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Method of simultaneously extracting microorganism macrogenome DNA and total DNA from sea precipitate

A marine sediment and metagenomic technology, applied in the field of extracting microbial metagenomic DNA and total RNA at the same time, can solve the problems of low biomass, low microbial content, and limited research and development, and achieve the effect of reducing shear

Inactive Publication Date: 2006-01-11
THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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Problems solved by technology

However, the ocean, especially the microbial content in the deep-sea environmental samples is very small. During the operation of the DNA extraction method in the prior art, due to the adsorption of clay to nucleic acid substances, degradation and loss in the extraction process, enzymes, inhibitors, etc. The co-extraction of impurities and the level of purification efficiency all affect the quality of the obtained nucleic acid substances, thus limiting the research and development. Therefore, it is necessary to establish a method for efficiently extracting nucleic acids from marine environments, especially deep-sea sediments. Nucleic acid substances in microorganisms
[0003] Many experimental methods have described how to extract microbial metagenomic DNA from soil or lake sediments (Wilson et al, 1997; Leff et al. Ultraclean TM Kits, but these methods are aimed at samples with a large amount of biomass or samples, and the obtained DNA has many differences in size and purity, and the DNA and RNA of microorganisms cannot be extracted at the same time
In the ocean, especially in deep sea sediments, the biomass is small, the inhibitor components are complex, and samples are not easy to obtain

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0038] 1. Marine sediments are collected and stored frozen at a temperature below -80°C for extraction;

[0039] 2. Add 1.5mL denaturing solution (4M guanidine isothiocyanate, 10mM Tris-HCl buffer solution (pH7.5), 1mM EDTA, 0.5% 2-mercaptoethanol) to 5g sample, mix well, add high temperature baked ( 180°C, 3h) sand, shake horizontally at 250r / min for 20min.

[0040] 3. Add 15mL nucleic acid extraction buffer (100mM sodium phosphate buffer (pH7.5), 100mM Tris-HCl (pH7.5), 100mM EDTA (pH8.0), 1.5M NaCl, 1% CTAB, 10% sucrose solution ); proteinase K (50 μg / mL), lysozyme (3 mg / mL), mix well and shake at 200 r / min for 30-50 min under the condition of 30-37 ° C.

[0041] 4. Add 1.5 mL of a solution containing 20% ​​SDS and 2% PVPP, incubate in a water bath at 65°C for 1 hour, invert and mix several times every 15 minutes, centrifuge, and transfer the supernatant to a new tube.

[0042]5. Add 5mL nucleic acid extraction buffer and 1mL SDS solution to the pellet, incubate in a 65°C...

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Abstract

A method for simultaneously extracting the macrogenomic DNA and total RNA of microbe from the sea sediment includes such steps as treating the sediment by modifying solution, adding sand, oscillating, adding proteinase and lysozyme, cracking adding SDS and PVPP solution, cracking further, adding extrant, extracting, depositing in isopropanol and purifying by nucleic acid adsortion resin.

Description

technical field [0001] The invention relates to a method capable of simultaneously extracting microbial metagenomic DNA and total RNA from marine sediments. Background technique [0002] A key problem in the research and development of microbial resources in the ocean, especially in the extreme environment of the deep sea, is that there are few types of microorganisms that can be artificially cultivated in samples, which directly affects the scope of resource research and development. The application of molecular biology technology based on nucleic acid substances overcomes the limitation of culture technology and provides a powerful means for the research and development of the ocean, especially the extreme environment of the deep sea. However, the ocean, especially the microbial content in the deep-sea environmental samples is very small. During the operation of the DNA extraction method in the prior art, due to the adsorption of clay to nucleic acid substances, degradatio...

Claims

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Application Information

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IPC IPC(8): C12P19/34
Inventor 曾润颖赵晶
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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