CN102851278A - Method for extracting deoxyribose nucleic acid (DNA) from callicarpa plant leaves - Google Patents
Method for extracting deoxyribose nucleic acid (DNA) from callicarpa plant leaves Download PDFInfo
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Abstract
The invention discloses a method for extracting DNA from callicarpa plant leaves. According to the method, the existing sodium dodecyl sulfate (SDS) extraction method is improved, the method is suitable for extracting DNA from fresh leaves or silica gel dried leaves of callicarpa plant, and the problems that the total DNA extracted through the existing SDS method carries a few proteins, and the purity of the DNA is low caused by elimination of impurities such as excessive flavonoids and condensed tannins in the callicarpa plant leaves are solved.
Description
Technical field
The invention belongs to the molecular biology of plants field, in particular to the DNA extraction method of the fresh blade of a kind of beautyberry plant and silica dehydrator blade.
Background technology
Verbenaceae beautyberry plant has more than 150 and plants, in domestic 49 kinds, wherein be rich in the secondary metabolic substds such as flavonoid, condensed tannin, polyose in most of kind blades and the root, have the effects such as hemostasis, the loose stasis of blood, anti-inflammatory, thus this to belong to many kind of plant are the traditional Chinese medicinal materialss of China.In addition, the real purple of this platymiscium cause and effect is string of beads pearl and gaining the name seemingly, to see orchard woods plant good autumn and winter, have ornamental value and DEVELOPMENT PROSPECT, but there are the problems such as plant type is at random, fruit is little, resistance is poor in some kind, therefore, in order to improve above-mentioned bad proterties, the ornamental plantation of developing this platymiscium is worth, and is necessary to carry out the breeding research of beautyberry plant, thereby obtains the better resource of gardens proterties.
Along with molecular biological development, utilize the molecular mark technology can greatly accelerate breeding process, improve breeding efficiency.And DNA extraction is prerequisite and the key of carrying out molecule marking research.The beautyberry plant is as medicinal plant, and its blade and root all contain more flavonoid, condensed tannin, and the existence of these secondary metabolites has had a strong impact on the purity that DNA extracts, and then has influence on follow-up DNA cloning work.Therefore, successfully to extract be the important prerequisite of carrying out the research of this platymiscium molecule marker and assistant breeding to the DNA of beautyberry plant.Have no report about beautyberry plant leaf DNA extraction method.
Electrophoresis result shows, often contain protein in the leaf DNA of the kinds such as this platymiscium Root of Girald Beautyberry that traditional DNA extraction method (such as the SDS method) extracts, Japanses beauty-berry, callicarpa kochiana, show as the point sample hole and present bright band under ultraviolet lamp, the DNA purity detecting shows A
260/280Less than 1.80, and owing to contain the impurity such as more flavonoid, condensed tannin in the blade, the existence of these materials has a great impact follow-up molecular marking technique.Therefore, be necessary the leaf DNA extracting method of beautyberry plant is improved.
Summary of the invention
For above-mentioned situation, in order to overcome the defective of prior art, the object of the invention is to provides a kind of method for extracting beautyberry plant leaf DNA by lot of experiments research.The method can effectively solve total DNA of traditional SDS extraction method extraction with a small amount of albumen, and eliminates the low problem of DNA purity that the impurity such as flavonoid too much in the beautyberry plant leaf, condensed tannin cause.
