Extracting method of total DNA of microorganisms in intestines of earthworms
A technology of intestinal microorganisms and extraction methods, which is applied in the field of extraction of total DNA of intestinal microorganisms in earthworms, can solve the problems of low extraction efficiency, affecting the extraction efficiency of total microbial DNA, and inability to overcome the interference of earthworm intestinal tracts, so as to improve the extraction efficiency, Improve the extraction efficiency and extraction quality, and overcome the effect of interference
Inactive Publication Date: 2016-08-03
NANJING UNIV
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Problems solved by technology
However, earthworm intestines contain a large amount of sediment, which seriously affects the extraction efficiency of total microbial DNA.
At present, there are few studies on the method of extracting the total DNA of earthworm intestinal microorganisms, and special kits for extracting feces are often used, such as: DNAStoolMiniKit, Qiagen, etc., these methods cannot overcome the interference caused by a large amount of sediment in the intestines of earthworms, and the extraction efficiency is low
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Embodiment 1
[0021] The method for extracting total DNA of earthworm gut microbes of the present invention includes the following steps:
[0022] Step 1. Place a piece of experimental earthworm on clean filter paper, collect the fresh feces of the earthworm, and store it in freezing at 4℃;
[0023] Step 2. After returning the earthworm to its original growth environment for 12 hours, place it on clean filter paper again to collect its fresh feces; mix the feces extracted twice and store them at 4°C for freezing;
[0024] Step 3. Take 0.2g of fresh earthworm feces (feces that are frozen and stored for no more than 24 hours) and place them in a centrifuge tube, add 20μL proteinase K and 100μL buffer, and use a shaker to shake for 2 minutes to mix, mix well and then at 56℃ Heat in a water bath for 60 minutes to obtain liquid phase A;
[0025] Step 4. Use the soil DNA extraction kit ( SpinKitforSoil kit) to extract DNA to obtain the total DNA of earthworm intestinal microorganisms;
[0026] Step 5. Us...
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The invention discloses an extracting method of total DNA of microorganisms in intestines of earthworms. According to the extracting method, on one hand, fresh excreta of the earthworms serves as a sample, and the sample is subjected to pretreatment; on the other hand, proteinase (digestion) K and a soil DNA extraction kit are combined for use; therefore, by means of the extracting method, the problem that due to the fact that an existing method cannot overcome sediment interference, the extracting efficiency of the total DNA of the microorganisms in the intestines of the earthworms is low is solved. According to the extracting method, the extracting efficiency and extracting quality of the total DNA of the microorganisms in the intestines of the earthworms are greatly improved, a total DNA strip obtained through 1% agarose gel electrophoresis detection is bright and clear, and therefore the condition of the microorganisms in the intestines of the earthworms can be reflected more accurately and comprehensively, and a foundation is laid for subsequent microorganism community structure and function research.
Description
Technical field [0001] The invention relates to a method for extracting total DNA of earthworm intestinal microorganisms, and belongs to the field of biotechnology science. Background technique [0002] Due to the unreasonable use of fertilizers and pesticides in agriculture and the discharge of industrial pollution, soil pollution has become an increasingly serious environmental problem in my country. As the most common group of soil animals, earthworms are important biomarkers for soil pollution detection and also important animals for soil bioremediation. As an important decomposer of the ecosystem, the ability of earthworms to digest and decompose organic matter in the soil is a strong guarantee for the material cycle of the ecosystem. The unique microbial community in the intestine of earthworms plays an important role in this process. In the research process of the intestinal microorganisms of earthworms, the extraction of total microbial DNA is particularly important. Ho...
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CPCC12N15/1003
Inventor 吴兵尹金宝张徐祥刘苏
Owner NANJING UNIV