Improved method for extracting total RNA in each growth period of pichia pastoris

A Pichia pastoris, growth stage technology, applied in the field of molecular biology research, can solve the problems of difficult extraction of total RNA from Pichia pastoris cells, and achieve stable and high-quality extraction results

Inactive Publication Date: 2013-03-13
JIANGNAN UNIV
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Problems solved by technology

[0004] The purpose of the present invention is to provide an improved method for extracting total RNA in each growth stage of Pichia pastoris, in order to solve t...

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  • Improved method for extracting total RNA in each growth period of pichia pastoris

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Embodiment 1X-33 and G

[0021] Example 1X-33 and the extraction of GS115 Pichia pastoris cells at 72h and 96h total RNA

[0022] Materials required for total RNA extraction:

[0023] (1) Materials required for Pichia pastoris cell disruption: porcelain mortar, liquid nitrogen, acid-washed glass beads (400nm), Trizol reagent.

[0024] (2) Shanghai Sangon UNIQ-10 Column Trizol Total RNA Extraction Kit consists of: Trizol Reagent, RPE Solution, DEPC-treated ddH 2 O, adsorption column and collection tube.

[0025] (3) Other reagents and consumables: absolute ethanol, chloroform, 1.5mL EP tube, pipette tip and EP tube pretreated with 0.1% DEPC. The specific steps of total RNA extraction are as follows:

[0026] (1) Take about 1×10 respectively 7 X-33 (72h and 96h) or GS115 (72h and 96h) cells were placed in a sterilized 1.5mL EP tube, quickly frozen by the liquid nitrogen method, and then stored in a -70°C refrigerator.

[0027] (2) Pre-cool the porcelain mortar with liquid nitrogen, and then pour 0....

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Abstract

The invention relates to the field of the molecular biology and provides an improved method for extracting total RNA in each growth period of pichia pastoris, aiming to solve the problem of difficult extraction of total RNA of pichia pastoris cells occuring in molecular research. The method is improved on the basis of Sangon UNIQ-10 column type Trizol total RNA extraction kit, and suitable for extraction of total RNA in each growth period of pichia pastoris in contrast with methods for extracting total RNA by using hot acid phenol; even for the stable-phase cells having thick cell walls, the method is still capable of successfully extracting total RNA and the extraction result is stable; moreover, the total RNA extracted by the method is good in quality, and has clear and tidy 28SrRNA and 18SrRNA strips in 2-time relationship; besides, the method is capable of meeting the purity requirement between 1.8 and 2.1 of OD260/OD280; and degradation hardly occurs; and therefore, the method meets the requirement of QRT-PCR (quantitative real-time polymerase chain reaction) measurement.

Description

technical field [0001] The invention relates to the field of molecular biology research, and is an improved method for extracting total RNA in each growth stage of Pichia pastoris. Background technique [0002] The expression system of Pichia pastoris is relatively perfect developed in recent years. It is widely used to express the methanolotrophic yeast expression system of exogenous protein. The overexpression of exogenous protein in Pichia pastoris, It is often accompanied by changes in the expression levels of other genes. By analyzing the differences in the expression levels of related genes, it is easy to find the key issues that affect the expression of foreign proteins, and the analysis of gene expression levels often involves the extraction of total RNA. [0003] Compared with other cells, Pichia pastoris has a stronger cell wall, especially Pichia pastoris cells in the stationary phase, which have thicker cell walls and poor permeability. These factors increase the...

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Application Information

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IPC IPC(8): C12N15/10
Inventor 刘立明龚香艺马冬玲王蒙冯斌
Owner JIANGNAN UNIV
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