CN103756994A - DNA extraction method for polysaccharide-rich plant dried leaves - Google Patents
DNA extraction method for polysaccharide-rich plant dried leaves Download PDFInfo
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Abstract
The present invention discloses a DNA extraction method for polysaccharide plant dried leaves. The DNA extraction method comprises dried leaf grinding, desaccharifying, cracking, extraction, precipitation, washing, detection and other steps, wherein desaccharifying is the key step, a pre-heated desaccharifying buffer solution is added before the nuclear membrane is not cracked and the genome DNA is not released, the leaf tissue solution is suspended under a 65 DEG C water bath condition so as to dissolve polysaccharides and other secondary metabolites in the buffer solution, and low speed centrifugation is adopted to remove the polysaccharides and other impurities, such that the final DNA obtained through precipitation has high concentration and high purity. The DNA extraction method is suitable for dried mature leaves of all cerasus plants and other polysaccharide-rich plants, wherein the concentration of the obtained DNA can be increased if the leaves are the young and tender leaves, and the more DNA with high quality can be obtained from the mature leaves.
Description
Technical field
The invention belongs to technical field of molecular biology, relate to a kind of dry blade DNA extraction method that is applicable to being rich in polyose plant, relate in particular to a kind of DNA extraction method of dry blade of applicable cherry platymiscium.
Background technology
The DNA that obtains high quality (high density, high purity, high integrality) is the first step of molecular biology experiment most critical, at present, develop several different methods, successfully from the histoorgans such as plant leaf, callus, tissue cultured seedling, fruit, phloem, extracted DNA.But vegetable material is owing to mostly containing miscellaneous secondary metabolites, in some cases, the difference of the inherent features such as the source of different plants or even one species plant tissue material, the difference of position extrinsic property and chemical composition, weave construction need to be selected diverse ways or do some special processing when extracting genomic dna.From being rich in polysaccharide, polyphenol, tannin, the method difficulty of extracting in the xylophyta of pigment and other secondary metabolites is just greater than most Cereal and greengrocery plant relatively, the DNA solution obtaining usually viscosity is large and even be gluey, it is common phenomenon during DNA of plants is extracted, major cause is polysaccharide, phenols in leaching process with DNA co-precipitation, form the sticky jelly of parcel DNA, be difficult to dissolve or produce brown stain, yielding poorly often appears in the DNA obtaining, of poor quality, easily degraded, DNA quality and purity have been had a strong impact on, can not being limited property endonuclease digestion, serious even can not carry out pcr amplification as template.So how the secondary metabolites such as high-efficient simple ground Polysaccharide removing, polyphenol, most important concerning extracting purifying DNA of plants.
Cherry platymiscium is typical perennial woody fruit tree crop, is rich in a large amount of aldehydes matters and polysaccharide in its mature leaf, and especially polysaccharide content is very abundant, and gained DNA crude extract is thickness jelly, mobility extreme difference, the carrying out of seriously limiting follow-up molecule experiment.Traditional method is all generally to take the tender blade not launching completely of children in spring to extract and adopt more fresh or freezing material at present, its this raw metabolic substd contained with respect to drying and ripening blade is less, and gained DNA reluctantly can be for the follow-up experiment use not high to DNA specification of quality.But owing to being subject to the impact of time and experiment progress, add extremely short and sampling sample time in the spring impact of distance, obtains young leaflet tablet difficulty larger, conventionally need get in summer the mature leaf of silica dehydrator.For dry mature leaf, adopt that traditional extracting method is difficult to desirable DNA.
Therefore, for cherry, belong to and other plant drying mature leaf is rich in the feature of polysaccharide, polyphenol and other secondary metabolite, the secondary substances such as high-efficient simple ground Polysaccharide removing, polyphenol, most important concerning extracting purifying DNA of plants, in the time of cannot obtaining other young tender materials such as spire for summer and autumn, be engaged in fruit tree genome research simultaneously new method is provided.
