Method for extracting high-molecular-weight genome from animal feces

An animal feces, high molecular weight technology, which is applied in the field of molecular biology of microorganisms, can solve the problems that the molecular weight of genomic DNA is difficult to reach more than 40kb, and the large fragment metagene library cannot be constructed, and the method is simple and convenient.

Inactive Publication Date: 2012-07-18
YUNNAN NORMAL UNIV
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Problems solved by technology

Although the genomic DNA extracted by these methods can meet the needs of microbial molecular ecological diversity research, molecular identification, and target gene amplification, the molecular weight of the obtained genomic DNA is generally difficult to reach more than 40kb, so it cannot be used for large-fragment metagenomic libraries. Construct

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  • Method for extracting high-molecular-weight genome from animal feces
  • Method for extracting high-molecular-weight genome from animal feces

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Embodiment

[0018] 1. Sample processing

[0019] (1) Under sterile conditions, weigh 1g of the feces sample of the Japanese slow loris, transfer it into a sterile centrifuge tube with a volume of 50ml, add 15ml of sterile PBS buffer, vortex fully, centrifuge at 100g for 15min, and discard the precipitate;

[0020] (2) Filter the supernatant with a sterilized cell filter with a pore size of 100 μm, wash the impurities trapped on the filter with sterile PBS buffer several times, and collect the filtrate;

[0021] (3) The filtrate was filtered with a sterile cell filter with a pore size of 70 μm, and the impurities trapped on the filter were washed with sterile PBS buffer several times, and the filtrate was collected;

[0022] (4) Filter the filtrate with a sterilized cell filter with a pore size of 40 μm, and wash the impurities trapped on the filter with sterile PBS buffer several times, collect the filtrate, and centrifuge at 5000g for 10 minutes to collect the bacterial precipitate;

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Abstract

The invention relates to a method for extracting high-molecular-weight genome from animal feces. The method comprises the steps of sample treatment, bacterial cell lysis and extraction of DNA (Deoxyribonucleic Acid). Specifically, the method comprises the steps of: dissolving an animal feces sample with aseptic PBS (Phosphate Buffered Saline) buffer, carrying out low-speed centrifuging and filtering to remove food residues in the feces, collecting bacterial cells, performing wall breaking on the bacteria by adopting lysozyme, decolorizing peptidase, protease and SDS (Sodium Dodecyl Sulfate), extracting with a mixture of phenol, chloroform and isoamyl alcohol as well as a mixture of chloroform and isoamyl alcohol, precipitating with isopropanol and sodium acetate, washing and drying, dissolving, and digesting RNA (Ribonucleic Acid) with RNAse (Ribonuclease). According to the invention, the method is simple in operation; and the DNA molecular weight of the extracted genome achieves more than 40kb, so that the researches on microorganism molecule ecological diversity, molecular identification, target gene amplification, macro genome library construction and the like are met.

Description

technical field [0001] The invention relates to the technical field of molecular biology of microorganisms, in particular to a method for extracting high molecular weight genomes from animal feces. Background technique [0002] The large and diverse microbial community in the gastrointestinal tract of animals is closely related to the feeding habits of animals, the immune function of the body, diseases and health, and has attracted more and more attention and has become a research hotspot. However, the destructive collection of gastrointestinal samples by traditional methods is greatly restricted, and it is not advisable for wild animals, so many studies use animal feces as research samples. [0003] At present, molecular biology technology has become the core technology for the research and development of animal gastrointestinal microbial resources, and the extraction of high-quality, complete and representative genomic DNA from fecal samples is the basis of the entire tech...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 许波杨富亚黄遵锡杨云娟李俊俊唐湘华
Owner YUNNAN NORMAL UNIV
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