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A kit and method for extracting genomic dna from evergreen woody plants

A woody plant and genome technology, applied in the extraction kit and field of evergreen woody plant genomic DNA, can solve the problems of different and complex plant tissue components, and achieve the prevention of combined browning, enhanced grinding effect, Increases the effect of purity and integrity

Active Publication Date: 2020-10-09
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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  • A kit and method for extracting genomic dna from evergreen woody plants
  • A kit and method for extracting genomic dna from evergreen woody plants
  • A kit and method for extracting genomic dna from evergreen woody plants

Examples

Experimental program
Comparison scheme
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 / L KAc, 0.1mol / L glucose, the mass fraction is 2% PVP, the mass fraction is 1.5% ascorbic acid and the volume fraction is 7% PEG6000; add β-mercaptoethanol before use, Make the volume fraction of β-mercaptoethanol in the rinse solution be 3%;

[0042] The lysate includes 2×CTAB, 0.2mol / L Tris-HCl, 0.03mol / L EDTA-Na2, 1.2mol / LNaCl, 0.4mol / L sorbitol, 0.02mol / L borax, and a mass fraction of 2% PVP , ascorbic acid with a mass fraction of 1.5%, 180 μg / mL proteinase K, 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 / L KAc, 0.15mol / L glucose, 3% PVP by mass fraction, 1% ascorbic acid by mass fraction and 8% PEG6000 by volume fraction; add β-mercaptoethanol before use , so that the volume fraction of β-mercaptoethanol in the rinse solution is 4%;

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

[0050] T...

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Abstract

The invention relates to an extraction kit and extraction method of evergreen woody plant genomic DNA, belonging to the field of genetic engineering. The kit includes rinsing solution, lysate, extract A, extract B, binding buffer, and column Buffer, elution buffer and adsorption column, also includes research aids; research aids include insoluble PVPP, quartz sand and ascorbic acid; rinse solution contains Tris‑HCl, EDTA‑Na2, NaCl, KAc, glucose, PVP, Ascorbic acid and PEG; components of lysate include CTAB, Tris‑HCl, EDTA‑Na2, NaCl, sorbitol, borax, PVP, ascorbic acid, and proteinase K. The method is flexible, fast, low-cost, and widely applicable, and can thoroughly remove secondary metabolites and other interfering substances in cells. The extracted genome has good quality, high purity, and less protein contamination.

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 Patents(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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