阅读说明：本技术 一种提取枣树叶片中的dna的方法 (Method for extracting DNA from jujube tree leaves ) 是由 李春丽 于 2018-08-16 设计创作，主要内容包括：本发明提供了一种提取枣树叶片中的DNA方法。本发明提供的提取枣树叶片中的DNA方法包括如下步骤：选取新鲜枣树嫩叶于冰箱内下暗处理、研磨、加入第一缓冲液、恒温水浴中、离心、加入酚、氯仿和异戊醇混合液、离心、加入氯仿、异戊醇混合液、离心、加入第二缓冲液、加入冷乙醇漂洗、加入第三缓冲液过夜溶解等步骤。使用本发明提供的方法提取的DNA纯度较高,浓度较大,本发明还提高了DNA提取的效率。(The invention provides a method for extracting DNA from jujube leaves. The method for extracting DNA from jujube tree leaves provided by the invention comprises the following steps: selecting fresh jujube tender leaves, performing dark treatment in a refrigerator, grinding, adding a first buffer solution, performing constant-temperature water bath, centrifuging, adding a mixed solution of phenol, chloroform and isoamyl alcohol, centrifuging, adding a mixed solution of chloroform and isoamyl alcohol, adding a second buffer solution, adding cold ethanol, rinsing, adding a third buffer solution, dissolving overnight and the like. The DNA extracted by the method provided by the invention has higher purity and higher concentration, and the invention also improves the efficiency of DNA extraction.)

1. A method for extracting DNA from jujube leaves comprises the following steps:

a) selecting fresh jujube tender leaves, performing dark treatment at 0-4 ℃ for 48h in a refrigerator, quickly weighing 0.1g of the fresh jujube tender leaves, grinding the fresh jujube tender leaves into fine powder in liquid nitrogen, quickly transferring the fine powder into a 1.5mL EP tube, adding 800 muL of a first buffer solution preheated at 65 ℃ and uniformly mixing the buffer solution, wherein the first buffer solution comprises 2.5mM PEX, 4% (g/mL) soluble PVP, 100mM Tris with the pH value of 7.5, 10mM EDTA with the pH value of 7.5, 2% (muL/mL) β -mercaptoethanol and 700mM NaCl;

b) incubating in a constant temperature water bath at 65 ℃ for 20min, and slightly inverting and uniformly mixing once every 5 min; taking out, centrifuging at 13000rpm for 5 min;

c) taking the supernatant, adding a mixed solution of phenol, chloroform and isoamylol with the same volume ratio of 25: 24: 1, turning upside down and mixing uniformly, and centrifuging at 13000rpm for 5 min;

d) taking the supernatant, adding a chloroform-isoamyl alcohol mixed solution with the same volume, wherein the volume ratio of chloroform to isoamyl alcohol in the chloroform-isoamyl alcohol mixed solution is 24: 1, reversing and uniformly mixing, and centrifuging at 13000rpm for 5 min;

e) transferring the supernatant to a 2mL EP tube added with 700. mu.L of precooled isopropanol, adding 150. mu.L of a second buffer solution, and slightly reversing the mixture up and down to mix the mixture, wherein the second buffer solution is 3M NaAC with the pH value of 5.2; centrifuging at 13000rpm at 4 deg.C for 10min, pouring off supernatant, and collecting precipitate;

f) adding 1mL of 70% cold ethanol into the precipitate, rinsing, repeating twice, and pouring off the ethanol to dry the precipitate at room temperature or in vacuum;

g) add 50. mu.L of a third buffer, RTE, containing 20ug/mL RNase at pH 8.0, and dissolve overnight at 4 ℃.

Technical Field

The invention relates to a DNA extraction method, in particular to a method for extracting DNA in jujube tree leaves.

Background

Xanthate, commonly known as xanthate because of its yellow color, is the most common collector for metal sulfide ore flotation. Xanthate of almost all varieties can be used as a collector for froth flotation. In recent years, with the rapid rise of nanotechnology, research on xanthate as a precursor for preparing sulfide nanoparticles has once more attracted attention to the traditional flotation collector. Pex (potassium ethyl xanthate) belongs to xanthate reagents, is a light yellow powder solid at normal temperature, is dark in color due to the existence of impurities, has pungent odor and certain toxicity, and therefore, care should be taken to protect the operator during operation.

It is known that the procedure is complicated to obtain high quality DNA from plant tissues or cells, especially when plants are used as raw materials. For plants with more polysaccharides, rigid cell walls, pigments or more chemical components with complex secondary metabolism, special consideration must be given to these components during the separation process, and it is more difficult to extract good DNA.

