CN110564722A - Method for extracting paphiopedilum genome DNA by using improved CTAB method - Google Patents
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 238000000605 extraction Methods 0.000 claims description 14
- 239000012043 crude product Substances 0.000 claims description 13
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 239000011536 extraction buffer Substances 0.000 claims description 12
- 239000003153 chemical reaction reagent Substances 0.000 claims description 11
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 10
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- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 claims description 6
- 239000003960 organic solvent Substances 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 238000001291 vacuum drying Methods 0.000 claims description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 5
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- 238000007400 DNA extraction Methods 0.000 abstract description 9
- 238000012216 screening Methods 0.000 abstract description 3
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Abstract
a method for extracting paphiopedilum genome DNA by using an improved CTAB method comprises the following steps: (1) collecting and screening paphiopedilum leaves, (2) ultra-low temperature grinding, (3) ultrasonic-assisted incubation, (4) resonance sound mixing and opacification, (5) precipitation preparation of DNA, and (6) refining and storing of DNA. The method for extracting paphiopedilum genome DNA obtains an ideal paphiopedilum genome DNA extraction method, and lays a foundation for the subsequent molecular genetics experimental study based on genome DNA.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and relates to a method for extracting paphiopedilum genome DNA by using an improved CTAB method.
Background
Paphiopedilum (also called cymbidium corruguatum Franch), orchidaceae perennial herbs, most of which are terrestrial species, and many hybrid varieties are one of the most popular cymbidium cultivated, and are mainly distributed in tropical and subtropical forests of asia, where the environment is warm, humid and semi-cloudy. The stem is very short; leaf strips or oblong needles, green or with reddish-brown patches. The flower is very peculiar, and the lip valve is in a pocket shape; the back calyx is developed and has various bright patterns; two lateral sepals are combined together; the petals are thick, the flower life is long, some flowers can be opened for more than 6 weeks, and the flowers can bloom in all seasons.
Paphiopedilum (Paphiopedilum) is an Orchidaceae plant (Orchidaceae), belongs to about 65 species, is distributed from tropical regions of asia to pacific islands, and has 18 species in China, and is produced in southwest and south China. The plant is famous for peculiar flower shape (such as lip is specially formed into a sacculus), and gorgeous flower color, has extremely high ornamental value, and has great development prospect in domestic and international markets.
In recent years, scientists have conducted a great deal of research on paphiopedilum from the aspects of biogenetic pollination, ecological conservation, plant geography, sterile seed germination and tissue culture technology, flower bud development, photo-biological characteristics, mycorrhiza and the like, while only a few reports are found on the research on paphiopedilum molecular biology and molecular genetics, and the research is mainly started from the aspects of screening paphiopedilum DNA barcode sequences and constructing and optimizing a molecular marker system, and no report is found on the comparative research on a paphiopedilum genome DNA extraction method at present. With the unprecedented development of modern biotechnology, molecular genetic improvement is certainly applied to germplasm resource protection and utilization of paphiopedilum, and high-quality genomic DNA extraction is a precondition for researching paphiopedilum from the molecular genetic level. Paphiopedilum leaves are tanned, the extraction of genome DNA is difficult, and the quality of the extracted DNA is not high.
The CTAB method is known as the cetyltrimethylammonium bromide method. CTAB is a cationic detergent which has the property of precipitating nucleic acids and acidic polysaccharides from solutions of low ionic strength; in a solution of high ionic strength (>0.7mol/L NaCl), CTAB forms complexes with proteins and polysaccharides, but does not precipitate nucleic acids; then extracting with organic solvent to remove protein, polysaccharide, phenols and other impurities, and precipitating with ethanol to separate out nucleic acid. In order to screen for a high-quality extraction method for obtaining paphiopedilum genomic DNA, it is necessary to design a method for extracting paphiopedilum genomic DNA using an improved CTAB method.
disclosure of Invention
The invention aims to provide a method for extracting paphiopedilum genome DNA by using an improved CTAB method.
