Efficient and economical soil microbial DNA extraction method

A soil microorganism and extraction method technology, which is applied in the field of rapid extraction of soil microorganism DNA, can solve the problems of poor method effect and high price of foreign kits, save reagent consumption, simplify the DNA collection process, and achieve high extraction efficiency. Effect

Active Publication Date: 2019-02-15
HEBEI UNIV OF TECH
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  • 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 foreign kits are expensive and domestic methods are not effective in the existing soil microbial DNA extraction process, and provide an efficient and economical soil microbial DNA extraction method

Method used

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  • Efficient and economical soil microbial DNA extraction method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1 (three repetitions)

[0028] The soil extraction kit Soil DNA Isolation Kit of mobio company was used to extract, and the method was as follows:

[0029] Add 2g soil to 15mL Bead Tube, add 0.25mLSR1 and 0.8mLSR2, add 2.5mL BeadSolution to Bead Tube, then add 3.5mL phenol: chloroform: isoamyl alcohol = 25:24:1 to the test tube, cover and vortex Blend until the layering disappears. Vortex at the maximum speed for 15 minutes, centrifuge at 2500g for 10 minutes at room temperature. After removal, carefully transfer the upper aqueous phase to a clean 15mL collection tube and discard the lower phenol. Add 1.5mL SR3 to the water phase, then vortex and mix well, incubate at 4°C for 10min, room temperature, centrifuge at 2500g for 10min. Transfer the supernatant to a new 15mL collection tube, add 5mL SR4 solution to the collection tube containing the supernatant, mix well and incubate at room temperature for 30min. Centrifuge at 2500g for 30min at room temperatu...

Embodiment 2

[0030] Embodiment 2 (cold-thaw method currently used)

[0031]Take 1 g of the ground soil powder, put it in a 10 mL centrifuge tube, add 2 mL of sodium phosphate buffer (0.12 mol / L, pH 8.0), mix it, put it on a shaker at 30 °C, and shake it at 150 r / min for 15 min. 8000r / min, centrifuge for 10min. Take the precipitate and repeat the above operation. Take the precipitate, add 1.5mL of lysis buffer I (0.15mol / LNaCl, 0.1mol / LEDTA, pH8.0) and 0.5mL of 50mg / L lysozyme, mix well, bathe in 37℃ water for 2h, and shake every 20-30min. Add 2mL of lysate II (0.1mol / LNaCl, 0.5mol / LTris-HCl, pH8.0, 10% SDS), freeze-thaw repeatedly 3 times, centrifuge at 8000r / min for 15min. The supernatant was mixed with an equal volume of phenol reagent (v (phenol): v (chloroform): v (isoamyl alcohol) = 25:24:1), centrifuged at 8000 r / min for 10 min. Repeat the previous step. Take the aqueous phase and mix it with an equal volume of chloroform and isoamyl alcohol mixture (v(chloroform:v(isoamyl alcoho...

Embodiment 3

[0033] Weigh 1 gram of soil, add 2ml of suspension buffer (sodium phosphate aqueous solution, 0.15mol / L, pH8.0), put 1 gram of glass beads with a particle size of 0.1mm into the extraction tube, and crack the soil under the friction of the glass beads All organisms in the medium include free DNA of G+ bacteria, yeasts, fungi, algae, nematodes, and even eubacterial spores, spores, animal and plant remains, etc.; add humic acid adsorbent 500 μl: it consists of nano-TiO 2 : 0.2M; activated carbon: 0.1% (mass percentage). The specific configuration process is: Weigh 3.2000g spectroscopically pure titanium dioxide into a container, add 12.8g ammonium sulfate, 32mL 98% sulfuric acid and weigh 0.2g activated carbon, heat to dissolve, cool to room temperature, add water to dilute to 200mL, shake well, and set aside . ) mixed fully and shaken for 1min; add 10μL of proteinase K (10mg / mL) solution, shake for 10min after mixing, then add 2ml of high-efficiency lysate (2% CTAB (mass perce...

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Abstract

The invention discloses an efficient and economical soil microbial DNA extraction method. The method comprises the following steps: step 1, obtaining a microbial suspension; weighing 1 g of soil, adding 2 ml of suspension Buffer C1, 1 g of glass beads having the particle diameter of 0.09-0.12 mm, 500 muL of humic acid adsorbent Buffer C2 and 10 muL of proteinase K (10 mg / mL), mixing and shaking; step 2, obtaining microbial DNA; step 3, obtaining crude DNA; placing the material obtained in the last step in a water bath of 60 DEG C while stirring, then centrifuging at a high speed of 4 DEG C, and collecting the first supernatant; and step 4, obtaining purified DNA. The method is based on the requirements of soil-contaminated microbial detection, can extract DNA containing no inhibiting factor from complex environmental samples such as soil and feces, and is high in extraction speed and accurate in analysis result.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to an extraction method for rapidly extracting soil microbial DNA. Background technique [0002] Due to industrial and agricultural production such as sewage irrigation, manure fertilization, and hospital wastewater discharge, the situation of microbial pollution in soil is becoming increasingly serious. Therefore, the detection of soil pollution microorganisms is particularly important. Soil microbial diversity analysis based on PCR technology can identify the types and contents of microorganisms in soil. However, due to the use of various fertilizers and straw returning to the farmland soil, the soil organic matter content is high and the humic acid is also high. Therefore, the total DNA of microorganisms extracted from the soil often has a certain amount of humic acid pollution. Humic acid has a great influence on PCR amplification, and a small amount of h...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1006
Inventor 李亮郭子渝崔忠信胡海诚常乐乐蒋雨萌
Owner HEBEI UNIV OF TECH
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