Summary of the invention
The object of the invention is to overcome cherry belong to and other be rich in the large problem of polyose plant drying mature leaf DNA extraction difficulty, a kind of DNA extraction method of applicable polyose plant drying blade is provided.It is material that the present invention adopts the silica dehydrator mature leaf of cherry local variety, cherry Wild plant, Nanking cherry and sweet cherry, extracts high-quality DNA.
Its technical scheme is:
A DNA extraction method for applicable polyose plant drying blade, comprises the following steps:
1) get the dry blade 0.5-1.0g of discolour silica gel, add PVP and V
cmortar in add liquid nitrogen and pulverize;
2) ground blade powder is proceeded in 2.0ml centrifuge tube, every pipe 0.2-0.4g, adds desaccharification damping fluid and the 10 μ l beta-mercaptoethanols of 1.5ml65 ℃ of preheating, after fully mixing at 65 ℃ of water-bath 10min;
3) the centrifugal 3min of 5000rpm at normal temperatures, abandons supernatant, collecting precipitation;
4) in precipitation, add 3% CTAB and 10 μ l beta-mercaptoethanols of 1ml65 ℃ of preheating, when 65 ℃ of water-bath 40min or 1h, every 5-10min, put upside down centrifuge tube so that liquid blending, the then centrifugal 10min of 12500rpm under normal temperature during this time;
5) get in the centrifuge tube that supernatant liquor proceeds to new 2.0ml, add isopyknic chloroform, jog extraction 5min, until chloroform phase solution changes color, the centrifugal 10min of 12500rpm at 8 ℃;
6) supernatant liquor proceeds in new 2.0ml centrifuge tube, repeating step 5);
7) get supernatant liquor approximately 600 μ l and proceed in new 1.5ml centrifuge tube, add the sodium-acetate of 2 times of volumes through the dehydrated alcohol of-20 ℃ of precoolings and the 3mol/L of 1/10 volume, put upside down and mix-20 placement 30min, the centrifugal 15min of 10000rpm, abandons supernatant, collecting precipitation;
8) in DNA precipitation, add 75% ethanol 800 μ l, jog is also placed 5min, outwells ethanol, so cleans twice, then cleans once with dehydrated alcohol, is placed in ventilation and spends the night and dry up;
9) DNA precipitation is dissolved with 50 μ l TE, is placed in-20 ℃ of Refrigerator stores standby;
10) get the DNA solution that 2 μ l dissolve, through 1% agarose gel electrophoresis, detect, and detect 260nm, the absorption value at 280nm place and the concentration of DNA on Eppendorf Biophotomete type nucleic acid protein analyser, and according to OD260/OD280 value, judge the purity of DNA.
Step 1) described in desaccharification damping fluid be 1mol/L sodium-chlor; 0.1mol/L Tris-HCl PH8.0; 0.02mol/L EDTAPH8.0; 0.4mol/L glucose.
Step 4) described in CTAB be 1.5mol/lNaCl; 0.1mol/L Tris-Hcl PH8.0; 0.02mol/LEDTAPH8.0; 2%PVP.
Wherein above-mentioned steps 1) described in grinding time add PVP and V
ceach 0.2-0.5g, material is dry can be selected not add when good, and grinding must be fully, quick.
Step 2) in adding before powder in 2.0ml centrifuge tube, can in centrifuge tube, add in advance the preheating in 65 ℃ of water-baths of desaccharification damping fluid, treat the material last centrifuge tube that adds rapidly of pulverizing, shake up water-bath.
Step 3) in centrifugal at normal temperature, centrifugal rotational speed is 3000-6000rpm, otherwise the heavy pipe end that is deposited to is difficult to disperse.
Step 4) in should fully shake up in time after adding CTAB lysate, be as much as possible suspension, in water-bath process, every the upper and lower jog of 3-5min once, guarantee that cracking is abundant.
