline solution, be vortexed washing 1min.
8. 2ml centrifuge tube is placed on magnetic frame, is inhaled after supernatant clarification and abandon supernatant.
9. repeating step 7-8.
Residual liquid is abandoned 10. inhaling after of short duration centrifugation, draught cupboard is dry.
11. adding 50-100 μ l deionized water, be vortexed elution 5min after being mixed with rifle piping and druming, obtains genomic DNA.
QPCR detection is carried out to the DNA of extraction, in which:
QPCR amplification condition are as follows: 95 DEG C, 2min；95 DEG C of 15s, 55 DEG C of 34s, totally 40 recycle.QPCR amplification be
The ABI 7500Real Time PCR System of ABI company is completed, and can also be completed in other amplification instruments.
The reaction system of qPCR are as follows: the system of 20 μ l includes:
The upstream and downstream primer sequence and probe sequence are respectively as follows:
Primer UP:5 '-CGAAATCGGTAGACGCTACG-3 '
Primer Down:5 '-TTCCATTGAGTCTCTGCACCT-3 '
Prober:5 '-GCAATCCTGAGCCAAATCC-3 '
Positive control is rice genome.
According to qPCR detection principle, when the DNA of extraction carries out qPCR detection, detection Ct value is recycled less than 35, then table
It is bright successfully to extract genomic DNA.Ct value is smaller, shows that the amount for extracting the genomic DNA obtained is more, purity is higher.
The cleaning solution of the present invention of embodiment 1 is extracted and is purified for timber DNA
Using cleaning solution of the present invention, the DNA in 100mg timber is extracted using silicagel column extraction method, including walk as follows
It is rapid:
1. weighing timber 100mg, liquid nitrogen is added, the grind into powder in mortar is immediately transferred into 2ml centrifuge tube.
2. lysate and 20 μ l Proteinase K, lid upper tube cap that 900 μ l65 DEG C preheating is added simultaneously obturage nozzle, whirlpool
Rotation oscillation 30s, is uniformly mixed, and 65 DEG C of water-bath 0.5h- are stayed overnight.
3. being cooled to room temperature, 125 μ l settling agents, ice bath 10min after vortex 1min is added.
4.12,000 × g is centrifuged 10min, takes 700 μ l of supernatant into new 2ml centrifuge tube, and 20 μ l combination liquid and 2 are added
The isopropanol of about 0.5 times of supernatant volume is added in μ l Carrier RNA after mixing, is vortexed and mixes 30s.
5. DNA is prepared pipe to be placed in new 2ml centrifuge tube, take the mixed liquor in 600 μ l steps 4 and is transferred to preparation pipe
In, 12,000 × g is centrifuged 1min.
6. abandoning filtrate, pipe will be prepared and put back into original 2ml centrifuge tube, mixed liquor remaining in step 4 is transferred to
It prepares in pipe, 12,000 × g is centrifuged 1min.
7. abandon filtrate, pipe will be prepared and put back into original 2ml centrifuge tube, be added 700 μ l cleaning solutions, 12,000 × g from
Heart 1min.
8. abandoning filtrate, pipe will be prepared and put back into original 2ml centrifuge tube, 700 μ l are added and remove saline solution, 12,000 × g from
Heart 1min.
9. repeating step 8.
10. abandoning filtrate, pipe will be prepared and put back into original 2ml centrifuge tube, 12,000 × g is centrifuged 1min.
11. DNA is prepared pipe to be placed in the 1.5ml centrifuge tube of another cleaning, add 100 μ l deionizations preparing periosteum center
Water is stored at room temperature 10min, and 12,000 × g is centrifuged 1min eluted dna, obtains genomic DNA.
Using cleaning solution of the present invention, the DNA in 100mg timber is extracted using nano magnetic microballon extraction method, including such as
Lower step:
1. weighing timber 100mg, liquid nitrogen is added, the grind into powder in mortar is immediately transferred into 2ml centrifuge tube.
2. the lysate and 20 μ lProteinase K of 900 μ l65 DEG C preheating is added, lid upper tube cap simultaneously obturages nozzle, is vortexed
30s is vibrated, is uniformly mixed, 65 DEG C of water-bath 0.5h- are stayed overnight.
3. being cooled to room temperature, 125 μ l settling agents, ice bath 10min after vortex 1min is added.
