Quick extracting method for large fragment sponge macro genome DNA

A technique of metagenomic and extraction method, which is applied in the field of rapid extraction of large fragments of sponge metagenomic DNA, can solve the problems of difficulty in extracting large fragments of DNA from sponge metagenomic, and achieves the effect of a simple method.

Inactive Publication Date: 2006-08-23
SHANGHAI JIAO TONG UNIV
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Problems solved by technology

[0004] The unique physiological structure of sponges and the huge amount of genetic information determine the difficulty of extracting large fragments of DNA from sponge metagenomics. At present, there is no convenient technology for extracting large fragments of DNA from sponge metagenomics.

Method used

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  • Quick extracting method for large fragment sponge macro genome DNA
  • Quick extracting method for large fragment sponge macro genome DNA

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Embodiment Construction

[0014] The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0015] Realize the technical route of the present invention such as figure 1 Said, first obtain the sponge sample, mechanically and chemically disperse the sponge tissue to obtain mixed cells, and then digest to break the cell wall to release DNA for the extraction of metagenomic DNA.

[0016] Specific embodiments of the present invention are as follows:

[0017] 1. Spongy tissue dispersion and mixed cell collection

[0018] (1) Cut out a little spongy tissue with a sterile scalpel, put it into a 5ml centrifuge tube with an appropriate amount of sterile calcium- and magnesium-free artificial seawater (CMFSW), stir it slightly with a large-caliber pipette tip, and blow it properly;

[0019] (2) Tear into 1-3mm with sterile blunt tweezers 3 Suspend the small pieces in the original suspension and blow them with a large-caliber...

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Abstract

The quick extracting process of large fragment sponge macro genome DNA includes the following steps: soaking sponge in artificial sea water without Ca and Mg, tearing the sponge into small blocks of 1-3 cu mm, soaking to disperse sponge cell and epicole microbes inside the solution, stilling, taking the mixed cell suspension from the upper layer; adding alkali wall-breaking buffer solution comprising EDTA and sodium dodecyl sarcosinate and Ca2+ containing proteinase K solution to digest cell wall; extracting DNA with the mixed solution of Tris-saturated phenol, chloroform and isoamyl alcohol and mixed solution of chloroform and isoamyl alcohol; adding sodium acetate to obtain DNA precipitate, washing with 70-75 % concentration alcohol ethanol solution, drying, dissolving in TE buffer solution, slaking RNA with RNase enzyme and detecting DNA in agar-sugar gel electrophoresis. The present invention is simple, fast and suitable for extracting sponge macro genome DNA with over 40000 bases.

Description

technical field [0001] The invention relates to a method for rapidly extracting large-segment sponge metagenomic DNA, which can be used for the construction of a metagenomic library and the screening of related functional genes or gene clusters. It belongs to the field of molecular technology of marine organisms. Background technique [0002] The sponge has a history of 600-800 million years ago and is a rather primitive multicellular animal. About 15,000 species of sponges are currently found, making up the second largest biomass in the ocean after corals. The sponge genome is large and mostly single-copy, with fewer introns. In addition, sponges are also rich in symbiotic microorganisms, covering archaea, bacteria, cyanobacteria, fungi and other microorganisms. Sponges are also one of the marine organisms with the most marine active substances discovered so far, which also reflects from the side the extremely rich genetic resources contained in the sponge. [0003] Met...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12N15/12
Inventor 李志勇吴杰
Owner SHANGHAI JIAO TONG UNIV
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