in order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a method for extracting paphiopedilum genome DNA by using an improved CTAB method comprises the following steps:
step 1: placing the paphiopedilum leaves into liquid nitrogen for quick freezing, then placing the quick-frozen paphiopedilum leaves at-80 ℃ for storage, and then crushing the paphiopedilum leaves by using a low-temperature ultrafine crusher at the temperature of-120 to-60 ℃ until the fineness is 300 to 500 meshes to obtain the frozen paphiopedilum leaf ultrafine powder;
Step 2: mixing the frozen paphiopedilum leaf superfine powder, 2% CTAB extraction buffer solution and beta-mercaptoethanol according to the mass ratio of 10:80:5, and incubating in water bath at 60-70 ℃ for 20 min; then, keeping the temperature of the water bath unchanged, and adopting the frequency of 25-35KHz and the power density of 0.4-0.5W/cm2Performing ultrasonic treatment for 10 min; the preparation method of the 2% CTAB extraction buffer solution comprises the following steps: mixing 20.00g of hexadecyl trimethyl ammonium bromide, 81.82g of NaCl, 100ml of Tris-HCl buffer solution with the concentration of 1M and the pH value of 8.0 and 40ml of EDTA solution with the concentration of 0.5M and the pH value of 8.0, fixing the volume to 1000ml, and uniformly stirring to obtain the product;
And step 3: cooling the incubated solution obtained in the step 1 to room temperature, mixing the incubated solution and an extraction reagent according to a mass ratio of 95:80, putting the mixture into a resonance acoustic mixer, and processing the mixture for 8 to 12min under the conditions of frequency of 80 to 120Hz and amplitude of 1.5 to 2.0mm to obtain a mixed emulsion; the extraction reagent is prepared by mixing phenol, chloroform and isoamylol according to the mass ratio of 25:24: 1;
and 4, step 4: centrifuging the mixed emulsion at 3.5-4.5 deg.C at 10000r/min for 10min, and collecting supernatant; mixing the supernatant with isopropanol 2 times of the volume of the supernatant, and standing the mixture for more than 30min at the temperature of between 20 ℃ below zero and 18 ℃ below zero; centrifuging at 12000r/min for 10min at 3.5-4.5 deg.C, removing the upper organic solvent, and collecting solid substance as DNA crude product;
And 5: mixing the DNA crude product with 75% ethanol 5-10 times the weight of the DNA crude product, stirring uniformly, standing for 5-10min, centrifuging at a rotation speed of 8000r/min for 5min at a temperature of 3.5-4.5 ℃, discarding supernatant, and collecting fixed substance as refined DNA; vacuum drying the refined DNA until the water content is lower than 5%, adding TE solution 1.5-2.0 times of the DNA, and dissolving to obtain DNA solution.
The preferable technical scheme is as follows: the temperature condition of the step 1 is-110 to-90 ℃.
the preferable technical scheme is as follows: the temperature of the incubation in step 2 was 65 ℃.
The preferable technical scheme is as follows: the vacuum drying conditions were: the vacuum gauge pressure is-0.09 MPa to-0.10 MPa, and the temperature is 36 ℃ to 38 ℃.
Due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:
1. The paphiopedilum leaf raw material is subjected to color sorting treatment, so that the defective leaves are removed, and the purity and efficiency of paphiopedilum genome DNA extraction are improved.
2. The ultrasonic-assisted CTAB extraction buffer solution is used for incubating the frozen paphiopedilum leaf superfine powder, so that the incubation effect is improved.
3. The invention improves the formula of the traditional CTAB extraction buffer solution (phenol, chloroform and isoamylol are mixed according to the mass ratio of 25:24: 1), and simultaneously adopts the resonance sound mixing mode to mix the incubated solution and the extraction reagent to prepare uniform mixed emulsion.
Drawings
FIG. 1 shows the electrophoresis of genomic DNA extraction of paphiopedilum castaneum (M: DL 2000 molecular weight standard; 1-3:3 groups of DNA replicates extracted by the method of the present invention).
FIG. 2 shows the electrophoresis of genomic DNA extraction of paphiopedilum purpureum (M: DL 2000 molecular weight standard; 1-3:3 groups of DNA replicates extracted by the method of the present invention).
