CN105018471A - Method for efficiently extracting edible fungus mycelium nutrient growth stage DNA - Google Patents
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Abstract
The invention provides a method which can be used for extracting edible fungus mycelium nutrient growth stage DNA. By means of the method, interference of culture mediums to the DNA extraction process can be effectively removed, the DNA extraction is high in efficiency and purity, and no PCR enzyme inhibitor is contained. The method used for extracting the edible fungus mycelium nutrient growth stage DNA comprises a pre-treatment phase and an extraction phase, wherein the pre-treatment phase comprises the following steps: 1, taking a culture medium covered with mycelium; 2, adding a buffer solution I, placing the culture medium onto a constant temperature shaking table, and shaking the table for 2-3 hours at the temperature of 35-40 DEC C and at the speed of 100-150 rpm; 3, taking the solution obtained in step 2, conducting low speed centrifugation for 10-15 minutes at the speed of 3000-4000 rpm, and collecting liquid supernatant; 4, adding a buffer solution II into sediment left in step 3 for washing for 2-5 times, and conducting the low speed centrifugation for 10-15 minutes at the speed of 3000-4000 rpm; taking and combing liquid supernatant obtained after washing of each time; 5, combining the centrifugated liquid supernatant obtained in step 3 and step 4, and conducting centrifugation for 10-15 minutes at the temperature of 4 DEG C and at the rotating speed of 1000-12000 rpm; discarding the liquid supernatant, and collecting sediment.
Description
Technical field
The invention belongs to molecular biology field of engineering technology, be specifically related to a kind of method of high efficiency extraction edible fungus mycelium vegetative growth phase DNA.
Technical background
Not only thing delicious food is fresh for edible mushrooms, and low-yield, lower fat, and the nutrient substances such as rich in proteins, food fibre and VITAMIN, developing into the 3rd based food outside the vegetable food that continues, animal food, i.e. fungus food.In addition, one of source of edible mushrooms or the multiple natural product such as functional polysaccharide, terpenoid, at raising immune function of human body, prevents and treats multiple chronic disease and anti-ageing aspect of waiting for a long time has significant effect.Therefore, edible mushrooms is more and more subject to the favor of consumers in general, and market demand constantly increases.Common edible mushrooms solid medium is generally that in the solid substance (wood chip, cotton seed hulls, crop material) of rich cellulose, xylogen, add appropriate aid nutrition material (wheat bran or rice bran) formulated with water again.
In recent years, although mushroom industry in China's development rapidly, country also strengthens year by year to mushroom industry supporting dynamics, and the present situation of fundamental research weakness does not also obtain the change of essence.Traditional research means cannot meet the demand of current edible mushrooms correlative study.And Protocols in Molecular Biology can break through the restriction of traditional research method, to greatly promote the research in the fields such as the molecular mechanism of Regulation Mechanism that edible mushrooms DNA molecular marker, edible fungi growth are grown, edible mushrooms environmental factor response, active matter of edible fungi and the qualification of anabolic molecular basis, edible mushrooms analysis of genetic diversity and edible fungus species thereof and breeding, and bases of all these researchs extract high-quality for the hereditary material DNA molecule in mycelium.
Conventional laboratory facilities from the mycelium liquid nutrient medium, the mycelium of PDA media surface or the sporophore of edible mushrooms, extract DNA, these extracting method comparative maturity.But, the growth of edible mushrooms on cultivation matrix (wood chip, cotton seed hulls, crop material, Chinese medicine slag) is divided into two stages: i.e. mycelium vegetative growth phase and the sporophore growth stage, and wherein mycelium vegetative growth phase is the of paramount importance stage in whole edible fungi growth growth course.At vegetative growth phase, mycelium and cultivation matrix are closely fitted together to, and mycelium effectively cannot be separated from cultivation matrix, cause DNA extraction difficulty, of poor quality, affect following amplification.The disappearance of edible fungi nutrition growth phase DNA extraction method, causes the data chain of whole edible fungi growth growth course Middle molecule Mechanism Study imperfect, have impact on carrying out in a deep going way of research.Therefore set up efficient, reliable edible fungus mycelium vegetative growth phase DNA extraction method and just seem particularly important.
