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Double-magnetic-particle intervened DNA extraction and purification method

A purification method and magnetic separation technology, applied in the field of DNA extraction and purification involving dual magnetic particles, can solve the problems of inability to use RNase, high RNase purification cost, poor stability, etc., to improve the DNA purification method, avoid operation and reagents. Good effect of waste, spheroidization and dispersion

Pending Publication Date: 2021-03-16
SOUTH CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As an enzyme substance, RNase has high purification cost, poor stability, and is difficult to preserve
More importantly, RNase is often derived from animal pancreas extracts, and RNase cannot be used in the extraction of medical-grade plasmid DNA

Method used

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  • Double-magnetic-particle intervened DNA extraction and purification method
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  • Double-magnetic-particle intervened DNA extraction and purification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The preparation of GO-MPs includes the following steps:

[0055] (1) Accurately weigh 2.4g of ferric chloride and dissolve it in 120mL of ethylene glycol for ultrasonic dissolution, then add 10.8g of sodium acetate and 4.5g of polyethylene glycol 4000 to fully dissolve to form a uniform dark yellow solution. Pack evenly into 4 Teflon autoclaves, and react at 200°C for 10h. After cooling to room temperature, the obtained PEG-MPs were separated by an external magnetic field, washed several times with ethanol and deionized water, and dried in an oven at 50°C under nitrogen protection.

[0056] (2) Take 50mg graphene oxide and ultrasonically disperse it in 30mL deionized water; meanwhile take 500mg PEG-MPs and ultrasonically disperse it in 20mL deionized water. Then the two were mixed and reacted in a water bath at 70° C. for 2 h under the protection of nitrogen. GO-MPs were separated by applying an external magnetic field, washed with ethanol and deionized water and drie...

Embodiment 2

[0062] The preparation of polyDOPA-MPs comprises the following steps:

[0063] (1) Accurately weigh 2.4g of ferric chloride and dissolve in 30mL of ethylene glycol and 90mL of diethylene glycol dual solvents. Then 9 g of sodium acrylate and 9 g of sodium acetate were added and stirred to form a dark yellow solution. Pack evenly into 4 Teflon autoclaves, and react at 200°C for 10h. The obtained product (COOH-MPs) was then washed several times with water and ethanol by magnetic separation, and dried at 50°C under nitrogen protection.

[0064] (2) Weigh 160 mg of dopa and dissolve in 160 mL of Tris-HCl (10 mM, pH 8.5) buffer; then weigh 40 mg of COOH-MPs and disperse in the dopa solution, and magnetically stir at room temperature (25° C.) for 24 h. After the reaction, the product polyDOPA-MPs was obtained by magnetic separation, washed with ethanol and deionized water three times, and finally dried at 50°C under nitrogen protection for later use.

[0065] The COOH-MPs and poly...

Embodiment 3

[0070] The extraction and purification method of E.coli DH5α genomic DNA comprises the following steps:

[0071] (1) the GO-MPs that embodiment 1 makes, the polyDOPA-MPs that embodiment 2 makes are respectively dispersed in Tris-HCl-EDTA buffer (10mmol / L Tris, 1mmol / L EDTA, pH value 5.8), The concentration is 100mg / mL; GO-MPs suspension and polyDOPA-MPs suspension are obtained respectively;

[0072] (2) Take 1.5mL E.coli DH 5α culture solution and centrifuge at 10000rpm for 1min to separate the bacteria from the culture medium. Then add 300 μL of lysate (25mmol / L Tris-HCl, 20mmol / L NaAc, 1mmol / L EDTA, 1% SDS, pH 7.8) to the cell pellet, and incubate in a water bath at 37°C for 1 hour. Oscillations from time to time. After the incubation is complete, add 100 μL NaCl (4.0 mol / L) to it and mix thoroughly, and centrifuge the mixture at 4°C (10,000 rpm) for 20 min;

[0073] (3) Take 200 μL supernatant (bacterial lysate), add 500 μL adsorption solution (10 mmol / L Tris-HCl and 1 m...

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Abstract

The invention discloses a double-magnetic-particle intervened DNA extraction and purification method. The method comprises the following steps: taking a sample lysis solution, adding an adsorption solution and a NaCl solution, uniformly mixing the mixture, adding a GO-MPs suspension, performing incubation, carrying out magnetic separation, and taking a supernatant; and adding a polyDOPA-MPs suspension, carrying out magnetic separation after incubation, discarding the supernatant, washing the obtained precipitate with an ethanol solution, adding an eluent, carrying out magnetic separation afterincubation, and taking the supernatant, namely the purified DNA. According to the method disclosed by the invention, the GO-MPs can selectively remove RNA from a mixed solution of DNA and RNA, and can replace RNase used in a traditional method, so that the RNA removal cost is reduced; and at the same time, the adsorption process is simple and rapid, and separation can be realized only through a magnetic field.

Description

technical field [0001] The invention relates to a DNA extraction and purification method involving double magnetic particles (graphene oxide modified magnetic particles GO-MPs and polydopa modified magnetic particles polyDOPA-MPs). Background technique [0002] High-quality DNA purification is the basis for many downstream operations including PCR, qPCR, and nucleic acid detection. Therefore, it is of great research value to develop low-cost, high-efficiency and high-quality DNA purification methods. [0003] The phenol-chloroform method is a traditional DNA extraction method, but this method has complicated steps, takes a long time, and requires the use of highly toxic reagents such as phenol and chloroform, which is very unfriendly to the environment and experimenters. [0004] The method based on solid phase extraction (SPE) avoids the application of toxic and harmful reagents, but still needs to use multiple centrifugation. The shearing force caused by centrifugation c...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/101
Inventor 贾丽付云海
Owner SOUTH CHINA NORMAL UNIVERSITY
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