FIG. 3 is an electrophoretogram of genomic DNA extraction from paphiopedilum longipetiolatum (M: DL 2000 molecular weight standard; 1-3:3 groups of DNA replicates extracted by the method of the present invention).
FIG. 4 shows the electrophoresis of genomic DNA extraction of paphiopedilum hirsutum (DL 2000 molecular weight standard; 1-3:3 groups of DNA replicates extracted by the patented method).
FIG. 5 shows the electrophoresis of genomic DNA extraction of paphiopedilum hirsutissimum (DL 2000 molecular weight standard; 1-3:3 groups of DNA replicates extracted by the patent method).
Detailed Description
The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.
Please refer to fig. 1-5. It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are only used for matching with the disclosure of the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions under which the present invention can be implemented, so that the present invention has no technical significance, and any structural modification, ratio relationship change, or size adjustment should still fall within the scope of the present invention without affecting the efficacy and the achievable purpose of the present invention. In addition, the terms "upper", "lower", "left", "right", "middle" and "one" used in the present specification are for clarity of description, and are not intended to limit the scope of the present invention, and the relative relationship between the terms and the terms is not to be construed as a scope of the present invention.
Example 1: method for extracting paphiopedilum genome DNA by using improved CTAB method
A method for extracting paphiopedilum genome DNA by using an improved CTAB method comprises the following technical steps.
(1) Collecting and screening paphiopedilum leaves
Collecting mature paphiopedilum leaves, cleaning with clear water to remove dirt on the leaves, and draining off water on the leaves for later use.
And (3) eliminating leaves with large color deviation, such as insect-damaged leaves, mildewed leaves, diseased leaves and the like by using CCD color sorting equipment. The CCD color sorting equipment has the minimum image identification area less than 0.01m2and the color selection precision is higher than 99.5%.
(2) Pulverizing at ultralow temperature
And placing the color-selected leaves into sufficient liquid nitrogen for quick freezing, and then placing the quick-frozen leaves into an ultra-low temperature refrigerator at minus 80 ℃ for storage for later use.
Taking out the leaves in an ultra-low temperature refrigerator, and crushing the leaves to 400 meshes of fineness at the temperature of 90 ℃ below zero by using low-temperature ultrafine crushing equipment to obtain the frozen paphiopedilum leaf ultrafine powder.
Preferably, the temperature of the low-temperature micronization is-100 ℃.
(3) Ultrasound assisted incubation
mixing the frozen paphiopedilum leaf superfine powder, 2% CTAB extraction buffer solution and beta-mercaptoethanol according to the mass ratio of 10:80:5, and incubating in a water bath at 65 ℃ for 20 min; then, the temperature of the water bath is kept constant, and the frequency is 30KHz and the power density is 0.45W/cm2the ultrasonic treatment of (3) is carried out for 10 min.
The preparation method of the 2% CTAB extraction buffer solution comprises the steps of mixing 20.00g of CTAB (cetyl trimethyl ammonium bromide), 81.82g of NaCl, 100ml of Tris-HCl (pH8.0) solution with the molar concentration of 1M and 40ml of EDTA (pH8.0) solution with the molar concentration of 0.5M, fixing the volume to 1000ml, and uniformly stirring to obtain the CTAB extraction buffer solution.
(4) Resonant acoustic hybrid opacification
And cooling the incubated solution to room temperature, mixing the incubated solution and the extraction reagent according to the mass ratio of 95:80, putting into resonance sound mixing, and processing for 10min under the conditions of frequency of 100Hz and amplitude of 1.8mm to obtain a mixed emulsion.
The formula of the extraction reagent is that phenol, chloroform and isoamylol are mixed according to the mass ratio of 25:24: 1.
(5) precipitation preparation of DNA
Centrifuging the mixed emulsion at 4.0 deg.C and 10000r/min for 10min, and collecting supernatant; mixing the supernatant with 2 times volume of isopropanol, and standing at-19 deg.C for more than 30 min; and centrifuging the mixed solution standing at the low temperature for 10min at the temperature of 4.0 ℃ and the rotating speed of 12000r/min, removing the upper organic solvent, and collecting solid substances, namely the crude DNA.