At present, from the mycelium liquid nutrient medium, existing a lot of report (as: the Zhang Y J of method of DNA is extracted in the mycelium of PDA media surface or the sporophore of edible mushrooms, Zhang S, Liu X Z, et al.Asimple method of genomic DNA extraction suitable for analysis of bulk fungalstrains [J] .Letters in applied microbiology, 2010, 51 (1): 114-118., hide gold flat, Lian Bin, Yuan Sheng. simple and easy to do edible fungus mycelium extracting genome DNA method [J]. Food science, 2005, 26 (3): 66-68., Tang Lianghua, Su Min, Zheng Danhua, Deng. the research [J] of edible mushrooms Total DNA extraction method. Fujian light textile, 2006 (1): 1-4.).But relevant mycelium vegetative growth phase, the method extracting DNA under mycelium and the tight admixture of cultivation matrix is also seldom studied.The people such as the Yu Changxia of rarely seen national edible mushrooms Engineering Technical Research Centre extract the report (Yu Changxia of DNA for biomass estimation of mushroom and needle mushroom vegetative growth phase, Cao Hui, Chen Mingjie, Deng. the structure [J] of mushroom 135 hypha biomass and DNA content typical curve thereof. edible mushrooms journal, 2010,17 (3): 1-7., Yu Changxia, Cao Hui, Chen Mingjie, Deng. the structure [J] of Flammulina velutipes mycelium biomass and its DNA content typical curve. edible mushrooms journal, 2013,20 (3): 6-9.).Owing to its objective is in order to characterising biological amount, do not require quality and the following amplification of DNA, therefore its method therefor is identical with conventional mycelium extracting method, does not do any improvement.
Summary of the invention
The object of this invention is to provide a kind of method that can be used for edible fungus mycelium vegetative growth phase DNA extraction.The method effectively can remove the interference of culture medium to DNA extraction process, has that DNA extraction efficiency is high, purity is high, not containing advantages such as PCR Taq enzyme inhibitor.
Term illustrates:
Rpm: the revolution of per minute, (revolutions per minute revolutions per).
PCR Taq enzyme inhibitor: many ionic surface active agents almost can suppress the activity of Taq enzyme as deoxycholamine acid sodium (0.06%) at much lower concentrations completely, sarcosyl (0.02%), sodium lauryl sulphate (0.01%), this kind of material is called PCR Taq enzyme inhibitor.
Technical scheme is as follows:
For a method for edible fungus mycelium vegetative growth phase DNA extraction, comprise pretreatment stage and extraction stage, it is characterized in that, pretreatment stage comprises the following steps:
1) get portion and be covered with mycelial substratum, slightly grind, put in a clean container;
2) add damping fluid I, being covered with mycelial substratum, and being placed in constant-temperature table to flooding, keep temperature 35 ~ 40 DEG C, shaking speed 100 ~ 150rpm reacts 2 ~ 3h;
3) get step 2) solution, 3000 ~ 4000rpm low-speed centrifugal, 10 ~ 15min, solution be divided into precipitation and supernatant liquor; Collect supernatant liquor;
4) step 3) in remaining precipitation, add damping fluid II and wash, 3000 ~ 4000rpm low-speed centrifugal, 10 ~ 15min, gets supernatant liquor; Washing process repeats 2 ~ 5 times, and the supernatant liquor got after each washing merges;
5) by step 3) and step 4) supernatant liquor merging, at rotating speed 10000 ~ 12000rpm, 4 DEG C of centrifugal 10 ~ 15min; Abandon supernatant liquor, collecting precipitation.
Preferably, step 2) in the add-on of damping fluid I be covered with mycelial substratum be advisable to flood, the volume ratio of damping fluid I and substratum is (1.5:1) ~ (4:1).The amount adding damping fluid I too much can cause the waste of damping fluid I, can't affect extraction effect; The amount adding damping fluid I crosses that I haven't seen you for ages causes mycelial cell structure to dissolve not exclusively, and then in mycelium, DNA release not exclusively, affects concentration and the efficiency of DNA extraction.Step 2) object be will will be attached to substratum mycelial cell structure dissolve, DNA wherein can fully be discharged; Under this object, those skilled in the art according to prompting of the present invention, to any adjustment of the add-on of damping fluid I, all within the scope of the present invention.The volume ratio of damping fluid I and substratum is the arbitrary combination of 1.5:1,1.8:1,2:1,2.2:1,2.4:1,2.6:1,2.8:1,3:1,3.5:1,4:1 or aforementioned proportion.
