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Bacterial lysis buffer, kit and method for extracting bacterial plasmid DNA

A bacterial plasmid and lysate technology, applied in the biological field, can solve the problems of higher requirements on reagent storage and transportation conditions, increase reagent costs, etc., and achieve the effect of meeting the needs of biological experiments, low cost, and good combination.

Active Publication Date: 2021-11-09
生工生物工程(上海)股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the addition of lysozyme and RNaseA will increase the cost of reagents, and also have higher requirements for the storage and transportation conditions of reagents

Method used

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  • Bacterial lysis buffer, kit and method for extracting bacterial plasmid DNA
  • Bacterial lysis buffer, kit and method for extracting bacterial plasmid DNA
  • Bacterial lysis buffer, kit and method for extracting bacterial plasmid DNA

Examples

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Effect test

Embodiment 1

[0068] A kit for extracting bacterial plasmid DNA, comprising cell lysate, washing solution A, washing solution B, eluent and silica gel membrane adsorption column; wherein:

[0069] The cell lysate consists of: 33mM citric acid, 17mM sodium citrate, 250mM sodium chloride, 3% Triton X-100 and 4M guanidine isothiocyanate;

[0070] Washing solution A consists of: 1M guanidine hydrochloride and 50% isopropanol;

[0071] Washing solution B consists of: 50mM Tris-HCl and 80% ethanol, pH 7.0;

[0072] The composition of the eluent is: 2.5mM Tris-HCl, pH 8.0;

[0073] Adsorption column: Sangon Bioengineering (Shanghai) Co., Ltd. nucleic acid purification adsorption column (including collection tube).

Embodiment 2

[0075] A kit for extracting bacterial plasmid DNA, comprising cell lysate, washing solution A, washing solution B, eluent and silica gel membrane adsorption column; wherein:

[0076] The cell lysate consists of: 27mM citric acid, 23mM sodium citrate, 250mM sodium chloride, 2% Triton X-100 and 4M guanidine isothiocyanate;

[0077] Washing solution A consists of: 1M guanidine hydrochloride and 50% isopropanol;

[0078] Washing solution B consists of: 50mM Tris-HCl and 80% ethanol, pH 7.0;

[0079] The composition of the eluent is: 2.5mM Tris-HCl, pH 8.0;

[0080] Adsorption column: Sangon Bioengineering (Shanghai) Co., Ltd. nucleic acid purification adsorption column (including collection tube).

Embodiment 3

[0082] A kit for extracting bacterial plasmid DNA, comprising cell lysate, washing solution A, washing solution B, eluent and silica gel membrane adsorption column; wherein:

[0083] The cell lysate consists of: 39mM citric acid, 11mM sodium citrate, 250mM sodium chloride, 2% Triton X-100 and 4M guanidine isothiocyanate;

[0084] Washing solution A consists of: 1M guanidine hydrochloride and 50% isopropanol;

[0085] Washing solution B consists of: 50mM Tris-HCl and 80% ethanol, pH 7.0;

[0086] The composition of the eluent is: 2.5mM Tris-HCl, pH 8.0;

[0087] Adsorption column: Sangon Bioengineering (Shanghai) Co., Ltd. nucleic acid purification adsorption column (including collection tube).

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Abstract

The invention provides a bacterial lysis buffer, a kit and a method for extracting bacterial plasmid DNA. The bacterial lysis buffer comprises the following components: 4-5M of protein denaturant, 100-500mM of sodium chloride, 2-3wt% of surfactant, 38-62mM of pH buffer and the balance of water; and the protein denaturant comprises at least one of guanidine isothiocyanate and guanidine hydrochloride. According to the invention, the synergistic effect of the surfactant and the protein denaturant (at least one of guanidine isothiocyanate and guanidine hydrochloride) is mainly used for cracking bacterial cells; the pH of the bacterial lysis buffer can be well adjusted through the pH buffer and the sodium chloride; the bacterial lysis buffer is simple in composition and low in cost, does not contain lysozyme and RNaseA, can be stably stored at room temperature for a long time, and has no influence on subsequent experiments; and the kit using the bacterial lysis buffer is high in extraction efficiency and high in speed when being used for extracting plasmid DNA, and can meet various biological experiment requirements.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a bacterial cell lysate, a kit and a method for extracting bacterial plasmid DNA. Background technique [0002] Plasmids are extrachromosomal genetic units that can replicate autonomously, including DNA molecules outside eukaryotic organelles and bacterial chromosomes. The vast majority of bacterial plasmid DNA is a double-stranded closed circular DNA molecule, often carrying antibiotic resistance genes, such as ampicillin resistance and chloramphenicol resistance. [0003] Plasmid DNA is a gene delivery tool, which is widely used in molecular biology research, involving various fields such as gene cloning, gene sequence analysis, gene vaccine and gene editing. Extracting plasmid DNA molecules from bacteria is the most basic operation in molecular biology experiments. At the same time, the efficiency, purity and quality of plasmid DNA molecular extraction are directly related to th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/101C12Q2527/119C12Q2527/125C12Q2523/113C12Q2523/308C12Q2523/32Y02A50/30
Inventor 赵斯斯薛茜陈辉芦丽亚万军飞
Owner 生工生物工程(上海)股份有限公司