Method for extracting total DNA (deoxyribonucleic acid) of mulberry rhizosphere soil microorganisms by adopting plurality of measures

A technology of soil microbes, mulberries, applied in the field of molecular ecology

Inactive Publication Date: 2012-08-22
INST OF ECONOMIC CROP HUBEI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The diversity and life activities of soil microorganisms in mulberry gardens are closely related to the formation and improvement of soil structure in mulberry gardens, the evolution of soil

Method used

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  • Method for extracting total DNA (deoxyribonucleic acid) of mulberry rhizosphere soil microorganisms by adopting plurality of measures
  • Method for extracting total DNA (deoxyribonucleic acid) of mulberry rhizosphere soil microorganisms by adopting plurality of measures
  • Method for extracting total DNA (deoxyribonucleic acid) of mulberry rhizosphere soil microorganisms by adopting plurality of measures

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] PVP pretreatment-CTAB-lysozyme-protease-SDS-repeated freeze-thaw method. Take 5g of soil sample and add 30mL of 20g / L sodium metaphosphate buffer solution (containing 10g / L PVP-K30; pH 8.5), oscillate on a shaking table for 15min, centrifuge at 8500r / min for 5min, take the precipitate, and repeat washing 3 times; take the precipitate, add 13.5mL DNA extraction solution I and a little quartz sand, vortex shaker for 3min; add 100mg / mL lysozyme 150μL, mix well, incubate at 37℃, 230r / min shaker for 1h; then add 20mg / mL proteinase K15μL, mix well , in a water bath at 37°C for 1h; add 1.5mL 10% SDS, and bathe in a water bath at 65°C for 1h; :24:1) extraction, 8500r / min centrifugation for 10min; take the supernatant, add an equal volume of chloroform / isoamyl alcohol (volume ratio 24:1) to extract again, 8500r / min centrifugation for 10min, take the supernatant, Add an equal volume of chloroform / isoamyl alcohol (volume ratio 24:1) to extract again, centrifuge at 8500r / min for 1...

Embodiment 2

[0053] PVP pretreatment-CTAB, CaCl2, BSA-lysozyme-protease-SDS-repeated freeze-thaw method Take 5g soil sample and add 20g / L sodium metaphosphate buffer (containing 10g / L PVP-K30; pH 8.5) 30mL, shaker Shake for 15 minutes, centrifuge at 8500r / min for 5 minutes, take the precipitate, and repeat washing 3 times; take the precipitate, add 13.5mL DNA extraction solution II and a little quartz sand, vortex shaker for 3min; add 100mg / mL lysozyme 150μL, mix well, 37 ℃, 230r / min shaker incubate for 1h; then add 20mg / mL proteinase K15μL, mix well, 37℃ water bath for 1h; add 1.5mL 10% SDS, 65℃ water bath for 1h; freeze-thaw 3 times, 8500r / min centrifuge 10min Get the supernatant, add an equal volume of phenol / chloroform / isoamyl alcohol (volume ratio 25:24:1) to extract, and centrifuge at 8500r / min for 10min; get the supernatant, add an equal volume of chloroform / isoamyl alcohol ( The volume ratio is 24:1) and extract again, centrifuge at 8500r / min for 10min, take the supernatant, add 0....

Embodiment 3

[0055] CTAB, PVP, CaCl2, BSA-lysozyme, protease-SDS-repeated freeze-thaw method. Take 5g soil sample, add 13.5mL DNA extraction solution III and a little quartz sand, vortex shaker for 3min; add 150μL 100mg / mL lysozyme, 15μL 20mg / mL proteinase K, mix well, incubate for 1h at 37℃, 230r / min shaker Add 15 μL of 20 mg / mL proteinase K, mix well, and bathe in water at 37°C for 1 hour; add 1.5 mL of 10% SDS, bathe in water at 65°C for 1 hour; Extract with phenol / chloroform / isoamyl alcohol (volume ratio 25:24:1), centrifuge at 8500r / min for 10 min; take the supernatant, add an equal volume of chloroform / isoamyl alcohol (volume ratio 24:1) to extract again, Centrifuge at 8500r / min for 10min, take the supernatant, add 0.7 times the volume of isopropanol and 0.1 times the volume of NaAc to the supernatant, place it overnight at -20°C, centrifuge at 8500r / min, 4°C for 20min, wash the precipitate with 70% ethanol, Centrifuge at 12500r / min for 10min, dry the precipitate at room temperature...

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Abstract

The invention discloses a method for extracting total DNA (deoxyribonucleic acid) of mulberry rhizosphere soil microorganisms by adopting a plurality of measures, which can achieve a good cell lysis effect by utilizing PVP (polyvinyl pyrrolidone) prewashing to remove humic acid, the synergy of CTAB (cetyltrimethyl ammonium bromide), lysozyme, protease and SDS (sodium dodecyl sulfate) to lyse cells and repetitively freezing and thawing the cells at the same time and utilizes PEG8000 to precipitate DNA, so that the purity of the obtained DNA is high. The adopted method is simple and convenient, and the DNA can be used in subsequent PCR (Polymerase Chain Reaction) analysis and DGGE (denaturing gradient gel electrophoresis) analysis without needing to be purified. The extracted DNA is so pure as to meet the requirement of direct subsequent molecular biology research, an extracted DNA fragment can be used in the amplification and DGGE map analysis of 16SrDNA, moreover, the specific band gel extraction and base sequencing of a DGGE band can be carried out, comparison with the sequence existing in an RDP (ribosomal database project) database can be carried out, or a new sequence probe can be established to identify unknown bacteria, so that the composition of a microbial community structure can be determined, and thereby laying a foundation for the future research of the community structure diversity and the ecological functions of the mulberry rhizosphere soil microorganisms.

Description

technical field [0001] The invention relates to the technical field of molecular ecology, in particular to a method for extracting total DNA of soil microorganisms in mulberry gardens. Background technique [0002] For a long time, the research on the diversity of soil microorganisms has been achieved through isolation and culture. However, the vast majority of microbial populations cannot be purely cultured, which has become a limiting factor in the study of soil microbial diversity. With the continuous deepening of the research on the structure of soil microbial communities, modern molecular biology research methods such as PCR-RFLP, PCR-SSCP, PCR-DGGE, etc. have avoided the shortcomings of traditional research methods that cannot fully obtain soil microbial diversity information, and can The diversity of soil microbial DNA can truly reflect the structure of the soil microbial population, and the efficient and high-quality extraction of the total DNA of the soil microbial...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
Inventor 邓文于翠胡兴明熊超叶楚华李勇彭波
Owner INST OF ECONOMIC CROP HUBEI ACADEMY OF AGRI SCI
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