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Improved centrifugal-column-method extraction reagent for free DNA (Deoxyribonucleic Acid) in peripheral blood and extraction method thereof
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An extraction method and peripheral blood technology, applied in the field of molecular biology, to achieve the effects of low operation difficulty, good promotion, and simple source of materials
Inactive Publication Date: 2017-05-31
SUZHOU ZHONGXIN BIOTECH
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[0005] Main purpose of the present invention is exactly for above present situation, solves the bottleneck problem that limits traditional centrifugal column method to extract peripheral blood free nucleic acid, provides a kind of improved centrifugal column method peripheral blood free DNA extraction reagent and extraction method thereof, and reagent composition used is simple, Easy to operate, low cost, no special equipment required, and can significantly reduce costs and increase nucleic acid extraction rate
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[0082] Proteinase K (Tiangen), QIAamp Mini spin column (Qiagen), Buffer AL (Qiagen), Buffer AW1 (Qiagen), Absolute Ethanol (Sinopharm), Buffer AW2 (Qiagen), Buffer AE (Qiagen), Qubit dsDNA HSAsay (Life Technologies).
[0083] Reagent I: the pH is 7.5, the solvent is ultrapure water, and the solute contains the following final concentration substances, yeast tRNA (PerkinElmer) 1ug / uL.
[0084] 2. Experimental operation process:
[0085] (1) Place the thawed plasma sample in a centrifuge Sigma (3K-15) pre-cooled to 4°C, and centrifuge at 16000×g at 4°C for 10 min.
[0087] (3) Use a pipette to carefully draw 1 mL of the supernatant of the centrifuged plasma sample into a 15-ml centrifuge tube added with proteinase K.
[0088] (4) Aspirate 1mL of buffer AL solution and 1uL of reagent I, add to the sample, and immediately vortex and mix for 15 seconds.
[0105] Proteinase K (Tiangen), QIAamp Mini spin column (Qiagen), Buffer AL (Qiagen), Buffer AW1 (Qiagen), Absolute Ethanol (Sinopharm), Buffer AW2 (Qiagen), Buffer AE (Qiagen), Qubit dsDNA HSAsay (Life Technologies).
[0106] Reagent I: the pH is 7.5, the solvent is ultrapure water, and the solute contains the following final concentration substances, yeast tRNA (PerkinElmer) 1ug / uL.
[0107] 2. Experimental operation process:
[0108] (1) Place the thawed plasma sample in a centrifuge Sigma (3K-15) pre-cooled to 4°C, and centrifuge at 16000×g at 4°C for 10 min.
[0109] (2) Pipette 100ul proteinase K to the bottom of a 15ml centrifuge tube.
[0110] (3) Use a pipette to carefully draw 1 mL of the supernatant of the centrifuged plasma sample into a 15-ml centrifuge tube added with proteinase K.
[0111] (4) Aspirate 1mL of buffer AL solution and 1uL of reagent I, add to the sample, and immediately vortex and mix for 15 seconds.
[0112] (5...
Embodiment 3
[0127] 1. Reagent preparation:
[0128] Proteinase K (Tiangen), QIAamp Mini spin column (Qiagen), Buffer AL (Qiagen), Buffer AW1 (Qiagen), Absolute Ethanol (Sinopharm), Buffer AW2 (Qiagen), Buffer AE (Qiagen), Qubit dsDNA HSAsay (Life Technologies).
[0129] Reagent I: the pH is 7.5, the solvent is ultrapure water, and the solute contains the following final concentration substances, yeast tRNA (PerkinElmer) 1ug / uL.
[0130] 2. Experimental operation process:
[0131] (1) Place the thawed plasma sample in a centrifuge Sigma (3K-15) pre-cooled to 4°C, and centrifuge at 16000×g at 4°C for 10 min.
[0132] (2) Pipette 100ul proteinase K to the bottom of a 15ml centrifuge tube.
[0133] (3) Use a pipette to carefully draw 1 mL of the supernatant of the centrifuged plasma sample into a 15-ml centrifuge tube added with proteinase K.
[0134] (4) Aspirate 1mL of buffer AL solution and 1uL of reagent I, add to the sample, and immediately vortex and mix for 15 seconds.
[0135] (5...
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Abstract
The invention discloses an improved centrifugal-column-method extraction reagent and an improved centrifugal-column-method extraction method for free DNA (Deoxyribonucleic Acid) in peripheral blood. The extraction reagent includes 0.2[mu]g / [mu]L to 5[mu]g / [mu]L of yeast tRNA (transfer Ribonucleic Acid); a rest part includes ultrapure water; moreover, the pH (potential of Hydrogen) value of the extraction agent for the DNA is 7.3 to 8.2. The extraction method comprises the following steps of mixing to-be-treated peripheral blood with protease K, then adding a first buffer solution and the extraction reagent into an obtained first mixture, uniformly mixing an obtained second mixture to form a digestive solution, adding absolute ethyl alcohol into the digestive solution, forming a digestive mixed solution, and centrifuging the digestive mixed solution to obtain the free DNA. By using the extraction reagent provided by the invention, the extraction efficiency can be improved; the extraction yield of an existing common centrifugal column method is increased by 3 to 10 times; thus, the replacement of a centrifugal-column-method kit which is high in price and is used for extracting the free DNA in the peripheral blood in a targeted manner is realized; the source of a material is simple; the cost is low; the extraction rate of a nucleic acid can be improved; problems that scientific research personnel encounter when extracting a free nucleic acid in the peripheral blood are greatly ameliorated; the extraction reagent and the extraction method have the advantages that the practicability is high and the popularization performance is good.
Description
technical field [0001] The invention relates to the technical field of molecular biology, more specifically to the technical field of DNA, in particular to an improved centrifugal column method for extracting free DNA from peripheral blood and an extraction method thereof. Background technique [0002] As we all know, most of the genetic material nucleic acid is located in the cell, but whether it is a healthy population or a diseased population, there is a small part of DNA located outside the cell, which is called cell-free DNA. Free DNA also exists in peripheral blood, called peripheral blood circulating DNA (Circulating DNA in plasma). [0003] In a 1987 paper, researchers studied the plasma of 37 patients with malignant tumors, and free DNA was isolated from 10 of the samples. The preliminarily identified plasma cell-free DNA is double-stranded, and the size range distribution is about 40bp-220bp. In the subsequent study, they made a basic judgment on the characterist...
Claims
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Application Information
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