Preferably, step 4) in the add-on of damping fluid II and the volume ratio of substratum be (1:3) ~ (1.5:1); As far as possible the object adding damping fluid II will the DNA be attached in the mycelium of substratum be rinsed to come out.Step 4) object be to rinse come out dissolving the DNA as far as possible, increase extraction efficiency, ensure the DNA concentration extracted.The add-on of damping fluid II is preferably the range intervals of the arbitrary combination of 1:3,1:2,1:1,1.5:1 or above-mentioned add-on.
Method for edible fungus mycelium vegetative growth phase DNA extraction of the present invention, the extraction stage can adopt prior art, also can adopt following optimum condition.
Preferably, the described method for edible fungus mycelium vegetative growth phase DNA extraction, is characterized in that, the extraction stage comprises the following steps:
1) abandon supernatant liquor, add stock solution I;
2) put into 30 ~ 45 DEG C of waters bath with thermostatic control, keep 0.5 ~ 2h;
3) add stock solution II, put in 30 ~ 45 DEG C of waters bath with thermostatic control, keep 0.5 ~ 2h;
4) stock solution III is added, mixing;
5) high salt CTAB solution is added, mixing;
6) 50 ~ 60 DEG C of water-bath 80 ~ 100min;
7) mixing solutions of phenol/chloroform/primary isoamyl alcohol is added; 8 word mixings, 5 ~ 20min;
8) 10000 ~ 15000rpm, 2 ~ 8 DEG C of centrifugal 5 ~ 20min;
9) get supernatant liquor, add chloroform: the mixed solution of primary isoamyl alcohol, 8 word mixings;
10) 10000 ~ 15000rpm, 2 ~ 8 DEG C of centrifugal 5 ~ 20min;
11) get supernatant liquor, add 700 ~ 900 μ L Virahols, 8 word mixings;
12) 10000 ~ 15000rpm, the centrifugal 5 ~ 20min of room temperature, abandons supernatant liquor;
13) ethanol purge 2 ~ 5 times are used;
14) 10000 ~ 15000rpm, the centrifugal 2 ~ 8min of room temperature;
15) abandon supernatant liquor, dry, add stock solution I;
16), in 2 ~ 6 DEG C of refrigerators, a night is dissolved;
17) preserve in-40 ~-10 DEG C of refrigerators.
Preferably, step 1) in, add 500 ~ 600 μ L stock solution I, more preferably 565 μ L stock solution I;
Preferably, step 2) in, 37 DEG C, water bath with thermostatic control 1h;
Preferably, step 3) in, add 40 ~ 60 μ L stock solution II, preferably add 50 μ L stock solution II, put into 37 DEG C of waters bath with thermostatic control.In order to obtain good extraction effect, proportionlity between each stock solution is extremely important, we are through great many of experiments, find that the volume ratio of stock solution I, stock solution II, stock solution III is (8 ~ 15): 1:(1.5 ~ 3) time, extraction effect is better; Preferred, the volume ratio of stock solution I, stock solution II, stock solution III is (10 ~ 13): 1:(1.7 ~ 2.5);
Preferably, step 4) in add 80 ~ 120 μ L stock solution III, preferably add 100 μ L stock solution III, mixing;
Preferably, step 5) in, add 60 ~ 100 μ L height salt CTAB solution, preferably add 80 μ L height salt CTAB solution, mixing;
Preferably, step 6) in, 55 DEG C of water-bath 90min;
Preferably, step 7) in, add 700 ~ 900 μ L phenol/chloroform/primary isoamyl alcohol mixed solutions; 8 word mixings, 10min; Phenol: chloroform: primary isoamyl alcohol volume ratio (20 ~ 30): (20 ~ 28): 1; Preferably, phenol: chloroform: primary isoamyl alcohol volume ratio 25: 24: 1.Preferably, 800 μ L phenol are added: chloroform: primary isoamyl alcohol mixed solution.
Preferably, step 8) in, 12000rpm, 4 DEG C of centrifugal 10min;
Preferably, step 9) in, add 700 ~ 900 μ L chloroforms: primary isoamyl alcohol mixed solution, 8 word mixings; Chloroform: primary isoamyl alcohol volume ratio 20 ~ 30: 1; Preferably, chloroform: primary isoamyl alcohol volume ratio 24: 1.Preferably, 800 μ L chloroforms are added: primary isoamyl alcohol mixed solution;
Preferably, step 10) in, 12000rpm, 4 DEG C of centrifugal 10min;
Preferably, step 11) in, add 700 ~ 900 μ L Virahols, 8 word mixings; Preferably, 800 μ L Virahols are added.
