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Plant bleeding sap RNA extracting method

An extraction method and a technology of wounding fluid, which are applied in the field of agriculture, can solve the problems of inability to meet the needs of reverse transcription, insufficient RNA quantity, and rapid RNA degradation, so as to meet the needs of reverse transcription, increase the amount of RNA obtained, and inhibit RNA. Degradation effect

Inactive Publication Date: 2015-11-11
JIANGSU POLYTECHNIC COLLEGE OF AGRI & FORESTRY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] At present, the methods for extracting RNA from plant tissues are very mature, but the effect of using these methods to extract RNA from grape, cucumber and other plant wound fluids is not satisfactory, and the following problems arise: 1) RNA cannot be extracted; 2) RNA in wound fluid degrades quickly ;3) The amount of RNA proposed is insufficient to meet the needs of reverse transcription

Method used

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  • Plant bleeding sap RNA extracting method
  • Plant bleeding sap RNA extracting method
  • Plant bleeding sap RNA extracting method

Examples

Experimental program
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Effect test

Embodiment 1

[0024] A method for extracting RNA from plant wound sap, comprising the following steps:

[0025] 1) Freeze-dry 5ml plant wound liquid to 1 / 10 of the original volume, add 1400μl SDS, and bathe in water at 65℃ for 30min;

[0026] 2) Add 300 μl of water-saturated phenol, vortex, then add 300 μl of chloroform, vortex and mix, and centrifuge at 13,000 rpm for 10 min at 4°C;

[0027] 3) Take 1200 μl of supernatant to a new centrifuge tube, add 400 μl of water-saturated phenol, vortex to mix, then add 400 μl of chloroform, vortex to mix, and centrifuge at 13,000 rpm for 10 min;

[0028] 4) Add 1,000 μl of supernatant to 500 μl of water-saturated phenol, vortex and mix, then add 500 μl of chloroform, vortex and mix, and centrifuge at 13,000 rpm for 10 min;

[0029] 5) Take 800 μl of the supernatant and add 800 μl of chloroform, vortex to mix, centrifuge at 13000 rpm for 10 min, take 700 μl of the supernatant and add an equal volume of isopropanol, and stand at -20°C for more than 5 ...

Embodiment 2

[0032] A method for extracting RNA from plant wound sap, comprising the following steps:

[0033] 1) Freeze-dry the plant wound liquid to 1 / 8 of the original volume, add 1300 μl of SDS, and bathe in water at 65°C for 25 minutes;

[0034] 2) Add 200 μl of water-saturated phenol, vortex, then add 200 μl of chloroform, vortex and mix, and centrifuge at 12,000 rpm for 8 minutes at 4°C;

[0035] 3) Take 1100μl supernatant to a new centrifuge tube, add 1 / 3 volume of water-saturated phenol, vortex mix, then add 1 / 3 volume of chloroform of the supernatant, vortex mix, centrifuge at 12000rpm for 8min;

[0036] 4) Take 900 μl of the supernatant and add 1 / 2 volume of water-saturated phenol to the supernatant, vortex to mix, then add 1 / 2 volume of chloroform to the supernatant, vortex to mix, and centrifuge at 12000rpm for 8min;

[0037] 5) Take 700 μl of the supernatant and add an equal volume of chloroform, vortex and mix well, centrifuge at 12,000 rpm for 10 minutes, take 600 μl of th...

Embodiment 3

[0040] A method for extracting RNA from plant wound sap, comprising the following steps:

[0041] 1) Freeze-dry the plant wound fluid to 1 / 9 of the original volume, add 1500 μl of SDS, and bathe in water at 65°C for 30 minutes;

[0042] 2) Add 400 μl of water-saturated phenol, vortex, then add 400 μl of chloroform, vortex and mix, and centrifuge at 12,500 rpm for 9 minutes at 4°C;

[0043] 3) Take 1300μl supernatant to a new centrifuge tube, add 1 / 3 volume of water-saturated phenol, vortex mix, then add 1 / 3 volume of chloroform of the supernatant, vortex mix, centrifuge at 12500rpm for 9min;

[0044] 4) Take 1100 μl of supernatant, add 1 / 2 volume of supernatant water-saturated phenol, vortex and mix, then add 1 / 2 volume of supernatant chloroform, vortex mix, centrifuge at 12500rpm for 9min;

[0045] 5) Take 900 μl of the supernatant and add an equal volume of chloroform, vortex to mix, centrifuge at 12,500 rpm for 10 minutes, take 800 μl of the supernatant and add an equal vo...

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Abstract

The invention discloses a plant bleeding sap RNA extracting method. The plant bleeding sap RNA extracting method comprises the following steps that freeze frying is conducted on plant bleeding sap, and SDS is added to perform water bathing; water saturated phenol is added and stirred, and then chloroform is added and stirred evenly for centrifugation; supernatant is taken into a novel centrifugation tube and added with water saturated phenol, even stirring is performed, then chloroform is added and stirred evenly for centrifugation; supernatant is taken and added with chloroform, even stirring is performed for centrifugation, and supernatant is taken and added with the same volume of isopropanol for standing; centrifugation is performed, the supernatant is poured off, 80% of ethyl alcohol is adopted for washing and precipitation, 100% of ethyl alcohol is adopted for rewashing, centrifugation and air drying are performed for RNA precipitation, and DEPC water is added. The plant bleeding sap RNA extracting method can inhibit degradation of the RNA in bleeding sap and improve the extracting efficiency of the RNA in bleeding sap. RNA extracting procedures are simplified, the obtained RNA amount is increased, and the amount of RNA extracted from the bleeding sap can meet the inverse transcription demand.

Description

technical field [0001] The invention relates to the technical field of agriculture, in particular to a method for extracting RNA from plant wound fluid. Background technique [0002] At present, the methods for extracting RNA from plant tissues are very mature, but the effect of using these methods to extract RNA from grape, cucumber and other plant wound fluids is not satisfactory, and the following problems arise: 1) RNA cannot be extracted; 2) RNA in wound fluid degrades quickly ; 3) The amount of RNA presented is not sufficient for reverse transcription. Contents of the invention [0003] Purpose of the invention: the purpose of the invention is to provide a method for extracting RNA from plant wound sap. [0004] Technical solution: In order to solve the above technical problems, the present invention provides a method for extracting RNA from plant wound fluid, comprising the following steps: [0005] 1) Freeze-dry the plant wound fluid to 1 / 8-1 / 10 of the original v...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 解振强王剑贾思振魏跃冯英娜刘叶琼
Owner JIANGSU POLYTECHNIC COLLEGE OF AGRI & FORESTRY
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