Soil DNA extracting method for evaluating diversity of microbial community of plant root system

A technology of microbial community and extraction method, applied in the field of extraction of microbial genomic DNA from root soil, can solve the problems of low DNA recovery rate, difficulty in removing humus in purification steps, etc., achieving low cost, reducing health damage, and applicability strong effect

Inactive Publication Date: 2010-05-19
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a method for extracting microbial genome DNA for evaluating the diversity of plant root soil microbial communities, to solve the problem that after the soil microbial cells are lysed, since humus has physicochemical properties similar to nucleic acids, the purification step There are problems such as difficult removal of humus and low DNA recovery

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] (1) Add 1.5 mL of sample soaking solution A to 0.5 g of watermelon root soil sample, vortex for 10 minutes, centrifuge at 10,000 rpm for 5 minutes, discard the supernatant, and repeat 3 times.

[0037] (2) Add 1.5 mL of sample immersion solution 2, place on ice for 5 minutes, vortex for 10 minutes, centrifuge at 10,000 rpm for 5 minutes, discard the supernatant, and repeat 3 times.

[0038] (3) Add 0.3 g of quartz sand and 1 glass bead with a diameter of 4 mm, and add 978 μl of phosphate buffer C (pH=8.0) to the sample. Vortex for 5 minutes.

[0039] (4) Add 122 μl of cell lysate D to the sample, and vortex for 5-20 minutes. Centrifuge at 13000rmp for 10min to precipitate debris.

[0040] (5) Transfer the supernatant to a clean centrifuge tube, add 250 μl of protein removal solution E to the tube, gently invert and mix 10 times by hand, leave at room temperature for 5 minutes, and centrifuge at 13,000 rpm for 5 minutes.

[0041] (6) Transfer the supernatant to a clea...

Embodiment 2

[0050] (1) Add 1.5mL sample soaking solution A to 0.5g strawberry root soil sample, vortex for 10 minutes, 10000 rpm, centrifuge for 5 minutes, discard the supernatant, repeat 3 times.

[0051] (2) Add 1.5 mL of sample immersion solution 2, place on ice for 5 minutes, vortex for 10 minutes, 10,000 rpm, centrifuge for 6 minutes, discard the supernatant, and repeat 3 times.

[0052] (3) Add 0.3 g of quartz sand and 1 glass bead with a diameter of 4 mm, and add 978 μl of phosphate buffer C (pH=8.0) to the sample. Vortex for 5 minutes.

[0053] (4) Add 122 μl of cell lysate D to the sample, and vortex for 5-20 minutes. Centrifuge at 13000rmp for 10min to precipitate debris.

[0054] (5) Transfer the supernatant to a clean centrifuge tube, add 250 μl of protein removal solution E to the tube, gently invert and mix 10 times by hand, leave at room temperature for 5 minutes, and centrifuge at 13,000 rpm for 5 minutes.

[0055] (6) Transfer the supernatant to a clean centrifuge tube...

Embodiment 3

[0064] (1) Add 1.5 mL of sample soaking solution A to 0.5 g of rice root soil sample, vortex for 10 minutes, 10,000 rpm, centrifuge for 5 minutes, discard the supernatant, and repeat 3 times.

[0065] (2) Add 1.5 mL of sample immersion solution 2, place on ice for 5 minutes, vortex for 10 minutes, 10,000 rpm, centrifuge for 7 minutes, discard the supernatant, and repeat 3 times.

[0066] (3) Add 0.3 g of quartz sand and 1 glass bead with a diameter of 4 mm, and add 978 μl of phosphate buffer C (pH=8.0) to the sample. Vortex for 5 minutes.

[0067] (4) Add 122 μl of cell lysate D to the sample, and vortex for 5-20 minutes. Centrifuge at 13000rmp for 10min to precipitate debris.

[0068] (5) Transfer the supernatant to a clean centrifuge tube, add 250 μl of protein removal solution E to the tube, gently invert and mix 10 times by hand, leave at room temperature for 5 minutes, and centrifuge at 13,000 rpm for 5 minutes.

[0069] (6) Transfer the supernatant to a clean centrifu...

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Abstract

The invention discloses a soil DNA extracting method for evaluating diversity of a microbial community of a plant root system. In the method, pretreatment is carried out on a sample before cell splitting, extracellular DNA and humic substances are removed, and the problems of difficult removal of the humic substances and low DNA recovery rate existing in a purification step are solved. In the extraction process of the method, phenol or chloroform is not used so as to reduce harm on the health of experimenters, obtain complete DNA and molecular fragments greater than 10kb and achieve high yield. OD260 / OD230 and OD260 / OD280 of the extracted soil microorganism DNA are close to standard values and can be directly applied to molecular operation so as to evaluate the diversity of the microbial community of the plant root system.

Description

technical field [0001] The invention relates to a DNA extraction method for evaluating the diversity of plant root soil microbial communities, in particular to an extraction method for extracting microbial genome DNA from root soil. Background technique [0002] Most soil microorganisms appear to be extremely adapted to their environment and are not cultureable under normal laboratory conditions. Extracting genomic DNA from the natural environment is a very useful method, which can be used to detect non-culturable microorganisms, track the behavior of some target strains or recombinant genes in the natural environment; it can also be used to reveal the relationship between plant rhizosphere soil microbial ecosystems Genetic diversity and its variation with the environment. This requires the extraction and purification of soil microbial genomic DNA from environmental samples. [0003] The main goal of genomic DNA extraction is to obtain the highest DNA recovery so that the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
Inventor 唐雪明王金斌赵凯谭芙蓉吴潇朱宏陶世如蒋玲曦王利刚刘华
Owner SHANGHAI ACAD OF AGRI SCI
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