Preferably, step 12) in, 13000rpm, the centrifugal 10min of room temperature, abandons supernatant liquor;
Preferably, step 13) in, with 75% ethanol purge three times;
Preferably, step 14) in, 12000rpm, the centrifugal 5min of room temperature;
Preferably, step 15) in, add 30 μ L stock solution I;
Preferably, step 16) in, in 4 DEG C of refrigerators, dissolve a night;
Preferably, step 17) in, preserve in-20 DEG C of refrigerators.
Damping fluid I:20mg/mL N,O-Diacetylmuramidase 500 μ L+20mg/mL Proteinase K 200 μ L+0.02 ~ 0.1mmol/L pH 6.0 ~ 7.0PBS buffer of pretreatment stage becomes 1000mL solution;
Damping fluid II:1mmol EDTA+10%SDS 10mL+0.02 ~ 0.1mmol/LpH 6.0 ~ 7.0PBS buffer of pretreatment stage becomes 100mL solution; EDTA is ethylenediamine tetraacetic acid (EDTA); SDS is sodium laurylsulfonate;
The stock solution I:1mmol EDTA+10mmol Tris-Hcl extracting DNA is mixed with 100mL solution; EDTA is ethylenediamine tetraacetic acid (EDTA); Tris-Hcl is three (methylol) aminomethane buffer solution;
The stock solution II:20mg/mL Proteinase K 20 μ L+10%SDS 10mL extracting DNA is mixed with 100mL solution; SDS is sodium laurylsulfonate;
The stock solution III:5mol NaCl+0.1%CTAB 10mL extracting DNA is mixed with 100mL solution; CTAB is cetyl trimethylammonium bromide;
High salt CTAB solution formula: 50mM Tris-HCl PH 8.0,4M NaCl, 1.8%CTAB, (this solution is known solution to 25mMEDTA PH 8.0, compound method is see Sue Porebski, L.Grant Bailey, Bernard R.Baum.Modification of a CTAB DNA extraction protocol for plantscontaining high polysaccharide and polyphenol components [J] .Plant MolecularBiology Reporter.March 1997, Volume 15, Issue 1, pp 8-15.)
In the present invention, if do not have specified otherwise, solvent is water.The part that the present invention does not describe in detail, all can adopt prior art.
Beneficial effect of the present invention is:
1, the present invention effectively can remove the interference of culture medium to DNA extraction process, is beneficial to the purity of mycelium DNA extraction.
2, the present invention has that DNA extraction efficiency is high, purity is high, not containing PCR Taq enzyme inhibitor, may be used for carrying out accurate molecular biology experiment.
3, the extract recipe composition that the present invention relates to is the conventional reagent that laboratory generally adopts, good economy performance.
4, the extracting solution that the present invention relates to is nontoxic, have no irritating odor, operational safety.
Table 1 the inventive method and ordinary method extract the contrast of DNA
Accompanying drawing explanation
Fig. 1 is the mycelium DNA agarose electrophoresis figure of embodiment 1
Fig. 2 is the mycelium DNA agarose electrophoresis figure of embodiment 2
Fig. 3 is the mycelium DNA agarose electrophoresis figure of embodiment 3
Embodiment
Embodiment 1
National edible mushrooms engineering popularization center of Shandong Academy of Agricultural Science, Institute of Agricultural Resources and Environment taken from by edible fungus mycelium sample, and the main component of cultivation matrix is cotton seed hulls, and edible fungus species is Pleurotus citrinopileatus.
