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Method for extracting total DNAs of soil microorganisms through PVP pretreatment

A soil microorganism, pretreatment technology, 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 mulberry soil microorganisms are closely related to the formation and improvement of mulberry soil structure, the evolution of soil fertility, the occurrence of mulberry root diseases, the supply of mulberry nutrients, and the growth and development of plants. However, no culture-free method has been applied so far. Report on Studying Soil Microbial Diversity in Mulberry Fields

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  • Method for extracting total DNAs of soil microorganisms through PVP pretreatment
  • Method for extracting total DNAs of soil microorganisms through PVP pretreatment
  • Method for extracting total DNAs of soil microorganisms through PVP pretreatment

Examples

Experimental program
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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 for 3min; add 150μL of 100mg / mL lysozyme, mix well, incubate at 37℃, 230r / min shaker for 1h; then add 15μL of 20mg / mL proteinase K, mix well, Add 1.5mL of 10% SDS in water bath at 37°C for 1h; add 1.5mL 10% SDS, bathe in water at 65°C for 1h; freeze-thaw three times, centrifuge at 8500r / min for 10min; take the supernatant, add an equal volume of phenol / chloroform / isoamyl alcohol (volume ratio 25: 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 cen...

Embodiment 2

[0054]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 0.7...

Embodiment 3

[0056] 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 DNAs (Deoxyribonucleic Acid) of soil microorganisms through PVP (Polyvinyl Pyrrolidone) pretreatment. Humic acid is removed through PVP pre-washing; CTAB (Cetyltrimethyl Ammonium Bromide), lysozyme, protease and SDS (Sodium Dodecyl Sulfonate) act together to perform lysis of cells and simultaneously freeze and thaw the cells repeatedly; therefore, an excellent cell lysis effect is achieved; and the DNAs are precipitated by utilizing isopropanol so that the purity of the obtained DNAs is high. The method is simple and convenient; and without being purified, the DNAs obtained by the method can be used for subsequent PCR (Polymerase Chain Reaction) analysis and DGGE (Denaturing Gradient Gel Electrophoresis) analysis. The purity of the extracted DNAs meets the requirement on directly performing subsequent molecular biological research; the extracted DNA segments can be subjected to 16SrDNA amplification and DGGE atlas analysis; glue cutting recovery and basic group sequencing can be performed on specific stripes of the DGGE band; and unknown bacteria can be identified through comparison with the conventional sequences in an RDP (Ribosomal Database Project) database or by establishing a new sequence probe; and therefore, the structural composition of the microbial community can be determined, a foundation can be laid for researching the structural diversity and ecological functions of the soil microorganisms in the rhizosphere of the mulberry in future.

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 truly reflects the structure of the soil microbial population, and the efficient and high-quality extraction of the total DNA of the soil microbial co...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
Inventor 胡兴明于翠邓文叶楚华李勇熊超彭波
Owner INST OF ECONOMIC CROP HUBEI ACADEMY OF AGRI SCI
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