Reagent for simultaneous extraction of DNA and RNA, extraction method and application

A technology of reagents and organic solvents, applied in the direction of DNA preparation, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of difficulty in extracting high-quality RNA, cumbersome operation steps, no reagents, etc., and achieve simple and fast operation methods with high purity and the effect of high concentration and low sample requirement

Active Publication Date: 2016-03-09
天津脉络医学检验有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The problem of the prior art that the present invention needs to solve is: existing reagent can only be used for DNA extraction or RNA extraction, and its operating procedure is loaded down with trivial details, does not have the reagent that can be used for DNA and RNA extraction simultaneously in existing reagent, simultaneously Facing difficulties in extracting high-quality RNA from a small number of samples

Method used

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  • Reagent for simultaneous extraction of DNA and RNA, extraction method and application
  • Reagent for simultaneous extraction of DNA and RNA, extraction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] (1) Processing of tissue samples

[0073] After 10 mg of the tissue sample to be tested (mouse liver tissue) was homogenized in liquid nitrogen, 1 ml of the reagent in Example 1 of Table 1 was added, shaken at 200 rpm for 5 minutes at room temperature, centrifuged at 12000 g for 15 minutes at room temperature, and separated into two layers. The upper layer of liquid is then used for RNA extraction, and the lower layer of liquid is then used for DNA extraction. The density of pure phenol is 1.07g / cm 3 , which is located in the lower layer of the aqueous phase when mixed with water. Chloroform makes the separation of the two solvents more obvious because chloroform is miscible with phenol and has a greater density of 1.47 g / cm 3 .

[0074] (2) RNA extraction:

[0075] (1) Carefully take out the upper layer liquid, add isopropanol drop by drop, shake slowly at room temperature 100rpm for 10 minutes, centrifuge at room temperature 12000g for 15 minutes, discard all the ...

Embodiment 2

[0085] (1) Processing of tissue samples

[0086] After 10 mg of the tissue sample to be tested (mouse liver tissue) was homogenized in liquid nitrogen, 1 ml of the reagent in Example 2 of Table 1 was added, shaken at 100 rpm at room temperature for 10 minutes, centrifuged at 15000 g at room temperature for 10 minutes, and separated into two layers. The upper layer of liquid is then used for RNA extraction, and the lower layer of liquid is then used for DNA extraction.

[0087] (2) RNA extraction

[0088] (1) Carefully remove the upper layer liquid, add isopropanol drop by drop, shake slowly at room temperature 200rpm for 15 minutes, centrifuge at room temperature 15000g for 10 minutes, and discard all the liquid.

[0089] (2) Add 1ml of 75% ethanol pre-cooled at 4°C, centrifuge at 15,000 g at room temperature for 10 minutes, blot all the liquid with a vacuum pump, open the centrifuge tube and place it at room temperature for 5 minutes.

[0090] (3) Add 10ul DEPC water to dis...

Embodiment 3

[0098] (1) Processing of tissue samples

[0099] After 10 mg of the tissue sample to be tested (mouse liver tissue) was homogenized in liquid nitrogen, 1 ml of the reagent in Example 3 of Table 1 was added, shaken at 200 rpm for 5 minutes at room temperature, centrifuged at 10,000 g for 20 minutes at room temperature, and separated into two layers. The upper layer of liquid is then used for RNA extraction, and the lower layer of liquid is then used for DNA extraction.

[0100] (2) RNA extraction:

[0101] (1) Carefully remove the upper liquid, add isopropanol drop by drop, shake slowly at room temperature at 50 rpm for 20 minutes, centrifuge at 10,000 g for 20 minutes at room temperature, and discard all the liquid.

[0102] (2) Add 1ml of 75% ethanol pre-cooled at 4°C, centrifuge at 10,000°C for 20 minutes at room temperature, blot all the liquid with a vacuum pump, open the centrifuge tube and place it at room temperature for 5 minutes.

[0103] (3) Add 10ul DEPC water to ...

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Abstract

The invention relates to the field of gene engineering, in particular to a reagent for simultaneous extraction of DNA and RNA, a method for simultaneously extracting the DNA and the RNA and application of the reagent. The reagent comprises a natural saccharide compound, a heavy metal salt compound, a neutral salt compound, a thiocyanic acid compound, an alcoholic compound, an organic solvent compound, a phenolic compound, N-lauroylsarcosine and a salt compound used for adjusting the acidity or alkalinity of a solution. The reagent for extracting the DNA and the RNA is optimized, protection to the DNA and the RNA is enhanced, the whole extraction process can be conducted at room temperature, cooling treatment does not need to be conducted, and the needs of follow-up second-generation sequencing and third-generation sequencing can be completely met. The operation method is simple and rapid, the quantity demand of samples is less, and the purity and the concentration of extracted DNA and RNA are high.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a reagent for simultaneous extraction of DNA and RNA, a method for simultaneous extraction of DNA and RNA, and applications of the reagent. Background technique [0002] RNA is a macromolecular substance in living cells and many viruses. Ribonucleotides are condensed into single-chain long chain molecules through phospholipid bonds. A ribonucleotide molecule is composed of phosphoric acid, ribose and base (A adenine , G guanine, C cytosine, U uracil). RNA is used in the synthesis of all proteins in living cells and carries the genetic information of many viruses. [0003] At present, sequencing is divided into different methods such as whole genome sequencing, exome sequencing, and specific region sequencing, which require different processing methods for samples. The same reagent cannot be used to extract DNA and RNA, and it can meet the needs of subsequent sequencing. Cont...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
CPCC12N15/1003C12Q1/6869
Inventor 刘鹏飞杨冰韩雪姬晓兵
Owner 天津脉络医学检验有限公司
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