The object of the present invention is achieved like this:
A kind of method for extracting beautyberry plant leaf DNA comprises the steps:
(1) in the sterilization centrifuge tube, adds lysate, for subsequent use, described lysate comprises EDTA 30-60mM, NaCl 60-120mM, 3-6wt% PVP, 1.5-2wt% SDS, 2-3wt% mercaptoethanol, the 0.7-1.2wt% RNAase of TrisHCl 80-120mM, the pH8.0 of following component: pH8.0;
(2) get the blade of beautyberry plant, in mortar, add liquid nitrogen and grind rapidly and be powder, then powder is changed in the described centrifuge tube, make the abundant mixing of sample and lysate;
(3) above-mentioned centrifuge tube is placed 62-68 ℃ of water-bath be incubated 15-30min, during shake centrifuge tube 2-4 time gently;
(4) take out centrifuge tube, it is 0.8-1.2M that adding KAc makes final concentration, then ice bath 15-30min;
(5) 10000-15000rpm is centrifugal under the room temperature;
(6) get supernatant liquor and put in another sterilization centrifuge tube, add isopyknic chloroform: the primary isoamyl alcohol mixing solutions, its proportioning is chloroform: the primary isoamyl alcohol volume ratio is 24:1, mixing, 6000-10000rpm is centrifugal under the room temperature;
(7) get supernatant liquor and place new sterilization centrifuge tube, repeating step (6) operation;
(8) get supernatant liquor after centrifugal and change in the new sterilization centrifuge tube, add the Virahol of-20 ℃ of lower precoolings of 2/3 volume, mixing places-20 ℃ of refrigerator 20-40min;
(9) 6-12 ℃ of lower 10000-15000rpm is centrifugal after the taking-up, abandons supernatant liquor, collecting precipitation;
(10) add 70%(v/v) ethanol rinsing precipitation, then 6-12 ℃ of lower 13000-18000rpm is centrifugal, abandons supernatant, collecting precipitation;
(11) repeating step (10) operation;
(12) abandon supernatant, get genomic dna.
Preferably, described method for extracting beautyberry plant leaf DNA comprises the steps:
(1) in the sterilization centrifuge tube, adds lysate, for subsequent use, described lysate comprises EDTA 45-50mM, NaCl 90-100mM, 4-5wt% PVP, 1.5-2wt% SDS, 2.3-2.7wt% mercaptoethanol, the 0.9-1.1wt% RNAase of TrisHCl 90-110mM, the pH8.0 of following component: pH8.0;
(2) get the blade of beautyberry plant, in mortar, add liquid nitrogen and grind rapidly and be powder, then powder is changed in the described sterilization centrifuge tube, turn upside down, make the abundant mixing of sample and lysate;
(3) above-mentioned centrifuge tube is placed 65 ℃ of water-baths be incubated 20min, during shake centrifuge tube 2-3 time gently;
(4) take out centrifuge tube, it is 0.95-1.05M that adding KAc makes final concentration, then ice bath 20 min;
(5) centrifugal 20 min of 13000 rpm under the room temperature;
(6) get supernatant liquor and put in another sterilization centrifuge tube, add isopyknic chloroform: the primary isoamyl alcohol mixing solutions, its proportioning is chloroform: the primary isoamyl alcohol volume ratio is 24:1, puts upside down gently centrifuge tube, mixing, the centrifugal 10min of 8000 rpm under the room temperature;
(7) get supernatant liquor and place new sterilization centrifuge tube, repeating step (6) operates once;
(8) get supernatant liquor after centrifugal and change in the new sterilization centrifuge tube, add the Virahol of-20 ℃ of lower precoolings of 2/3 volume, put upside down gently mixing, place-20 ℃ of refrigerator 30 min;
(9) take out rear 9 ℃ of lower centrifugal 10 min of 13000 rpm, abandon supernatant liquor, collecting precipitation;
(10) add 70%(v/v) ethanol rinsing precipitation, then 9 ℃ of lower centrifugal 15 min of 15000 rpm abandon supernatant, collecting precipitation;
(11) repeating step (10) operation once;
(12) abandon supernatant, get genomic dna.
Further preferably, described method for extracting beautyberry plant leaf DNA, wherein the described plant leaf of step (2) should be removed petiole, vein part, gets the mesophyll part, and its consumption is about 1cm
2Area, the about 20mg of quality.
Further preferably, described method for extracting beautyberry plant leaf DNA, wherein said beautyberry plant is Callicarpa dichotoma (Lour.) K.Koch, Root of Girald Beautyberry, Japanses beauty-berry, callicarpa kochiana, Root or leaf of Bigleaf Beautyberry, leaf of Whitehairy Beautyberry, callicarpa pedunculata, Shortstipe Beautyberry, callicarpa nudiflora.