Get and be covered with mycelial substratum 20g, slightly grind, in 100mL triangular flask; Adding appropriate DNA damping fluid I, being covered with mycelial substratum to flooding; The volume ratio of damping fluid I and substratum is 3:1; Put into shaking table, 35 DEG C, 100rpm shakes 2h; Get solution, 3000rpm, centrifugal 10min; Take out supernatant liquor; Residue precipitation, add damping fluid II and wash, the volume ratio of damping fluid II and substratum is 1:1,3000rpm, centrifugal 10min; Get supernatant liquor; Washing process repeats 5 times; Repeatedly centrifugal supernatant liquor is merged, 12000rpm, 4 DEG C of centrifugal 10min; Abandon supernatant liquor, precipitation adds 565 μ L stock solution I; Put 37 DEG C of waters bath with thermostatic control and keep 1h; Add 50 μ L stock solution II, put 37 DEG C of waters bath with thermostatic control and keep 1h; Add 100 μ L stock solution III, mixing; Add 80 μ L height salt CTAB solution, mixing; 55 DEG C of water-bath 90min; Add 800 μ L phenol chloroform isoamyl alcohols; 8 word mixings, 10min; 12000rpm, 4 DEG C of centrifugal 10min; Get supernatant liquor, add 800 μ L chloroform isoamyl alcohols, 8 word mixings; 12000rpm, 4 DEG C of centrifugal 10min; Get supernatant liquor, add 800 μ L Virahols, 8 word mixings; 13000rpm, the centrifugal 10min of room temperature, abandons supernatant liquor; With 75% ethanol purge three times; 12000rpm, the centrifugal 5min of room temperature; Abandon supernatant liquor, dry, add 30 μ L stock solution I; In 4 DEG C of refrigerators, dissolve a night; Preserve in-20 DEG C of refrigerators.
Get 1 μ L DNA, dilute 50 times, carry out quality examination with ultraviolet spectrophotometer.Get 5 μ L DNA, detect DNA band with agarose gel electrophoresis.
It is 510 μ g/mL, OD that detection obtains DNA concentration
260/ OD
280be 1.89.Its agarose electrophoresis detected result shows, and DNA band is more clear bright, sees Fig. 1.Detected result shows that DNA extraction total amount is high, purity is high.Use known detection method.
Embodiment 2
Edible fungus mycelium sample is from this laboratory Chinese medicine slag edible fungus culturing test, and the main component of cultivation matrix is Chinese medicine slag (root of large-flowered skullcap and sweet Stevia), and edible fungus species is flat mushroom.
Get and be covered with mycelial substratum 50g, slightly grind, in 250mL triangular flask; Adding appropriate damping fluid I, being covered with mycelial substratum to flooding bacterium block; The volume ratio of damping fluid I and substratum is 2:1; Put in 40 DEG C of constant-temperature tables, 150rpm shakes 3h; Get solution, 4000rpm, centrifugal 15min; Get supernatant liquor; Residue precipitation, add damping fluid II and wash, the volume ratio of damping fluid II and substratum is 1:2,4000rpm, centrifugal 15min; Get supernatant liquor; Washing process repeats 3 times; Repeatedly centrifugal supernatant liquor is merged, 12000rpm, 4 DEG C of centrifugal 15min; Abandon supernatant liquor, add 600 μ L stock solution I; Put into 37 DEG C of constant temperature water bath 1.5h; Add 60 μ L stock solution II, put into 37 DEG C of constant temperature water bath 2h; Add 100 μ L stock solution III, mixing; Add 100 μ L height salt CTAB solution, mixing; 55 DEG C of water-bath 90min; Add 800 μ L phenol chloroform isoamyl alcohols; 8 word mixings, 10min; 14000rpm, 4 DEG C of centrifugal 10min; Get supernatant liquor, add 800 μ L chloroform isoamyl alcohols, 8 word mixings; 14000rpm, 4 DEG C of centrifugal 10min; Get supernatant liquor, add 800 μ L Virahols, 8 word mixings; 14000rpm, the centrifugal 10min of room temperature, abandons supernatant liquor; With 75% ethanol purge three times; 14000rpm, the centrifugal 5min of room temperature; Abandon supernatant liquor, dry, add 30 μ L stock solution I; In 4 DEG C of refrigerators, dissolve a night; Preserve in-20 DEG C of refrigerators.
Get 1 μ L DNA, dilute 50 times, carry out quality examination with ultraviolet spectrophotometer.Get 5 μ L DNA, detect DNA band with agarose gel electrophoresis.
It is 626 μ g/mL, OD that detection obtains DNA concentration
260/ OD
280be 1.85.Its agarose electrophoresis detected result shows, and DNA band is more clear bright, sees Fig. 2.Detected result shows that DNA extraction total amount is high, purity is high.Use known detection method.