4.12,000 × g is centrifuged 10min, takes 700 μ l of supernatant into new 2ml centrifuge tube, and 20 μ l magnetic beads, 2 μ are added
LCarrier RNA is vortexed and the isopropanol of about 1.25 times of supernatant volumes is added after mixing, is vortexed and mixes 10min.
5. 2ml centrifuge tube is placed on magnetic frame, is inhaled after supernatant clarification and abandon supernatant.
6. adding 700 μ l cleaning solutions, be vortexed washing 1min.
7. 2ml centrifuge tube is placed on magnetic frame, is inhaled after supernatant clarification and abandon supernatant.
8. 700 μ l is added to remove saline solution, be vortexed washing 1min.
9. 2ml centrifuge tube is placed on magnetic frame, is inhaled after supernatant clarification and abandon supernatant.
10. repeating step 8-9.
Residual liquid is abandoned 11. inhaling after of short duration centrifugation, draught cupboard is dry.
12. adding 50-100 μ l deionized water, be vortexed elution 5min after being mixed with rifle piping and druming, obtains genomic DNA.
Embodiment 2 purifies tamarind tree plant genomic DNA
Tamarind tree plant genomic DNA is extracted, and is washed using cleaning solution of the present invention, qPCR method pair is utilized
The DNA of extraction is detected, and wherein the recipe ingredient of cleaning solution includes:
Guanidine hydrochloride: 3.5M
Disodium ethylene diamine tetraacetate: 20mM
Trishydroxymethylaminomethane: 35mM
Hydrochloric acid: 0.6%
2- fourth oxyethanol: 10%
PH:6.8-7.2
As a result such as Fig. 1, show that the Ct value of the present embodiment is 26.81, i.e., cleaning solution of the present invention can effectively remove plant
The impurity such as polyphenol, polysaccharide in object, the final plant genome DNA for obtaining high-purity.
Embodiment 3 purifies the genomic DNA of different Caesalpiniaceae plant samples
Caesalpiniaceae difference plant genome DNA is extracted, is washed using cleaning solution of the present invention, the pH value of cleaning solution is
6.8-7.2.Cleaning solution is specifically formulated as follows, comprising:
As a result such as Fig. 2, when showing that cleaning solution of the present invention can effectively remove the extraction of different Caesalpiniaceae Plant Genomes
The impurity pollution such as polysaccharide polyphenol, the final plant genome DNA for obtaining high-purity.
The effect of the different washing formula of liquid of embodiment 4
Tamarind tree plant genomic DNA is extracted by silicagel column method described in embodiment 1, is washed using cleaning solution of the present invention
It washs (experimental group), and the cleaning solution (control group) of itself and commercialization currently on the market is compared, the washing of control group 1
Liquid from Qiagen brand, control group 2 cleaning solution from the cleaning solution of Omega brand, control group 3 from Tiangeng brand, right
It is formulated according to the cleaning solution of group 4 from the classical of " fine works molecular biology experiment guide " (the 5th edition, Science Press).This implementation
Example experimental group cleaning solution formula include:
Guanidine hydrochloride: 1.5M
Disodium ethylene diamine tetraacetate: 5mM
Trishydroxymethylaminomethane: 15mM
Hydrochloric acid: 0.2%
2- fourth oxyethanol: 5%
PH:6.8-7.2
As a result such as Fig. 3, show cleaning solution of the present invention when carrying out Caesalpiniaceae plant genome DNA and extracting, Ct
Minimum shows that it has significant effect in terms of improving genome purity.
5 tamarind tree plant different parts extracting genome DNA of embodiment
Using nano magnetic microballon extraction method described in embodiment 1, the tissue gene group DNA of tamarind tree different parts, institute are extracted
Stating washing formula of liquid includes:
Guanidine hydrochloride: 4.5M
Disodium ethylene diamine tetraacetate: 70mM
Trishydroxymethylaminomethane: 80mM
Hydrochloric acid: 0.9%
2- fourth oxyethanol: 18%
PH:6.8-7.2
As a result as Fig. 4, tissue gene group DNA of the cleaning solution of the present invention for Caesalpiniaceae plant different parts are purified
In, the pollution of polysaccharide polyphenol can be effectively removed, the purity of purifying gained genomic DNA is improved.
6 tamarind tree plant genomic DNA Different Extraction Method of embodiment is equipped with the measure of merit of cleaning solution of the present invention
Tamarind tree plant genomic DNA is extracted using different extracting methods, is then equipped with of the present invention wash
Liquid (experimental group) is washed, tests its purification effect, and it is compared with the cleaning solution (control group) in original method.It surveys respectively
Try CTAB method, the PTB method, Low pH extraction with high salts method, Qiagen RNA isolation kit of improvement.