(6) purification and storage of DNA
Mixing the DNA crude product with 75% ethanol with the mass fraction of 8 times of the mass, stirring uniformly, standing for 7min, centrifuging for 5min at the temperature of 4.0 ℃ and the rotating speed of 8000r/min, discarding the supernatant, and collecting the fixed substance, namely the refined DNA.
Placing the refined DNA in an environment with the vacuum gauge pressure of-0.095 MPa and the temperature of 37 ℃, and drying in vacuum until the water content is lower than 5%; then, a 1.6-fold mass of TE (containing 2000U/mL RNaseA) solution was added thereto to dissolve the DNA solution. The DNA solution was stored at-19 ℃ for further use.
Quality detection of paphiopedilum genomic DNA extracted by the method
Five paphiopedilum plants including paphiopedilum castaneum (p.malipoense), paphiopedilum purpureum (p.purpurpurputum), paphiopedilum longipetaloides (p.dianthus), paphiopedilum harderianum (p.micranthumum) and paphiopedilum hirsutum (p.hirsutissimum) are taken as test materials, the method of the embodiment is used for extracting the genomic DNA of the five paphiopedilum plants respectively, and the quality detection is carried out on the 5 genomic DNAs.
and (3) respectively detecting the integrity and the purity of the genome DNA of paphiopedilum armeniacum by using an agarose gel electrophoresis method and a spectrophotometer method. After electrophoresis of 2.5. mu.L of the DNA sample in 1% agarose gel, the integrity of the sample was observed by imaging with a gel imaging system (Bio-rad, Universal Hood II, USA), and 1.0. mu.L of the DNA sample was subjected to an ultraspectrophotometer (Thermo NanoDrop 2000, USA) to determine A of the DNA sample260/A280Absorbance ratio and concentration.
(1) Integrity results analysis of genomic DNA of five paphiopedilum species:
The electrophoresis picture shows that the genomic DNA of the five paphiopedilum is subjected to 1% agarose gel electrophoresis and gel imaging detection, and the paphiopedilum genomic DNA bands extracted by the method of the embodiment are clear and visible, are single and bright and have no dispersion phenomenon, which indicates that the integrity of the sample DNA is better.
(2) Purity results of five paphiopedilum genomic DNAs were analyzed:
The purity of the DNA can usually be determined by its A260/A280the ratio is judged, generally regarded as A260/A280When the ratio is between 1.6 and 1.9, the DNA purity is higher, and the requirement of subsequent molecular biology experiments can be met. The method of the invention extracts the genome DNA of five paphiopedilum and the purity result analysis is as follows: extracted paphiopedilum majus genomic DNA, A260/A280The ratio is 1.685; extracted paphiopedilum purpureum genomic DNA, A260/A280The ratio is 1.790; extracted paphiopedilum longipetiolum genome DNA, A260/A280the ratio is 1.655; extracted paphiopedilum harderi genomic DNA, A260/A280The ratio is 1.870; extracted paphiopedilum hirsutissimum genomic DNA, A260/A280The ratio was 1.825.
in conclusion, the method extracts paphiopedilum genome DNA, A260/A280The ratio is between 1.655 and 1.870, and the electrophoresis gel sample hole of the sample DNA has no bright spot phenomenon (lanes 1 to 3 in figures 1 to 5), which shows that the integrity and purity of the extracted DNA are better and no impurity is polluted.
