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High-efficiency extraction method of peanut leaf DNA

An extraction method and leaf technology, applied in the field of high-efficiency extraction of peanut leaf DNA, can solve the problems of high extraction cost, low extraction efficiency, and low cost, and achieve the effect of good DNA extraction quality, high extraction efficiency, and simple operation

Inactive Publication Date: 2018-03-23
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are two main techniques for extracting peanut tissue DNA: 1. Using biological kits, this method has high extraction quality, but the kits are expensive and the extraction cost is high; 2. Traditional CTAB method, this method is low in cost, but the extraction The effect is not good and cannot meet the requirements of subsequent experiments
At the same time, the above two methods require the use of liquid nitrogen during the sample grinding process, resulting in slow grinding speed and low extraction efficiency, and the quality of DNA will be greatly reduced due to the oxidation of phenolic substances in the leaves.

Method used

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  • High-efficiency extraction method of peanut leaf DNA

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Experimental program
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Effect test

Embodiment 1

[0022] A method for efficiently extracting peanut leaf DNA, comprising the steps of:

[0023] (1) Collection of peanut leaf samples: take about 0.2 g of fresh young leaves, and quickly put them into a 2ml centrifuge tube for cryopreservation;

[0024] (2) Grinding of peanut leaves: at room temperature, add 300ul 2wt% CTAB solution (add 1wt% β-mercaptoethanol before use) to a centrifuge tube, add steel balls, use a tissue grinder 60HZ to grind the sample for 60s (60s can grind the leaves Grind it fully while avoiding sample oxidation to the greatest extent), and then add 600ul CTAB solution (add 1wt% β-mercaptoethanol before use), and put it in a -20°C refrigerator for long-term storage;

[0025] (3) Water bath: put the sample solution obtained in step (2) into 65°C water for 30 minutes, and shake the sample solution once every 5-10 minutes;

[0026] (4) Use chloroform / isoamyl alcohol (volume ratio 24:1) solution for the first extraction: add an equal volume (900ul) of chlorof...

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Abstract

The invention belongs to the technical field of crops, and more specifically relates to a high-efficiency extraction method of peanut leaf DNA. The high-efficiency extraction method comprises following steps: peanut leaf samples are collected; CTAB solution is added into the collected peanut leaf samples in two steps; samples are smashed; the smashed peanut leaf samples are introduced into a waterbath pot for a certain time period of water bath; a chloroform / isoamyl alcohol solution is adopted for a first time of extraction; a phenol / chloroform / isoamyl alcohol solution is adopted for a secondtime of extraction; after extraction, an obtained supernate is collected, precipitation of DNA is carried out, and obtained DNA is washed and stored. The high-efficiency extraction method is capableof realizing high efficiency extraction of peanut leaf DNA at room temperature, and is especially suitable for extraction of DNA of large amounts of samples; extraction cost is relatively; extractiontime is short; batch extraction of a large amount of DNA can be realized; the quality of extracted DNA is basically the same with that of DNA extracted using DNA kits. Operation of the high-efficiencyextraction method is simple, and extraction cost is reduced by half or more of that of extraction using kits.

Description

technical field [0001] The invention belongs to the technical field of crops, and in particular relates to a method for efficiently extracting peanut leaf DNA. Background technique [0002] Peanut tissue DNA extraction is the first step in peanut genetic research and molecular biology experiments. The quality and efficiency of DNA extraction are directly related to the success or failure of subsequent experiments. Leaf tissue is the most commonly used material for DNA extraction. Phenolic compounds are one of the most diverse and widely distributed secondary metabolites in peanut leaves. These substances tend to interact with nucleic acids to form stable irreversible complexes when cells are broken. At present, there are two main techniques for extracting peanut tissue DNA: 1. Using biological kits, this method has high extraction quality, but the kits are expensive and the extraction cost is high; 2. Traditional CTAB method, this method has low cost, but the extraction Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 陈伟刚黄莉姜慧芳罗怀勇陈玉宁周小静刘念雷永
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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