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A kind of preparation method of amino magnetic nanoparticles and its application in dna extraction

A magnetic nanoparticle and magnetic nanoparticle technology, applied in the fields of nanomaterials and molecular biology, can solve the problems of low DNA purity, DNA damage, toxic reagents, etc., and achieve the effects of strong DNA binding ability, easy operation and low price.

Active Publication Date: 2022-06-24
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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  • A kind of preparation method of amino magnetic nanoparticles and its application in dna extraction
  • A kind of preparation method of amino magnetic nanoparticles and its application in dna extraction
  • A kind of preparation method of amino magnetic nanoparticles and its application in dna extraction

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Experimental program
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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: To remove oxygen dissolved in the solvent, 100 mL of deionized water was degassed with nitrogen for 30 minutes; 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 500 rpm to make the reaction occur. The temperature was gradually raised to 80 °C to ensure that the salt components in the reaction system were completely dissolved; then, 1 M NaOH solution was slowly added to the iron salt mixture, and at the same time, the stirring speed was increased to 1000 rpm, and at pH about 12, The solution gradually turned black, and kept for 30 minutes to ensure the reaction was complete; finally, the solid-liquid separation was carried out with a...

Embodiment 2

[0050] Determination of binding buffers for EDA-SHT-MNPs to isolate and extract DNA

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

[0052] The binding buffer was set to a gradient with the concentrations of PEG8000 and NaCl, respectively, so that the concentration (w / v) of PEG8000 was 0%, 10%, 20%, 30%, 40% and 50%, and the concentration of NaCl was 0 mol / L, 0.15625mol / L, 0.3125mol / L, 0.625mol / L, 1.25mol / L, 2.5mol / L and 5mol / L, used for subsequent binding and separation experiments of plasmid DNA.

[0053] Pipette 21.6 μg of magnetic nanomaterials (EDA-SHT-MNPs) into a 1.5 mL EP tube, and after magnetic separation, discard the supernatant. 60 μL of the above-mentioned binding buffers containing different PEG concentrations and NaCl concentrations were added to the above-mentioned EP tube, and...

Embodiment 3

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

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

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

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Abstract

The invention provides a preparation method of amino magnetic nanoparticles and its application in DNA extraction. The preparation method of amino magnetic nanoparticles comprises the following steps: (1) Fe 3 o 4 Preparation of magnetic nanoparticles; (2) Fe coated with sodium hydrogen tartrate 3 o 4 Preparation of magnetic nanoparticles; (3) ethylenediamine-modified sodium hydrogen tartrate-coated Fe 3 o 4 Preparation of Magnetic Nanoparticles. The synthesis process of the amino magnetic nanoparticles prepared by the present invention has no high temperature and high pressure, and no three wastes are produced, so it has the characteristics of green environmental protection and simple process; compared with traditional commercial kits, the required raw materials are easy to obtain and the price is relatively cheap, and The synthesis steps are simple and easy to operate; the surface has amino groups and is in a positively charged state, which is conducive to the combination with negatively charged DNA. In the application of DNA extraction, magnetic nanoparticles show strong DNA binding ability and high extraction efficiency. , and the extraction steps are simple, avoiding the use of toxic reagents such as chloroform.

Description

technical field [0001] The invention relates to nanomaterials and molecular biology, in particular to a preparation method of amino magnetic nanoparticle 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 also become the primary task in the upstream of biological research, and the quality of the products obtained by separation and purification will directly affect the 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. The traditional DNA extraction methods mainly include cesium chloride ultracentrifugation, ion exchange resin method, polyethylene glycol precipitation method, boiling lysis method and alkaline lys...

Claims

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Application Information

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Patent Type & Authority Patents(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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