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Preparation method of amino magnetic nanoparticles and application thereof in DNA (desoxyribonucleic acid) extraction

A magnetic nanoparticle and magnetic nanoparticle technology, applied in the fields of nanomaterials and molecular biology, can solve problems such as low DNA purity, DNA damage, and toxic reagents, and achieve the effect of simple process, easy operation, and low price

Active Publication Date: 2019-12-10
ANHUI UNIVERSITY OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional DNA extraction methods mainly include cesium chloride ultracentrifugation method, ion exchange resin method, polyethylene glycol precipitation method, boiling lysis method and alkali lysis method, etc. The reagent is poisonous
At the same time, conventional commercial nucleic acid extraction methods are cumbersome and costly. In addition, multiple high-speed centrifugation is usually required, which will cause certain damage to DNA.

Method used

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  • Preparation method of amino magnetic nanoparticles and application thereof in DNA (desoxyribonucleic acid) extraction
  • Preparation method of amino magnetic nanoparticles and application thereof in DNA (desoxyribonucleic acid) extraction
  • Preparation method of amino magnetic nanoparticles and application thereof in DNA (desoxyribonucleic acid) extraction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Preparation and Characterization of EDA-SHT-MNPs

[0042] A preparation method of amino magnetic nanoparticles, comprising the following steps:

[0043] (1) Fe 3 o 4 Preparation of magnetic nanoparticles: In order to remove dissolved oxygen in the solvent, 100 mL of deionized water was degassed with nitrogen for 30 min; then, FeSO was added to it 4 ·7H 2 O and FeCl 3 ·6H 2 O, to ensure Fe 2+ and Fe 3+ The ratio of ions is 2:1, and magnetic heating and stirring are performed at a stirring speed of 500rpm to make it react. The temperature was gradually raised to 80°C to ensure that the salt components in the reaction system were completely dissolved; subsequently, a 1M NaOH solution was slowly added to the iron salt mixture, and at the same time the stirring speed was increased to 1000rpm, at a pH of about 12, The solution gradually turns black and is kept for 30 minutes to ensure that the reaction is complete; finally, use a magnet for solid-liquid separation to ...

Embodiment 2

[0050] Determining the binding buffer for EDA-SHT-MNPs to separate and extract DNA

[0051] In this example, the influence of the two main components PEG8000 and NaCl in the binding buffer on the separation and capture of plasmid DNA by EDA-SHT-MNPs is mainly discussed. The specific experimental steps are as follows:

[0052] Binding buffer solution is set gradient with the concentration of PEG8000 and NaCl respectively, makes the concentration (w / v) of PEG8000 be 0%, 10%, 20%, 30%, 40% and 50%, the concentration of NaCl is 0mol / L, 0.15625mol / L, 0.3125mol / L, 0.625mol / L, 1.25mol / L, 2.5mol / L and 5mol / L are used for subsequent plasmid DNA binding and separation experiments.

[0053] Pipette 21.6 μg of magnetic nanomaterials (EDA-SHT-MNPs) into a 1.5mLEP tube, and discard the supernatant after magnetic separation. Add 60 μL of the above-mentioned binding buffer containing different PEG concentrations and NaCl concentrations to the above-mentioned EP tube, shake and mix well, so t...

Embodiment 3

[0058] EDA-SHT-MNPs and commercial plasmid DNA extraction kits were used to extract plasmid DNA from bacteria

[0059] In this example, taking the extraction of the transformed Flag-Hakai plasmid in bacteria as an example, the effects of DNA extraction from EDA-SHT-MNPs and a commercial plasmid DNA extraction kit (Axygen) were compared.

[0060] The procedure for extracting DNA with a commercial kit is as follows: take 1 mL of overnight cultured bacterial solution, centrifuge at 12,000×g for 1 min, and discard the supernatant. Add 125 µL of buffer S1 to suspend the bacterial pellet. Then add 125 μL of buffer S2, gently and fully turn up and down 4-6 times until a clear solution is formed. Add 175 μL buffer S3, mix gently 6-8 times, and centrifuge at 12000×g for 10 min. Transfer the centrifuged supernatant to a preparation tube, centrifuge at 12,000×g for 1 min, and discard the filtrate. Put the preparation tube back into the EP tube, add 250 μL buffer W1, centrifuge at 1200...

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Abstract

The invention provides a preparation method of amino magnetic nanoparticles and application thereof in DNA (desoxyribonucleic acid) extraction. The preparation method of the amino magnetic nanoparticles comprises the following steps: (1) preparing Fe3O4 magnetic nanoparticles; (2) preparing sodium bitartrate coated Fe3O4 magnetic nanoparticles; and (3) preparing ethidene diamine modified sodium bitartrate coated Fe3O4 magnetic nanoparticles. The amino magnetic nanoparticles prepared by using the preparation method is free of high temperature or high pressure in the synthesis process, are freeof waste, and thus have the characteristics of being green and environment and simple in process; compared with a conventional commercial kit, the nanoparticles are easy in raw material obtaining, lowin price, simple in synthesis step and easy to operate; and the nanoparticles have amino groups on surfaces, are at positive charge states and are beneficial to combination with DNA with negative charges, in the DNA extraction application, the magnetic nanoparticles have good DNA combination capabilities and high extraction efficiency, extraction steps are simple and convenient, and toxic reagents such as chloroform can be avoided.

Description

technical field [0001] The invention relates to nanomaterials and molecular biology, in particular to a method for preparing amino magnetic nanoparticles and its application in DNA extraction. Background technique [0002] DNA is an important research object in biological research. With the deepening of related research on its structure and function, DNA separation and purification technology has become the primary task of the upstream of biological research, and the quality of the products obtained by separation and purification will directly affect subsequent experiments. Research result. Therefore, the development of new and efficient DNA separation and purification technology has become the core research content in the field of biological separation and purification. Traditional DNA extraction methods mainly include cesium chloride ultracentrifugation method, ion exchange resin method, polyethylene glycol precipitation method, boiling lysis method and alkali lysis metho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/22B01J20/28C12N15/10B01J20/30
CPCB01J20/06B01J20/22B01J20/28009C12N15/1013
Inventor 刘姿马亮陈学明刘祥吴雨晴朱音谛
Owner ANHUI UNIVERSITY OF TECHNOLOGY
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