CN110804612A - Method for extracting trace and high-quality dendrobium officinale genome DNA - Google Patents
Method for extracting trace and high-quality dendrobium officinale genome DNA Download PDFInfo
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- CN110804612A CN110804612A CN201911328021.5A CN201911328021A CN110804612A CN 110804612 A CN110804612 A CN 110804612A CN 201911328021 A CN201911328021 A CN 201911328021A CN 110804612 A CN110804612 A CN 110804612A
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Abstract
The invention discloses a method for extracting trace and high-quality dendrobium officinale genome DNA, and belongs to the technical field of biology. The method is improved on the basis of extracting DNA by a conventional CTAB method, and a step of removing polysaccharide secondary metabolites is firstly carried out before cell lysis by the CTAB method, so that the polysaccharide secondary metabolites can be effectively removed; 5mol/L NaCl with the volume of 1/10 is added before DNA is precipitated by isopropanol, and polysaccharide is further removed, so that high-quality genome DNA is obtained. The method is simple, convenient and quick, the cost is low, and the obtained genome DNA has excellent quality.
Description
Technical Field
The patent belongs to the technical field of biology, and particularly relates to a method for extracting trace and high-quality dendrobium officinale genome DNA.
Background
Dendrobium officinale Kimura et Migo (A. am)Dendrobium candidumWall, ex Lindl) is a medicinal plant of Dendrobium of Orchidaceae, and its effective components are herba Dendrobii polysaccharide and alkaloid. The content of polysaccharide in Dendrobium officinale is high, many physicochemical properties are similar to those of DNA, when DNA is precipitated in the process of extracting Dendrobium officinale DNA by conventional CTAB method, polysaccharide is precipitated at the same time, and serious shadow is causedThe requirements of genetic engineering operations cannot be met depending on the quality, yield and purity of DNA. A simple, convenient and high-quality extraction method of the dendrobium officinale genome DNA is needed.
Disclosure of Invention
Aiming at the problems, the invention provides a method for extracting the trace and high-quality dendrobium officinale genome DNA. The method is simple, convenient and rapid, and can effectively remove Dendrobium officinale polysaccharide in the DNA extraction process to obtain high-quality Dendrobium officinale genome DNA meeting genetic engineering operation.
In order to achieve the purpose, the invention adopts the following technical scheme:
a reagent for extracting dendrobium officinale genome DNA comprises the following components:
solution A: 0.15mol/L NaCl, 20mmol/L EDTA, 100mmol/L Tris-HCl with pH8.0, 0.2% mercaptoethanol (now added when used);
solution B: 2% CTAB, 1.4mol/L NaCl, 20mmol/L EDTA, 100mmol/L pH8.0Tris-HCl, 0.2% mercaptoethanol (now added);
solution C: chloroform: the volume ratio of isoamyl alcohol is 24: 1;
solution E: 5mol/L NaCl;
solution F: isopropyl alcohol;
solution G: 75% alcohol;
solution H: 10 mmol/L pH7.4Tris-HCl, 1 mmol/L EDTA.
Solution A, B, E, H was used after autoclaving.
A method for extracting the genomic DNA of the dendrobium officinale by using the reagent for extracting the genomic DNA of the dendrobium officinale comprises the following steps:
(1) grinding 100mg of fresh dendrobium officinale leaves by using liquid nitrogen, and transferring the powder to a 2mL centrifuge tube;
(2) adding 2mL of solution A, slightly shaking and uniformly mixing, and incubating for 5min at 65 ℃;
(3) centrifuging at 2000rpm for 3min, and collecting precipitate;
(4) adding 2mL of solution A, slightly shaking, resuspending and mixing uniformly, and incubating for 5min at 65 ℃;
(5) centrifuging at 2000rpm for 3min, and collecting precipitate;
(6) adding 1mL of solution B preheated at 65 ℃, incubating for 15min at 65 ℃, taking out and cooling to room temperature;
(7) adding the solution C with the same volume, gently mixing uniformly, and centrifuging at 5000rpm for 10 min;
(8) taking the supernatant, adding the solution C with the same volume, gently mixing uniformly, and centrifuging at 5000rpm for 10 min;
(9) taking the supernatant, adding solution E with the volume of 1/10, gently mixing uniformly, adding solution F with the same volume, and gently mixing uniformly;
(10) centrifuging at 5000rpm for 10min, and discarding the supernatant;
(11) washing the precipitate with solution G for 2 times, centrifuging at 5000rpm for 5min, and drying the DNA precipitate;
(12) 50 μ L of solution H was added to dissolve the DNA.
