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Regeneration method of silicon matrix nucleic acid purification column

A purification column and matrix technology, which is applied in the field of nucleic acid purification in molecular biology, can solve the problems of unfavorable ecological environment protection, low temperature conditions, and cumbersome operation in the later discharge of the treatment solution, so as to restore the nucleic acid binding ability, prolong the processing time, and reduce the cost. Effect

Pending Publication Date: 2021-08-20
广西产研院生物制造技术研究所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Tagliavia et al. (2009), Jiang Wangsheng et al. (Chinese patent 200910220752.8), Han Han (Chinese patent 201010149814.3) and Xiong Minghua et al. (2015) mainly use lye or acid to sequentially treat nucleic acid purification columns that contaminate genomic DNA, which can effectively remove the nucleic acid on the column residues, but these methods use higher concentrations of corrosive strong acids and bases (NaOH, H 2 SO 4 , HCl) components and 65°C water bath conditions, there are potential safety hazards, and the later discharge of the treatment liquid is not conducive to ecological protection
Liu Chuanqing et al. (2014) put the used DNA purification column in 0.01mol / L phosphate buffer at pH 2.4, and treated it at 4°C for 6 days. No consideration is given to the contamination of nucleic acid residues to the extraction of other nucleic acids, and the treatment method requires low temperature conditions and takes a long time
Chinese patent (patent number: 201110294478.6) discloses a siliceous membrane centrifugal adsorption column regeneration solution and its use method. The treatment method has mild conditions and is ecologically friendly. Nucleic acid purification column

Method used

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  • Regeneration method of silicon matrix nucleic acid purification column
  • Regeneration method of silicon matrix nucleic acid purification column
  • Regeneration method of silicon matrix nucleic acid purification column

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0017] Add 40g of citric acid (anhydrous citric acid) into 2000mL of deionized water, stir until completely dissolved, then add 0.5mL of Trition X-100, stir well, and prepare a solution containing 2% (W / V) citric acid and 0.025 % (V / V) TritionX-100 solution A; Weigh 1.58g of Tris-HCl (trishydroxymethylaminomethane hydrochloride) and add it to 2000mL deionized water, fully stir until completely dissolved, and adjust the pH to 8.0 to obtain 5mM Tris-HCl (pH 8.0) buffer was solution B.

[0018] The method of the present invention handles and regenerates the silicon matrix nucleic acid purification column, and its operation steps are as follows:

[0019] Use 75% alcohol (or absolute ethanol) to wipe off the markers on the used nucleic acid purification column, separate the purification column from its external collection tube, soak the purification column in 2 times the volume of A solution, and place it at room temperature for 20r Gently stir for 10-15 hours under the condition ...

example 2

[0021] Add 60g of citric acid (anhydrous citric acid) to 2000mL of deionized water, stir well until it is completely dissolved, then add 1.0mL of Trition X-100, stir well, and prepare to contain 3% (W / V) citric acid and 0.05 % (V / V) TritionX-100 solution A; Weigh 3.16g of Tris-HCl (trishydroxymethylaminomethane hydrochloride) and add it to 2000mL deionized water, fully stir until completely dissolved, and adjust the pH to 8.0 to obtain 10mM Tris-HCl (pH 8.0) buffer is solution B.

[0022] Solution A and solution B were prepared according to this example, and the used silica matrix nucleic acid purification column was regenerated according to the operation steps of Example 1.

example 3

[0024] Add 80g of citric acid (anhydrous citric acid) into 2000mL of deionized water, stir well until it is completely dissolved, then add 2.0mL of Trition X-100, stir well, and prepare to contain 4% (W / V) citric acid and 0.1 % (V / V) TritionX-100 solution A; Weigh 3.16g of Tris-HCl (trishydroxymethylaminomethane hydrochloride) and add it to 2000mL deionized water, fully stir until completely dissolved, and adjust the pH to 8.0 to obtain 10mM Tris-HCl (pH 8.0) buffer is solution B.

[0025] Solution A and solution B were prepared according to this example, and the used silica matrix nucleic acid purification column was regenerated according to the operation steps of Example 1.

[0026] In order to further describe the method of the present invention better, the inventor took the cloned Escherichia coli putative oxidoreductase (putative oxidoreductase) gene yqhD as the experimental object, and carried out PCR product respectively by using a new purification column and a purifica...

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Abstract

The invention discloses a regeneration method of a silicon matrix nucleic acid purification column. The regeneration method specifically comprises the following steps that the purification column is soaked in a solution A, and is slightly stirred at room temperature for 10-15 hours; then the purification column is washed twice with tap water; the purification column is soaked in a solution B to be balanced to be neutral; and the purification column is soaked in purified water, sterilized at 121 DEG C for 15 minutes, and dried in a drying oven at 60 DEG C for later use. According to the regeneration method of the silicon matrix nucleic acid purification column, a large number of purification columns can be treated at a time, the purification columns are regenerated five times, and the nucleic acid adsorption rate is kept at about 95%. The regeneration method is simple to operate, has the advantages of economy, high efficiency, environmental protection and the like, and is suitable for regeneration and reutilization of the silicon matrix nucleic acid purification columns commonly used in the market.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid purification in molecular biology, and relates to a method for regenerating an experimental silica-based nucleic acid purification column, in particular to a method with simple operation, mild conditions, and convenience for large-scale treatment and regeneration of such purification columns method. Background technique [0002] Nucleic acid is the carrier and transmitter of biological genetic information and the most basic operation object of molecular biology. Nucleic acid extraction and purification are important components of molecular biology experiments. The classic phenol-chloroform method to extract and purify nucleic acid is not only cumbersome, but also uses toxic substances such as phenol and chloroform, and is also polluted by various impurities. Centrifugal nucleic acid purification columns are usually a centrifugal structure consisting of an outer collection tube and an inner p...

Claims

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Application Information

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IPC IPC(8): C12N15/10B01D15/20
CPCC12N15/101B01D15/203
Inventor 韦旭钦胡雪玲吴睿彭小玉阮恒李广曹志强王则奋周培林李略
Owner 广西产研院生物制造技术研究所有限公司