Method for extracting total RNA

An extraction method and centrifuge tube technology, applied in DNA preparation, recombinant DNA technology and other directions, can solve problems such as high toxicity, pollute the environment, and unsatisfactory effects, and achieve the effects of reducing harm, simple operation steps, and preventing pollution.

Inactive Publication Date: 2011-07-13
杨军
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Problems solved by technology

[0002] At present, there are many methods for extracting total RNA from common plant materials, mainly SDS-Phenol method, CTAB method, Trizol method, guanidine isothiocyanate method, hot boric acid method, etc. These methods all use organic solvents in the extraction process (Balanced phenol, chloroform) to extract and remove organic substances such as protein, DNA, polysaccharides, polyphenols, etc. in the material, because most of the organic solvents have strong carcinogenicity, high toxicity, and seriously pollute the environment
There are also some RNA extraction methods that use acidic phenol extraction to remove protein and DNA, but the effect is not ideal

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Embodiment Construction

[0024] Embodiments of the present invention will be further described below in conjunction with the accompanying drawings.

[0025] Solution and reagent preparation:

[0026] ① Cell lysate

[0027] SDS 2% (w / v)

[0028] Sodium citrate 68 mmol / L

[0029] Citric acid 132 mmol / L

[0030] EDTA 1mmol / L

[0031] Spermidine Trihydrochloride 0.5 g / L (w / v)

[0032] ② Protein-DNA Precipitation Solution

[0033] NaCl 4 mol / L

[0034] Sodium citrate 16 mmol / L

[0035] Citric acid 32 mmol / L

[0036] ③ Lithium chloride solution (LiCl) concentration is 8 mol / L

[0037] ④ The concentration of Guanidine Hydrochloride is 10% (w / v)

[0038] ⑤ DNase Ⅰ solution diluted with DEPC water, the concentration of DNase Ⅰ solution is 0.5 units / μl

[0039] ⑥ 75% ethanol (prepared), isopropanol, liquid nitrogen and sterilized 0.1% DEPC water (referred to as DEPC water).

[0040] see figure 1 : The specific steps of the total RNA extraction method are:

[0041] (1) Take 25 mg of aerospace mutag...

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Abstract

The invention relates to an optimized method for extracting total RNA of a plant material, which comprises the steps of: 1, grinding a fresh plant material into fine powder under a liquid nitrogen condition, and transferring to a centrifuge tube containing a cell lysing solution; 2, adding a protein-DNA settling solution in the centrifuge tube in the step 1; 3, transferring supernate to a new centrifuge tube, and adding isopropanol; 4, discarding the supernate, washing sediments with alcohol, drying in air, and adding a DNA enzyme I solution diluted with DEPC (diethyl pyrocarbonate) water; 5, adding the DEPC water and a guanidine hydrochloride solution; 6, transferring the supernate to the new centrifuge tube, and adding LiCl solution; and 7, centrifuging, washing with alcohol, drying in air, and dissolving in the DEPC water for storing at low temperature. The invention has the advantages of simple operation steps, convenience of operation and no need of extracting the total RNA with conventional organic solvents (phenol and chloroform), thereby preventing polluting the environment without hurting a human body.

Description

technical field [0001] The invention relates to an optimized method for extracting total RNA from plant materials. Background technique [0002] At present, there are many methods for extracting total RNA from common plant materials, mainly including SDS-Phenol method, CTAB method, Trizol method, guanidine isothiocyanate method, hot boric acid method, etc. These methods all use organic solvents in the extraction process. (Balanced phenol, chloroform) to extract and remove organic substances such as protein, DNA, polysaccharides, polyphenols, etc. in the material, because most of the organic solvents have strong carcinogenicity, high toxicity, and seriously pollute the environment. In some RNA extraction methods, acidic phenol extraction is used to remove protein and DNA, but the effect is not satisfactory. We urgently need a low-cost, simple and convenient operation, no pollution to the environment, little harm to the human body, and no organic solvent extraction at all, wh...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 马西豹杨军刘滋茂张思楠
Owner 杨军
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