The invention has the advantages that:
the content of the dendrobium officinale polysaccharide is high, the physicochemical property of the dendrobium officinale polysaccharide is similar to that of DNA, when the DNA is precipitated in the process of extracting the dendrobium officinale DNA by the conventional CTAB method, the polysaccharide is also precipitated at the same time, and the quality of the DNA is seriously influenced. The invention is improved on the basis of extracting DNA by a conventional CTAB method, and a step of removing the secondary polysaccharide metabolite is firstly carried out before the CTAB cell lysis, so that the secondary polysaccharide metabolite can be effectively removed; 5mol/L NaCl with the volume of 1/10 is added before DNA is precipitated by isopropanol, and polysaccharide is further removed, so that high-quality genome DNA is obtained. The method is simple, convenient and quick, the cost is low, and the obtained genome DNA has excellent quality.
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FIG. 1 is a graph showing the effect of removing polysaccharides by the method of the present invention and a conventional CTAB method. A and B are DNA supernatant extracted from dendrobium officinale by a conventional CTAB method; c and D are DNA supernatant of dendrobium officinale extracted by the method.
FIG. 2 is an agarose gel electrophoresis image of DNA extracted by the method of the present invention.
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the following examples are only examples of the present invention and do not represent the scope of the present invention defined by the claims.
Example 1
A reagent for extracting dendrobium officinale genome DNA comprises the following components:
solution A: 0.15mol/L NaCl, 20mmol/L EDTA, 100mmol/L Tris-HCl with pH8.0, 0.2% mercaptoethanol (now added when used);
solution B: 2% CTAB, 1.4mol/L NaCl, 20mmol/L EDTA, 100mmol/L pH8.0Tris-HCl, 0.2% mercaptoethanol (now added);
solution C: chloroform: the volume ratio of isoamyl alcohol is 24: 1;
solution E: 5mol/L NaCl;
solution F: isopropyl alcohol;
solution G: 75% alcohol;
solution H: 10 mmol/L pH7.4Tris-HCl, 1 mmol/L EDTA.
Solution A, B, E, H was used after autoclaving.
A method for extracting the genomic DNA of the dendrobium officinale by using the reagent for extracting the genomic DNA of the dendrobium officinale comprises the following steps:
(1) grinding 100mg of fresh dendrobium officinale leaves by using liquid nitrogen, and transferring the powder to a 2mL centrifuge tube;
(2) adding 2mL of solution A, slightly shaking and uniformly mixing, and incubating for 5min at 65 ℃;
(3) centrifuging at 2000rpm for 3min, and collecting precipitate;
(4) adding 2mL of solution A, slightly shaking, resuspending and mixing uniformly, and incubating for 5min at 65 ℃;
(5) centrifuging at 2000rpm for 3min, and collecting precipitate;
(6) adding 1mL of solution B preheated at 65 ℃, incubating for 15min at 65 ℃, taking out and cooling to room temperature;
(7) adding the solution C with the same volume, gently mixing uniformly, and centrifuging at 5000rpm for 10 min;
(8) taking the supernatant, adding the solution C with the same volume, gently mixing uniformly, and centrifuging at 5000rpm for 10 min;
(9) taking the supernatant, adding solution E with the volume of 1/10, gently mixing uniformly, adding solution F with the same volume, and gently mixing uniformly;
(10) centrifuging at 5000rpm for 10min, and discarding the supernatant;
(11) washing the precipitate with solution G for 2 times, centrifuging at 5000rpm for 5min, and drying the DNA precipitate;
(12) 50 μ L of solution H was added to dissolve the DNA.
Extracting the genome DNA of the dendrobium officinale by a conventional CTAB method as a control.
The extraction method of example 1 and the comparative conventional CTAB method are shown in figure 1 for extracting the genome DNA supernatant of Dendrobium officinale Kimura et Migo. As can be seen from FIG. 1, the genomic DNA supernatant (FIGS. 1A and 1B) extracted by the conventional CTAB method of comparative example 1 is turbid and contains impurities such as polysaccharides; the dendrobium officinale genome DNA supernatant (shown in figure 1C and figure 1D) extracted by the method of the embodiment 1 of the invention is clear, and the removal effect of impurities such as polysaccharide is good.
