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PCR purified reagent and method of purifying PCR product using same

A product and reagent technology, applied in the field of biological reagents, can solve environmental waste and other problems, and achieve the effects of protecting the environment, reducing garbage, saving social resources and research funds

Inactive Publication Date: 2018-10-12
WUHAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The technical problem to be solved by the present invention is: how to provide a PCR purification reagent to solve the problem of environment and waste caused by the one-time use of the existing PCR purification kit

Method used

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  • PCR purified reagent and method of purifying PCR product using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] The preparation of embodiment 1 DNA binding solution

[0021] DNA binding solution contains 3M sodium chloride (NaCl) and 30% (v / v) ethanol.

[0022] Take 50 ml of DNA Binding Solution as an example, the preparation method is as follows:

[0023] (1) Weigh 8.775 grams of NaCl in an electronic balance, and place the weighed NaCl in a 50 ml sterile centrifuge tube;

[0024] (2) Add 35 ml of distilled water to the centrifuge tube, and heat to dissolve NaCl in a 50°C water bath;

[0025] (3) Measure 15 milliliters of absolute ethanol and place it in a 50 milliliter sterile centrifuge tube;

[0026] (4) Add absolute ethanol to 35 ml of NaCl solution with a 5 ml pipette gun, cover the centrifuge tube and tighten the centrifuge tube, invert the centrifuge tube several times to thoroughly mix the NaCl solution and ethanol;

[0027] (5) Repeat step (4) once;

[0028] (6) Change to a 1ml pipette gun, absorb absolute ethanol, and continue to add it to the NaCl and ethanol mixt...

Embodiment 2

[0029] Embodiment 2 configuration of washing liquid

[0030] The washing solution contained 10 mM Tris.HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 80% (v / v) ethanol.

[0031] Take 100ml washing liquid as an example below, the preparation method is as follows:

[0032] (1) Prepare 1M Tris.HCl (pH 8.0) mother liquor and 0.5M EDTA mother liquor according to conventional methods;

[0033] (2) Weigh 0.585 gram of NaCl in electronic balance, place in 250 milliliter reagent bottle;

[0034] (3) Add 1 ml of 1M Tris.HCl (pH8.0) mother solution and 200 microliters of 0.5M EDTA mother solution to the reagent bottle;

[0035] (4) Add 18.5 milliliters of distilled water to completely dissolve NaCl in the reagent bottle;

[0036] (5) Measure 80 ml of absolute ethanol with a graduated cylinder, add it to the reagent bottle, mix thoroughly with the solution, and seal it with a cap.

experiment example

[0038] In order to determine the effect of the reagents of the present invention in DNA purification, the PCR product (LIF DNA, about 600bp) was purified using regeneration columns in Qiagen and Axygen PCR purification kits and the reagents of the present invention. The preparation method of the regenerated silicon column refers to the patent application number: 201710437190.7, and the title of the invention is: method for rapid regeneration of silicon column. At the same time, it was compared with the purification effect of the original silicon column and reagents of Qiagen and Axygen PCR purification kits.

[0039] Utilize reagent of the present invention and regeneration silicon column to complete PCR product purification method as follows:

[0040] (1) Amplify and synthesize 50 microliters of PCR products according to conventional PCR methods;

[0041] (2) Transfer the PCR product to a 1.5 milliliter sterile centrifuge tube, add 5 times the volume of the DNA binding solut...

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Abstract

The invention discloses a PCR purified reagent and a method of purifying a PCR product using same. The PCR purified reagent is prepared from a DNA binding buffer and a cleaning solution, wherein the DNA binding buffer is prepared from 3mol / L of sodium chloride and alcohol with volume concentration of 30%, and the cleaning solution is prepared from 10mmol / L of Tris.HCl, 100mmol / L of NaCl, 1mmol / L of EDTA, and alcohol with volume concentration of 80%, and pH of Tris.HCl is 8.0. The PCR purified reagent can be used for purifying DNA successfully without affecting DNA purification output. If the PCR purified reagent is used with a recycled silicon column, fewer disposable commercial kits are used, less laboratory rubbish is generated, the environment is protected, and social resources and usecosts are saved.

Description

technical field [0001] The invention belongs to the field of biological reagents, in particular to a PCR purification reagent and a method for purifying PCR products using the reagent. Background technique [0002] Gene cloning is one of the greatest inventions of the 20th century, widely used in many research fields including biology, medicine, agriculture, biopharmaceuticals, etc. Gene cloning is the use of replication-capable vectors or plasmids to connect the obtained target gene or DNA (deoxyribonucleic acid) fragments with the vector in vitro to form a recombinant vector and transform it into a host bacterium for replication. The process of obtaining a large amount of recombinant DNA through steps such as plasmids. Gene cloning includes a series of experimental steps, including the amplification and purification of the target gene, restriction enzyme digestion reaction and agarose gel electrophoresis, DNA gel extraction and ligation reaction, bacterial transformation,...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1006
Inventor 邓文生王娟赵沙沙
Owner WUHAN UNIV OF SCI & TECH
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