Step 5) in during extracting only with chloroform, add phenol effect bad on the contrary, fully shake up after adding chloroform.
Step 6) in can according to how many definite extracting number of times of the protein layer between centrifugal latter two liquid phase of extracting, until centre does not have protein layer.
Step 7) in precipitation all dehydrated alcohols are minimum will add 2 times of volumes, if the darker words of precipitated liquid color are added sodium-acetate in right amount, can effectively remove color.
Step 8) in 75% ethanol of washing precipitation at least want 800 μ l, washing times can according to circumstances increase, and finally with absolute ethanol washing, dries up.
Compared with prior art, beneficial effect of the present invention is:
It is material extraction DNA that the present invention adopts the cherry platymiscium drying and ripening blade that polysaccharide content is a lot, adopts extracting method flow process of the present invention can effectively remove the polysaccharide in leaf tissue, and the DNA purity obtaining is high, OD260/OD280=1.76-1.87; OD260/OD230=1.93-2.16, integrity and good fluidity, can be dissolved in rapidly water or TE, can not separate out precipitation after dissolving, and solution is thickness not, and 0.6% agarose gel electrophoresis detects clip size and concentrates between 40K-60K.
1, the method for the invention adopts dry mature leaf for extracting material, compared to just came with fresh or freezing children is tender in the past, the method is loose to extracting the requirement of material, can whenever the drawing materials of leaf growth, and the genomic dna of acquisition can meet subsequent experimental requirement.
2, the inventive method is before suspension cracking, adopt desaccharification damping fluid water-bath suspension leaf tissue solution, can make polysaccharide before nuclear membrane cracking fully be dissolved in the middle of damping fluid with other this raw metabolic substd, by low-speed centrifugal, can make nucleus and other component precipitation, and polysaccharide and other this raw metabolic substd are together removed.With respect to the loss of the more effective control of method DNA of removing polysaccharide in when precipitation.
3, adopt method of the present invention can shorten extraction time to 2.5 hour, experiment flow is simplified relatively.
Accompanying drawing explanation
Fig. 1 is the total gene DNA with the cherry platymiscium of traditional method for extracting, through 1.0% agarose gel electrophoresis detected result;
Fig. 2 is the total genomic dna with the cherry platymiscium of method extraction of the present invention, through 1.0% agarose gel electrophoresis detected result.
Embodiment
Below in conjunction with the drawings and specific embodiments, technical scheme of the present invention is described further.
A DNA extraction method for applicable polyose plant drying blade, comprises the following steps:
1) get the dry cherry of discolour silica gel, sweet cherry (red light), wild Chinese cherry and the each 0.5-1.0g of Nanking cherry blade, add 0.3gPVP and V
cmortar in add liquid nitrogen and pulverize rapidly, if grind insufficiently, increase liquid nitrogen grinding number of times;
2) ground blade powder is proceeded to rapidly in 2.0ml centrifuge tube, each sample 3 is managed, and the about 0.2-0.4g of every pipe adds desaccharification damping fluid (the 1mol/L sodium-chlor of 1.5ml65 ℃ of preheating; 0.1mol/L Tris-HCl PH8.0; 0.02mol/L EDTAPH8.0; 0.4mol/L glucose; ) and 10 μ l beta-mercaptoethanols, after fully mixing at 65 ℃ of water-bath 10min;
3) the centrifugal 3min of 5000rpm at normal temperatures, abandons supernatant, collecting precipitation;
4) in precipitation, add 3% CTAB (1.5mol/lNaCl of 1ml65 ℃ of preheating; 0.1mol/LTris-Hcl PH8.0; 0.02mol/LEDTAPH8.0; 2%PVP; 3%CTAB) He 10 μ l beta-mercaptoethanols, 65 ℃ of water-bath 1h, put upside down centrifuge tube so that liquid blending, the then centrifugal 10nin of 12500rpm under normal temperature every 5min therebetween;
5) get in the centrifuge tube that supernatant liquor proceeds to new 2.0ml, add isopyknic chloroform, jog extraction 5min, until chloroform phase solution changes color, the centrifugal 10min of 12500rpm at 8 ℃;
6) supernatant liquor proceeds in new 2.0ml centrifuge tube, repeating step 5) (can repeatedly repeat);
7) getting supernatant liquor approximately 600 μ l proceeds in new 1.5ml centrifuge tube, add the sodium-acetate of 2 times of volumes through the dehydrated alcohol of-20 ℃ of precoolings and the 3mol/L of 1/10 volume, put upside down gently and mix-20 placement 30min, the centrifugal 15min of 10000rpm, abandon supernatant, collecting precipitation;
8) in DNA precipitation, add 75% ethanol 800 μ l, jog is also placed 5min, outwells gently ethanol, so cleans twice, then cleans once with dehydrated alcohol, is placed in ventilation and spends the night and dry up;
9) DNA precipitation is dissolved with 40 μ l TE, is placed in-20 ℃ of Refrigerator stores standby.
