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Kit for extracting microbial genome DNA from soil and its method

A technology of microorganisms and kits, which is applied in the field of kits for extracting microbial genomic DNA, which can solve the problems of high impurities in DNA samples and low degree of cell lysis, and achieve the effects of comprehensive information, less loss of genomic DNA, and low cost

Inactive Publication Date: 2008-08-13
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a kit and method for rapidly and efficiently extracting microbial genomic DNA from soil samples, so as to solve the problem of DNA samples obtained by conventional extraction methods when using molecular means to study soil microbial communities. PCR inhibitors (mainly humic substances) and other issues

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Make the soil microbial genome DNA extraction kit according to the following formula:

[0043] Reagent A: immersion solution, components: 200mM Tris, 100mM EDTA, 100mM NaCl, 2mM sodium citrate, 10mM CaCl 2 , 0.5% Tween-80, 0.5% glycerin, pH8.5.

[0044] Reagent B: pretreatment solution containing 70% ethanol.

[0045] Reagent C: lysate, containing 100mM Tris (pH 8.0), 50mM EDTA, 8% SDS (pH 8.0).

[0046] Reagent D: RNAase A with a content of 30 mg / ml.

[0047] Reagent E: 100mM Al 2 (SO 4 ) 3

[0048] Reagent F: 200g / L CTAB.

[0049] Reagent G: DNA linking solution, containing 10M GuSCN (guanidine isothiocyanate), 300mM Tris, adjusted to pH 5.8.

[0050] Reagent H: DNA washing solution, 50mM Tris (pH 7.5), mixed with absolute ethanol 3:7 (V / V).

[0051] Reagent K: DNA eluent, 10mM Tris (pH 8.0).

[0052] DNA adsorption column: silica gel column

[0053] Microbial genomic DNA was extracted according to the following procedure:

[0054] 1. Take 1g of mangrove s...

Embodiment 2

[0068] Various reagents were prepared according to the following formula:

[0069] Reagent A: immersion solution, components: 100mM Tris, 80mM EDTA, 100mM NaCl, 2mM sodium citrate, 10mM CaCl 2 , 0.4% Tween-80, 0.5% glycerol (pH8.5).

[0070] Reagent B: pretreatment solution containing 70% ethanol.

[0071] Reagent C: lysate, containing 100 mM Tris, 100 mM EDTA, 5% SDS (pH 8.0).

[0072] Reagent D: RNAase A with a content of 20 mg / ml.

[0073] Reagent E: 100mM Al 2 (SO 4 ) 3

[0074] Reagent F: 200g / L CTAB.

[0075] Reagent G: DNA linking solution, containing 7M GuSCN, 200mM Tris, adjusted pH to 5.8.

[0076] Reagent H: DNA washing solution, 50mM Tris (pH 7.5), mixed with absolute ethanol 3:7 (V / V).

[0077] Reagent K: for DNA eluent, ddH 2 O.

[0078] DNA adsorption column: silica gel column

[0079] Microbial genomic DNA was extracted according to the following procedure:

[0080] 1. Take 1g of vegetable field soil and add 1.5ml of reagent A, mash it thoroughly w...

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Abstract

The invention relates to an agent box to distill microorganism gene group DNA from earth and the distilling method. The agent box includes agent A(immersion fluid), agent B(pretreatment fluid), agent C(cracking fluid), and agent D that is made up of RNA enzyme A, agent E Al2(SO4)3, agent F hexadecyl trimethyl amine, and agent G(DNA cleaning fluid). The distilling method includes the following steps: dipping the sample, taking pretreatment to the sample, cracking the microorganism, removing humus, taking DNA coupling, removing protein impurity, adhering DNA, washing DNA, and taking elution to DNA. The advantages of the invention are rapid, little DNA loss, little harmful to researchers, high yield, and low cost, etc.

Description

technical field [0001] The invention relates to a kit for extracting microbial genome DNA from various soil samples, and further relates to a method for using the kit to extract microbial genome DNA. Background technique [0002] Modern soil microecology research requires the extraction of microbial genomic DNA in soil and use it for downstream molecular operations, such as PCR, SSCP, DGGE, AFLP, etc. However, compared with purely cultured microorganisms, there are some difficulties in the extraction of soil microbial DNA, and the DNA obtained by conventional methods is difficult to meet the requirements of downstream molecular operations. This is mainly caused by the following two reasons. [0003] (1) The soil composition is complex and contains a variety of PCR inhibitors, such as heavy metals, polysaccharides, polyphenols, humus, etc. Among them, because humic substances have similar properties to DNA, they can co-precipitate with DNA in the routine extraction of DNA, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C12N15/10
Inventor 罗鹏胡超群王青柏
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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