Further preferably, described method for extracting beautyberry plant leaf DNA, the blade of wherein said beautyberry plant is fresh blade or silica dehydrator blade.
The beautyberry plant leaf DNA extraction method that the present invention relates to is compared with traditional SDS extraction method, has the following advantages and significant progress:
(1) final extract because group DNA purity is high, effectively overcome traditional CT AB method and extracted and contain the problems such as albumen, DNA purity are lower, guaranteed carrying out smoothly of follow-up molecular marking technique.
(2) extraction time shortens to 4-5h, has improved working efficiency.
(3) required sample size is few, and the DNA yield is high, only needs the 20mg blade, and the DNA of extraction can satisfy the consumption of molecule marker.
(4) improve according to the characteristics of beautyberry plant, be the universal method that is fit to beautyberry plant leaf DNA extraction, be particularly useful for Callicarpa dichotoma (Lour.) K.Koch, Root of Girald Beautyberry, Japanses beauty-berry, callicarpa kochiana, Root or leaf of Bigleaf Beautyberry, leaf of Whitehairy Beautyberry, callicarpa pedunculata, Shortstipe Beautyberry, this platymiscium leaf DNA such as callicarpa nudiflora is extracted.In addition, the present invention is equally applicable to the plant leaf DNA extraction that other albumen is difficult for removal and contains the secondary metabolic substds such as flavonoid, condensed tannin.
(5) be applicable to adopt the beautyberry plant leaf of silica gel method kept dry, the long-distance transportation that has solved nonlocal sampling causes the problem of sample fails.
Embodiment
Form is described in further detail foregoing of the present invention again by the following examples, but this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following embodiment, all technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
The extraction of embodiment one fresh leaf DNA
The lysis buffer composition is: TrisCl 100 mM, pH8.0; EDTA 50mM, pH8.0; NaCl 100mM; 5% PVP, it is for subsequent use to configure 121 ℃ of sterilizations of rear high temperature 20min;
It is for subsequent use to add 500 μ L lysis buffers, the SDS of 50 μ L 20%, 14 μ L mercaptoethanols, 5 μ L RNAase in 1.5 mL sterilization centrifuge tube; Get the about 1cm of the fresh blade of Callicarpa dichotoma (Lour.) K.Koch
2, in mortar, add liquid nitrogen and grind rapidly and be powder, afterwards with powder transfer to above-mentioned centrifuge tube, turn upside down, make the abundant mixing of sample and lysate; Above-mentioned centrifuge tube is placed 65 ℃ of water-bath insulation 20 min, shake centrifuge tube 2-3 time gently therebetween; Take out centrifuge tube, add the KAc of 150 μ L 5M, then ice bath 20 min; Centrifugal 20 min of 13000 rpm under the room temperature; Get supernatant liquor and put in the sterilization centrifuge tube, add isopyknic chloroform: the primary isoamyl alcohol mixing solutions, its proportioning is chloroform: the primary isoamyl alcohol volume ratio is 24:1, puts upside down gently centrifuge tube, mixing, centrifugal 10 min of 8000 rpm under the room temperature; Get supernatant liquor and place the new aseptic centrifuge tube of 1.5 mL, repeat the equal-volume chloroform: the primary isoamyl alcohol extracting once; Get supernatant liquor after centrifugal and change in the 1.5 new mL centrifuge tubes, add the Virahol of-20 ℃ of lower precoolings of 2/3 volume, put upside down gently mixing, place-20 ℃ of refrigerator 30min; Take out rear 9 ℃ of lower centrifugal 10 min of 13000 rpm, abandon supernatant liquor, collecting precipitation; Add the 70%(volume ratio) ethanol 1 mL rinsing precipitation, then 9 ℃ of lower centrifugal 15 min of 15000 rpm abandon supernatant, collecting precipitation; Repeat 70% ethanol rinsing once; Abandon supernatant, centrifuge tube is upside down on the thieving paper, air-dry, get air-dry thing, with the dissolving of 50 μ L TE damping fluids, namely get high-quality genomic dna, through being directly used in pcr amplification after the dilution.