10) get the DNA solution that 2 μ l dissolve, through 1% agarose gel electrophoresis, detect, as shown in Figure 2, be followed successively by from left to right each 3 of cherry, sweet cherry, wild Chinese cherry, Nanking cherry.
11) get after 50 times of DNA solution dilutions that 2 μ l dissolve, on EppendorfBiophotomete type nucleic acid protein analyser, detect 260nm, the absorption value at 280nm place and the concentration of DNA, and according to OD260/OD280 and OD260/OD230 value, judge the purity of DNA, as shown in table 1.
Table 1
| ? | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| (ng/ μ l) for concentration | 102.4 | 89.3 | 39.3 | 95.9 | 44.4 | 91.9 | 65.2 | 50.7 | 48.6 | 78.4 | 54.4 | 57.4 |
| OD260/OD280 | 1.82 | 1.81 | 1.76 | 1.87 | 1.84 | 1.87 | 1.82 | 1.85 | 1.86 | 1.87 | 1.83 | 1.80 |
| OD260/OD230 | 2.11 | 2.02 | 1.98 | 1.94 | 2.16 | 1.90 | 1.98 | 1.96 | 1.99 | 2.11 | 1.98 | 2.04 |
[0050]table 2
| Medicine final concentration | Join 1L aequum | Join 100ml aequum |
| 1mol/L sodium-chlor | 58.3g | 5.83g |
| 0.1mol/L?Tris-HCl?PH8.0 | 100ml | 10ml |
| 0.02mol/L?EDTA?PH8.0 | 40ml | 4ml |
| 0.4mol/L glucose | 72g | 7.2g |
The preparation of desaccharification damping fluid is as shown in table 2.
3%CTAB buffer formulation is as shown in table 3:
Table 3
| Medicine final concentration | Join 1L aequum | Join 100ml aequum |
| 1.4mol/L?NaCl | 81.62g | 8.162g |
| 0.1mol/L?Tris-HCl?PH8.0 | 100ml | 10ml |
| 0.02mol/L?EDTA?PH8.0 | 40ml | 4ml |
| 2％PVP | 20g | 2g |
| 3％CTAB | 30g | 3g |
Note: Tris-HCl and EDTA are first made into respectively mother liquor, Tris-HCl mother liquor: 1M (PH=8); EDTA mother liquor: 0.5M (PH=8).
If without desaccharification damping fluid processing before cracking, in the DNA of last precipitation, also have very many polysaccharose substances, cause whole DNA solution to be thickness glue-like, mobility extreme difference, agarose gel electrophoresis shows that DNA quality is very low, as shown in Figure 1.Adopt method of the present invention, effectively Polysaccharide removing, the quality of the DNA of extraction is higher.
The above; it is only preferably embodiment of the present invention; protection scope of the present invention is not limited to this; anyly be familiar with those skilled in the art; in the technical scope disclosing in the present invention, the simple transformation of the technical scheme that can obtain apparently or equivalence are replaced and are all fallen within the scope of protection of the present invention.
