Method for extracting potato tuber total RNA (ribonucleic acid) by means of improved Trizol

Potato cubes, fresh technology applied in the field of molecular biology to solve problems such as unsatisfactory results

Inactive Publication Date: 2014-01-29
SICHUAN AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Since its inception, the Trizol method has been widely used in the RNA extraction of tissues due to its simple operation and short time-consuming, but it is not effective for potato tubers rich in starch, polysaccharides, polyphenols and many secondary metabolites. ideal

Method used

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  • Method for extracting potato tuber total RNA (ribonucleic acid) by means of improved Trizol
  • Method for extracting potato tuber total RNA (ribonucleic acid) by means of improved Trizol

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Embodiment Construction

[0025] (1) Take about 0.1g of fresh tubers, add liquid nitrogen to grind to powder, quickly transfer to a 1.5mL centrifuge tube, add 1mL Trizol reagent, mix well, and let stand at 25°C for 5min; (2) 4°C, 12000r / Centrifuge for 5 min, pour the supernatant directly into a new 1.5mL centrifuge tube; (3) Add 200 μl chloroform to the supernatant collected in step (2), mix by inverting, let stand at room temperature for 5 min, centrifuge at 4°C, 12000r / min 15min; (4) Put the supernatant collected in step (3) into a new 1.5mL centrifuge tube, add an equal volume of isopropanol, mix up and down, and precipitate at -20°C for 30min; (5) 4°C, 12000r / min Centrifuge for 10 min, discard the supernatant, add 1 mL of 75% ethanol to wash the precipitate, centrifuge at 8000 r / min at 4°C for 5 min, discard the supernatant, and dry; (6) Add 500 μl DEPC water to dissolve the dried precipitate in step (5), and then add 500 μl Phenol / chloroform / isoamyl alcohol (25:24:1), mix by inverting, centrifuge...

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Abstract

The invention provides a method for extracting potato tuber total RNA (ribonucleic acid) by means of improved Trizol. The method is characterized by comprising the steps of adding phenol/chloroform/isoamyl alcohol (25:24:1) for performing primary extraction after extracting RNA by the regular Trizol (Invitrogen Company), then transferring liquid supernatant to a new tube, adding 0.7 time of isopropyl alcohol in comparison with the volume of the liquid supernatant and 0.2 time of 1mol/L sodium acetate in comparison with the total volume to precipitate RNA, respectively washing the obtained RNA once with 1mL of 3mol/L sodium acetate (pH=5.2) and 1mL of 75% alcohol, finally, drying, and dissolving with DEPC (diethylpyrocarbonate) water. Such method for extracting potato tuber total RNA by means of improved Trizol has high yield and high capacity; moreover, the obtained total RNA has excellent quality and high purity, and can meet the requirements of molecular biology researches, such as inverse transcription, Northern hybridization, gene in vitro translation, cDNA (complementary deoxyribonucleic acid) library construction and rapid amplification of cDNA ends.

Description

1. Technical field [0001] The invention belongs to the field of molecular biology, in particular to a method for extracting potato tuber RNA. 2. Background technology [0002] Potato (Solaum tuberosum L.) is an important food crop, industrial raw material and health food in the world, especially tubers are closely related to humans. After the potato genome sequence is determined, it is very important to study the functions of genes in tubers. High-purity and high-quality RNA is the necessary prerequisite and key for molecular biology research such as Northern hybridization, gene translation in vitro, cDNA library construction, and rapid amplification of cDNA ends. [0003] Potato tubers contain a lot of starch, polysaccharides, polyphenols, and many secondary metabolites, which will seriously affect the quality of their total RNA extraction. After liquid, the dry powder quickly absorbs water and turns into a gel, which cannot be separated from the liquid phase even through ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 李立芹王西瑶王伟杨先泉倪苏刘帆
Owner SICHUAN AGRI UNIV
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