Nucleic acid rapid-purifying method and kit

A kit and nucleic acid technology, applied in chemical instruments and methods, biochemical equipment and methods, organic chemistry, etc., can solve the problems of low nucleic acid extraction efficiency, high salt content of nucleic acid samples, and additional purification steps

Inactive Publication Date: 2014-05-14
EMERGING THERAPEUTICS SHANGHAI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, solid-liquid separation often still requires centrifugation or suction filtration, which is cumbersome and difficult to automate
[0009] In summary, there are still some deficiencies in the existing nucleic acid separation technology: for example, the use of protease digestion process increases the time of nucleic acid extraction, which needs to be stored at a specific temperature; cell sample lysis and binding are carried out step by step, adding purification steps; elution The post-nucleic acid sample has high salt content, which is not conducive to the downstream molecular biology experiment operation; the nucleic acid extraction efficiency is not high, and the impurities such as protein are high

Method used

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  • Nucleic acid rapid-purifying method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] The preparation of embodiment 1 different surface modification magnetic microspheres

[0114] 1) Preparation of aminated silica magnetic microspheres: Weigh an appropriate amount of commercially available silanol-based silica magnetic microspheres, add absolute ethanol, water, concentrated ammonia water, and finally add 3'-aminopropyltriethoxysilane, After mixing, stir and react at room temperature for 3 hours, and wash the product with absolute ethanol and distilled water in sequence to obtain magnetic microspheres with amino groups bonded to the surface.

[0115] 2) Preparation of carboxylated silica magnetic microspheres: Weigh an appropriate amount of commercially available silanol-based silica magnetic microspheres, add absolute ethanol, water, concentrated ammonia water, and finally add 3'-glycidyloxypropyl trimethoxy base silane, stirred and reacted at room temperature for 3 hours after mixing, and the product was washed with absolute ethanol and distilled water ...

Embodiment 2

[0116] Embodiment 2 purifies nucleic acid with kit

[0117] The specific configuration examples of the kit for extracting DNA described in this kit include:

[0118] Magnetic microspheres: the self-made carboxylated silica magnetic microspheres

[0119] Lysis binding solution: 4M guanidine hydrochloride, 2% Triton X-100, 0.1% SDS, 0.01% mercaptoethanol, 0.1M NaCl, 0.6M LiCl, 10mM Tris-HCl (pH5.5), 1mM EDTA, 25% isopropanol

[0120] Wash solution I: 200mM NaCl solution, 0.8M LiCl, 70% ethanol, 50mM Tris buffer (pH6.5)

[0121] Wash solution II: 70% ethanol

[0122] Eluent: 1mM EDTA, 10mM Tris-HCl (pH8.0)

[0123] Purification steps:

[0124] 1) Take 200 μL of anticoagulant blood in a 1.5ml centrifuge tube, add 750 μL of lysis and adsorption solution, shake and mix to lyse the cells evenly, and let stand for 5 minutes;

[0125] 2) Add 1 mg of magnetic microspheres and shake gently for 10 minutes;

[0126] 3) Adsorb the magnetic microspheres with a magnetic separation rack,...

Embodiment 3

[0132] Example 3 Comparison of Several Surface Modified Magnetic Microspheres Purifying Whole Blood Genomic DNA

[0133] Sample: Anticoagulated blood of mice treated with sodium heparin

[0134] Material:

[0135] Lysis binding solution: 4M guanidine hydrochloride, 2% Triton X-100, 0.1% SDS, 0.01% mercaptoethanol, 0.1M NaCl, 0.6M LiCl, 10mM Tris-HCl (pH5.5), 1mM EGTA, 25% isopropanol

[0136] Wash solution I: 100mM NaCl solution, 0.8M LiCl, 70% ethanol, 50mM Tris buffer (pH6.5)

[0137] Wash solution II: 70% ethanol

[0138] Eluent: 1mM EDTA, 10mM Tris-HCl (pH8.0)

[0139] Magnetic Microspheres:

[0140] Magnetic microspheres A: commercially available silanol-based silica magnetic microspheres

[0141] Magnetic Microspheres B: Aminated Silica Magnetic Microspheres

[0142] Magnetic Microspheres C: Carboxylated Silica Magnetic Microspheres

[0143] Using the experimental procedure in Example 2 to purify genomic DNA, the results are as follows:

[0144]

[0145] The r...

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Abstract

The invention discloses a nucleic acid rapid-purifying method and kit. Specifically, the present invention discloses a reagent combination used for rapid purification of nucleic acids from a nucleic acid-containing sample, the kit prepared on the basis of the reagent combination, and the nucleic acid rapid-purifying method on the basis of the reagent combination or the kit. The method of the invention has the advantages of high nucleic acid yield, high nucleic acid purity, rapidness, room temperature operation, no need of protease digestion process and the like, and is suitable for analysis or extraction and purification of the nucleic acids of various downstream experiments.

Description

technical field [0001] The invention relates to a method and a kit for rapidly purifying nucleic acid from nucleic acid-containing samples. The method can quickly and easily extract nucleic acid, and is particularly suitable for extracting nucleic acid substances from various samples, such as whole blood, cells, and tissues. Background technique [0002] As the carrier of genetic information, nucleic acid is the material basis of gene expression. In addition to playing a very important role in the normal growth, development, and reproduction of organisms, it is also related to abnormal conditions of life, such as tumor occurrence, radiation, etc. Injuries, genetic diseases, etc. are also closely related. Therefore, nucleic acid separation and purification is a very important link in molecular biology and medical research. [0003] According to the separation principle, the existing separation technologies include phenol-chloroform method, salting-out technology, ion exchang...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C07H1/06
CPCC12N15/1013C12Q1/6806C12Q2527/125C12N15/1006
Inventor 李莉李永梅府宇雷程亮储丹
Owner EMERGING THERAPEUTICS SHANGHAI CO LTD
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