The extraction of embodiment second silica gel method kept dry leaf DNA
Field sample is preserved: the young leaflet tablet 3-4 sheet of field acquisition callicarpa kochiana, pack in the 50mL centrifuge tube, with the space between the particles filled blade of blue silica gel and the centrifugal tube wall, note making blade fully by the silica gel particle embedding as far as possible, then screw the centrifuge tube lid, in 1-2 days, silica gel takes back before becoming pink indoorly, blade taken out put into the sky centrifuge tube, put into-70 ℃ of refrigerators and preserve.
The lysis buffer composition is: TrisCl 100 mM, pH8.0; EDTA 50mM, pH8.0; NaCl 100mM; 5% PVP, it is for subsequent use to configure 121 ℃ of sterilizations of rear high temperature 20min;
DNA extraction: DNA extraction adds 500 μ L lysis buffers, the SDS of 50 μ L 20%, 14 μ L mercaptoethanols, 5 μ L RNAase in 1.5 mL sterilization centrifuge tube for subsequent use; From Ultralow Temperature Freezer, get the about 1cm of the dry blade of Loquat Leaf
2, in mortar, add liquid nitrogen and grind rapidly and be powder, afterwards with powder transfer to above-mentioned centrifuge tube, turn upside down, make the abundant mixing of sample and lysate; Above-mentioned centrifuge tube is placed 65 ℃ of water-bath insulation 20 min, shake centrifuge tube 2-3 time gently therebetween; Take out centrifuge tube, add the KAc of 150 μ L 5M, then ice bath 20 min; Centrifugal 20 min of 13000 rpm under the room temperature; Get supernatant liquor and put in the sterilization centrifuge tube, add isopyknic chloroform: the primary isoamyl alcohol mixing solutions, its proportioning is chloroform: the primary isoamyl alcohol volume ratio is 24:1, puts upside down gently centrifuge tube, mixing, centrifugal 10 min of 8000 rpm under the room temperature; Get supernatant liquor and place the new aseptic centrifuge tube of 1.5 mL, repeat the equal-volume chloroform: the primary isoamyl alcohol extracting once; Get supernatant liquor after centrifugal and change in the 1.5 new mL centrifuge tubes, add the Virahol of-20 ℃ of lower precoolings of 2/3 volume, put upside down gently mixing, place-20 ℃ of refrigerator 30min; Take out rear 9 ℃ of lower centrifugal 10 min of 13000 rpm, abandon supernatant liquor, collecting precipitation; Add the 70%(volume ratio) ethanol 1 mL rinsing precipitation, then 9 ℃ of lower centrifugal 15 min of 15000 rpm abandon supernatant, collecting precipitation; Repeat 70% ethanol rinsing once; Abandon supernatant, centrifuge tube is upside down on the thieving paper, air-dry, get air-dry thing, with the dissolving of 50 μ L TE damping fluids, namely get high-quality genomic dna, through being directly used in pcr amplification after the dilution.
Claims (5)
1. a method that is used for extracting beautyberry plant leaf DNA comprises the steps:
(1) in the sterilization centrifuge tube, adds lysate, for subsequent use, described lysate comprises EDTA 30-60mM, NaCl 60-120mM, 3-6wt% PVP, 1.5-2wt% SDS, 2-3wt% mercaptoethanol, the 0.7-1.2wt% RNAase of TrisHCl 80-120mM, the pH8.0 of following component: pH8.0;
(2) get the blade of beautyberry plant, in mortar, add liquid nitrogen and grind rapidly and be powder, then powder is changed in the described centrifuge tube, make the abundant mixing of sample and lysate;
(3) above-mentioned centrifuge tube is placed 62-68 ℃ of water-bath be incubated 15-30min, during shake centrifuge tube 2-4 time gently;
(4) take out centrifuge tube, it is 0.8-1.2M that adding KAc makes final concentration, then ice bath 15-30min;
(5) 10000-15000rpm is centrifugal under the room temperature;
(6) get supernatant liquor and put in another sterilization centrifuge tube, add isopyknic chloroform: the primary isoamyl alcohol mixing solutions, its proportioning is chloroform: the primary isoamyl alcohol volume ratio is 24:1, mixing, 6000-10000rpm is centrifugal under the room temperature;
(7) get supernatant liquor and place new sterilization centrifuge tube, repeating step (6) operation;
(8) get supernatant liquor after centrifugal and change in the new sterilization centrifuge tube, add the Virahol of-20 ℃ of lower precoolings of 2/3 volume, mixing places-20 ℃ of refrigerator 20-40min;
(9) 6-12 ℃ of lower 10000-15000rpm is centrifugal after the taking-up, abandons supernatant liquor, collecting precipitation;
(10) add 70%(v/v) ethanol rinsing precipitation, then 6-12 ℃ of lower 13000-18000rpm is centrifugal, abandons supernatant, collecting precipitation;
(11) repeating step (10) operation;
(12) abandon supernatant, get genomic dna.
