CN112029763A - Method for rapidly extracting high-quality genome DNA from stem tip of dragon fruit - Google Patents
Method for rapidly extracting high-quality genome DNA from stem tip of dragon fruit Download PDFInfo
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Abstract
The invention relates to the technical field of DNA extraction, and discloses a method for quickly extracting high-quality genome DNA from stem tips of pitaya. The method overcomes the interference of viscous substances in the pitaya stem material on DNA extraction, and solves the problems that the DNA is easy to degrade and the extracted DNA has more impurities in the process of extracting the pitaya DNA; the method has the advantages of few operation steps, long operation time, high extraction efficiency, high purity, high integrity and high extraction yield of the DNA, and the quality and the quantity of the obtained DNA meet the requirements of constructing a genome sequencing library.
Description
Technical Field
The invention relates to the technical field of DNA extraction, in particular to a method for quickly extracting high-quality genome DNA from stem tips of pitaya.
Background
Hylocereus or Serpentis (Selenicerus) belonging to Cactaceae (Cactaceae) is a health-care type special tropical fruit, originally produced in the central and south America, and is currently planted in provinces such as Guangxi, Guangdong, Guizhou, Hainan, Fujian and Yunnan on a large scale. The pitaya has the characteristic of tropical desert, the drought resistance of plant stems and tendrils is strong, the viscosity of stem meat is high, and the pitaya is rich in secondary metabolites such as polysaccharide, flavone, polyphenol, amino acid, phytol, vitamin, phytosterol and the like.
The molecular biology research of the dragon fruit becomes a research hotspot in recent years, the acquisition of high-quality DNA of the dragon fruit is the first key link for developing the molecular biology research, and the stem and the vine of the dragon fruit are extremely sticky, so that the high-quality DNA is difficult to extract from the dragon fruit. At present, researchers develop a plurality of extraction methods of pitaya DNA, such as a trace CTAB method of primula forbesii, a stem DNA extraction method of Xiaguafei and a plant polysaccharide polyphenol DNA extraction kit sold in the market. Multiple tests show that the existing method has the problems of less total amount of extracted DNA, blockage of an adsorption column or complex steps, long time consumption and the like, and the DNA quality is low, the concentration is low, and the library construction requirement of high-throughput sequencing is difficult to meet. Therefore, the inventor develops a method which is suitable for preparing a dragon fruit DNA library and can quickly extract the high-quality DNA of the dragon fruit.
Disclosure of Invention
Based on the above problems, the present invention provides a method for rapidly extracting high quality genomic DNA from stem tips of dragon fruits, and the present invention provides
In order to solve the technical problems, the invention provides a method for quickly extracting high-quality genome DNA from stem tips of pitaya, which comprises the following steps:
s1: putting stem tip tissues of 0.1-0.2 g of pitaya stem and tendril into an EP (EP) tube, shearing, adding 1mLCTAB lysate, and grinding for 2min by using a tissue grinder;
s2: the sample ground in the step S1 is put into a water bath condition of 65 ℃ for cracking for 60min, and is uniformly mixed for 2-3 times in the process;
s3: centrifuging the solution processed in the step S2 at 12000rpm for 5min, sucking 800 μ L of supernatant, transferring into a new centrifuge tube, adding 200 μ L of 20% PVP40, mixing uniformly, adding 300 μ L of Tris saturated phenol and 300 μ L of chloroform respectively, shaking vigorously and mixing uniformly, standing at room temperature for 10min, centrifuging at 12000rpm for 10min, and sucking the supernatant;
s4: continuously repeating the operation method in the step 3 for the supernatant obtained in the step S3 twice to obtain a final supernatant;
s5: adding chloroform into the final supernatant obtained in the step S4, shaking vigorously and mixing uniformly, and standing at room temperature for 10min, wherein the volume ratio of the final supernatant to the chloroform is 1: 1;
s6: centrifuging the solution treated in the step S5 at 12000rpm for 10min, sucking the supernatant, adding 1/10 times of volume of 3M NaAC and 0.8 times of volume of isopropanol into the supernatant to precipitate DNA;
s7: centrifuging the sample treated in the step S6 at 12000rpm for 10min, taking the precipitate, washing the precipitate twice with 70% ethanol, drying in the air, and dissolving DNA with 200-500 mu L of TE containing RNase.
Further, the CTAB lysate in step S1 contains 1% mercaptoethanol.
Compared with the prior art, the invention has the beneficial effects that: the method overcomes the interference of viscous substances in the pitaya stem material on DNA extraction, and solves the problems that the DNA is easy to degrade and the extracted DNA has more impurities in the process of extracting the pitaya DNA; the method has the advantages of few operation steps, long operation time, high extraction efficiency, high purity, high integrity and high extraction yield of the DNA, and the quality and the quantity of the obtained DNA meet the requirements of constructing a genome sequencing library.
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FIG. 1 is an electrophoretogram of DNA extraction mass of an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.
