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Extracting method for total DNA of termite alcohol sample enteric microorganism genome

A technology of intestinal microorganisms and extraction methods, which is applied in the field of extraction of total DNA of intestinal microorganism genomes from termite alcohol samples, can solve the problems that the intestinal tract cannot be directly stripped, the extraction of total intestinal microbial DNA is increased, and cannot be applied, and achieves the preservation of abundant DNA. Degree and diversity, improve the effect of quality and integrity

Inactive Publication Date: 2018-04-13
GUANGDONG INST OF APPLIED BIOLOGICAL RESOURCES
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Problems solved by technology

Among them, the cell wall of Gram-positive bacteria is usually difficult to be directly broken by cell lysate, and a large number of Gram-negative bacteria parasitize the protoflagellates, making the commonly used genomic DNA extraction kits unsuitable
In addition, termite samples collected in the field are often soaked in alcohol, and the intestines cannot be directly stripped, which adds great difficulty to the extraction of the total DNA of intestinal microorganisms

Method used

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  • Extracting method for total DNA of termite alcohol sample enteric microorganism genome
  • Extracting method for total DNA of termite alcohol sample enteric microorganism genome
  • Extracting method for total DNA of termite alcohol sample enteric microorganism genome

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Embodiment 1

[0021] 1. Take 30 1-year-old Coptotermes worker ants soaked in 95% alcohol by volume, absorb the surface alcohol with filter paper, and air-dry for 5 minutes, then transfer to 2mL 20mM Tris-EDTA (20mM Tris-HCl, 1mM EDTA, pH 8.0) buffer solution, soak in an ice bath for 2 hours until the space between the termite epidermis and intestinal tract is filled with buffer solution and appears translucent, and the worker ants after soaking and softening are obtained;

[0022] 2. Soak the softened worker ants in Tris-EDTA solution, first remove the head and chest at the position of the second pair of feet under the dissecting microscope, then tear the epidermis from the abdomen, and finally peel off the complete solidified tissue blocks of the midgut and hindgut ( figure 1 ), transfer this tissue block into 700 μL 20mM Tris-EDTA (20mM Tris-HCl, 1mM EDTA, pH8.0) buffer;

[0023] 3. Use steel balls to vibrate the homogenizer to rupture intestinal epidermal cells, the vibration frequency ...

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Abstract

The invention discloses an extracting method for a total DNA of a termite alcohol sample enteric microorganism genome. Termite tissues soaked with alcohol are softened again by a Tris-EDTA solution, intestinal tissue epidermis is oscillated and broken at low speed by a tissue homogenizer to release enteric microorganisms, cell walls of bacteria are treated by lysozyme, microbial cells are fully split with the aid of protease K and SDS, DNA is released fully, and finally, the extracting method is used with a DNA extracting universal kid. Therefore, by the extracting method, the problems that byan existing method, complete intestinal structures are difficultly obtained from termite alcohol samples, due to diversity of intestinal microbes, splitting of microbial cells is incomplete, and DNAreleasing efficiency is low are solved. By the extracting method, abundance and diversity of termite alcohol sample enteric microorganisms are retained to a maximum degree, and therefore, quality andcompleteness of extraction of total DNA of communities are improved.

Description

Technical field: [0001] The invention belongs to the field of DNA extraction, and in particular relates to a method for extracting the total DNA of intestinal microbial genomes of termite alcohol samples. Background technique: [0002] Termites are the most important carbon cycle organisms in the earth's ecological environment, and their utilization rate of lignocellulose can reach more than 97%. For termites to be able to have such an efficient lignocellulosic digestive system, the microbial symbionts in their hindgut play a crucial role. The study of termite intestinal microbial community is the only way to elucidate its digestive mechanism and the construction of in vitro biomimetic digestive system. Most of these intestinal microorganisms cannot be purified and cultured in vitro under the current technical conditions. Therefore, molecular biology techniques based on DNA analysis are an important means to study the intestinal microbial community of termites. [0003] Us...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 曾文慧钟俊鸿李秋剑刘炳荣胡少芳
Owner GUANGDONG INST OF APPLIED BIOLOGICAL RESOURCES
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