2. described method for extracting beautyberry plant leaf DNA according to claim 1 is characterized in that comprising the steps:
(1) in the sterilization centrifuge tube, adds lysate, for subsequent use, described lysate comprises EDTA 45-50mM, NaCl 90-100mM, 4-5wt% PVP, 1.5-2wt% SDS, 2.3-2.7wt% mercaptoethanol, the 0.9-1.1wt% RNAase of TrisHCl 90-110mM, the pH8.0 of following component: pH8.0;
(2) get the blade of beautyberry plant, in mortar, add liquid nitrogen and grind rapidly and be powder, then powder is changed in the described sterilization centrifuge tube, turn upside down, make the abundant mixing of sample and lysate;
(3) above-mentioned centrifuge tube is placed 65 ℃ of water-baths be incubated 20min, during shake centrifuge tube 2-3 time gently;
(4) take out centrifuge tube, it is 0.95-1.05M that adding KAc makes final concentration, then ice bath 20 min;
(5) centrifugal 20 min of 13000 rpm under the room temperature;
(6) get supernatant liquor and put in another sterilization centrifuge tube, add isopyknic chloroform: the primary isoamyl alcohol mixing solutions, its proportioning is chloroform: the primary isoamyl alcohol volume ratio is 24:1, puts upside down gently centrifuge tube, mixing, the centrifugal 10min of 8000 rpm under the room temperature;
(7) get supernatant liquor and place new sterilization centrifuge tube, repeating step (6) operates once;
(8) get supernatant liquor after centrifugal and change in the new sterilization centrifuge tube, add the Virahol of-20 ℃ of lower precoolings of 2/3 volume, put upside down gently mixing, place-20 ℃ of refrigerator 30 min;
(9) take out rear 9 ℃ of lower centrifugal 10 min of 13000 rpm, abandon supernatant liquor, collecting precipitation;
(10) add 70%(v/v) ethanol rinsing precipitation, then 9 ℃ of lower centrifugal 15 min of 15000 rpm abandon supernatant, collecting precipitation;
(11) repeating step (10) operation once;
(12) abandon supernatant, get genomic dna.
3. described method for extracting beautyberry plant leaf DNA according to claim 1 and 2, it is characterized in that: the described plant leaf of step (2) should be removed petiole, vein part, gets the mesophyll part, and its consumption is about 1cm
2Area, the about 20mg of quality.
4. described method for extracting beautyberry plant leaf DNA according to claim 1 and 2, it is characterized in that: described beautyberry plant is Callicarpa dichotoma (Lour.) K.Koch, Root of Girald Beautyberry, Japanses beauty-berry, callicarpa kochiana, Root or leaf of Bigleaf Beautyberry, leaf of Whitehairy Beautyberry, callicarpa pedunculata, Shortstipe Beautyberry, callicarpa nudiflora.
5. described method for extracting beautyberry plant leaf DNA according to claim 1 and 2, it is characterized in that: the blade of described beautyberry plant is fresh blade or silica dehydrator blade.
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Application publication date: 20130102 |