An agarose gel electrophoresis chart of the genomic DNA of Dendrobium officinale extracted by the method of the embodiment 1 of the invention is shown in FIG. 2. The results of fig. 2 show that: the genome DNA of the dendrobium officinale extracted by the method has A260/A280 in the range of 1.8-2.0, A260/230 more than 2.0, no impurity pollution and good integrity.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (2)
1. A reagent for extracting dendrobium officinale genome DNA is characterized by comprising the following components:
solution A: 0.15mol/L NaCl, 20mmol/L EDTA, 100mmol/L Tris-HCl with pH8.0, 0.2% mercaptoethanol;
solution B: 2% CTAB, 1.4mol/L NaCl, 20mmol/L EDTA, 100mmol/L pH8.0Tris-HCl, 0.2% mercaptoethanol;
solution C: chloroform: the volume ratio of isoamyl alcohol is 24: 1;
solution E: 5mol/L NaCl;
solution F: isopropyl alcohol;
solution G: 75% alcohol;
solution H: 10 mmol/L pH7.4Tris-HCl, 1 mmol/L EDTA;
solution A, B, E, H was used after autoclaving, 0.2% mercaptoethanol was added as is.
2. The method for extracting the genomic DNA of the dendrobium officinale by using the reagent for extracting the genomic DNA of the dendrobium officinale according to claim 1 is characterized by comprising the following steps of:
(1) grinding 100mg of fresh dendrobium officinale leaves by using liquid nitrogen, and transferring the powder to a 2mL centrifuge tube;
(2) adding 2mL of solution A, slightly shaking and uniformly mixing, and incubating for 5min at 65 ℃;
(3) centrifuging at 2000rpm for 3min, and collecting precipitate;
(4) adding 2mL of solution A, slightly shaking, resuspending and mixing uniformly, and incubating for 5min at 65 ℃;
(5) centrifuging at 2000rpm for 3min, and collecting precipitate;
(6) adding 1mL of solution B preheated at 65 ℃, incubating for 15min at 65 ℃, taking out and cooling to room temperature;
(7) adding the solution C with the same volume, gently mixing uniformly, and centrifuging at 5000rpm for 10 min;
(8) taking the supernatant, adding the solution C with the same volume, gently mixing uniformly, and centrifuging at 5000rpm for 10 min;
(9) taking the supernatant, adding solution E with the volume of 1/10, gently mixing uniformly, adding solution F with the same volume, and gently mixing uniformly;
(10) centrifuging at 5000rpm for 10min, and discarding the supernatant;
(11) washing the precipitate with solution G for 2 times, centrifuging at 5000rpm for 5min, and drying the DNA precipitate;
(12) 50 μ L of solution H was added to dissolve the DNA.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111440788A (en) * | 2020-04-09 | 2020-07-24 | 武汉菲沙基因信息有限公司 | Efficient extraction method of dendrobium officinale genome DNA |
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| CN103756994A (en) * | 2013-03-21 | 2014-04-30 | 四川农业大学 | DNA extraction method for polysaccharide-rich plant dried leaves |
| CN106636075A (en) * | 2017-03-13 | 2017-05-10 | 成都育芽科技有限公司 | Method for extracting genome DNA of dendrobium officinale |
| CN109439652A (en) * | 2018-11-21 | 2019-03-08 | 上海派森诺生物科技股份有限公司 | A kind of extracting method of the plant genome DNA rich in polysaccharide |
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Patent Citations (3)
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|---|---|---|---|---|
| CN103756994A (en) * | 2013-03-21 | 2014-04-30 | 四川农业大学 | DNA extraction method for polysaccharide-rich plant dried leaves |
| CN106636075A (en) * | 2017-03-13 | 2017-05-10 | 成都育芽科技有限公司 | Method for extracting genome DNA of dendrobium officinale |
| CN109439652A (en) * | 2018-11-21 | 2019-03-08 | 上海派森诺生物科技股份有限公司 | A kind of extracting method of the plant genome DNA rich in polysaccharide |
Non-Patent Citations (4)
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| 彭锐等: ""石斛总DNA的提取及鉴定"", 《中国中药杂志》 * |
| 曾杰等: ""顽拗植物类群的总DNA制备"", 《植物学报》 * |
| 李玉奇等主编: "《食品生物化学实验》", 30 September 2018, 西南交通大学出版社 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111440788A (en) * | 2020-04-09 | 2020-07-24 | 武汉菲沙基因信息有限公司 | Efficient extraction method of dendrobium officinale genome DNA |
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