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Improved method for extracting total RNA in special vegetable tender leaves

A technology for characteristic vegetables and tender leaves, applied in the field of molecular biology, can solve the problems of wasting tender leaf materials and bursting pipes, etc.

Inactive Publication Date: 2015-02-25
SUGARCANE RES INST FUJIAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problem of degradation, tube explosion and the need to prepare liquid nitrogen and waste more tender leaf materials in the process of extracting total RNA from young leaves of characteristic vegetables such as geranium and okra by the traditional Trizol method

Method used

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  • Improved method for extracting total RNA in special vegetable tender leaves

Examples

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Effect test

Embodiment 1

[0017] (1) Take 0.1g of tender vegetable leaves, wash and dry them with ultrapure water, put them in a mortar after autoclaving and cooling, and put them together with grinding rods in a -80°C refrigerator for 4 hours;

[0018] (2) Put on gauze gloves and take out the mortar containing the tender leaves of special vegetables from the refrigerator, quickly grind the tender leaves for 10 seconds with a grinding rod, then use a pipette gun to draw 1ml Trizol into the mortar, and continue to grind the jelly until it is completely dissolved ;

[0019] (3) Pipette 0.5ml of the Trizol mixture containing target RNA into two 1.5ml centrifuge tubes from the mortar, and let stand at room temperature for 2 minutes;

[0020] (4) Add 0.2ml chloroform to each tube, vortex and mix well, let stand for 2min, centrifuge at 12000g for 10min at 4°C, and take the supernatant;

[0021] (5) Add 0.5ml of ice-cold isopropanol, mix gently, let stand for 10min, centrifuge at 12000g at 4°C for 10min, dis...

Embodiment 2

[0024] (1) Take 0.3g of special vegetable tender leaves, wash and dry them with ultrapure water, put them in a mortar after autoclaving and cooling, and put them together with grinding rods in a -80°C refrigerator for 5 hours;

[0025] (2) Put on gauze gloves and take out the mortar containing the tender leaves of special vegetables from the refrigerator, quickly grind the tender leaves for 12 seconds with a grinding rod, then use a pipette to draw 3ml Trizol into the mortar, and continue to grind the jelly until it is completely dissolved ;

[0026] (3) Use a pipette gun to pipette 1ml of the Trizol mixture containing target RNA into three 1.5ml centrifuge tubes from the mortar, and let stand at room temperature for 2 minutes;

[0027] (4) Add 0.2ml chloroform to each tube, vortex and mix well, let stand for 2min, centrifuge at 12000g for 10min at 4°C, and take the supernatant;

[0028] (5) Add 0.5ml of ice-cold isopropanol, mix gently, let stand for 10min, centrifuge at 120...

Embodiment 3

[0031] (1) Take 0.6g of tender vegetable leaves, wash and dry them with ultrapure water, put them in a mortar after autoclaving and cooling, and put them together with grinding rods in a -80°C refrigerator for 6 hours;

[0032] (2) Put on gauze gloves and take out the mortar containing the tender leaves of special vegetables from the refrigerator, quickly grind the tender leaves for 15 seconds with a grinding rod, then use a pipette gun to draw 6ml Trizol into the mortar, and continue to grind the jelly until it is completely dissolved ;

[0033] (3) Use a pipette gun to draw 1ml of the Trizol mixture containing the target RNA from the mortar into six 1.5ml centrifuge tubes, and let stand at room temperature for 2 minutes;

[0034] (4) Add 0.2ml chloroform to each tube, vortex and mix well, let stand for 2min, centrifuge at 12000g for 10min at 4°C, and take the supernatant;

[0035] (5) Add 0.5ml of ice-cold isopropanol, mix gently, let stand for 10min, centrifuge at 12000g a...

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Abstract

The invention provides an improved method for extracting total RNA in special vegetable tender leaves. The improved method for extracting the total RNA in the special vegetable tender leaves comprises the following steps: putting an autoclaved and cooled mortar, an autoclave and cooled grinding rod and special vegetable tender leaves into a refrigerator at the temperature of -80 DEG C for more than 4 hours, taking the mortar, the grinding rod and the special vegetable tender leaves out, quickly grinding for about 10s, then sucking right amount of Trizol into the mortar by adopting a pipette, continuously grinding and mixing until jelly is completely dissolved, then sucking the dissolved mixture into a 1.5ml centrifugal tube by adopting a pipette, and then washing, precipitating and carrying out DEPCH2O dissolution, thus the target product, namely the total RNA in special vegetable blades, is obtained. The improved method for extracting the total RNA in the special vegetable tender leaves has the advantages that the problem that RNA is degraded easily (when being placed in the air for a too long time) or a centrifugal tube filled with Trizol mixture bursts (liquid nitrogen is not completely volatilized) as a medicine spoon is used for transferring plant tissue blades smashed by liquid nitrogen into Trizol in the traditional Trizol method for extracting the total RNA in plant tissue blades is solved, the liquid nitrogen is not required to be prepared, and no loss of the ground special vegetable tender leaf powder is produced.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and more specifically relates to an improved method for extracting total RNA from tender leaves of characteristic vegetables. Background technique [0002] In recent years, with the development of molecular biotechnology. RNA functional analysis has become an important basis for modern molecular biology research, so high-quality total RNA extraction is very important for functional gene molecular biology research. At present, there are many methods for extracting total RNA from plant materials, such as guanidine isothiocyanate method, phenol method, SDS method, lithium chloride precipitation method, CTAB method and commercial Trizol kit method. At present, the Trizol extraction method for total RNA is more common. However, when the traditional Trizol method (including other methods) is used to extract total RNA from plant leaves, it is necessary to grind the target plant tissue materi...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 张少平邱珊莲郑开斌刘荣章林碧珍
Owner SUGARCANE RES INST FUJIAN ACAD OF AGRI SCI
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