A kind of kit and extraction method for rapid extraction of bacterial plasmid DNA by magnetic bead method

A bacterial plasmid and extraction method technology, applied in the field of molecular biology, can solve problems such as long time consumption and poor binding ability, and achieve the effect of reducing nucleic acid loss, fragment integrity, and promoting aggregation and precipitation

Active Publication Date: 2022-07-05
WUHAN TEXTILE UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] However, the above extraction methods or the magnetic beads in the kit have poor binding ability to DNA, and the process of magnetic beads adsorbing DNA takes a long time

Method used

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  • A kind of kit and extraction method for rapid extraction of bacterial plasmid DNA by magnetic bead method
  • A kind of kit and extraction method for rapid extraction of bacterial plasmid DNA by magnetic bead method

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0046] Embodiment 1 provides a kit for rapidly extracting bacterial plasmid DNA by magnetic bead method, the kit includes a lysis reagent set for lysing bacteria and an extraction reagent set for extracting plasmid DNA;

[0047] The lysis reagent group is composed of bacterial suspension, lysis solution and neutralization solution. The resuspension is an aqueous solution containing 15 mM ethylenediaminetetraacetic acid and 20 mM tris hydrochloride, with a pH of 7.5; the lysing solution is an aqueous solution containing 0.3 M sodium hydroxide and 2% sodium dodecyl sulfate. Aqueous solution; the neutralization solution is an aqueous solution containing 4M potassium acetate and 3M acetic acid, with a pH of 5.0;

[0048] The extraction reagent group is composed of a magnetic bead binding solution, a washing solution, and an eluent. The washing solution is an isopropanol aqueous solution or an ethanol aqueous solution with a volume fraction of 80%; the eluent is distilled water. ...

Embodiment 2

[0057] The difference from Example 1 is that the preparation ratio of the magnetic bead binding solution is different, and the others are the same as those in Example 1, which will not be repeated here.

[0058] In this embodiment, the magnetic bead binding solution is a mixed magnetic bead solution consisting of 0.15M sodium acetate, 2wt% dithiothreitol, 3M guanidine isothiocyanate and 3mL of silanol magnetic bead suspension with a size of 700nm , wherein the magnetic bead suspension is prepared by mixing magnetic beads and isopropanol according to a mass ratio of 1:20, and the pH of the solution is 7.5.

Embodiment 3

[0060] The difference from Example 1 is that the preparation ratio of the magnetic bead binding solution is different, and the others are the same as those in Example 1, which will not be repeated here.

[0061] In this embodiment, the magnetic bead binding solution is a mixed magnetic bead solution comprising 0.3% sodium acetate, 3wt% dithiothreitol, 3M guanidine isothiocyanate, and 3mL of a suspension of silicon carboxyl-based magnetic beads with a size of 1000 nm, The magnetic bead suspension is prepared by mixing magnetic beads and isopropanol according to a mass ratio of 1:10, and the pH of the solution is 8.5.

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Abstract

The invention provides a kit and an extraction method for rapidly extracting bacterial plasmid DNA by a magnetic bead method. The kit includes a lysis reagent set for lysing bacteria and an extraction reagent set for extracting plasmid DNA; the extraction reagent set consists of a magnetic bead binding solution, a washing solution, and an elution solution. In the method for rapid extraction of plasmid DNA based on the kit provided by the present invention, magnetic beads with silanol or silyl carboxyl groups modified on the surface, 0.15-0.45M sodium acetate, 1-3wt% dithiothreitol and 3- The mixture of 5M guanidine isothiocyanate is used as a composite system of magnetic bead binding solution, which greatly enhances the binding ability of plasmid DNA and magnetic beads, improves the extraction efficiency, and accelerates the extraction speed. The adsorption process of magnetic beads to DNA only needs 1 In addition, the extracted DNA has high purity and complete fragments; at the same time, the reagents are non-toxic and have little environmental pollution, and can be used to replace most similar products on the market.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a kit and an extraction method for rapidly extracting bacterial plasmid DNA by a magnetic bead method. Background technique [0002] In the prior art, the extraction method of bacterial plasmid DNA generally includes steps such as bacterial lysis, DNA aggregation and precipitation, impurity removal, and DNA purification and separation. Traditional extraction methods mainly include column method and CTAB cracking method. These methods are cumbersome and time-consuming, which is not conducive to large-scale extraction. In addition, organic solvents such as chloroform and phenol may be used during the operation, resulting in greater pollution. [0003] The magnetic bead method bacterial plasmid DNA extraction kit utilizes the specific adsorption of surface-modified magnetic beads and DNA molecules, and adopts magnetic nano-separation technology, which has the advantages of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
CPCC12N15/1013C12Q2527/125
Inventor 王栋钟亚平鲁振坦
Owner WUHAN TEXTILE UNIV
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