A method for extracting high molecular weight genomic DNA from environmental samples

An environmental sample, high molecular weight technology, applied in the field of molecular biology, to achieve simple operation, avoid mechanical shear damage, and good repeatability

Inactive Publication Date: 2011-12-07
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In order to solve the deficiencies in existing environmental sample genome extraction methods, the present invention provides a method for extracting high molecular weight genomic DNA from environmental samples

Method used

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  • A method for extracting high molecular weight genomic DNA from environmental samples
  • A method for extracting high molecular weight genomic DNA from environmental samples

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Embodiment Construction

[0024] Take activated sludge with a wet weight of about 1g, add 1mL of 1% low-melting point agarose kept at 50°C, mix well, and cool to solidify;

[0025] Put the gel piece into 10mL wall-breaking buffer, incubate at 37°C for 24h, and keep shaking at 50rpm horizontally during the period;

[0026] Pour off the wall-breaking buffer, add 10mL of digestion buffer, incubate at 37°C for 24h, and keep shaking at 50rpm horizontally during the period;

[0027] Pour off the digestion buffer, add 100mL washing solution to wash three times, and then wash once with 4°C electrophoresis buffer;

[0028] Cut the gel block digested by breaking the wall into a size of 8mm in length, 6mm in width and 2mm in height, insert it into the spotting hole of 0.7% low-melting point agarose gel, and seal the spotting hole with 0.7% low-melting point agarose gel After opening the gap, electrophoresis at a voltage of 5v / cm in the electrophoresis buffer for 4 hours at low temperature; after rotating the 0.7...

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Abstract

The invention belongs to the technical field of molecular biology, and relates to a method for extracting a high molecular weight genome DNA from an environmental sample. The method of the invention comprises steps of embedding, wall breaking, digestion, electrophoresis, gel recovery and dissolving. An environmental sample is suspended by a fluid suspension, added with low melting point agarose and cooled at room temperature for solidification; a gel block is put in a wall breaking buffer, incubated, and kept vibrating horizontally; the wall breaking buffer is discharged, and a digestion buffer is added to the gel block, incubated, and kept vibrating horizontally; the digestion buffer is discharged, and the gel block is washed by a washing liquid and then washed by an electrophoretic buffer; after wall breaking and digestion, the gel block is cut into small blocks and inserted into a sample application hole of the low melting point agarose gel; gap of the sample application hole is sealed by the low melting point agarose gel, and an electrophoresis is carried out in an electrophoretic buffer; the gel containing DNA is cut off after dyeing, and the DNA is recovered through an electroelution; and the DNA obtained after the electroelution is dried and dissolved in an aseptic deionized water or a TE solution, and a dissolving solution is the extracted DNA. The method of the invention has good repeatability, and operations are simple, so as to completely satisfy molecular operations of database establish, etc.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and relates to a method for extracting genome DNA from environmental samples. Background technique [0002] Various microorganisms play an extremely important role in the cycle of material elements and life processes in various environments on the earth, but because there are a large number of uncultivated microorganisms in the environment, the research on all microorganisms in the environment is more meaningful. At present, the study of all microorganisms in the environment has gone deep to the molecular level, mainly through various metagenomic techniques. Such as molecular cloning, RFLP analysis, DGGE analysis, library construction, etc., the development of these methods and technologies have put forward higher requirements for the size of DNA fragments. Generally speaking, to construct a genomic library, the initial DNA length must be more than 40kb, otherwise there are very few eff...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 李旭东郝纯杨俊仕刘庆华
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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