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Method for extracting RNA from cotton tissue

A cotton and tissue technology, applied in chemical instruments and methods, preparation of sugar derivatives, sugar derivatives, etc., can solve the problems of easy loss of RNA, can not meet the molecular biology research of cotton, etc., and achieve low price and ensure integrity. Effect

Inactive Publication Date: 2007-02-14
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The guanidine thiocyanate generally adopted in the existing technology of extracting RNA from cotton tissue--phenol extraction--chloroform extraction order is not suitable for the extraction of cotton tissue RNA, adopts prior art, in phenol extraction RNA is easily lost during the process, which cannot meet the molecular biology research of cotton

Method used

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  • Method for extracting RNA from cotton tissue
  • Method for extracting RNA from cotton tissue
  • Method for extracting RNA from cotton tissue

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Embodiment 1: utilize cotton root to extract RNA sample

[0039] (1) Take 2 grams of fresh cotton root, wash it with water and grind it into powder with liquid nitrogen; quickly transfer the sample to a centrifuge tube, and add 20 ml of pre-cooled RNA extraction buffer (pH6.0 ) (containing 0.5% PVP4000) and 200 microliters of β-mercaptoethanol, upside down to mix evenly, add 2 ml of sodium acetate, mix evenly;

[0040] (2) Add an equal volume of chloroform solution, mix upside down to make it a uniform phase, place on ice for 15 minutes, centrifuge at 12000×g for 15 minutes at 4°C, and recover the supernatant;

[0041] (3) Take the supernatant into a new tube, add an equal volume of frozen (-20°C) isopropanol to precipitate RNA, place it in a -20°C refrigerator for 30 minutes, and centrifuge at 10,000×g for 15 minutes at 4°C;

[0042] (4) Discard the supernatant, dissolve the precipitate completely with 3ml RNA extraction buffer, then add 3ml of acid phenol solution (p...

Embodiment 2

[0048] Embodiment 2: utilize cotton true leaf to extract RNA sample

[0049] (1) Take 1 gram of fresh cotton true leaves, grind it into powder with liquid nitrogen; quickly transfer the sample to a centrifuge tube, and add 15 ml of pre-cooled RNA extraction buffer (pH6.5) (containing 2 % PVP4000) and 200 microliters of β-mercaptoethanol, upside down to mix evenly, add 1 ml of sodium acetate, mix evenly;

[0050] (2) Add an equal volume of chloroform solution, mix upside down to make it a uniform phase, place on ice for 15 minutes, centrifuge at 12000×g for 15 minutes at 4°C, and recover the supernatant;

[0051] (3) Take the supernatant from step (2) into a new tube, add an equal volume of frozen (-20°C) isopropanol to precipitate RNA, and place it in a -20°C refrigerator for 1 hour, 4°C, 12000×g Centrifuge for 15 minutes to recover the precipitate;

[0052] (4) Discard the supernatant, dissolve the precipitate completely with 5ml RNA extraction buffer, then add 5ml of acid ...

Embodiment 3

[0058] Embodiment 3: extract RNA sample from cotton callus

[0059] (1) Get 0.3 grams of fresh cotton callus and put it into a glass homogenizer, and add 3 milliliters of pre-cooled RNA extraction buffer (pH6.3) (containing 0.5% PVP4000), grind it, and add 0.24 milliliters of Sodium acetate, mix well, pour into the centrifuge tube;

[0060] (2) Add an equal volume of chloroform solution, mix upside down to make it a uniform phase, place on ice for 10 minutes, centrifuge at 12000×g for 10 minutes at 4°C, and recover the supernatant;

[0061] (3) Take the supernatant from step (2) into a new tube, add an equal volume of frozen (-20°C) isopropanol to precipitate RNA, and place it in a -20°C refrigerator for 0.5 hours, 4°C, 10000×g Centrifuge for 10 minutes to recover the precipitate;

[0062] (4) Discard the supernatant, dissolve the precipitate thoroughly with 0.5ml RNA extraction buffer, then add 0.5ml of acid phenol solution (pH 4.7-5.2), mix up and down, ice bath for 10 min...

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Abstract

A process for extracting RNA from cotton tissue includes such steps as extracting in guanidine thiocyanate-chloroform solution, dissolving the coarse extract in the cracking liquid containing the polyvinyl pyrrolidone 4000 and beta-mercaptoethanol, and purifying by water-saturated phenol.

Description

technical field [0001] The invention relates to the technical field of cotton molecular biology. Specifically, it relates to a method for extracting and purifying RNA used in molecular biology from cotton tissue. Background technique [0002] RNA extraction is the basis for molecular biology research. The acquisition of high-quality RNA is of great significance for functional genomics and genetics research. There are many extraction methods for plant RNA, the existing methods mainly include guanidinium thiocyanate-phenol-chloroform extraction and purification method, phenol-SDS method and CTAB method, etc., wherein guanidine thiocyanate-phenol-chloroform The extraction and purification method is widely used in the extraction of RNA from various animals and plants, and is an effective and economical method for extracting RNA. Cotton is a kind of shrub woody plant. Its tissues and organs are rich in polyphenols, polysaccharides and secondary metabolites. It is difficult to ...

Claims

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Application Information

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IPC IPC(8): C07H21/02C07H1/08
Inventor 张献龙朱龙付涂礼莉聂以春郭小平曾范昌刘迪秋
Owner HUAZHONG AGRI UNIV
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