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Method for rapidly extracting microbial genomes from soil sample

A technology for soil microorganisms and soil samples, applied in the field of rapid extraction of microbial genomes in soil samples, can solve the problems of analyzing the impact of biological analysis, and achieve the effects of high DNA integrity, time saving, and simple reagents

Inactive Publication Date: 2016-03-30
ANHUI YUANDA MACHINERY MFG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Inorganic salts and organic matter in the soil need to be separated and processed in advance; in addition, the residual humic acid and heavy metals in the DNA extraction process will also affect the later analytical biological analysis

Method used

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  • Method for rapidly extracting microbial genomes from soil sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Soil sample: the soil around the wheat root system in the wheat field, when the wheat was mature and the sickle was about to open.

[0032] The specific process of separating and extracting the soil microbial genome is as follows:

[0033] (1) Grind and pulverize 2g of soil sample in liquid nitrogen;

[0034] (2) Put the ground sample in a 10ml centrifuge tube, add genome extraction buffer (including 0.2MNaCl, 0.05MEDTA, 0.15Tris-HCl and 1mg / ml lysozyme), shake for 3min, mix well, and place in a 37°C water bath Incubate in medium for 30min;

[0035] (3) Add proteinase K solution and SDS solution to the solution to a final concentration of 0.02 mg / ml and 0.2%, mix slightly, and bathe in 55°C water for 30 minutes;

[0036] (4) Centrifuge at 10,000 rpm for 5 minutes at 4°C, and collect the supernatant;

[0037] (5) The supernatant was extracted with an equal volume of phenol at room temperature for 5 minutes;

[0038] (6) Centrifuge at 10,000 rpm for 5 minutes at 4°C, ...

Embodiment 2

[0046] Soil sample: the soil around the corn root system in a cornfield, and the sampling time is the jointing stage of corn growth.

[0047] The specific process of separating and extracting the soil microbial genome is as follows:

[0048] (1) Grind and pulverize 5g of soil sample in liquid nitrogen;

[0049] (2) Put part of the ground sample in a 10ml centrifuge tube, add genome extraction buffer (including 0.2MNaCl, 0.05MEDTA, 0.15Tris-HCl and 2mg / ml lysozyme), shake for 3min, mix well, and store at 37°C Incubate in a water bath for 30 minutes;

[0050](3) Add proteinase K solution and SDS solution to the solution to a final concentration of 0.03 mg / ml and 0.3%, mix slightly, and bathe in 55°C water for 30 minutes;

[0051] (4) Centrifuge at 10,000 rpm for 10 minutes at 4°C, and collect the supernatant;

[0052] (5) The supernatant was extracted with an equal volume of phenol at room temperature for 5 minutes;

[0053] (6) Centrifuge at 10,000 rpm for 10 minutes at 4°C...

Embodiment 3

[0061] Soil sample: the soil around the tomato root system in the tomato planting greenhouse, and the sampling time is the flowering and fruit-setting period of tomato growth.

[0062] The specific process of separating and extracting the soil microbial genome is as follows:

[0063] (1) Grind and pulverize 1g of soil sample in liquid nitrogen;

[0064] (2) Put part of the ground sample in a 10ml centrifuge tube, add genome extraction buffer (including 0.2MNaCl, 0.05MEDTA, 0.15Tris-HCl and 2mg / ml lysozyme), shake for 3min, mix well, and store at 37°C Incubate in a water bath for 30 minutes;

[0065] (3) Add proteinase K solution and SDS solution to the solution to a final concentration of 0.05mg / ml and 0.5%, mix slightly, and bathe in 55°C water for 30 minutes;

[0066] (4) Centrifuge at 12,000 rpm for 10 minutes at 4°C, and collect the supernatant;

[0067] (5) The supernatant was extracted with an equal volume of phenol at room temperature for 5 minutes;

[0068] (6) Cen...

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Abstract

The invention discloses a method for rapidly extracting microbial genomes from a soil sample. The method comprises the steps that the soil sample is ground and smashed in liquid nitrogen, a genome extraction buffer solution is added to the smashed sample, and incubation is conducted for 30 min in a water bath of 37 DEG C; a protease K solution and an SDS solution are added, water bath is conducted for 30 min at 55 DEG C, centrifugation is conducted, and precipitation is discarded, supernatant is collected and extracted for the first time through phenol; centrifugation is conducted, supernatant is collected and extracted for the second time through mixed liquid of phenol, chloroform and isoamyl alcohol, centrifugation is conducted, and supernatant is collected; ethyl alcohol or isopropyl alcohol is added to the supernatant, total liquid is subjected to centrifugation and passes through a DNA recovery and purification column, high-speed centrifugation is conducted on the recovery and purification column again, drying is conducted, DNA eluant is added, and a microbial genome sample is obtained after centrifugation is conducted. The yield of the extracted microbial genomes can reach 80 micrograms / gram, the integrity of DNA is high, the size is 23 kb or above, pollution of RNA or protein does not exist, and OD260 / OD280 is close to the standard value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a rapid extraction method of microbial genomes in soil samples. technical background [0002] The microorganisms in the soil are a comprehensive system. There are about 10 to the eleventh power of microorganisms per gram of soil (wet weight), and more than 99% of them are not cultivable. The traditional analysis of soil microbial community is to use microbial counting, isolation culture, enrichment culture, and through the physiological and biochemical analysis of microbial metabolites to determine the types of microorganisms, so as to determine the situation of harmful and beneficial microorganisms in the soil, and then targeted Carry out the construction of advantageous and beneficial soil microbial ecological community. However, conventional microbial isolation and culture techniques have great limitations, because the types of microorganisms that can be isolated by c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 高宇
Owner ANHUI YUANDA MACHINERY MFG
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