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Extraction kit and method for genome DNA of evergreen woody plants

A woody plant and genome technology is applied in the extraction kit and extraction field of the genomic DNA of evergreen woody plants, which can solve the problems of different, complex plant tissue components, etc., to prevent binding browning, enhance grinding effect, Effect of increasing purity and integrity

Active Publication Date: 2017-11-07
HENAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although DNA extraction from organisms has become a routine technique in molecular biology laboratories, the composition of plant tissues is complex and varies among different plant materials, especially perennial evergreen woody plants such as conifers , Magnoliaceae plants, etc., because of their thick cell walls and vigorous metabolism, the cells contain a large amount of secondary metabolites such as polysaccharides and polyphenols, and the surface of the leaves is leathery, so they are often called "stubborn plants" by molecular biology researchers. (recalcitrant plant), how to remove these impurities to obtain high-quality DNA is the primary problem facing this type of plant research

Method used

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  • Extraction kit and method for genome DNA of evergreen woody plants
  • Extraction kit and method for genome DNA of evergreen woody plants
  • Extraction kit and method for genome DNA of evergreen woody plants

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Take fresh and healthy juniper (Cedpressaceae) leaves, put them in a sealed ice box and bring them back to the laboratory, wash them with distilled water to remove the dirt on the surface of the leaves and dry them, remove the petioles and veins from the leaves, cut them into pieces, and put 50mg into the liquid Add liquid nitrogen to a nitrogen pre-cooled sterilized mortar, and add research aids, and quickly grind to a fine powder to obtain a sample powder. During the grinding process, the pestle is repeatedly ground in a clockwise and counterclockwise direction to ensure that the sample is ground thoroughly. Put the sample powder in a 2mL centrifuge tube, add 1mL of rinse solution, vortex and mix well, then process on ice for 5min, centrifuge at 8,000rpm for 5min, and discard the supernatant; repeat this step once; where, the rinse solution is added with β- Mercaptoethanol, so that the volume fraction of β-mercaptoethanol in the rinse solution is 2%. Add 1 mg of activ...

Embodiment 2

[0040] Get fresh and healthy leaves of Arborvitae (Cedraceae) and use a method similar to that of Example 1 to extract the genomic DNA of Arborvitae, the difference being that 70 mg of it is cut and ground, and the research aid used includes 0.05 g of insoluble PVPP, 0.2g quartz sand and 0.09g ascorbic acid;

[0041] The rinsing liquid comprises 0.1mol / L Tris-HCl, 0.09mol / L EDTA-Na 2 , 0.8mol / L NaCl, 1mol / LKAc, 0.1mol / L glucose, a mass fraction of 2% PVP, a mass fraction of 1.5% ascorbic acid and a volume fraction of 7% PEG6000; before use, add β-mercaptoethanol to make The volume fraction of β-mercaptoethanol in the rinse solution is 3%;

[0042] The lysate includes 2×CTAB, 0.2mol / L Tris-HCl, 0.03mol / L EDTA-Na2, 1.2mol / L NaCl, 0.4mol / L sorbitol, 0.02mol / L borax, and the mass fraction is 2% PVP, ascorbic acid with a mass fraction of 1.5%, proteinase K at 180 μg / mL, pH 8.0; add β-mercaptoethanol before use, so that the volume fraction of β-mercaptoethanol in the lysate is 4%;...

Embodiment 3

[0047] Take fresh and healthy cedar (Pinaceae) leaves, and use a method similar to that of Example 1 to extract the genomic DNA of Arborvitae, the difference being that 80 mg of it is chopped and ground, and the research aid used includes 0.1 g of insoluble PVPP , 0.18g quartz sand and 0.08g ascorbic acid;

[0048] The rinse solution includes 0.1mol / L Tris-HCl, 0.08mol / L EDTA-Na 2 , 0.7mol / L NaCl, 0.8mol / LKAc, 0.15mol / L glucose, the mass fraction is 3% PVP, the mass fraction is 1% ascorbic acid and the volume fraction is 8% PEG6000; add β-mercaptoethanol before use, Make the volume fraction of β-mercaptoethanol in the rinse solution be 4%;

[0049] The lysate includes 3×CTAB, 0.3mol / L Tris-HCl, 0.05mol / L EDTA-Na 2 , 1.4mol / L NaCl, 0.3mol / L sorbitol, 0.03mol / L borax, PVP with a mass fraction of 2.5%, ascorbic acid with a mass fraction of 2%, 160 μg / mL proteinase K, pH8.0; add β before use -mercaptoethanol, so that the volume fraction of β-mercaptoethanol in the rinse solutio...

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Abstract

The invention relates to an extraction kit and method for genome DNA of evergreen woody plants and belongs to the field of genetic engineering. The extraction kit comprises a rinsing liquor, a lysate, a leaching liquor A, a leaching liquor B, a binding buffer liquor, a column buffer liquor, an elution buffer liquor and an adsorption column, and further comprises a grinding assisting agent, wherein the grinding assisting agent comprises insoluble PVPP, quartz sand and ascorbic acid; the rinsing liquor comprises Tris-HCl, EDTA-Na2, NaCl, KAc, glucose, PVP, ascorbic acid and PEG; and the lysate comprises CTAB, Tris-HCl, EDTA-Na2, NaCl, sorbitol, borax, PVP, ascorbic acid and protease K. The extraction method is flexible and quick in operation, low in cost and wide in application range, and can radically remove secondary metabolites and other interfering substances in cells, and the extracted genome is good in quality, high in purity and less in protein pollution.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and relates to the extraction of plant genome DNA, in particular to an extraction kit and extraction method of evergreen woody plant genome DNA. Background technique [0002] Evergreens are plants that have green leaves throughout the year. Due to the characteristics of evergreen in all seasons, this kind of plants can effectively improve the quality of urban landscaping, enhance the beauty of landscaping, and at the same time play an important role in maintaining the balance of the ecosystem, so they are often used as the first choice for greening. There are many species of evergreen woody plants with tall trees and straight and round stems. They are important raw materials for construction, papermaking and board production. Plants of some families and genera are indispensable and precious materials for studying the origin, development and evolution of plants, and their scientific re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1006C12N15/101
Inventor 李红英杨雪王少博周洲李雪萍
Owner HENAN UNIV OF SCI & TECH
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