Embodiment 3
Edible fungus mycelium sample is from this laboratory Chinese medicine slag edible fungus culturing test, and the main component of cultivation matrix is Chinese medicine slag (radix paeoniae rubrathe and Flos Carthami), and edible fungus species is Pleurotus eryngii.
Get and be covered with mycelial substratum 20g, slightly grind, in 100mL triangular flask; Add appropriate DNA damping fluid I, to flooding bacterium block; The volume ratio of damping fluid I and substratum is 1.8:1; Put in 37 DEG C of constant-temperature tables, 120rpm shakes 3h; Get solution, 3500rpm, centrifugal 12min; Get supernatant liquor; Residue precipitation, add damping fluid II and wash, the volume ratio of damping fluid II and substratum is 1:1,3500rpm, centrifugal 12min; Get supernatant liquor; Washing process repeats 4 times; Repeatedly centrifugal supernatant liquor is merged, 11000rpm, 4 DEG C of centrifugal 12min; Abandon supernatant liquor, add 560 μ L stock solution I; Put into water-bath, 37 DEG C, water bath with thermostatic control 0.5h; Add 50 μ L stock solution II, put into water-bath 37 DEG C, water bath with thermostatic control 1h; Add 100 μ L stock solution III, mixing; Add 80 μ L height salt CTAB solution, mixing; 55 DEG C of water-bath 90min; Add 800 μ L phenol chloroform isoamyl alcohols; 8 word mixings, 10min; 12000rpm, 4 DEG C of centrifugal 10min; Get supernatant liquor, add 800 μ L chloroform isoamyl alcohols, 8 word mixings; 12000rpm, 4 DEG C of centrifugal 10min; Get supernatant liquor, add 800 μ L Virahols, 8 word mixings; 13000rpm, the centrifugal 10min of room temperature, abandons supernatant liquor; With 75% ethanol purge three times; 12000rpm, the centrifugal 5min of room temperature; Abandon supernatant liquor, dry, add 30 μ L stock solution I; In 4 DEG C of refrigerators, dissolve a night; Preserve in-20 DEG C of refrigerators.
Get 1 μ L DNA, dilute 50 times, carry out quality examination with ultraviolet spectrophotometer.Get 5 μ L DNA, detect DNA band with agarose gel electrophoresis.
It is 609 μ g/mL, OD that detection obtains DNA concentration
260/ OD
280be 1.86.Its agarose electrophoresis detected result shows, and DNA band is more clear bright, sees Fig. 3.Detected result shows that DNA extraction total amount is high, purity is high.Use known detection method.
Adopt the method for embodiment 1, use different cultivation matrixes and edible fungus species to test, the results are shown in Table 2.We find, cultivation matrix and edible fungus species are on extraction not impact, and the present invention is applicable to various cultivation matrix and edible fungus species.
The DNA extraction effect comparison of table 2 different cultivation matrix culturing edible fungus bacterial classification
Claims (12)
1., for a method for edible fungus mycelium vegetative growth phase DNA extraction, comprise pretreatment stage and extraction stage, it is characterized in that, pretreatment stage comprises the following steps:
1) get portion and be covered with mycelial substratum, slightly grind, put in a clean container;
2) add damping fluid I, being covered with mycelial substratum, and being positioned on shaking table to flooding, keep temperature 35 ~ 40 DEG C, shaking speed 100 ~ 150rpm shakes 2 ~ 3h;
3) get step 2) solution, 3000 ~ 4000rpm low-speed centrifugal, 10 ~ 15min, solution be divided into precipitation and supernatant liquor; Collect supernatant liquor;
4) step 3) in remaining precipitation, add damping fluid II and wash, 3000 ~ 4000rpm low-speed centrifugal, 10 ~ 15min, gets supernatant; Washing process repeats 2 ~ 5 times, and the supernatant liquor got after each washing merges;
5) by step 3) and step 4) centrifugal supernatant liquor is brought together, at rotating speed 10000 ~ 12000rpm, 4 DEG C of centrifugal 10-15min; Abandon supernatant liquor, collecting precipitation.
2., as claimed in claim 1 for the method for edible fungus mycelium vegetative growth phase DNA extraction, it is characterized in that, step 2) in the volume ratio of damping fluid I and substratum be (1.5:1) ~ (4:1).