The washing formula of liquid of the present embodiment experimental group includes:
Guanidine hydrochloride: 3.5M
Disodium ethylene diamine tetraacetate: 20mM
Trishydroxymethylaminomethane: 35mM
Hydrochloric acid: 0.6%
2- fourth oxyethanol: 10%
PH:6.8-7.2
DNA extracted using the CTAB method of improvement, the CTAB method of the improvement is referring to Wang Guanlin, Fang Hongjun.Plant gene work
Journey (second edition), Beijing Science Press, 2002.744.Wherein the cleaning solution of experimental group is using washing described in the present embodiment
Liquid, the cleaning solution of control group 1 are original cleaning solution in the books.
Using PTB method extract DNA, wherein PTB method reagent include: PTB (bromination N- phenyl acetamide), EDTA, Proteinase K,
Phenol chloroform, chloroform isoamyl alcohol, dehydrated alcohol, ammonium acetate, TE.PTB method operating procedure, which includes: (1), impregnates wood powder with EDTA
48h makes timber demineralization.(2) after demineralization, add Proteinase K and PTB solution in 65 degree of water-bath 12h.(3) add phenol after the water bath is over
Chloroform is primary.(4) supernatant is taken after being centrifuged, it is primary that chlorination imitates isoamyl alcohol extraction.(5) step 4 is repeated.(6) anhydrous second is added
Alcohol and ammonium acetate, storage 12h precipitates DNA in -20 degree refrigerators.(7) be centrifuged DNA precipitating washed 2 times with cleaning solution and
It is stood overnight in 80% ethyl alcohol.(8) the DNA precipitating for being centrifuged completely, uses TE solution dissolving DNA after dry.Wherein experimental group
Cleaning solution is 80% ethyl alcohol using cleaning solution described in the present embodiment, the cleaning solution of control group 2.
Using Low pH extraction with high salts method extract DNA, wherein Low pH extraction with high salts method reagent include: CTAB, 5M NaCl, EDTA, Tris,
35% ethyl alcohol, cleaning solution, dehydrated alcohol and TE.Low pH extraction with high salts method operating procedure includes: (1) in wood powder plus CTAB is extracted
Liquid, 65 degree of water-bath 2h after mixing.(2) it is centrifuged after the water bath is over plus after the mixing of 35% ethyl alcohol.(3) supernatant is taken, phenol chloroform is added
Extracting is primary.(4) supernatant is taken after being centrifuged, it is primary that chlorination imitates isoamyl alcohol extraction.(5) step 4 is repeated.(6) supernatant is taken, adds 5M's
NaCl and dehydrated alcohol precipitate DNA after mixing, DNA are collected by centrifugation.(7) DNA precipitating is washed with cleaning solution.(8) TE is used after drying
Dissolving DNA.Wherein the cleaning solution of experimental group is 70% ethyl alcohol using cleaning solution described in the present embodiment, the cleaning solution of control group 3.
DNA is extracted using Qiagen RNA isolation kit, the entitled DNeasy Plant Mini Kit of kit and needs make
With the QIAshreder Maxi spin column in DNeasy Plant Maxi Kit.Wherein the cleaning solution of experimental group uses
Cleaning solution described in the present embodiment, the cleaning solution of control group 4 are the cleaning solution provided in Qiagen kit.
As a result such as Fig. 5, show using cleaning solution of the present invention, it is mentioned from different Caesalpiniaceae plant genome DNAs
It takes method to be used cooperatively, effect more better than cleaning solution in original method can be obtained.
Application of the cleaning solution of the present invention of embodiment 7 in the purifying of variety classes Plant Genome
The present embodiment is by cleaning solution of the present invention respectively to broad-leaved yellow wingceltis (leguminous plant), the maple that comes into leaves (Aceraceae plant), Asia
Numb (flax family (Linaceae) plant), vomiting nut (loganiaceae plant), false yucca (Agavaceae plants), wide yulan (Magnoliacea plant), folder
Bamboo peach (apocynaceae plant), Chinese ilex (emotionally section plant) xylem DNA purified.
