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Kit and method for quickly extracting RNA (ribonucleic acid) of soil microorganisms

A soil microorganism and kit technology, applied in the biological field, can solve the problem of high price of the kit, and achieve the effect of saving extraction time, fewer steps, and maintaining integrity

Inactive Publication Date: 2015-01-21
FUJIAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are many methods for soil microbial RNA extraction at home and abroad, which are mainly divided into two categories, one is to extract RNA based on a special solution, and the other is based on a spin column. However, most of these kits are relatively expensive and Multiple trials are required to successfully extract soil microbial RNA, which imposes a large economic burden on small laboratories for soil microbial RNA research

Method used

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  • Kit and method for quickly extracting RNA (ribonucleic acid) of soil microorganisms
  • Kit and method for quickly extracting RNA (ribonucleic acid) of soil microorganisms

Examples

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Effect test

Embodiment 1

[0035] A kit for rapidly extracting soil microbial RNA, the specific steps are as follows:

[0036] (1) Raw material: fresh rice rhizosphere soil, fresh corn rhizosphere soil, fresh sugarcane rhizosphere soil.

[0037] (2) Pretreatment of soil samples: Weigh 0.1-1g of soil into a 2ml centrifuge tube with a balance, add 50-200μl Buffer A, 300-600μl sterile water, 300-600μl Buffer B, and vortex for 30- 60s; add 50-200μl Buffer C, 100-500μl Buffer D, vortex for 30-60s; centrifuge at 8000-10000g for 1-5min, remove the supernatant. The pre-treatment is mainly to remove soil impurities such as humus and prevent the impact of impurities on downstream experiments.

[0038] (3) Extraction of soil microbial RNA: add 200-400μl Buffer D, 0.1-1g 0.5-1mm glass beads, 200-400μl Buffer E, 200-400μl Buffer F, vortex for 1-3min, 10000-12000g at 4°C Centrifuge for 2-5min. Pipette 500-800ul of the supernatant into a 1.5ml centrifuge tube, add 500-800ul of phenol:chloroform:isoamyl alcohol (25:...

Embodiment 2

[0052] A method for rapidly extracting soil microbial RNA, comprising the steps of:

[0053] Pretreatment of soil samples: Take 0.1g of soil in a 2ml centrifuge tube, add 50μl Buffer A, 300μl sterile water, 30μl Buffer B, vortex for 30s, add 50μl Buffer C, 100μl Buffer D, vortex for 30s , centrifuge at 8000g for 1min, and remove the supernatant to obtain precipitate A;

[0054] The Buffer A is a 0.5-2M Tris-Cl buffer solution with a pH of 5;

[0055] The Buffer B is 0.1M Al 2 (SO 4 ) 3 solution;

[0056] The Buffer C is 2M NaOH solution;

[0057] The Buffer D is 0.1M Tris-Cl buffer solution with pH=7;

[0058] Extraction of soil microbial RNA: add 200μl Buffer D, 0.1g of 0.5-1mm glass beads to the precipitate A, 200μl Buffer E, 200μl Buffer F, vortex and shake for 1min, centrifuge at 10000g for 2-5min at 4°C, and take 50ul Transfer the supernatant to a 1.5ml centrifuge tube, add 500 μl of a mixture of phenol, chloroform and isoamyl alcohol, the ratio of volume to part o...

Embodiment 3

[0067] A method for rapidly extracting soil microbial RNA, the specific steps are as follows:

[0068] (1) Raw material: fresh rice rhizosphere soil, fresh corn rhizosphere soil, fresh sugarcane rhizosphere soil.

[0069] (2) Pretreatment of soil samples: Weigh 0.5g of different soil samples into 2ml centrifuge tubes with a balance, and each sample has 3 replicates, add 100μl Buffer A, 690μl sterile water, 210μl Buffer B, and vortex Shake for 30s; add 100μl Buffer C and 300μl Buffer D, vortex for 30s; centrifuge at 8000g for 2min, remove the supernatant.

[0070] (3) Extraction of soil microbial gene RNA: add 325 μl Buffer D, 0.5 g 0.5 mm glass beads, 325 μl Buffer E, 325 μl Buffer F, vortex for 1 min, and centrifuge at 11000 g for 2 min at 4°C. Pipette 750ul of the supernatant into a 1.5ml centrifuge tube, add 750ul of phenol:chloroform:isoamyl alcohol (25:24:1), vortex for 30s, and centrifuge at 16000g at 4°C for 1min. Transfer the supernatant to a 1.5ml centrifuge tube, a...

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Abstract

The invention relates to the biotechnical field and particularly relates to a kit and a method for quickly extracting RNA (ribonucleic acid) of soil microorganisms. According to the kit and the method for quickly extracting RNA of the soil microorganisms disclosed by the invention, aluminum hydroxide is firstly utilized to remove various impurities in the soil, and then a RNA adsorption column is utilized to adsorb the RNA of the soil microorganisms after glass beads are used to crush cells of the soil microorganisms, so that a great deal of extracting time is saved. The method disclosed by the invention is short in used time, less in steps, relatively convenient and quick, and capable of keeping the integrity of RNA.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a kit and method for rapidly extracting soil microbial RNA. Background technique [0002] There are many kinds of microorganisms in the soil, and the number is extremely large. One gram of fertile soil usually contains hundreds of millions to billions of microorganisms, and each gram of barren soil also contains millions to tens of millions of microorganisms. Generally speaking, the more fertile the soil, The more types and numbers of microorganisms. In addition, the number of microorganisms in the soil surface or cultivated layer and near the plant roots is also high. Accelerated organisms in soil mainly include bacteria, fungi, actinomycetes, algae and protozoa. Among the microorganisms in the soil, bacteria are the most numerous, and bacteria account for 70% to 90% of the total amount of soil microorganisms, and there are many types. [0003] In the study of soil microorganisms...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 王清水余彦
Owner FUJIAN NORMAL UNIV