Example (b):
the embodiment provides a method for quickly extracting high-quality genomic DNA from a stem tip of a dragon fruit, which comprises the following steps:
s1: putting 0.1-0.2 g of stem tip tissue of the stem and tendril of the dragon fruit into a 2mL EP tube, shearing, adding 1mL of CTAB lysate, and grinding for 2min by using a tissue grinder, wherein the CTAB lysate contains 1% mercaptoethanol;
s2: the sample ground in the step S1 is put into a water bath condition of 65 ℃ for cracking for 60min, and is uniformly mixed for 2-3 times in the process;
s3: centrifuging the solution processed in the step S2 at 12000rpm for 5min, sucking 800 μ L of supernatant, transferring into a new 2mL centrifuge tube, adding 200 μ L of 20% PVP40, mixing uniformly, adding 300 μ L of saturated phenol and 300 μ L of chloroform respectively, shaking vigorously and mixing uniformly, standing at room temperature for 10min, centrifuging at 12000rpm for 10min, and sucking the supernatant;
s4: continuously repeating the operation method in the step 3 for the supernatant obtained in the step S3 twice to obtain a final supernatant;
s5: adding chloroform into the final supernatant obtained in the step S4, shaking vigorously and mixing uniformly, and standing at room temperature for 10min, wherein the final supernatant: the volume ratio of chloroform is 1: 1;
s6: centrifuging the solution treated in the step S5 at 12000rpm for 10min, sucking the supernatant, adding 1/10 times of 3M NaAc and 0.8 times of isopropanol into the supernatant to precipitate DNA;
s7: centrifuging the sample treated in the step S6 at 12000rpm for 10min, taking the precipitate, washing the precipitate twice with 70% ethanol, drying in the air, and dissolving DNA with 200-500 mu L of TE containing RNase.
Referring to fig. 1, fig. 1 is an electrophoresis chart of DNA extraction quality detection extracted by the extraction method of this embodiment, and the following table shows the extraction quality detection results of the extraction method of this embodiment:
as can be seen from the electrophorogram and the extraction quality data, the content of the extracted DNA is high; in addition, the extraction method of the embodiment overcomes the defects of the existing extraction methods as follows: the stem DNA extraction method for the fast summer flying is complicated in steps, but the extraction method of the embodiment is simple in steps, saves time and is high in extraction rate; the micro CTAB method of the male residuum has the advantages of low extracted DNA content, incapability of meeting the requirement of library building and easiness in degradation, and the extraction method of the embodiment has simple steps, high extracted DNA content and can meet the requirement of library building.
The above is an embodiment of the present invention. The embodiments and specific parameters in the embodiments are only for the purpose of clearly illustrating the verification process of the invention and are not intended to limit the scope of the invention, which is defined by the claims, and all equivalent structural changes made by using the contents of the specification and the drawings of the present invention should be covered by the scope of the present invention.
Claims (2)
1. A method for rapidly extracting high-quality genomic DNA from stem tips of pitaya is characterized by comprising the following steps of:
s1: putting stem tip tissues of 0.1-0.2 g of pitaya stem and tendril into an EP (EP) tube, shearing, adding 1mLCTAB lysate, and grinding for 2min by using a tissue grinder;
s2: the sample ground in the step S1 is put into a water bath condition of 65 ℃ for cracking for 60min, and is uniformly mixed for 2-3 times in the process;
s3: centrifuging the solution processed in the step S2 at 12000rpm for 5min, sucking 800 μ L of supernatant, transferring into a new centrifuge tube, adding 200 μ L of 20% PVP40, mixing uniformly, adding 300 μ L of Tris saturated phenol and 300 μ L of chloroform respectively, shaking vigorously and mixing uniformly, standing at room temperature for 10min, centrifuging at 12000rpm for 10min, and sucking the supernatant;
s4: continuously repeating the operation method in the step 3 for the supernatant obtained in the step S3 twice to obtain a final supernatant;
s5: adding chloroform into the final supernatant obtained in the step S4, shaking vigorously and mixing uniformly, and standing at room temperature for 10min, wherein the volume ratio of the final supernatant to the chloroform is 1: 1;
s6: centrifuging the solution treated in the step S5 at 12000rpm for 10min, sucking the supernatant, adding 1/10 times of volume of 3M NaAC and 0.8 times of volume of isopropanol into the supernatant to precipitate DNA;
s7: centrifuging the sample treated in the step S6 at 12000rpm for 10min, taking the precipitate, washing the precipitate twice with 70% ethanol, drying in the air, and dissolving DNA with 200-500 mu L of TE containing RNase.
2. The method for rapidly extracting high-quality genomic DNA from pitaya stem tips as claimed in claim 1, wherein the CTAB lysate in the step S1 contains 1% mercaptoethanol.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111876515A (en) * | 2020-07-30 | 2020-11-03 | 广西壮族自治区农业科学院 | Construction method of dragon fruit germplasm resource SSR fingerprint database and identification system |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101985618A (en) * | 2009-12-28 | 2011-03-16 | 陈珂 | Method for extracting genome DNA of Jatropha curcas energy plant |
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- 2020-08-28 CN CN202010882713.0A patent/CN112029763A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101985618A (en) * | 2009-12-28 | 2011-03-16 | 陈珂 | Method for extracting genome DNA of Jatropha curcas energy plant |
Non-Patent Citations (3)
| Title |
|---|
| 农全东 等: "火龙果茎基因组DNA提取方法改良", 《植物学报》 * |
| 武志江 等: "利用荧光标记SSR构建火龙果种质资源分子身份证", 《中国南方果树》 * |
| 韦茜 等: "CTAB法提取火龙果基因组DNA的试验研究", 《安徽农业科学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111876515A (en) * | 2020-07-30 | 2020-11-03 | 广西壮族自治区农业科学院 | Construction method of dragon fruit germplasm resource SSR fingerprint database and identification system |
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