Example 2: method for extracting paphiopedilum genome DNA by using improved CTAB method
A method for extracting paphiopedilum genome DNA by using an improved CTAB method comprises the following steps:
Step 1: placing the paphiopedilum leaves into liquid nitrogen for quick freezing, then placing the quick-frozen paphiopedilum leaves at-80 ℃ for storage, and then crushing the paphiopedilum leaves by using a low-temperature ultrafine crusher at-120 ℃ until the fineness is 300 meshes to obtain the frozen paphiopedilum leaf ultrafine powder;
Step 2: mixing the frozen paphiopedilum leaf superfine powder, 2% CTAB extraction buffer solution and beta-mercaptoethanol according to the mass ratio of 10:80:5, and incubating in water bath at 60 ℃ for 20 min; then, the temperature of the water bath is kept constant, and the frequency of 25KHz and the power density of 0.4W/cm are adopted2performing ultrasonic treatment for 10 min; the preparation method of the 2% CTAB extraction buffer solution comprises the following steps: mixing 20.00g of hexadecyl trimethyl ammonium bromide, 81.82g of NaCl, 100ml of Tris-HCl buffer solution with the concentration of 1M and the pH value of 8.0 and 40ml of EDTA solution with the concentration of 0.5M and the pH value of 8.0, fixing the volume to 1000ml, and uniformly stirring to obtain the product;
And step 3: cooling the incubated solution obtained in the step 1 to room temperature, mixing the incubated solution and an extraction reagent according to a mass ratio of 95:80, putting the mixture into a resonance acoustic mixer, and processing for 8min under the conditions of frequency of 80Hz and amplitude of 1.5mm to obtain a mixed emulsion; the extraction reagent is prepared by mixing phenol, chloroform and isoamylol according to the mass ratio of 25:24: 1;
and 4, step 4: centrifuging the mixed emulsion at 3.5 deg.C at 10000r/min for 10min, and collecting supernatant; mixing the supernatant with 2 times of isopropanol, and standing at-20 deg.C for more than 30 min; centrifuging at 12000r/min for 10min at 3.5 deg.C, removing the upper organic solvent, and collecting the solid substance as DNA crude product;
And 5: mixing the DNA crude product with 75% ethanol in 5 times of the mass of the DNA crude product, stirring uniformly, standing for 5min, centrifuging at a rotation speed of 8000r/min for 5min at the temperature of 3.5 ℃, removing supernatant, and collecting fixed substances, namely refined DNA; the purified DNA was vacuum-dried to a water content of 5%, and then a TE solution 1.5 times the mass of the DNA was added thereto and dissolved to obtain a DNA solution.
The preferred embodiment is: the vacuum drying conditions were: the gauge pressure is-0.09 MPa and the temperature is 36 ℃.
Example 3: method for extracting paphiopedilum genome DNA by using improved CTAB method
A method for extracting paphiopedilum genome DNA by using an improved CTAB method comprises the following steps:
Step 1: placing the paphiopedilum leaves into liquid nitrogen for quick freezing, then placing the quick-frozen paphiopedilum leaves at-80 ℃ for storage, and then crushing the paphiopedilum leaves by using a low-temperature ultrafine crusher at-60 ℃ until the fineness is 500 meshes to obtain the frozen paphiopedilum leaf ultrafine powder;
Step 2: mixing the frozen paphiopedilum leaf superfine powder, 2% CTAB extraction buffer solution and beta-mercaptoethanol according to the mass ratio of 10:80:5, and incubating in 70 ℃ water bath for 20 min; then, the temperature of the water bath is kept constant, and the frequency of 35KHz and the power density of 0.5W/cm are adopted2Performing ultrasonic treatment for 10 min; the preparation method of the 2% CTAB extraction buffer solution comprises the following steps: mixing 20.00g of hexadecyl trimethyl ammonium bromide, 81.82g of NaCl, 100ml of Tris-HCl buffer solution with the concentration of 1M and the pH value of 8.0 and 40ml of EDTA solution with the concentration of 0.5M and the pH value of 8.0, fixing the volume to 1000ml, and uniformly stirring to obtain the product;
and step 3: cooling the incubated solution obtained in the step 1 to room temperature, mixing the incubated solution and an extraction reagent according to a mass ratio of 95:80, putting the mixture into a resonance acoustic mixer, and processing for 12min under the conditions of 120Hz frequency and 2.0mm amplitude to obtain a mixed emulsion; the extraction reagent is prepared by mixing phenol, chloroform and isoamylol according to the mass ratio of 25:24: 1;
And 4, step 4: centrifuging the mixed emulsion at 4.5 deg.C at 10000r/min for 10min, and collecting supernatant; mixing the supernatant with 2 times of isopropanol, and standing at-18 deg.C for more than 30 min; centrifuging at 4.5 deg.C at 12000r/min for 10min, removing upper organic solvent, and collecting solid substance as DNA crude product;
And 5: mixing the DNA crude product with 75% ethanol 5-10 times the weight of the DNA crude product, stirring, standing for 10min, centrifuging at 8000r/min for 5min at 4.5 deg.C, discarding supernatant, and collecting fixed substance as refined DNA; the purified DNA was vacuum-dried to a water content of 4.5%, and then 2.0 times by mass of TE solution was added thereto and dissolved to obtain a DNA solution.
