Method for extracting vibrio parahaemolyticus total RNA

A technology of Vibrio hemolyticus and Vibrio bacteria, which is applied in the field of extracting total RNA of Vibrio parahaemolyticus, can solve the problems of difficult extraction of high-quality RNA, extraction of Vibrio parahaemolyticus RNA, and short half-life, achieving shortened precipitation time, The reagent is simple and the integrity is good

Inactive Publication Date: 2010-07-14
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Bacterial RNA has the characteristics of less content, short half-life, and easy degradation, so it is more difficult to extract high-quality RNA

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Follow the steps below to extract total RNA from Vibrio parahaemolyticus:

[0025] 1. Precipitate bacteria: Take 1ml of Vibrio parahaemolyticus solution in a 1.5ml centrifuge tube, centrifuge at 12000rpm for 1min at 4°C;

[0026] 2. Crack the cells and dissolve the nucleic acid, protein, polysaccharide and other substances in the cells: Discard the supernatant, freeze the bacteria with liquid nitrogen, grind them into powder, and transfer the powder to a 1.5ml centrifuge tube (operate on liquid nitrogen to ensure low temperature ), add 800ul Trizol, shake for 1min, let stand on ice for 3min, shake repeatedly 3 times;

[0027] 3. Remove impurities such as proteins and polysaccharides: add 200ul of chloroform / isoamyl alcohol with a mixing ratio of 24:1; centrifuge at 12000rpm for 5min; transfer 500ul of the supernatant to a 1.5ml centrifuge tube, add pre- Cold isopropanol, precipitate at room temperature for 10 minutes to fully precipitate, be careful not to absorb the m...

Embodiment 2

[0031] Follow the steps below to extract total RNA from Vibrio parahaemolyticus:

[0032] 1. Precipitate bacteria: Take 1ml of Vibrio parahaemolyticus solution in a 1.5ml centrifuge tube, centrifuge at 12000rpm for 1min at 4°C;

[0033] 2. Crack the cells and dissolve the nucleic acid, protein, polysaccharide and other substances in the cells: Discard the supernatant, freeze the bacteria with liquid nitrogen, grind them into powder, and transfer the powder to a 1.5ml centrifuge tube (operate on liquid nitrogen to ensure low temperature ), add 800ul Trizol, shake for 1min, let stand on ice for 3min, shake repeatedly 3 times;

[0034] 3. Remove impurities such as proteins and polysaccharides: add 200ul of chloroform / isoamyl alcohol with a mixing ratio of 24:1; centrifuge at 12000rpm for 5min; transfer 500ul of the supernatant to a 1.5ml centrifuge tube, add pre- Cold isopropanol, place at -20°C for 1 hour to fully precipitate, be careful not to absorb the middle layer, otherwis...

Embodiment 3

[0038] Follow the steps below to extract total RNA from Vibrio parahaemolyticus:

[0039] 1. Precipitate bacteria: Take 1ml of Vibrio parahaemolyticus solution in a 1.5ml centrifuge tube, centrifuge at 12000rpm for 1min at 4°C;

[0040] 2. Crack the cells and dissolve the nucleic acid, protein, polysaccharide and other substances in the cells: Discard the supernatant, freeze the bacteria with liquid nitrogen, grind them into powder, and transfer the powder to a 1.5ml centrifuge tube (operate on liquid nitrogen to ensure low temperature ), add 800ul Trizol, shake for 1min, let stand on ice for 3min, shake repeatedly 3 times;

[0041] 3. Remove impurities such as proteins and polysaccharides: add 200ul of chloroform / isoamyl alcohol with a mixing ratio of 24:1; centrifuge at 12000rpm for 5min; transfer 500ul of the supernatant to a 1.5ml centrifuge tube, add pre- Cold isopropanol, place at -20°C for more than 1 hour to fully precipitate, be careful not to absorb the middle layer...

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PUM

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Abstract

The invention provides a method for extracting vibrio parahaemolyticus total RNA, which is characterized by comprising the following steps of: (1) putting 1ml of vibrio parahaemolyticus liquid in a centrifuge tube to be centrifuged; (2) removing supernatant, quickly freezing thallus with liquid nitrogen, grinding into powder, transferring the powder into the centrifuge tube, adding Trizol, shaking for 1min, standing on ice for 3min, and repeatedly shaking for three times; (3) adding chloroform/isoamylol, and centrifuging; (4) transferring 500ul of supernatant into the centrifuge tube, adding 500ul of isopropanol, standing to fully precipitate and centrifuging; (5) carefully removing supernatant, washing with 70 percent of ethanol, and centrifuging; and (6) carefully absorbing supernatant as far as possible, inversely standing on a ultra-clean table to dry alcohol, adding DEPC water for dissolving, and storing under the temperature of 80 DEG C below zero. Since adopting the technical scheme, the invention has the advantages and the beneficial effects that the method can extract total RNA with high concentration, high purity and good integrity in the short three hours, and the used reagent is simple and has low cost.

Description

technical field [0001] The invention relates to a method for extracting total RNA of vibrio parahaemolyticus. Background technique [0002] Vibrio parahaemolyticus (Vibrio parahaemolyticus) is a Gram-negative polymorphic bacillus or slightly curved Vibrio, which widely exists in seawater and grows well in a medium containing 3% to 3.5% salt concentration, so it is also called pathogenic Halobacteria. Vibrio parahaemolyticus is a common pathogenic bacteria in aquatic products, pickled vegetables, cooked meat, poultry meat and poultry eggs. or undercooked food can cause food poisoning, so it is of great significance to carry out biological research on Vibrio parahaemolyticus. Isolation and extraction of high-quality RNA is the basis for subsequent molecular biology research such as PCR, Real-time PCR, gene cloning, and cDNA library establishment. Bacterial RNA has the characteristics of less content, short half-life, and easy degradation, so it is more difficult to extract ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 陈星潘迎捷赵勇孙晓红
Owner SHANGHAI OCEAN UNIV
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