Washing formula of liquid described in the present embodiment is as follows:
Guanidine hydrochloride: 3.5M
Disodium ethylene diamine tetraacetate: 20mM
Trishydroxymethylaminomethane: 35mM
Hydrochloric acid: 0.6%
2- fourth oxyethanol: 10%
PH:6.8-7.2
DNA is extracted using 1 the method for embodiment, and is equipped with cleaning solution described in the present embodiment, extracts result such as Fig. 6 institute
Show.Cleaning solution of the present invention can improve the extraction effect of variety classes Plant Genome extraction.
Cleaning solution of the present invention can remove the pollution such as polysaccharide, the polyphenol of different plants during Genomic Purification extensively.
Claims (5)
1. cleaning solution is extracting the application on DNA, which is characterized in that the cleaning solution includes guanidine hydrochloride, ethylenediamine tetra-acetic acid
Disodium, trishydroxymethylaminomethane, hydrochloric acid and 2- fourth oxyethanol, pH 6.8-7.2, and in DNA extraction process described in addition
Cleaning solution, with Polysaccharide removing and polyphenol simultaneously.
2. application according to claim 1, which is characterized in that the concentration of the guanidine hydrochloride is 1M-5M, ethylenediamine tetra-acetic acid
The concentration of disodium is 1mM-50mM, the concentration of trishydroxymethylaminomethane is 10mM-100mM, the concentration of hydrochloric acid is 0.1%-
The concentration of 1%, 2- fourth oxyethanol is 5%-20%.
3. application according to claim 1, which is characterized in that the concentration of guanidine hydrochloride is 3.5M, ethylenediamine tetra-acetic acid two
Na concn is 20mM, and trishydroxymethylaminomethane concentration is 35mM, and concentration of hydrochloric acid 0.6%, 2- fourth oxyethanol concentration is 10%.
4. according to claim 1 to application described in one of 3, which is characterized in that the DNA is to be mentioned using silicagel column extraction method
The DNA taken the or DNA extracted using nano magnetic microballon extraction method；Wherein
Application of the cleaning solution in the DNA extraction method of silicagel column, comprising the following steps:
(1) a certain amount of wood sample is weighed, liquid nitrogen is milled；
(2) lysate is added and Proteinase K is cracked；
(3) column combination liquid was added in centrifuging and taking supernatant；
(4) DNA is prepared pipe to be placed in new 2ml centrifuge tube, takes the solution in step 3 and be transferred to and prepares in pipe, 12,000
× g is centrifuged 1min；
(5) filtrate is abandoned, pipe will be prepared and put back into original 2ml centrifuge tube, 700 μ l cleaning solutions, 12,000 × g centrifugation is added
1min；
(6) filtrate is abandoned, pipe will be prepared and put back into original 2ml centrifuge tube, 700 μ l are added and go saline solution, 12,000 × g centrifugation
1min；
(7) step 6 is repeated；
(8) filtrate is abandoned, pipe will be prepared and put back into original 2ml centrifuge tube, 12,000 × g is centrifuged 1min；
(9) DNA is prepared pipe to be placed in the 1.5ml centrifuge tube of another cleaning, adds 100 μ l deionized waters preparing periosteum center,
It is stored at room temperature 10min, 12,000 × g is centrifuged 1min eluted dna, obtains genome；
Application of the cleaning solution in the DNA extraction method of nanometer magnetic microsphere, comprising the following steps:
(1) a certain amount of wood sample is weighed, liquid nitrogen is milled；
(2) lysate is added and Proteinase K is cracked；
(3) centrifuging and taking supernatant is added magnetic bead and combines liquid；
(4) centrifuge tube is placed on magnetic frame, is inhaled after supernatant clarification and abandons supernatant；
(5) plus 700 μ l cleaning solutions, vortex wash 1min；
(6) 2ml centrifuge tube is placed on magnetic frame, is inhaled after supernatant clarification and abandons supernatant；
(7) plus 700 μ l remove saline solution, and be vortexed washing 1min；
(8) 2ml centrifuge tube is placed on magnetic frame, is inhaled after supernatant clarification and abandons supernatant；
(9) step 7-8 is repeated；
(10) it is inhaled after of short duration centrifugation and abandons residual liquid, draught cupboard is dry；
(11) add 50-100 μ l deionized water, be vortexed elution 5min after being mixed with rifle piping and druming, acquisition genomic DNA.
5. according to claim 1 to application described in one of 3, which is characterized in that extraction is DNA in Caesalpiniaceae plant.
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| 一种从富含次生物质的植物中提取RNA的方法;李大力;《南京理工大学学报》;20011031;第25卷(第5期);第548页第1段、第4段第2-3行 * |
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