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Plant disease bacteria total DNA extracting method

A plant disease and extraction method technology, applied in the field of molecular biology experiments, can solve the problems of extraction liquid pollution, purity, concentration, low yield, unstable results, etc., to prevent phenol residues, improve DNA yield and purity, and experiment The result is a stable effect

Pending Publication Date: 2019-10-18
湖州三零科技股份有限公司
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  • Abstract
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Problems solved by technology

At present, there are many methods for extracting genomic DNA of plant pathogenic bacteria, but they are easily polluted by impurities such as extracts, and the extraction purity, concentration, and yield are not high, which makes the results of subsequent sequencing experiments unstable. Therefore, the present invention is proposed.

Method used

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Embodiment

[0036] Embodiment: 1. Take the thalline sample and mix it in LB+0.4% Gly liquid medium for 48 hours, then use a 50 mL sterilized centrifuge tube at 4000 rpm to collect the thalli. Wash the cells with 15 mL of STE buffer to obtain a suspension.

[0037] 2. Take 0.7g suspension, add 15mL STE buffer, vortex and mix; weigh lysozyme powder and dissolve it in the suspension to make the final concentration 3mg / mL, mix well; put in 37℃, 200rpm constant temperature incubator The mixture was reacted for 3.5h to obtain the mixed solution I.

[0038] 3. After the reaction, the bacterial solution is in the form of a uniform powder. Add 200 μL of 10 mg / mL proteinase K and 1.5 mL of 20% SDS at the same time. After mixing, put it in a 55 ° C water bath for more than 1 hour, and shake it every 10 minutes.

[0039] 4. Add 3mL of 5M NaCl and mix thoroughly so that the final concentration of NaCl is 0.8M. Add 1.95mL CTAB / NaCl solution, mix thoroughly and place in a water bath at 68°C for 10min....

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Abstract

The invention relates to the technical field of molecular biology experiments, in particular to a plant disease bacteria total DNA extracting method. The plant disease bacteria total DNA extracting method comprises the steps that a bacteria sample is subjected to culture by using a fluid medium, dissolving by using lysozyme, DNA separation by using a CTAB / NaCl solution and multiple chloroform andphenol-chloroform extraction to obtain an extracting solution containing DNA, and impurities in the extracting solution are removed to obtain the disease bacteria total DNA. The DNA extracting methodis good in cell wall breaking effect, the phenol-chloroform layered extraction rate is high, and the method is especially suitable for extracting the DNA of pathogenic bacteria of fruits and vegetables, such as rice, wheat, tomato and cucumber.

Description

technical field [0001] The invention relates to the technical field of molecular biology experiments, in particular to a method for extracting total DNA of plant pathogenic bacteria. Background technique [0002] Modern biotechnology provides an important means to further understand the nature of life activities and make better use of its laws to serve human beings. However, DNA isolation is the first and very important step in the successful use of this technology. The quality of DNA directly determines the success or failure of subsequent experiments. At present, there are many methods for extracting genomic DNA of plant pathogenic bacteria, but they are easily contaminated by impurities such as extracts, and the extraction purity, concentration, and yield are not high, which makes the results of subsequent sequencing experiments unstable. Therefore, the present invention is proposed. Contents of the invention [0003] The object of the present invention is to provide a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 王琦旻
Owner 湖州三零科技股份有限公司
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