Novel method for extracting recalcitrant plant tissue DNA

A plant tissue, stubborn technology, applied in the field of DNA extraction, can solve the problems of limited amount of DNA extraction from pear tissue, low DNA purity, cumbersome operation, etc., and achieve the effect of less extraction steps, high DNA yield and more impurities

Inactive Publication Date: 2016-04-20
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
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AI Technical Summary

Problems solved by technology

The existing traditional CTAB method and kit extraction method have disadvantages in the extraction of the above-mentioned pear tissue DNA: the traditional CTAB and its improved method are cumbersome to operate, and the purity of the obtained DNA is low, and the extraction amount of the above-mentioned pear tissue DNA is very limited. , low efficiency; the kit method is only suitable for the extraction of a small amount of DNA

Method used

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  • Novel method for extracting recalcitrant plant tissue DNA
  • Novel method for extracting recalcitrant plant tissue DNA
  • Novel method for extracting recalcitrant plant tissue DNA

Examples

Experimental program
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Effect test

Embodiment 1

[0039] (1) Take 0.6 g of pear pulp tissue samples and place them in a mortar pre-cooled with liquid nitrogen, and quickly grind them to a powdery state. During the grinding process, the DNA is prevented from being degraded, and liquid nitrogen is continuously added during the grinding process. Transfer the ground powder into a 2mLEP tube, add 600μL sodium laurate extraction buffer, and shake repeatedly and slowly;

[0040] (2) Add 600 μL of a mixture of phenol, chloroform and isoamyl alcohol (25:24:1), shake repeatedly and slowly, centrifuge at 4°C, 12000 rpm for 20 minutes, and absorb 1200 μL of the supernatant;

[0041] (3) Add 840 μL of isopropanol to the supernatant obtained in step (2), slowly turn it upside down several times, let it settle for 1 hour, centrifuge at 4°C, 12000 rpm for 5 minutes, and pour off the supernatant;

[0042] (4) Wash the precipitate with 75% ethanol, gently shake and wash repeatedly, centrifuge at 10,000 rpm at 4°C for 10 minutes, discard the su...

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Abstract

The invention discloses a novel method for extracting recalcitrant plant tissue DNA. In this method, an extract buffer for use is sodium laurate extract buffer. The method has the advantages that an anionic surfactant good in biological degradability, sodium laurate, is used as an extract buffer, secondary substances such as polysaccharides, pigments and polyphenols present in pear tissues can be eluted, the method does not require water bath and thereby is time efficient and simple to perform, degradation of DNA in an extraction process is maximally reduced, DNA harvest rate is high, harvested DNA is complete, and the defect that the quantity of DNA extracted with a kit is low, DNA extraction by CTAB (cetyl trimethyl ammonium bromide) extraction takes a long time with excess impurity is overcome.

Description

technical field [0001] The invention belongs to the technical field of DNA extraction, and relates to a new method for extracting DNA from recalcitrant plant tissues. Background technique [0002] DNA is the basic genetic material and the carrier of genetic information. A certain amount and high-quality DNA samples are the basis for restriction enzyme digestion, gene cloning, molecular hybridization, genetic polymorphism analysis and genomics and other molecular biology research. Therefore, obtaining high-quality and high-quantity DNA is extremely important. Due to the differences in the source, location, shape and other external properties of different plants or even the same type of plant tissue materials, as well as the differences in the internal characteristics such as chemical composition and tissue structure, there are obvious differences in the difficulty of DNA extraction. The difficulty of extracting DNA from most cereals and vegetable plants is relatively lower ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 殷豪吴潇张绍铃齐开杰吴俊
Owner NANJING AGRICULTURAL UNIVERSITY
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