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Kit for magnetic bead method for bacterial genome DNA extraction and extraction method thereof

A genome and kit technology, applied in the field of molecular biology, can solve the problems of high price, insufficient interaction between magnetic beads and buffer, and achieve the effects of stable and reliable quality, increased extraction efficiency, and high purity

Active Publication Date: 2014-12-17
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, well-known foreign biotechnology companies, such as Qiagen, Promega, Amresco, etc. have successively developed magnetic bead nucleic acid extraction kits or magnetic bead high-throughput nucleic acid extraction instruments. The nucleic acid is of high quality and the fragments are complete, but the price is expensive; the magnetic bead method nucleic acid extraction kit developed by well-known domestic biotechnology companies, such as Tiangen, Kangwei Century, and Jinmag, has built-in magnetic beads that are polydisperse magnetic Although the magnetic response speed of beads is very fast, the sedimentation speed of the magnetic beads is also very fast, which leads to insufficient interaction between the magnetic beads and the buffer, which has also become a major factor restricting the development of its products

Method used

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  • Kit for magnetic bead method for bacterial genome DNA extraction and extraction method thereof
  • Kit for magnetic bead method for bacterial genome DNA extraction and extraction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1, monodisperse Fe 3 O 4 SiO 2 - Preparation of AEAPS nanomagnetic beads.

[0031] (1) Monodisperse iron ferric oxide nanospheres were prepared by solvothermal method. The specific preparation process was as follows: Weighed 2.0 g of sodium acetate and 0.25 g of ferric chloride hexahydrate, respectively, and added them to 50 ml of ethylene glycol solution. After magnetic stirring for 1 hour, the system was transferred to a reaction kettle, and reacted at 100° C. for 10 hours. After the reaction was completed, the resulting black solution was centrifuged, and washed 3 times with deionized water and ethanol successively to obtain monodisperse ferric oxide nanospheres (Fe 3 o 4 ), the resulting product was dried in a 60°C oven for use;

[0032](2) Monodisperse silica-coated Fe3O4 nanospheres were prepared by the improved Stäber method. The specific preparation process was as follows: 0.1 g of Fe3O4 nanospheres prepared in step (1) were weighed ...

Embodiment 2

[0034] Embodiment 2: application embodiment.

[0035] (1) Take 1ml of fresh Shigella culture solution and place it in a 2.0ml EP tube, centrifuge at 12000 rpm for 1min, collect the bacteria, add 200ul buffer A, beat and mix well, and resuspend the bacteria;

[0036] (2) Add 200ul buffer B to the EP tube in step (1), incubate in a 56°C water bath for 10min;

[0037] (3) Add 200ul of buffer C to the EP tube incubated in step (2), beat and mix, and let stand at room temperature for 3 minutes;

[0038] (4) Add 30ul of magnetic bead suspension D to the EP tube after step (3), beat and mix well, place the EP tube on the magnetic stand, let it stand for 30s, and carefully remove the liquid when the magnetic beads are completely absorbed;

[0039] (5) Remove the EP tube in step (4) from the magnetic stand, add 500ul of buffer E, beat and mix, place the EP tube on the magnetic stand, let it stand for 30s, and carefully remove the liquid when the magnetic beads are completely absorbe...

Embodiment 3

[0045] Embodiment 3: application embodiment.

[0046] (1) Take 1ml of fresh Staphylococcus aureus culture solution and place it in a 2.0ml EP tube, centrifuge at 12000 rpm for 1min, collect the bacteria, add 200ul buffer A, 20μl lysozyme (20mg / ml), vortex and mix well, and EP Tubes were incubated in a 37°C water bath for 1 hour, and mixed 3 times during the period;

[0047] (2) Add 200ul buffer B to the EP tube in step (1), incubate in a 56°C water bath for 10min;

[0048] (3) Add 200ul of buffer C to the EP tube incubated in step (2), beat and mix, and let stand at room temperature for 3 minutes;

[0049] (4) Add 30ul magnetic bead suspension D to the EP tube after step (3), place the EP tube on the magnetic stand, let it stand for 30s, and carefully remove the liquid when the magnetic beads are completely absorbed;

[0050] (5) Remove the EP tube in step (4) from the magnetic stand, add 500ul of buffer E, beat and mix, place the EP tube on the magnetic stand, let it stan...

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Abstract

The invention discloses a kit for a magnetic bead method for bacterial genome DNA extraction and an extraction method thereof. The kit includes the following seven components: a buffer solution A, a buffer solution B, a buffer solution C, a magnetic bead suspension solution D, a buffer solution E, a buffer solution F and a buffer solution G. composition of the magnetic bead suspension solution D is 50mg of monodisperse Fe3O4 @ SiO2 AEAPS nanometer magnetic beads in each 200 mmol / L sodium chloride aqueous solution. The extraction method for the kit comprises six steps: thallus re-suspension, cracking, nucleic acid deposition, magnetic bead adsorption, washing and elution. The kit provided by the invention adopts efficient monodisperse nano magnetic beads combined with a unique buffer system, so that the extracted bacterial genome DNA has large fragment, high purity and stable and reliable quality, and can meet the requirements of the follow-up experiments.

Description

[0001] technical field [0002] The invention belongs to the technical field of molecular biology, and relates to a kit for extracting bacterial genome DNA by a magnetic bead method and an extraction method thereof. Background technique [0003] In molecular biology techniques, whether it is the establishment of gene libraries, enzyme digestion, molecular cloning or genome-wide association analysis, it is necessary to extract DNA templates from samples. The concentration, purity, and integrity of the primary structure of the extracted DNA directly affect subsequent research, so the quality of DNA is the primary factor for the success of genetic research. [0004] Among the traditional DNA extraction methods, the spin column method and the phenol-chloroform method are the two most important methods. Although the quality of the extracted DNA is high, the detection time is long and labor-intensive. Toxic reagents such as chloroform caused great harm to the operator and the eco...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 郝荣章宋宏彬李杨赵荣涛许金坤卢晓董世彪邱少富王勇贾雷立李鹏谢靖王立贵吴志豪
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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