Protein, phenol and tannin in various tissues of jujube treeHigh contents of pigment and polysaccharide, especially for the tender tissue growing vigorously. When the content of various substances such as protein contained in plant tissues is high, the extraction quality of genome DNA is seriously influenced, the phenomenon that the DNA is sticky is easy to appear, a gun head is difficult to absorb, and various enzymes completely or partially lose activity when acting on the DNA. For the ISSR molecular marker technology, the DNA fingerprint technology which is rapid, simple, reliable and sensitive and can provide rich genome information is based on the PCR technology, so that the ISSR is easily influenced by the PCR (such as template concentration, primers, temperature, Taq DNA enzyme and Mg)²⁺Concentration, etc.). If the extracted DNA is degraded or the purity is not enough, the experiment has no result or the result is unstable.

So far, methods for extracting DNA from jujube trees have been published. The method mainly utilizes SDS or CTAB reagent, generally CTAB is extracted and purified in a plurality of steps, DNA is seriously lost, and the amount of extracted DNA is relatively small; although the SDS method is simple, the purity of extraction is generally not sufficient, and it is difficult to extract high-purity genomic DNA particularly for jujube species having complex components.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention provides a method for extracting DNA in jujube tree leaves by using a PEX method.

The method for extracting DNA in jujube tree leaves by using a PEX method comprises the following steps of 1) selecting fresh jujube tree leaves, conducting dark treatment at 0-4 ℃ in a refrigerator for 48 hours, quickly weighing 0.1g of the fresh jujube tree leaves, grinding the fresh jujube tree leaves into fine powder in liquid nitrogen, quickly transferring the fine powder into a 1.5mL EP tube, adding 800 μ L of a first buffer solution preheated at 65 ℃ for mixing uniformly, wherein the first buffer solution comprises 2.5mM of PEX, 4% (g/mL) of soluble PVP, 100mM of Tris, 7.5 pH, 10mM of EDTA, 7.5 pH, 2% (μ L/mL) of β -mercaptoethanol and 700mM of NaCl, b) conducting constant temperature water bath at 65 ℃ for 20min, conducting once every 5min, taking out the mixture, conducting centrifugation at 13000rpm for 5min, c) adding supernatant, adding phenol, chloroform and isoamyl alcohol, rinsing the mixture, adding the supernatant, precipitating the supernatant, adding the isoamyl alcohol, adding the supernatant, rinsing the supernatant, adding the supernatant, the supernatant is washed, the supernatant is added with the concentration of 2 μ L of the concentration of the supernatant is 150 μ L of the supernatant is 1 μ L of the supernatant, the supernatant is 1 μ L of the supernatant is 1.5 μ L of the supernatant, the supernatant is added with the supernatant, the supernatant is added with 2 μ L of the supernatant, the supernatant is added with the concentration of the supernatant is 1.5 mM of the concentration of the supernatant is 1.5 μ L of the supernatant is 1.5 mM of the supernatant, the supernatant is 1.5 min, the supernatant is changed.

Drawings

FIG. 1 shows the results of 0.8% agarose gel electrophoresis of the extracted DNA;

FIG. 2 shows the mass spectrum of DNA detected by ISSR molecular marker technique amplified by primer UBC-807;

FIG. 3 shows the mass spectrum of DNA detected by ISSR molecular marker technique amplified by primer UBC-811.

Wherein, No. 1-10 is material, M is molecular mass standard

Detailed Description

In the embodiment, the following steps are adopted to extract the DNA in the jujube tree leaves:

a) selecting fresh jujube tender leaves, carrying out dark treatment at 0-4 ℃ in a refrigerator for 48h, quickly weighing 0.1g of the fresh jujube tender leaves, grinding the fresh jujube tender leaves into fine powder in liquid nitrogen, quickly transferring the fine powder into a 1.5mL EP tube, adding 800 μ L of a first buffer solution preheated at 65 ℃ to mix uniformly, wherein the first buffer solution comprises 2.5mM PEX, 4% (4 g in each 100 mL), 100mM Tris with the pH value of 7.5, 10mM EDTA with the pH value of 7.5, 2% (2 μ L in each 100 mL) β -mercaptoethanol and 700mM NaCl, b) incubating in a constant-temperature water bath at 65 ℃ for 20min, slightly inverting and mixing once every 5min, taking out, centrifuging at 13000rpm for 5min at room temperature, c) taking the supernatant, adding equal volume of phenol, chloroform and isoamylol into the supernatant, wherein the phenol, the ratio of chloroform to isoamylol is 25: 24: 1 (volume ratio), slightly inverting and mixing uniformly mixing up and then 13000rpm for 5min, d) adding the supernatant of the chloroform and isoamylol, rinsing the supernatant, wherein the supernatant is added into a third buffer solution, the supernatant of the supernatant, the supernatant of the supernatant is added with the supernatant of 2 μ L of the supernatant, the supernatant of.

