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Method for rapidly extracting and purifying total RNA of cyanobacteria

A cyanobacterial and fast technology, applied in the biological field, can solve the problems of low extraction efficiency and poor RNA purity, and achieve the effects of simple operation, high purity and good integrity

Inactive Publication Date: 2020-02-18
INST OF URBAN ENVIRONMENT CHINESE ACAD OF SCI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to solve the shortcomings of low extraction efficiency and poor RNA purity of existing cyanobacteria total RNA extraction methods, and to provide a rapid extraction method that can obtain high-purity cyanobacteria total RNA

Method used

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  • Method for rapidly extracting and purifying total RNA of cyanobacteria
  • Method for rapidly extracting and purifying total RNA of cyanobacteria
  • Method for rapidly extracting and purifying total RNA of cyanobacteria

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Embodiment one: the extraction of Microcystis aeruginosa RNA (chlorophyll a=10 μ g / L), comprises the following steps:

[0029] a) Collect algae cells: Take 10 mL of algae liquid into a 50 mL centrifuge tube, centrifuge at 6000 g for 1 min, and remove the supernatant;

[0030] b) Clean the algae cells: add 0.9% NaCl 10 mL, mix with the turbine for 10 s, suspend the algae cells, centrifuge again at 6000 g for 1 min, and remove the supernatant;

[0031] c) Pretreatment 1: quickly transfer the algae cell samples to liquid nitrogen for 1 min, take out the algae cell mass, grind with liquid nitrogen for 3 min, and grind to powder;

[0032] d) Pretreatment 2: Transfer all algae powder to a 1.5 mL centrifuge tube (DEPC water treatment, RNase-free), add 100 μL lysozyme solution (final concentration 3 mg / L), and mix with a turbine for 30 s;

[0033] e) Pre-purification: add DNase (8 μL, concentration 1 U / μl) and RNase inhibitor (3 μL, concentration 40 U / μl) to the cyanobacteria ...

Embodiment 2

[0037] Embodiment two: the extraction of Microcystis aeruginosa RNA (chlorophyll a=20 μ g / L), comprises the following steps:

[0038] a) Collect algae cells: Take 10 mL of algae liquid into a 50 mL centrifuge tube, centrifuge at 6000 g for 1 min, and remove the supernatant;

[0039] b) Clean the algae cells: add 0.9% NaCl 10 mL, mix with the turbine for 10 s, suspend the algae cells, centrifuge again at 6000 g for 1 min, and remove the supernatant;

[0040] c) Pretreatment 1: quickly transfer the algae cell samples to liquid nitrogen for 1 min, take out the algae cell mass, grind with liquid nitrogen for 3 min, and grind to powder;

[0041] d) Pretreatment 2: Transfer all algae powder to a 1.5 mL centrifuge tube (DEPC water treatment, RNase-free), add 100 μL lysozyme solution (concentration: 4 mg / L), and mix with a turbine for 30 s;

[0042] e) Pre-purification: add DNase (9 μL, concentration 1 U / μl) and RNase inhibitor (4 μL, concentration 40 U / μl) to the cyanobacteria suspe...

Embodiment 3

[0046] Embodiment three: the extraction of Microcystis aeruginosa RNA (chlorophyll a=40 μ g / L), comprises the following steps:

[0047] a) Collect algae cells: Take 10 mL of algae liquid into a 50 mL centrifuge tube, centrifuge at 6000 g for 1 min, and remove the supernatant;

[0048] b) Clean the algae cells: add 0.9% NaCl 10 mL, mix with the turbine for 10 s, suspend the algae cells, centrifuge again at 6000 g for 1 min, and remove the supernatant;

[0049] c) Pretreatment 1: quickly transfer the algae cell samples to liquid nitrogen for 1 min, take out the algae cell mass, grind with liquid nitrogen for 3 min, and grind to powder;

[0050] d) Pretreatment 2: Transfer all algae powder to a 1.5 mL centrifuge tube (DEPC water treatment, RNase-free), add 100 μL lysozyme solution (concentration: 4 mg / L), and mix with a turbine for 30 s;

[0051] e) Pre-purification: add DNase (10 μL, concentration 1 U / μl) and RNase inhibitor (5 μL, concentration 40 U / μl) to the cyanobacteria susp...

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Abstract

The present invention relates to a method for rapidly extracting and purifying total RNA of cyanobacteria. The method is characterized in that a two-step pretreatment method and a one-step pre-purification method are invented on the basis of a column type bacteria RNA extraction method, so that the total RNA of the cyanobacteria is rapidly extracted and purified. The method is suitable for extracting the total RNA of the cyanobacteria. The method is simple in operation and time-saving, and can finish extraction and purification of the RNA of the cyanobacteria in only 30 min, and the extractedRNA is high in purity (without DNA pollution) and good in integrity, and can be used for subsequent molecular biological experiments.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for rapidly extracting and purifying cyanobacteria RNA. Background technique [0002] Cyanobacteria, also known as cyanobacteria, belong to prokaryotes and are one of the oldest organisms on earth. Among algae, cyanobacteria are also the simplest and most primitive single-celled organisms. In recent years, with the intensification of eutrophication in water bodies, cyanobacteria blooms have become increasingly serious, posing a serious threat to human life and production and aquatic ecosystems. [0003] RNA is an important genetic material in cyanobacteria cells, and it is the key to study its physiological and biochemical functions. The isolation and acquisition of high-purity and complete RNA is the key to molecular biology experiments such as RT-PCR, qRT-PCR, and Northern hybridization. [0004] At present, the method of extracting RNA from column bacteria is widely used...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 于鑫李曦李晶晶陈辉叶成松
Owner INST OF URBAN ENVIRONMENT CHINESE ACAD OF SCI
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