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Method for extracting metagenome DNA from endosymbiotic bacterium mycetocyte of bemisia tabaci

A technology of metagenomics and symbiotic bacteria, which is applied in the field of extracting metagenomic DNA, can solve the problems of difficult extraction, and achieve the effect of easy implementation, economical cost saving and simple implementation

Inactive Publication Date: 2014-12-10
YANAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to provide a method for extracting metagenomic DNA from endosymbiont cells of Bemisia tabaci, which solves the problem in the prior art that it is difficult to extract high-purity DNA with a molecular size of more than 30kb from a small amount of microbial samples. The Problem with Microbial Metagenomic DNA

Method used

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  • Method for extracting metagenome DNA from endosymbiotic bacterium mycetocyte of bemisia tabaci

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] step 1:

[0055] The endosymbiont bacteria cell extract containing Bemisia tabaci was used denaturing solution (denaturing solution consisted of: 4M guanidine isothiocyanate, pH 7.0 10mM Tris-HCl buffer, 1mM EDTA, volume final concentration of 0.5 % 2-mercaptoethanol) treatment, the wet weight-volume ratio of the whitefly endosymbiont bacterium cell extract to the denaturation solution is 1g: 1mL to obtain a mixed solution;

[0056] Step 2:

[0057] Add nucleic acid extraction buffer (nucleic acid extraction buffer consists of: 10mM sodium phosphate buffer with pH 7.5, 10mM Tris-HCl buffer with pH 7.5, 10mM Tris-HCl buffer with pH 8.0) in the sample processed in step 1. EDTA, the NaCl of 1.5M, the CTAB that mass final concentration is 1%, the sucrose that mass final concentration is 10%) and enzyme (enzyme is proteinase K and lysozyme use simultaneously, wherein the amount that adds proteinase K is after adding proteinase K Final concentration reaches 30 μ g / mL, and t...

Embodiment 2

[0067] step 1:

[0068] The endosymbiont bacterial cell extract containing Bemisia tabaci was denatured solution (denaturation solution is composed of: 6M guanidine isothiocyanate, pH is 7.5 10mM Tris-HCl buffer solution, 1mM EDTA, volume final concentration is 0.5 % 2-mercaptoethanol) treatment, the wet weight-volume ratio of the whitefly endosymbiont bacterium cell extract to the denaturation solution is 1g: 2mL to obtain a mixed solution;

[0069] Step 2:

[0070] Add nucleic acid extraction buffer (nucleic acid extraction buffer consists of: 10mM sodium phosphate buffer with pH 7.5, 10mM Tris-HCl buffer with pH 7.5, 10mM Tris-HCl buffer with pH 8.0) in the sample processed in step 1. EDTA, the NaCl of 1M, the CTAB that mass final concentration is 1%, the sucrose that mass final concentration is 5%) and enzyme (enzyme is that proteinase K and lysozyme are used simultaneously, and wherein the amount of adding proteinase K is that after adding proteinase K, its final Concen...

Embodiment 3

[0080] step 1:

[0081] The endosymbiont bacteria cell extract containing Bemisia tabaci was used denaturing solution (denaturing solution consisted of: 5M guanidine isothiocyanate, pH 8.0 10mM Tris-HCl buffer, 1mM EDTA, the volume final concentration was 0.5 % 2-mercaptoethanol) treatment, the wet weight-volume ratio of the whitefly endosymbiont bacterium cell extract to the denaturation solution is 1g: 1.5mL to obtain a mixed solution;

[0082] Step 2:

[0083] Add nucleic acid extraction buffer (nucleic acid extraction buffer consists of: 10mM sodium phosphate buffer with pH 7.5, 10mM Tris-HCl buffer with pH 7.5, 10mM Tris-HCl buffer with pH 8.0) in the sample processed in step 1. EDTA, the NaCl of 2M, the CTAB that mass final concentration is 1%, the sucrose that mass final concentration is 8%) and enzyme (enzyme is that proteinase K and lysozyme are used simultaneously, and wherein the amount of adding proteinase K is its final after adding proteinase K Concentration re...

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Abstract

The invention discloses a method for extracting metagenome DNA from endosymbiotic bacterium mycetocyte of bemisia tabaci. The method comprises the following steps: treating a mycetocyte extract containing the endosymbiotic bacteria of the bemisia tabaci by using a denaturing solution, adding protease and lysozyme to crack, adding an SDS solution to treat, adding an extraction agent, precipitating the extracted supernate by using isopropanol and finally purifying by using a nucleic acid adsorption resin to obtain the DNA. The method for directly extracting the metagenome DNA, disclosed by the invention, is simple in whole process, needs no valuable drugs and is relatively economic and practical; by means of the method, high-purity microbiological metagenome DNA with the molecular size above 30kb can be extracted from the endosymbiotic bacterium mycetocyte of the bemisia tabaci; and the method is particularly suitable for the extraction of the metagenome DNA from endosymbiotic bacterium extracts of insects and is also suitable for the extraction of the metagenome DNA from some other microbiological samples at the same time.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a method for extracting metagenomic DNA from a small amount of microbial samples, in particular to a method for extracting metagenomic DNA from endosymbiotic bacterial cells of whitefly whitefly. Background technique [0002] Endosymbionts are closely related to insect host biology in nature, and the interaction between whitefly and its endosymbiotic bacteria has attracted extensive attention. prevention and treatment. However, the "bottleneck" of the current research on the function of endosymbionts is that in vitro pure culture cannot be achieved, so little is known about the function of endosymbionts in Bemisia tabaci. Removal of endosymbiotic bacteria in insects is an indirect method for the study of endosymbiont function, but heat treatment, lysozyme treatment, and antibiotic treatment are largely inapplicable. The application of molecular biology technology based ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 赵瑞华贺晓龙
Owner YANAN UNIV
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