Simple extraction method for DNAs of microbes in river environment sample

A technology of microorganisms and microbial cells, which is applied in the fields of environmental science and bioengineering, can solve the problems of small amount of DNA, many steps of microbial DNA extraction, long operation time, etc., and achieve a strong practical effect

Inactive Publication Date: 2013-03-27
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the problem that the microbial DNA extraction steps in the existing river environment samples are various, the operation time is long, the reagents used are expensive, and the amount of extracted DNA is small and the purity is not enough, which cannot accurately reflect the diversity and diversity of microorganisms in the river environment samples. A simple extraction method for microbial DNA in river environment samples is provided to solve problems such as population abundance

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  • Simple extraction method for DNAs of microbes in river environment sample
  • Simple extraction method for DNAs of microbes in river environment sample
  • Simple extraction method for DNAs of microbes in river environment sample

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Example 1: Extraction of microbial DNA in Tianjin Haihe environmental samples

[0044] 1. Extraction of microbial DNA in sediment samples

[0045] (1) Lysis of microbial cells in sediment samples:

[0046] Weigh 1 g of bottom sludge into a 10 mL sterile centrifuge tube, add 2 mL of DNA Extraction Buffer, vortex for 10 minutes, add 1 mL of SDS with a mass concentration of 20%, and vortex thoroughly. Place the centrifuge tube in liquid nitrogen for 30 seconds, then place it in a 65°C water bath for 20 minutes; repeat this operation 1-2 times. The samples were taken out and centrifuged at 8000rpm for 15 minutes. Transfer the supernatant to another 10mL sterile centrifuge tube for later use.

[0047] Add 2mL DNA Extraction Buffer and 0.5ml 50mg / mL lysozyme to the precipitate, and vortex until mixed. Water bath at 37°C for 2 hours, add 1 mL of SDS with a mass concentration of 20%, bathe in water at 65°C for 30 minutes, gently shake the centrifuge tube 2-3 times in the mi...

Embodiment 2

[0063] Embodiment 2: Extraction of microbial DNA in different river environment samples

[0064] 1. Extraction of microbial DNA from sediment samples of the Hunhe River in Liaoning

[0065] (1) Lysis of microbial cells in sediment samples:

[0066] Weigh 1 g of bottom sludge into a 10 mL sterile centrifuge tube, add 2 mL of DNA Extraction Buffer, vortex for 10 minutes, add 1 mL of SDS with a mass concentration of 20%, and vortex thoroughly. Place the centrifuge tube in liquid nitrogen for 30 seconds, then place it in a 65°C water bath for 20 minutes, and repeat this operation 1-2 times. The samples were taken out and centrifuged at 8000rpm for 15 minutes. Transfer the supernatant to another 10mL sterile centrifuge tube for later use. Add 2mL DNA Extraction Buffer and 0.5ml 50mg / mL lysozyme to the precipitate, and vortex until mixed. Water bath at 37°C for 2 hours, add 1 mL of SDS with a mass concentration of 20%, bathe in water at 65°C for 30 minutes, gently shake the cent...

Embodiment 3

[0082] Embodiment 3: the extraction of other types of microbial DNA

[0083] (1) Soil microbial cell lysis in Tianjin area:

[0084] Weigh 1 g of soil into a 10 mL sterile centrifuge tube, add 2 mL of DNA Extraction Buffer, vortex for 10 minutes, add 1 mL of SDS with a mass concentration of 20%, and vortex thoroughly. Place the centrifuge tube in liquid nitrogen for 30 seconds, then place it in a 65°C water bath for 20 minutes, and repeat this operation 1-2 times. The samples were taken out and centrifuged at 8000rpm for 15 minutes. Transfer the supernatant to another 10mL sterile centrifuge tube for later use. Add 2mL DNA Extraction Buffer and 0.5ml 50mg / mL lysozyme to the precipitate, and vortex until mixed. Water bath at 37°C for 2 hours, add 1 mL of SDS with a mass concentration of 20%, bathe in water at 65°C for 30 minutes, gently shake the centrifuge tube 2-3 times in the middle, and centrifuge at 8000rpm for 15 minutes. Mix the supernatant with the previous supernat...

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Abstract

The invention relates to a simple extraction for the DNAs of microbes in a river environment sample. The method disclosed by the invention comprises: dividing the river environment sample into a water sample and a bottom sediment sample; and extracting the DNAs of the microbes in the water sample and bottom sediment sample respectively. The method can also be used for extracting any environment sample alone. In the invention, by combining a liquid nitrogen repeated freezing and thawing process, a sodium dodecyl sulfate (SDS) process, a lysozyme process and the like, the microbial cells in theenvironment sample can be fully lysed to fully release DNA; and analysis on bacterial diversity by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique indicates theextraction method can effectively reflect the integrity and diversity of microbes in the river environment sample.

Description

technical field [0001] The invention relates to a simple extraction of microbial DNA in river environment samples. The method uses biotechnology means to process the environment samples and belongs to the technical fields of environmental science and bioengineering. Background technique [0002] Environmental samples are a very complex system, generally composed of a variety of organisms and complex chemical components, such as river sediment is formed by the combination of ecosystems composed of microorganisms such as bacteria and micro-animals such as protozoa and metazoa, and colloidal substances floc particles. Therefore, environmental samples have the characteristics of diverse flora structures, complex flora functions, and may contain DNA hybridization and PCR amplification inhibitors such as humic acid, organic matter, and heavy metals. In the past, the extracted river sediment DNA contained many impurities such as humic acid, which would seriously affect the subsequ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
Inventor 毛大庆任君罗义张宏杰
Owner NANKAI UNIV
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