Extraction method of mangrove plant DNA

An extraction method, the technology of mangrove plants, which is applied in the field of plant DNA extraction, can solve the problems of high-demand operations that cannot be carried out smoothly, high operating technical requirements, and genomic DNA loss, so as to prevent precipitation, high purity, and remove polysaccharides. Effect

Inactive Publication Date: 2010-12-15
ZHEJIANG MARICULTURE RES INST
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  • Application Information

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Problems solved by technology

However, due to the particularity of mangrove plants, there are also some difficulties in the research at the molecular level, one of which is the DNA extraction of mangrove plants, because: mangrove plants are famous for their rich tannins, and their cell walls contain A large number of substances such as tannins and polysaccharides, in the process of nucleic acid extraction, the existence of a large amount of tannins and polysaccharides makes them form co-precipitation with DNA, thus affecting the purity of nucleic acids, although the DNA extraction methods of general land plants are very mature , such as the common classic CTAB method, SDS method, etc., and these methods cannot deal with the problem of polysaccharides well, so that relatively pure DNA cannot be obtained. At present, the most commonly used method for removing polysaccharides at home and abroad is to use the classic CsCl Ultracentrifugation method, but this method has high requirements for experimental equipment and operating technology, and is expensive. In addition, some researchers use purification methods such as column filtration. Although genomic DNA is purified well, it is easy to The loss and breakage of genomic DNA cannot meet the high requirements of DNA operations such as enzyme digestion and connection. In addition, there are few reports on how to remove tannins during the DNA extraction process; if further development of population genetics, systematic Evolution, molecular marker-assisted breeding, etc., due to the large number of experimental samples that need to be processed, the cumbersome and expensive defects of the above-mentioned physical or chemical methods are even more unfavorable for experimental research, and DNA has high requirements. Operations such as molecular cloning and genetic modification cannot be carried out smoothly

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  • Extraction method of mangrove plant DNA
  • Extraction method of mangrove plant DNA

Examples

Experimental program
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Effect test

Embodiment 1

[0030] (1), put 0.1g of leaves into a mortar, add a small amount of quartz sand, fully grind into powder with liquid nitrogen, add 0.5ml of preheated 2% CTAB extract to dissolve, 2% CTAB extract includes 2% CTAB, 0.1 TrisHCl of mol / L, EDTA of 20mmol / L, NaCl of 1.4mol / L and 1% β-mercaptoethanol, the pH value of described TrisHCl is 8.0, then add 50 μ l 2% PVP, pack into 1.5ml centrifuge tube, total The volume is about 0.5ml;

[0031] (2) Put it in a water bath at 55°C for 60 minutes, add 2 μl RNase, keep warm at 37°C for 1 hour, then add about 0.5ml of chloroform-isoamyl alcohol mixture and mix, chloroform and isoamyl alcohol in the chloroform-isoamyl alcohol mixture The ratio of isoamyl alcohol is 24:1, and it is centrifuged at 8000 rpm for 15 minutes at room temperature;

[0032](3), take the supernatant and transfer it to a centrifuge tube, add 1 / 2 volume of preheated 2% CTAB extract, the 2% CTAB extract is consistent with the previous step (1), then add an equal volume of ...

Embodiment 2

[0036] (1), take 0.1g of fresh tender leaves and put them into a mortar, add a small amount of quartz sand, fully grind them into powder with liquid nitrogen, add 0.5ml of preheated 2% CTAB extract to dissolve, 2% CTAB extract includes 2% CTAB , the TrisHCl of 0.1mol / L, the EDTA of 20mmol / L, the NaCl of 1.4mol / L and the β-mercaptoethanol of 1%, the pH value of described TrisHCl is 8.0, add 50 μ l 2% PVP again, pack into 1.5ml centrifuge tube , the total volume is about 0.5ml;

[0037] (2) Place in a water bath at 55°C for 45 minutes, add 2 μl of RNase, incubate at 37°C for 1.5 hours, then add about 0.5ml of chloroform-isoamyl alcohol mixture and mix, chloroform and isoamyl alcohol in the chloroform-isoamyl alcohol mixture The ratio of isoamyl alcohol is 24:1, and it is centrifuged at 12000 rpm for 10 minutes at room temperature;

[0038] (3), take the supernatant and transfer it to a centrifuge tube, add 1 / 2 volume of preheated 2% CTAB extract, the 2% CTAB extract is consiste...

Embodiment 3

[0042] (1), put 0.2g fresh leaves into a mortar, add a small amount of quartz sand, fully grind into powder with liquid nitrogen, add 1ml of preheated 2% CTAB extract to dissolve, 2% CTAB extract includes 2% CTAB, 0.1 TrisHCl of mol / L, the EDTA of 20mmol / L, the NaCl of 1.4mol / L and the β-mercaptoethanol of 1%, the pH value of described TrisHCl is 8.0, then add 100 μ l 2% PVP, pack into 5ml centrifuge tube, total volume About 1ml;

[0043] (2) Put it in a water bath for 80 minutes at 60°C, add 6 μl RNase, keep warm at 37°C for 2 hours, then add about 1 ml of chloroform-isoamyl alcohol mixture and mix, chloroform and isoamyl alcohol in the chloroform-isoamyl alcohol mixture The proportioning ratio of amyl alcohol is 24:1, and it is centrifuged at 10,000 rpm for 10 minutes at room temperature;

[0044] (3), take the supernatant and transfer it to a centrifuge tube, add 1 / 2 volume of preheated 2% CTAB extract, the 2% CTAB extract is consistent with the previous step (1), then add...

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Abstract

The invention relates to an extraction method of mangrove plant DNA, comprising the following steps: (1) taking a leaf to grind, adding preheated 2% of CTAB extract to dissolve, and then adding 2 % of PVP by volume; (2) after water bath, adding a small quantity of RNase for heat preservation, then adding chloroform-isoamylol mixed liquor, and carrying out high-speed centrifugation at normal temperature; (3) putting supernate into a centrifuge tube, adding the preheated 2% of CTAB extract, adding chloroform-isoamylol mixed liquor, and mixing and carrying out high-speed centrifugation at normal temperature; (4) taking supernate and then putting into the centrifuge tube, adding NaCl solution and cold isopropanol, mixing, and putting and precipitating DNA at low temperature; (5) carrying out high-speed centrifugation at low temperature, abandoning the supernate, recovering the precipitate, washing by ethanol, air-drying, adding TE buffer solution, and storing at low temperature. The invention can effectively remove polysaccharide and tannin produced in the DNA extraction process, and the obtained mangrove plant has high DNA content and high purity.

Description

technical field [0001] The invention relates to a plant DNA extraction method, in particular to a DNA extraction method for mangrove plants rich in polysaccharides and tannic acid. Background technique [0002] Mangrove is a rare woody viviparous plant that grows in tidal flats and shoals at the junction of tropical and subtropical land and sea. It is a special ecosystem that transitions from land to sea. There are 23 families, 34 genera, and 81 species of mangrove plants in the world. There are 13 families and 24 species of mangrove trees in my country, mainly distributed in Hainan, Guangdong and the coastal areas of Fujian. Because mangroves grow at the junction of land and sea, they are natural barriers for the land, which is of great significance to reducing the damage caused by natural disasters such as typhoons; the mangrove ecosystem also has the function of absorbing and accumulating heavy metal elements, removing organochlorine pesticides, and improving environmenta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 陈少波丁文勇仇建标陈骁刘伟成艾为明谢起浪李尚鲁王成义王广银郑春芳池伟黄丽
Owner ZHEJIANG MARICULTURE RES INST
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