The preferred embodiment is: the vacuum drying conditions were: the vacuum gauge pressure is-0.10 MPa, and the temperature is 38 ℃.
The foregoing is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting thereof in any way, and any modifications or variations thereof that fall within the spirit of the invention are intended to be included within the scope thereof.
Claims (4)
1. A method for extracting paphiopedilum genome DNA by using an improved CTAB method is characterized by comprising the following steps: comprises the following steps:
step 1: placing the paphiopedilum leaves into liquid nitrogen for quick freezing, then placing the quick-frozen paphiopedilum leaves at-80 ℃ for storage, and then crushing the paphiopedilum leaves by using a low-temperature ultrafine crusher at the temperature of-120 to-60 ℃ until the fineness is 300 to 500 meshes to obtain the frozen paphiopedilum leaf ultrafine powder;
Step 2: mixing the frozen paphiopedilum leaf superfine powder, 2% CTAB extraction buffer solution and beta-mercaptoethanol according to the mass ratio of 10:80:5, and incubating in water bath at 60-70 ℃ for 20 min; then, keeping the temperature of the water bath unchanged, and adopting the frequency of 25-35KHz and the power density of 0.4-0.5W/cm2Performing ultrasonic treatment for 10 min; the 2% CTAB extraction bufferThe preparation method comprises the following steps: mixing 20.00g of hexadecyl trimethyl ammonium bromide, 81.82g of NaCl, 100ml of Tris-HCl buffer solution with the concentration of 1M and the pH value of 8.0 and 40ml of EDTA solution with the concentration of 0.5M and the pH value of 8.0, fixing the volume to 1000ml, and uniformly stirring to obtain the product;
And step 3: cooling the incubated solution obtained in the step 1 to room temperature, mixing the incubated solution and an extraction reagent according to a mass ratio of 95:80, putting the mixture into a resonance acoustic mixer, and processing the mixture for 8 to 12min under the conditions of frequency of 80 to 120Hz and amplitude of 1.5 to 2.0mm to obtain a mixed emulsion; the extraction reagent is prepared by mixing phenol, chloroform and isoamylol according to the mass ratio of 25:24: 1;
And 4, step 4: centrifuging the mixed emulsion at 3.5-4.5 deg.C at 10000r/min for 10min, and collecting supernatant; mixing the supernatant with isopropanol 2 times of the volume of the supernatant, and standing the mixture for more than 30min at the temperature of between 20 ℃ below zero and 18 ℃ below zero; centrifuging at 12000r/min for 10min at 3.5-4.5 deg.C, removing the upper organic solvent, and collecting solid substance as DNA crude product;
And 5: mixing the DNA crude product with 75% ethanol 5-10 times the weight of the DNA crude product, stirring uniformly, standing for 5-10min, centrifuging at a rotation speed of 8000r/min for 5min at a temperature of 3.5-4.5 ℃, discarding supernatant, and collecting fixed substance as refined DNA; vacuum drying the refined DNA until the water content is lower than 5%, adding TE solution 1.5-2.0 times of the DNA, and dissolving to obtain DNA solution.
2. the method for extracting genomic DNA of paphiopedilum according to claim 1, wherein the method comprises the steps of: the temperature condition of the step 1 is-110 to-90 ℃.
3. The method for extracting genomic DNA of paphiopedilum according to claim 1, wherein the method comprises the steps of: the temperature of the incubation in step 2 was 65 ℃.
4. The method for extracting genomic DNA of paphiopedilum according to claim 1, wherein the method comprises the steps of: the vacuum drying conditions were: the vacuum gauge pressure is-0.09 MPa to-0.10 MPa, and the temperature is 36 ℃ to 38 ℃.
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