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Method for extracting DNA of Pholidota yunnanensis Rolfe

An extraction method and a technology of putting leaves on fruits, which are applied in the field of plant DNA extraction, can solve the problems of poor DNA quality, difficulty in accurate identification, difficulty in meeting identification, etc., and achieve high DNA extraction rate, ensure integrity, and improve solubilization effect Effect

Pending Publication Date: 2021-12-03
贵州光正制药有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Judging from the collected wild resources of the upper leaves of the fruit, it has complex diversity, and it is difficult to identify them accurately only from the morphological identification. Therefore, the use of biological identification can improve the accuracy of identification, while the existing DNA The extraction method, the quality of the obtained DNA is not good, it is difficult to meet the follow-up identification

Method used

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  • Method for extracting DNA of Pholidota yunnanensis Rolfe
  • Method for extracting DNA of Pholidota yunnanensis Rolfe
  • Method for extracting DNA of Pholidota yunnanensis Rolfe

Examples

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Effect test

Embodiment 1

[0032] A method for extracting leaf DNA from fruit, comprising the steps of:

[0033] (1) Put the upper leaf of the fruit in a polyacrylamide solution with a volume concentration of 10% according to the mass ratio of 1:1.3 and stir for 10 minutes. solution, and then ground into powder after being quick-frozen in liquid nitrogen; the volume concentration of the Tween 20 solution is 40%;

[0034] (2) Pour the powder of the upper leaves of the fruit into a cold EP tube, and add a DNA extraction solution with a temperature of 55°C. After shaking well, take a water bath at 55°C for 30 minutes, and turn it upside down 3 times during the water bath; the DNA extraction solution is 2 %CTAB, 0.08% (V / V) β-mercaptoethanol, 0.01% sodium dodecylbenzenesulfonate, 1.1mol / LNaCl;

[0035] (3) After the EP tube was taken out and cooled to room temperature, the extraction solution was added and mixed upside down, centrifuged at 12000×g for 5 min, and the supernatant A was taken; the volume rati...

Embodiment 2

[0040] A method for extracting leaf DNA from fruit, comprising the steps of:

[0041] (1) Put the upper leaf of the fruit in a polyacrylamide solution with a volume concentration of 30% according to the mass ratio of 1:1.8 and stir for 20 minutes. After taking it out and washing it twice, spray Tween 40 with 0.5% of the upper leaf mass on the surface. solution, and then ground into powder after liquid nitrogen quick-freezing; the volume concentration of the Tween 40 solution is 50%;

[0042] (2) Pour the powder of the upper leaves of the fruit into a cold EP tube, and add a DNA extraction solution with a temperature of 60°C. After shaking well, take a water bath at 60°C for 60 minutes, and turn it upside down 5 times during the water bath; the DNA extraction solution is 3 %CTAB, 0.1% (V / V) β-mercaptoethanol, 0.04% sodium dodecylbenzenesulfonate, 1.3mol / LNaCl;

[0043] (3) After the EP tube was taken out and cooled to room temperature, the extraction solution was added and mix...

Embodiment 3

[0048] A method for extracting leaf DNA from fruit, comprising the steps of:

[0049] (1) Put the upper leaves of the fruit in a polyacrylamide solution with a volume concentration of 15% according to the mass ratio of 1:1.4 and stir for 18 minutes. After taking it out and washing it twice, spray the surface with 0.4% of the upper leaves of the fruit. 20 solution, and then ground into powder after being quick-frozen in liquid nitrogen; the volume concentration of the Span-20 solution is 48%;

[0050] (2) Pour the powder of the upper leaves of the fruit into a cold EP tube, and add a DNA extraction solution with a temperature of 58°C. After shaking well, take a water bath at 58°C for 50 minutes, and turn it upside down 4 times during the water bath; the DNA extraction solution is 2.5 %CTAB, 0.08% (V / V) β-mercaptoethanol, 0.04% sodium dodecylbenzenesulfonate, 1.2mol / LNaCl;

[0051] (3) After the EP tube was taken out and cooled to room temperature, the extraction solution was a...

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Abstract

The invention belongs to the technical field of plant DNA extraction, and particularly relates to a method for extracting DNA of Pholidota yunnanensis Rolfe. The method comprises the following steps: (1) putting Pholidota yunnanensis Rolfe into a polyacrylamide solution, stirring for 10-20 minutes, taking out, washing, spraying a nonionic surfactant solution on the surface, quickly freezing with liquid nitrogen, and grinding into powder; (2) adding a DNA extraction solution into the powder, and carrying out water bath extraction; (3) adding an extraction solution, uniformly mixing, centrifuging, and taking supernate A; (4) adding a chloroform / isoamyl alcohol mixture into the supernate A, uniformly mixing, centrifuging, and taking supernate B; and (5) adding absolute ethyl alcohol into the supernate B, uniformly mixing, standing, centrifuging, separating out precipitate, and washing with water. The method provided by the invention has characteristics of high DNA extraction rate and few impurities, and meets requirements of subsequent DNA molecular identification.

Description

technical field [0001] The invention belongs to the technical field of plant DNA extraction, and in particular relates to a method for extracting DNA from upper leaves of fruits. Background technique [0002] The upper leaf of the fruit is a variety recorded in the "Quality Standards for Traditional Chinese Medicinal Materials and Ethnic Medicinal Materials in Guizhou Province" (2003 edition). Phlegm and cough, the effect of promoting qi and relieving pain, used for tuberculosis cough, hemoptysis, chronic bronchitis, chronic pharyngitis, hernia pain, sore swelling and pain embolism. [0003] Judging from the collected wild resources of the upper leaves of the fruit, it has complex diversity, and it is difficult to identify them accurately only from the morphological identification. Therefore, the use of biological identification can improve the accuracy of identification, while the existing DNA The extraction method, the quality of the obtained DNA is not good, it is diffic...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/6806
CPCC12N15/1003C12Q1/6806C12Q2523/32C12Q2527/125
Inventor 杨建波杨佳红张旭罗云杨艳张道琴
Owner 贵州光正制药有限责任公司