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Method for efficiently extracting tropical plant DNA

A tropical plant, high-efficiency technology, applied in DNA preparation, recombinant DNA technology, etc., can solve the problems of high environmental damage and low safety of extraction process

Inactive Publication Date: 2011-11-23
INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The patent application number is 200910095919.2, which proposes a method of simultaneously extracting DNA and RNA from plants such as yams by means of CTAB. The reagents of this method contain harmful substances such as β-mercaptoethanol, and toxic substances such as chloroform and phenol are also used. Harmful substances have been removed, which is harmful to the environment and the safety of the extraction process is low

Method used

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  • Method for efficiently extracting tropical plant DNA
  • Method for efficiently extracting tropical plant DNA
  • Method for efficiently extracting tropical plant DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The extraction process of the DNA of Chrysanthemum chrysanthemum provided by this implementation is as follows:

[0031] (1) Take Chrysanthemum chrysanthemum leaves as raw materials, collect the young leaves, mature leaves and old leaves of the same plant and mix them into one sample, store them in liquid nitrogen immediately, and then use a sterile grinding rod to place them in a liquid nitrogen container. The leaves were ground into powder in a mortar, and about 100 mg of tissue powder was taken from each sample for DNA extraction, and the remaining tissue powder was stored in a -80°C refrigerator for later use;

[0032](2) Preheat the lysis buffer of the present invention at 65°C;

[0033] (3) Add about 100 mg of ground powder into a 2.0 mL sterile EP tube, immediately add 1.0 mL of the lysis buffer of the present invention, and mix well;

[0034] (4) Put the above lysis buffer in a 65°C water bath for 30 minutes, and mix it every 5 minutes;

[0035] (5) Centrifug...

Embodiment 2

[0041] In this embodiment, the leaves of the king coconut tree are used as raw materials, and young leaves, mature leaves and old leaves of the same plant are collected and mixed into a sample, which is immediately stored in liquid nitrogen. Afterwards, the leaves were ground to a powder using sterile grinding rods in a mortar filled with liquid nitrogen. About 100 mg of tissue powder was taken from each sample for DNA extraction. The remaining tissue powders were stored in a -80°C refrigerator for future use.

[0042] The extraction steps are as follows:

[0043] (1) Preheat the lysis buffer of the present invention at 60°C,

[0044] (2) Put about 1 g of fresh plant leaves into a mortar filled with liquid nitrogen and quickly grind them to powder;

[0045] (3) Add about 100 mg of ground powder into a 2.0 mL sterile EP tube, immediately add 2.0 mL of the lysis buffer of the present invention, and mix well;

[0046] (4) Water bath at 60°C for 40 minutes, and mix well every ...

Embodiment 3

[0053] In this embodiment, the leaves of the papaya tree are used as raw materials, and young leaves, mature leaves and old leaves of the same plant are collected and mixed into a sample, which is immediately stored in liquid nitrogen. Afterwards, the leaves were ground to a powder using sterile grinding rods in a mortar filled with liquid nitrogen. About 100 mg of tissue powder was taken from each sample for DNA extraction. The remaining tissue powders were stored in a -80°C refrigerator for future use.

[0054] The extraction steps are as follows:

[0055] (1) Preheat the lysis buffer of the present invention at 70°C;

[0056] (2) Put about 1 g of fresh plant leaves into a mortar filled with liquid nitrogen and quickly grind them to powder;

[0057] (3) Add about 100 mg of ground powder into a 2.0 mL sterile EP tube, immediately add 1.5 mL of the lysis buffer of the present invention, and mix well;

[0058] (4) 70°C water bath for 20 minutes, and mix once every 5 minutes...

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Abstract

The invention discloses a method for efficiently extracting tropical plant DNA. The method comprises the following steps: preparing leaves, roots, stems or fruits of a tropical plant, crushing, placing in a sterile container, adding a cracking buffer solution used in the invention, uniformly mixing; performing the water bath reaction for 20-40 min when adjusting the temperature to 60-70 DEG C, centrifuging mixed solution obtained after the reaction, taking supernatant, adding the isopropanol to deposit DNA, centrifuging, abandoning the supernatant, taking the precipitate, washing 2-5 times byusing an organic solvent, centrifuging after each washing, abandoning the supernatant, drying the precipitate obtained after centrifugation to obtain the total DNA extracted from the tropical plant. The extraction method has security and friendliness because no toxic or harmful component such as beta-mercaptoethanol, chloroform and phenol is used; and the extraction method has the advantages of universality, high efficiency, security and convenience.

Description

technical field [0001] The invention relates to a method for extracting DNA, in particular to a method for efficiently extracting DNA from tropical plants. Background technique [0002] High-quality DNA is an important guarantee for molecular biology research. So far, many methods for extracting DNA have been proposed, such as the method based on chemical solution extraction and the method of column adsorption extraction (An Zewei et al., 2009). However, different materials will have specific requirements for DNA extraction methods, especially for some recalcitrant and rare materials (Hasan, et al, 2008; Rogers and Bendich, 1985; Tang, et al , 2009). Tropical plants are rich in cellulose, polysaccharides, polyphenols, proteins, fats and other substances that make it difficult to lyse cells and isolate and purify nucleic acids, thus increasing the difficulty of DNA extraction (Wang, et al, 2008). At present, although researchers have reported some methods for extracting DN...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 黄启星王旭初孔华郭运玲郭安平
Owner INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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