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Extraction method for South American wedelia chinensis total ribonucleic acid (RNA)

A technology of wedge chrysanthemum and centrifuge tube, applied in DNA preparation, recombinant DNA technology and other directions, can solve the problems of less research at the molecular level and the like

Inactive Publication Date: 2012-09-19
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on Wedelia is mainly focused on growth and development, reproductive characteristics, medicinal value and the physiological and biochemical mechanism of allelopathy to some other plants, but there are few studies on its molecular level.

Method used

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  • Extraction method for South American wedelia chinensis total ribonucleic acid (RNA)
  • Extraction method for South American wedelia chinensis total ribonucleic acid (RNA)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] 1. Preparation of experimental drugs:

[0024] Prepare RNA extraction buffer (100mL): 10mL 1M Tris-HCl pH8.0, 4mL 500mM EDTA pH8.0, 2% (mass volume percentage concentration) CTAB, 0.14mol NaCl, 2%~3% (volume percentage Concentration) β-mercaptoethanol, 0.5mol dithiothreitol (DTT), 2%~4% (mass volume percent concentration) polyvinylpyrrolidone (PVP).

[0025] 8mol / L LiCl (prepared with 0.1% DEPC water)

[0026] TE buffer (prepared with 0.1% DEPC water)

[0027] 3mol / LNaAc (prepared with 0.1% DEPC water)

[0028] 70%~80% ethanol (prepared with 0.1% DEPC water after sterilization)

[0029] 2. Handling of experimental supplies:

[0030] All ceramic glassware, centrifuge tubes and pipette tips used in the experiment were soaked in 0.1% DEPC water at 37°C overnight, and then autoclaved at 120°C for more than 1 hour. Dry at 65°C for later use. The electrophoresis tank, gel plate and sampling comb were soaked in 2.5%~3.5% hydrogen peroxide.

[0031] Experimental st...

Embodiment 2

[0045] 1. Preparation of experimental drugs:

[0046] Prepare RNA extraction buffer (100mL): 10mL 1M Tris-HCl pH8.0, 4mL 500mM EDTA pH8.0, 2% (mass volume percentage concentration) CTAB, 0.14mol NaCl, 2%~3% (volume percentage Concentration) β-mercaptoethanol, 0.5mol dithiothreitol (DTT), 2%~4% (mass volume percent concentration) polyvinylpyrrolidone (PVP).

[0047] 8mol / L LiCl (prepared with 0.1% DEPC water)

[0048] TE buffer (prepared with 0.1% DEPC water)

[0049] 3mol / LNaAc (prepared with 0.1% DEPC water)

[0050] 70%~80% ethanol (prepared with 0.1% DEPC water after sterilization)

[0051] 2. Handling of experimental supplies:

[0052] All ceramic glassware, centrifuge tubes and pipette tips used in the experiment were soaked in 0.1% DEPC water at 37°C overnight, and then autoclaved at 120°C for more than 1 hour. Dry at 65°C for later use. The electrophoresis tank, gel plate and sampling comb were soaked in 2.5%~3.5% hydrogen peroxide.

[0053] Experimental st...

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Abstract

The invention relates to an extraction method for a South American wedelia chinensis total ribonucleic acid (RNA) and belongs to the technical field of biology. The method includes grinding a fresh South American wedelia chinensis material to be fine powders by liquid nitrogen, performing a warm bath at the temperature of 65DEG C for 30 minutes, adding equivoluminal chloroforms and evenly mixing, centrifuging at the low temperature of 4DEG C, subjecting an obtained mixture to a precipitation by adding a lithum chloride solution, centrifuging, abandoning supernatant, dissolving the precipitation by tellurium, adding equivoluminal chloroforms, centrifuging, extracting the supernatant to a new centrifuge tube, adding diethyl ether, centrifuging, extracting the supernatant, repeating a diethyl ether operation for two times, adding natrium aceticum and absolute ethyl alcohol for precipitating the RNA, centrifuging, abandoning supernatant, washing the RNA by 70% of ethanol to obtain the precipitation, drying the obtained precipitation at the room temperature, dissolving by aseptic diethylpyrocarbonate (DEPC) water, and preserving for a reservation at the temperature of 80 DEG C below zero. According to the extraction method for the South American wedelia chinensis total ribonucleic acid (RNA), the experiment processes are simple, the demands for equipment and experiment conditions are low, the result is accurate, effective, the repeatability is good, and the method has an important guiding and practical significance for extracting the total RNA of plant tissues which are rich in phenols and polysaccharides.

Description

technical field [0001] The invention relates to an optimized method for extracting total RNA from Compositae plants, which is suitable for extracting total RNA from plant tissues rich in polysaccharides and phenolic compounds such as Wedelia chinensis, and belongs to the field of biotechnology. Background technique [0002] Extracting high-quality RNA from plant tissues is a necessary prerequisite and key to the study of plant molecular biology. Many plant tissues are rich in secondary metabolites such as polysaccharides and phenols, which cause RNA to oxidize, brown and degrade during the extraction process, which is not conducive to the isolation and purification of RNA. For plant materials with low content of common polysaccharides and phenols, Trizol-based products can be extracted, but for some plant tissues rich in polysaccharides, polyphenols and secondary metabolites, high-quality RNA cannot be obtained by ordinary extraction methods, often A large amount of polysac...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 田远飞杜道林黄萍薛永来付卫国杨冉
Owner JIANGSU UNIV
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