Claims (4)
1. a DNA extraction method for applicable polyose plant drying blade, is characterized in that, comprises the following steps:
1) get the dry blade 0.5-1.0g of discolour silica gel, add PVP and V
cmortar in add liquid nitrogen and pulverize;
2) ground blade powder is proceeded in 2.0ml centrifuge tube, every pipe 0.2-0.4g, adds desaccharification damping fluid and the 10 μ l beta-mercaptoethanols of 1.5ml65 ℃ of preheating, after fully mixing at 65 ℃ of water-bath 10min;
3) the centrifugal 3min of 5000rpm at normal temperatures, abandons supernatant, collecting precipitation;
4) in precipitation, add 3% CTAB and 10 μ l beta-mercaptoethanols of 1ml65 ℃ of preheating, when 65 ℃ of water-bath 40min or 1h, every 5-10min, put upside down centrifuge tube so that liquid blending, the then centrifugal 10min of 12500rpm under normal temperature during this time;
5) get in the centrifuge tube that supernatant liquor proceeds to new 2.0ml, add isopyknic chloroform, jog extraction 5min, until chloroform phase solution changes color, the centrifugal 10min of 12500rpm at 8 ℃;
6) supernatant liquor proceeds in new 2.0ml centrifuge tube, repeating step 5);
7) get supernatant liquor approximately 600 μ l and proceed in new 1.5ml centrifuge tube, add the sodium-acetate of 2 times of volumes through the dehydrated alcohol of-20 ℃ of precoolings and the 3mol/L of 1/10 volume, put upside down and mix-20 placement 30min, the centrifugal 15min of 10000rpm, abandons supernatant, collecting precipitation;
8) in DNA precipitation, add 75% ethanol 800 μ l, jog is also placed 5min, outwells ethanol, so cleans twice, then cleans once with dehydrated alcohol, is placed in ventilation and spends the night and dry up;
9) DNA precipitation is dissolved with 50 μ l TE, is placed in-20 ℃ of Refrigerator stores standby;
10) get the DNA solution that 2 μ l dissolve, through 1% agarose gel electrophoresis, detect, and detect 260nm, the absorption value at 280nm place and the concentration of DNA on Eppendorf Biophotomete type nucleic acid protein analyser, and according to OD260/OD280 value, judge the purity of DNA.
2. the DNA extraction method of applicable polyose plant drying blade according to claim 1, its special card is, step 1) described in desaccharification damping fluid be 1mol/L sodium-chlor; 0.1mol/L Tris-HCl PH8.0; 0.02mol/L EDTAPH8.0; 0.4mol/L glucose.
3. the DNA extraction method of applicable polyose plant drying blade according to claim 1, is characterized in that step 4) described in CTAB be 1.5mol/lNaCl; 0.1mol/L Tris-Hcl PH8.0; 0.02mol/L EDTAPH8.0; 2%PVP.
4. the DNA extraction method that is applicable to polyose plant drying blade according to claim 1-3 any one, is characterized in that, described polyose plant is cherry platymiscium.
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| CN104152435A (en) * | 2014-07-24 | 2014-11-19 | 四川农业大学 | High-efficiency extraction method for trace cherry leaf DNA (deoxyribonucleic acid) |
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| CN109880822A (en) * | 2019-03-12 | 2019-06-14 | 中国林业科学研究院亚热带林业研究所 | A kind of idesia high quality DNA extracting method |
| CN110229810A (en) * | 2019-06-24 | 2019-09-13 | 福建省农业科学院果树研究所 | A kind of method of high efficiency extraction olive blade nucleic acid substances |
| CN110804612A (en) * | 2019-12-20 | 2020-02-18 | 福建省农业科学院亚热带农业研究所（福建省农业科学院蔗麻研究中心） | Method for extracting trace and high-quality dendrobium officinale genome DNA |
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