The extracted DNA was analyzed by the following experiment.

1. And identifying the concentration and purity of the extracted DNA.

mu.L of each of the extracted DNAs was subjected to 0.8% agarose gel electrophoresis and detected by a gel imager (UVP in USA), and the results are shown in FIG. 1.

The result shows that the extracted 10 jujube tree DNAs are neat and single, and basically have no scattered bands, which indicates that the integrity of the extracted DNAs is better, most sample application holes of the materials are clearly visible (a few pore channels have tailing phenomena such as 3 rd, 6 th and 9 th), and the content of impurities of the DNAs is preliminarily indicated to be less.

The obtained DNA was further tested for concentration and purity by a micro-UV spectrophotometer (Thermo nanodrop 2000), and the concentration and A of the DNA were read₂₆₀/A₂₈₀And A₂₆₀/A₂₃₀The ratio of (a) to (b). (Table 1).

A₂₆₀/A₂₈₀And A₂₆₀/A₂₃₀The ratio of (a) to (b) is used to evaluate the purity of the sample. For pure DNA samples, A is typical₂₆₀/A₂₈₀Is greater than 1.8, A₂₆₀/A₂₃₀Is greater than 2.0. When A is₂₆₀/A₂₈₀A ratio of less than 1.8 indicates the effect of the presence of protein or phenolic material; if A₂₆₀/A₂₃₀A ratio of (A) to (B) of less than 2.0 indicates that the sample is contaminated with carbohydrates (sugars), salts or organic solventsThe sample was further purified.

TABLE 1 sample DNA purity and concentration

As can be seen from Table 1, the concentrations of the extracted DNA samples of jujube trees were high, and A was found in most of the materials₂₆₀/A₂₈₀Ratio greater than 1.8, A₂₆₀/A₂₃₀Is greater than 2. Thus, the method has good purification effect on protein or phenolic substances in the jujube tree material, and has good purification effect on A of partial materials₂₆₀/A₂₃₀The ratio is less than 2.0, which indicates that the removal effect of carbohydrate (sugar), salt or organic solvent pollution is poor, and individuals think that the complete removal is difficult probably because of the high sugar content in several jujube tree materials, but the later ISSR molecular marker experiment verification result shows that the analysis of the experiment is not influenced basically.

2. ISSR molecular marker application analysis for extracted jujube tree DNA

The extracted DNA is uniformly diluted to the used concentration by deionized water according to the requirements of an ISSR-PCR reaction system, and is reserved at 4 ℃ (the storage time under the condition is too long).

ISSR-PCR reaction System: 10 × Buffer (Mg)²⁺plus), 200. mu. mol/L dNTP, 0.2. mu. mol/L primer, 3 ng/. mu.L template, 0.04U/. mu.L Taq DNA polymerase, complement ddH₂O to 20. mu.L.

The PCR amplification reaction program is as follows: 94 ℃, 5min, 1 cycle; 94 ℃ for 45 s; 50 ℃ for 45 s; 72 ℃, 90s, 35 cycles; finally, extending for 10min at 72 ℃; storing at 4 ℃. The PCR products were separated by electrophoresis on a 1% agarose gel and photographed by scanning with a gel image analyzer. And further evaluating the DNA quality according to the definition and polymorphism of the fingerprint. The results are shown in FIG. 2.

As seen from FIG. 2, two ISSR primers UBC-807 and UBC-811 are screened out by the preliminary experiment, and according to the verification of the system amplification result, each material has corresponding polymorphism bands (even if the purity of part of the material is not high), the result is stable and reliable, and the ISSR primers UBC-807 and UBC-811 are effective experimental result graphs and are consistent with the preliminary test results. Indicating that the extracted DNA was significant.

Compared with a CTAB and SDS extraction method, the PEX method used by the invention is simpler and time-saving, the extraction amount is large, and the extracted DNA can meet the requirements of ISSR-PCR experiments. And the PEX reagent is generally used as a chemical reagent and is relatively low in price, which is undoubtedly a good choice for researchers who research a large number of samples (such as plant tissues and population separation). The invention is a widely used plant DNA extraction method, the extracted DNA has higher purity and higher concentration, and the DNA extraction efficiency is improved. Individuals believe that this extraction method is not only applicable to jujube trees, but also can be generalized to the study of other species such as high-sugar materials. If the extraction result is still unsatisfactory by the standard extraction method, the NaCl concentration can be properly increased, or the purification is continued by adding phenol, chloroform and isoamylol until the satisfactory effect is achieved.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.