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Kit and method for extracting bacterial plasmids by alkali lysis method

A technology of bacterial plasmids and kits, applied in biochemical equipment and methods, microbial measurement/inspection, DNA preparation, etc., can solve the problems of inability to use sequencing and enzyme digestion, short time-consuming, easy failure, etc., to meet the requirements of sequencing and enzyme digestion operations, reducing contact time, and long storage time

Active Publication Date: 2022-06-24
HUNAN UNIV OF SCI & ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the traditional SDS alkaline lysis method (J. Sambrook (Sambrook.J.), D.W. Russell, Huang Peitang et al. translation. Molecular Cloning Experiment Guide [M]. Science Press, 2008) when extracting the plasmid, the solution The concentration of I glucose is low and no RNase enzyme is added, and no protein inhibitor is added to solution III, and phenol-chloroform extraction is required to remove the protein. The extraction takes a long time, and the extracted plasmid is less, which may be degraded and contains a large amount. Bacterial RNA cannot be used for operations such as sequencing and enzyme digestion
Various plasmid extraction kits currently on the market, due to the use of silica gel membrane technology or magnetic bead technology, take a short time to extract plasmids, but require the use of biological agents such as RNase and lysozyme, which are prone to failure and short storage time, so for some Waste of reagents and loss of money for laboratories with few experimental staff and insufficient funds

Method used

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  • Kit and method for extracting bacterial plasmids by alkali lysis method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] 1. The composition of reagents for extracting bacterial plasmids:

[0032] Bacterial suspension: 50mmol / L glucose, 10mmol / L EDTA, 20mmol / L Tris-HCl, 1.5mol / LLiCl, 0.5mol / L NH 4 Ac, solution pH 8.0;

[0033] Bacterial lysate: 0.2mol / L NaOH, 1% SDS;

[0034] Plasmid renaturation solution: 0.8mol / L KAc and 3.5mol / L guanidine isothiocyanate, pH 4.8;

[0035] Washing solution: 70% ethanol in water.

[0036] 2. The method of extracting the recombinant plasmid and pBI121-EGFP plasmid connecting pGM-T and the target gene with the reagent described in step 1:

[0037] 1) Take 1 mL of Escherichia coli DH5α culture solution in a 1.5 mL centrifuge tube, centrifuge at 12,000 r / min for 1 min, remove the supernatant, and retain the precipitate;

[0038] 2) Add 150 μL of bacterial suspension to the centrifuge tube and place it on a shaker to fully suspend the bacterial pellet;

[0039] 3) Add 250 μL of bacterial lysate to the bacterial suspension, invert the centrifuge tube 5 time...

Embodiment 2

[0049] 1. The composition of reagents for extracting bacterial plasmids:

[0050] Bacterial suspension: 120mmol / L glucose, 30mmol / L EDTA, 30mmol / L Tris-HCl, 3.0mol / LLiCl, 1.5mol / L NH 4 Ac, solution pH 8.0;

[0051] Bacterial lysate: 0.2mol / L NaOH, 1% SDS;

[0052] Plasmid renaturation solution: 3mol / L KAc and 4.5mol / L guanidine isothiocyanate, pH 4.8;

[0053] Washing solution: 80% ethanol in water.

[0054] 2. The method of extracting the recombinant plasmid and pBI121-EGFP plasmid connecting pGM-T and the target gene with the reagent described in step 1:

[0055] 1) Take Escherichia coli DH5α culture medium in a 1.5mL centrifuge tube, centrifuge at 12000r / min for 1min, remove the supernatant, and keep the precipitate; then add 1.5mL of Escherichia coli DH5α culture medium to the same centrifuge tube, centrifuge at 12000r / min for 1min , remove the supernatant and keep the precipitate;

[0056] 2) Add 370 μL of bacterial suspension to the centrifuge tube and place it on a...

Embodiment 3

[0067] 1. The composition of reagents for extracting bacterial plasmids:

[0068] Bacterial suspension: 100mmol / L glucose, 20mmol / L EDTA, 25mmol / L Tris-HCl, 2.5mol / LLiCl, 1.0mol / L NH 4 Ac, solution pH 8.0;

[0069] Bacterial lysate: 0.2mol / L NaOH, 1% SDS;

[0070] Plasmid renaturation solution: 1.0mol / L KAc and 4.0mol / L guanidine isothiocyanate, pH 4.8;

[0071] Washing solution: 80% ethanol in water.

[0072] 2. The method of extracting the recombinant plasmid and pBI121-EGFP plasmid connecting pGM-T and the target gene with the reagent described in step 1:

[0073] 1) Take 1.5mL of Escherichia coli DH5α culture medium in a 1.5mL centrifuge tube, centrifuge at 12000r / min for 1 min, remove the supernatant, and keep the precipitate; then add 1.5mL of Escherichia coli DH5α culture medium to the same centrifuge tube, 12000r / min Centrifuge for 1 min, remove the supernatant, and keep the precipitate; collect 5 mL of the E. coli DH5α culture solution by the same operation;

[0...

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Abstract

The invention relates to a kit and a method for extracting bacterial plasmids by an alkali lysis method, and belongs to the technical field of biology. According to the kit for extracting the bacterial plasmids by the alkali lysis method, inorganic salts LiCl and NH4Ac are added into bacterial suspension, so that RNA (Ribonucleic Acid) in a bacterial lysis solution is easy to precipitate and remove, the preservation time of the extraction solution is long, and the required expenditure is low; the glucose concentration is increased in the bacterial lysis solution, and high-concentration guanidine isothiocyanate is added in the plasmid renaturation solution, so that the degradation of the plasmids can be prevented, and the integrity of the extracted plasmids is ensured. Meanwhile, the silica gel membrane technology is adopted, the time needed for collecting plasmids in the solution is short, the contact time of the plasmids and the solution is shortened, small plasmids can be extracted, large plasmids can also be extracted, and the requirements for sequencing, enzyme digestion and other operations of molecular biology experiments can be met.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a kit for extracting bacterial plasmids by an alkaline lysis method and a method thereof. Background technique [0002] Plasmid is a kind of self-replicating circular DNA molecule in bacteria independent of chromosome, which can be used for gene preservation and replication, enzyme digestion, construction and transformation of recombinant vector and other researches. The quality of plasmids is closely related to the progress of molecular biology research, and plasmids with good quality are conducive to the smooth progress of molecular biology research. [0003] The extraction of plasmids in Escherichia coli generally includes the steps of lysing bacteria, denaturing proteins and nucleic acids, renaturing plasmids, removing impurities, and precipitating plasmids. Due to the traditional SDS alkaline lysis method (J. Sambrook.J., written by D.W. Russell, translated by Huang Peitang et ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003C12Q2527/125C12Q2523/32Y02A50/30
Inventor 黄国文
Owner HUNAN UNIV OF SCI & ENG
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