Kit for rapidly extracting sludge microbial genome DNA and extracting method

A kit and technology for microorganisms, which are applied in the field of rapid extraction of microbial genome DNA from sludge and the field of extraction, can solve the problem that the extraction method has not been reported too much.

Inactive Publication Date: 2015-04-29
FUJIAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The key to analyzing sludge microorganisms with molecular biology methods is the extraction of DNA, but there have not been many reports on the extraction methods of microbial DNA in sludge

Method used

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  • Kit for rapidly extracting sludge microbial genome DNA and extracting method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The kit for rapid extraction of sludge microbial genomic DNA consists of a DNA adsorption column and the following working solution:

[0047] Working solution A: from the final concentration of 0.1mol / L trishydroxymethylaminomethane (Tris), the final concentration of 0.1mol / L ethylenediaminetetraacetic acid (EDTA), the final concentration of 0.1mol / L sodium phosphate (Na 3 PO 4 ), a final concentration of 1.5mol / L sodium chloride (NaCl) and a final concentration of 1% cetyltrimethylammonium bromide (CTAB), pH=8.0;

[0048] Working solution B: sodium dodecyl sulfate (SDS) with a mass concentration of 20%;

[0049] Working solution C: the volume ratio is a mixture of phenol: chloroform: isoamyl alcohol=25:24:1;

[0050] Working solution D: the volume ratio is the mixed solution of chloroform:isoamyl alcohol=24:1;

[0051] Precipitating agent: mass concentration 70% ethanol;

[0052] Protein-removing solution: composed of guanidine hydrochloride at a final concentratio...

Embodiment 2

[0056] The kit for rapid extraction of sludge microbial genomic DNA consists of a DNA adsorption column and the following working solution:

[0057] Working solution A: from the final concentration of 0.3mol / L trishydroxymethyl aminomethane (Tris), the final concentration of 0.3mol / L ethylenediaminetetraacetic acid (EDTA), the final concentration of 0.13mol / L sodium phosphate (Na 3 PO 4 ), a final concentration of 1mol / L sodium chloride (NaCl) and a final concentration of 1.5% cetyltrimethylammonium bromide (CTAB), pH=7.0;

[0058] Working solution B: sodium dodecyl sulfate (SDS) with a mass concentration of 10%;

[0059] Working solution C: the volume ratio is a mixture of phenol: chloroform: isoamyl alcohol=25:24:1;

[0060] Working solution D: the volume ratio is the mixed solution of chloroform:isoamyl alcohol=24:1;

[0061] Precipitating agent: mass concentration 75% ethanol;

[0062] Protein-removing solution: composed of guanidine hydrochloride at a final concentrat...

Embodiment 3

[0066] The kit for rapid extraction of sludge microbial genomic DNA consists of a DNA adsorption column and the following working solution:

[0067] Working solution A: from the final concentration of 0.5mol / L trishydroxymethylaminomethane (Tris), the final concentration of 0.5mol / L ethylenediaminetetraacetic acid (EDTA), the final concentration of 0.5mol / L sodium phosphate (Na 3 PO 4 ), a final concentration of 2mol / L sodium chloride (NaCl) and a final concentration of 2% cetyltrimethylammonium bromide (CTAB), pH=9.0;

[0068] Working solution B: sodium dodecyl sulfate (SDS) with a mass concentration of 30%;

[0069] Working solution C: the volume ratio is a mixture of phenol: chloroform: isoamyl alcohol=25:24:1;

[0070] Working solution D: the volume ratio is the mixed solution of chloroform:isoamyl alcohol=24:1;

[0071] Precipitating agent: mass concentration 80% ethanol;

[0072] Protein-removing solution: composed of guanidine hydrochloride at a final concentration ...

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Abstract

The invention discloses a kit for rapidly extracting sludge microbial genome DNA and an extracting method. The sludge microbial genome DNA is extracted by the following steps: breaking sludge microbial cells, removing impurity proteins, adsorbing DNA by using a DNA adsorption column, and removing impurities and eluting the DNA. The conventional method for extracting the sludge microbial genome DNA often uses a method for precipitating the genome by using anhydrous ethanol, the operating time is above 12 hours, and the genome DNA is easily degraded. The kit mainly uses a silicone mould adsorption column for absorbing the genome DNA, and then uses a specific protein-removing solution and a specific bleaching solution for removing the proteins and other impurities; the kit can extract the sludge microbial genome DNA within 4 hours, so that the kit is more convenient and quicker.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a kit and an extraction method for rapidly extracting the genome DNA of sludge microorganisms. Background technique [0002] The metabolic activities of microorganisms in the sludge oxidize and decompose the organic matter in the water into inorganic matter, thereby achieving purification. The microorganisms in the sludge are mainly composed of bacteria, which can account for about 90-95% of the total weight of microorganisms in the sludge. Bacteria play an important role in the purification of organic pollutants. The microorganisms in the sludge mainly act as decomposers, which can decompose organic matter into inorganic matter, restore them to nature, and provide inorganic nutrients for plant survival. [0003] The traditional method of isolating and cultivating microorganisms is the earliest method used in the study of sludge microbial diversity. However, the bacteri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 王清水余彦
Owner FUJIAN NORMAL UNIV
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