Method for simultaneously extracting DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) from lily tissue

A lily and tissue technology, applied in the field of plant molecular biology, can solve the problems of time-consuming, only for a single DNA or RNA, and low separation efficiency, and achieve the effect of short time-consuming and easy operation

Inactive Publication Date: 2012-11-14
KUNMING UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0005] Many plant tissues are rich in polysaccharides, polyphenols and secondary metabolites, making nucleic acid extraction difficult
At present, the extraction of nucleic acid from these plant tissues is generally only aimed at a single DNA or RNA, and a method is rarely used to extract DNA and RNA at the same time
When the plant tissue is limited or rare, it is difficult to extract DNA or RNA to meet the needs of the experiment
Lily tissue is rich in polysaccharides, polyphenols and secondary metabolites, especially the polysaccharide content in lily scales is extremely high, which brings great difficulties to the extraction of nucleic acid from lily tissue
The existing methods for synchronously extracting plant DNA and RNA have disadvantages such as time-consuming, complicated operation, and low separation efficiency, and the extraction effect on lily tissue DNA and RNA is not good

Method used

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  • Method for simultaneously extracting DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) from lily tissue
  • Method for simultaneously extracting DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) from lily tissue
  • Method for simultaneously extracting DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) from lily tissue

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Embodiment 1: the method for extracting RNA and DNA simultaneously from Lily of the Minjiang River scale, concrete operation is as follows:

[0035] (1) Take 0.8g of young young scales of wild Minjiang lily quick-frozen in liquid nitrogen, grind them into powder with liquid nitrogen, transfer the sample powder to a centrifuge tube quickly, add 12mL of pre-cooled guanidine isothiocyanate extraction buffer, and shake fully Mix well and place on ice for 12min, then add 1.2mL 3mol L -1 Sodium acetate (pH5.0), mixed upside down; the composition of guanidine isothiocyanate extraction buffer: 4.5mol L -1 Guanidine isothiocyanate, 25mmol L -1 Sodium citrate, 0.5% (w / v) sodium lauryl sarcosine, 3% (w / v) soluble polyvinylpyrrolidone, 1 mL of β-mercaptoethanol per 100 mL of extraction buffer before use;

[0036] (2) Add 12 mL of chloroform to the sample mixture obtained in step (1), shake vigorously to mix, place on ice for 5 minutes, then centrifuge at 12000g at 4°C for 10 min...

Embodiment 2

[0048] Embodiment 2: the method for extracting RNA and DNA simultaneously from the leaf of Lilium siberia, concrete operation is as follows:

[0049] (1) Take 1 g of the young leaves of Oriental lily variety Siberia quick-frozen in liquid nitrogen, grind them into powder with liquid nitrogen, transfer the sample powder to a centrifuge tube quickly, add 12 mL of pre-cooled guanidine isothiocyanate extraction buffer, and shake fully Mix well and place on ice for 15min, then add 1.2mL 3mol L -1 Sodium acetate (pH4.5), mixed upside down; the composition of guanidine isothiocyanate extraction buffer: 4.5mol L -1 Guanidine isothiocyanate, 25mmol L -1 Sodium citrate, 0.5% (w / v) sodium lauryl sarcosine, 3% (w / v) soluble polyvinylpyrrolidone, 1 mL of β-mercaptoethanol per 100 mL of extraction buffer before use;

[0050] (2) Add 12 mL of chloroform to the sample mixture obtained in step (1), shake vigorously to mix, place on ice for 5 minutes, then centrifuge at 12000g at 4°C for 10 ...

Embodiment 3

[0062] Embodiment 3: from the method for simultaneously extracting RNA and DNA from Tongjiang lily root, concrete operations are as follows:

[0063] (1) Take 0.5g of the young Lily of Tongjiang root that was quick-frozen in liquid nitrogen, grind it into powder with liquid nitrogen, transfer the sample powder to a centrifuge tube quickly, add 6.5mL of pre-cooled guanidine isothiocyanate extract, and shake fully Mix well and place on ice for 10min, then add 0.65mL 3mol L -1 Sodium acetate (pH5.5), mixed upside down; the composition of guanidine isothiocyanate extraction buffer: 4.5mol L -1 Guanidine isothiocyanate, 25mmol L -1 Sodium citrate, 0.5% (w / v) sodium lauryl sarcosine, 3% (w / v) soluble polyvinylpyrrolidone, 1 mL of β-mercaptoethanol per 100 mL of extraction buffer before use;

[0064] (2) Add 6.5 mL of chloroform to the sample mixture obtained in step (1), shake vigorously and mix well, place on ice for 5 minutes, then centrifuge at 12000g at 4°C for 10 minutes...

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Abstract

The invention discloses a method for simultaneously extracting DNA (deoxyribonucleic acid) and RNA from lily tissue. The method comprises the following steps of: obtaining total nucleic acid solution through crude extraction and purification of the same steps from a lily tissue sample; and then sequentially selectively precipitating RNA and DNA, thereby obtaining high-quality DNA and RNA. The method disclosed by the invention is short in time, economic and fast, and good in stability; and the extracted nucleic acid is high in quality. The method has the characteristics that high-concentration potassium acetate is utilized for precipitating polysaccharides twice, so that the polysaccharides in the lily sample can be effectively removed; and in the DNA and RNA separation process, the RNA is selectively precipitated by means of the synergistic effect of lithium chloride and absolute ethyl alcohol and then the DNA is precipitated by using sodium acetate and isopropanol; and therefore, the efficiency of the DNA-RNA separation is high, the loss of the nucleic acid is low and the precipitation time is greatly shortened. The method is suitable for simultaneously extracting the DNA and the RNA from the lily tissue containing rich polysaccharides and other secondary metabolites.

Description

technical field [0001] The invention belongs to the technical field of plant molecular biology, in particular to a method for simultaneously extracting DNA and RNA from lily tissues, in particular to a method for simultaneously extracting DNA and RNA from lily scales. Background technique [0002] Extracting high-quality DNA and RNA is one of the most basic tasks in molecular biology experimental research. It is necessary to use a simple and efficient nucleic acid extraction method to improve experimental efficiency. At present, the extraction methods of plant tissue DNA mainly include CTAB method, SDS method, high-salt and low-osmosis method, etc. The main methods of RNA extraction include guanidine isothiocyanate-phenol-chloroform method (one-step method), CTAB method, SDS method and Thermal boric acid method, etc. The steps of these extraction methods are basically similar. First, the cell wall of plant cells is fully broken, and then the protein in the nucleoprotein com...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 刘迪秋饶健刘亚龙李红丽葛锋陈朝银
Owner KUNMING UNIV OF SCI & TECH
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