Method for extracting DNA from acidic mine water bed mud in coal mine

An extraction method and acid technology, applied in the field of molecular biology, can solve the problems of affecting PCR molecular biology operations, low extraction efficiency, and many humic acids in the extraction products.

Inactive Publication Date: 2010-02-17
HENAN INST OF ENG
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Problems solved by technology

[0005] The purpose of the present invention is to apply biochemistry and molecular biology technology to provide a method for efficiently extracting and purifying the DNA of acidic mine bottom mud in coal mines, so as to solve the problem that conventional DNA extraction kits are not efficient in extracting DNA from the acidic mine bottom mud of coal mines. Carry out subsequent DNA purification work and extract more humic acid substances, which will affect the follow-up PCR and other molecular biological operations, so as to be better applied to the molecular ecology research on the diversity of acid mine bottom mud in coal mines

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0013] 1. Collection of microorganisms in coal mine acid mine mud samples

[0014] Select the coal mine acidic mine bottom mud site, take the coal mine acidic mine bottom mud by five-point sampling method, add 100ml of phosphate buffer eluent containing Tween (Tween 80 or 40) pH5.0-7.0, 37 ° C constant temperature oscillator Oscillate for 30 minutes, ultrasonically oscillate for 6 minutes, shake vigorously every two minutes, collect the suspension after ultrasonic oscillation, centrifuge at 1000g for 10 minutes, and collect the supernatant.

[0015] 2. Membrane filter bacteria:

[0016] The 0.5 μm membrane was filtered under reduced pressure to collect the membrane.

[0017] 3. Rupture of cells

[0018] Add 4.5mL lysis buffer, 20μL 20mM helicase, 20μL 20mM lysozyme, 37°C, 250rpm constant temperature shaking for 30 minutes, remove and add 20μL 20mM Proteinase K, 37°C, 250rpm constant temperature shaking for 30 minutes, add 0.5mL 20% SDS ( Sodium dodecylsulfonate), bathed in ...

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Abstract

The invention relates to a method for extracting DNA from acidic mine water bed mud in a coal mine. Buffer solution elution, supersonic oscillation and membrane filtration for thallus collection are adopted in the method, and cell lysis is performed to extract DNA. The extracted DNA can be applied to such molecular biology technologies as PCR amplification, DGGE and TRFLP and the like for analyzing microbial communities in the acidic mine water bed mud in the coal mine without purification. The method has the advantages of being simple and rapid, and being capable of truly reflecting diversityof different microbial populations in the acidic mine water bed mud in the coal mine.

Description

technical field [0001] The invention relates to the extraction and purification of DNA, in particular to a method for extracting DNA from acid mine bottom mud in coal mines, and belongs to the technical field of molecular biology. technical background [0002] During the mining process of coal mines, due to changes in the environment of sulfides (mainly pyrite) contained in coal-bearing strata, redox reactions occur spontaneously, which can lead to acid mine drainage. Acid mine drainage has the characteristics of low pH value and high salinity, and is highly soluble and erosive. This kind of mine drainage can carry a large amount of heavy metals and harmful chemicals into the environment. Some harmful substances in coal mine acid wastewater will also be deposited in the river sediment, and the long-term release of various pollutants in the sediment will also affect the water quality of the downstream water body. At present, many studies have been carried out on the influenc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/04C12N15/10
Inventor 张建云崔树军谷立坤赵保生廉有轩金维平吴烨武秀琴张庆甫刘道伟
Owner HENAN INST OF ENG
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