3., as claimed in claim 1 for the method for edible fungus mycelium vegetative growth phase DNA extraction, it is characterized in that, step 4) in the volume ratio of damping fluid II and substratum be (1:3) ~ (1.5:1).
4. the method for edible fungus mycelium vegetative growth phase DNA extraction as described in any one of claims 1 to 3, is characterized in that, the extraction stage comprises the following steps:
1) abandon supernatant liquor, add stock solution I;
2) put into 30 ~ 45 DEG C of waters bath with thermostatic control, keep 0.5 ~ 2h;
3) add stock solution II, put in 30 ~ 45 DEG C of waters bath with thermostatic control, keep 0.5 ~ 2h;
4) stock solution III is added, mixing;
5) high salt CTAB solution is added, mixing;
6) 50 ~ 60 DEG C of water-bath 80 ~ 100min;
7) mixing solutions of phenol/chloroform/primary isoamyl alcohol is added; 8 word mixings, 5 ~ 20min;
8) 10000 ~ 15000rpm, 2 ~ 8 DEG C of centrifugal 5 ~ 20min;
9) get supernatant liquor, add the mixed solution of chloroform/primary isoamyl alcohol, 8 word mixings;
10) 10000 ~ 15000rpm, 2 ~ 8 DEG C of centrifugal 5 ~ 20min;
11) get supernatant liquor, add 700 ~ 900 μ L Virahols, 8 word mixings;
12) 10000 ~ 15000rpm, the centrifugal 5 ~ 20min of room temperature, abandons supernatant liquor;
13) ethanol purge 2 ~ 5 times are used;
14) 10000 ~ 15000rpm, the centrifugal 2 ~ 8min of room temperature;
15) abandon supernatant liquor, dry, add stock solution I;
16), in 2 ~ 6 DEG C of refrigerators, a night is dissolved;
17) preserve in-40 ~-10 DEG C of refrigerators.
5. as claimed in claim 4 for the method for edible fungus mycelium vegetative growth phase DNA extraction, it is characterized in that, the volume ratio of stock solution I, stock solution II, stock solution III is (8 ~ 15): 1:(1.5 ~ 3); Preferred, the volume ratio of stock solution I, stock solution II, stock solution III is (10 ~ 13): 1:(1.7 ~ 2.5).
6. step is as claimed in claim 4 for the method for edible fungus mycelium vegetative growth phase DNA extraction, it is characterized in that, step 5) in, add 60 ~ 100 μ L height salt CTAB solution, mixing; Step 6) in, 55 DEG C of water-bath 90min.
7., as claimed in claim 4 for the method for edible fungus mycelium vegetative growth phase DNA extraction, it is characterized in that, step 7) in, add 700 ~ 900 μ L phenol/chloroform/primary isoamyl alcohol mixed solutions; 8 word mixings, 10min; Phenol: chloroform: primary isoamyl alcohol volume ratio (20 ~ 30): (20 ~ 28): 1; Preferably, phenol: chloroform: primary isoamyl alcohol volume ratio 25: 24: 1.Preferably, 800 μ L phenol are added: chloroform: primary isoamyl alcohol mixed solution.
8., as claimed in claim 4 for the method for edible fungus mycelium vegetative growth phase DNA extraction, it is characterized in that, step 9) in, add 700 ~ 900 μ L chloroform/primary isoamyl alcohol mixed solutions, 8 word mixings; Chloroform: primary isoamyl alcohol volume ratio 20 ~ 30: 1; Preferably, chloroform: primary isoamyl alcohol volume ratio 24: 1.
9., as claimed in claim 4 for the method for edible fungus mycelium vegetative growth phase DNA extraction, it is characterized in that, step 11) in, add 700 ~ 900 μ L Virahols, 8 word mixings.
10., as claimed in claim 4 for the method for edible fungus mycelium vegetative growth phase DNA extraction, it is characterized in that, step 13) in, with 75% ethanol purge three times.
11., as claimed in claim 4 for the method for edible fungus mycelium vegetative growth phase DNA extraction, is characterized in that, step 14) in, 12000rpm, the centrifugal 5min of room temperature.
12., as claimed in claim 4 for the method for edible fungus mycelium vegetative growth phase DNA extraction, is characterized in that, step 15) in, add 30 μ L stock solution I; Step 16) in, in 4 DEG C of refrigerators, dissolve a night.
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