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Method for extracting large numbers of DNA with magnetic particulate

A magnetic particle, a large number of technologies, applied in the direction of DNA preparation, recombinant DNA technology, etc., can solve the problems of unsatisfactory extraction of a large amount of DNA, lower yield, inconvenient operation, etc., to shorten the time of DNA extraction, shorten the time, and avoid damage Effect

Inactive Publication Date: 2010-06-23
SHANXI LIFEGEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Usually, the purification of 50-500 μl whole blood can be scaled down or scaled up according to the volume of the original blood sample, but when purifying genomic DNA from a large number of samples (such as 1-10ml) blood, simple scale-up will cause inconvenience in operation , lower yield, lower purity and other problems, but cannot meet the needs of a large amount of DNA extraction

Method used

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  • Method for extracting large numbers of DNA with magnetic particulate
  • Method for extracting large numbers of DNA with magnetic particulate
  • Method for extracting large numbers of DNA with magnetic particulate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] (1) Pretreatment of magnetic particles: transfer 1 ml of magnetic particles into a 10 ml centrifuge tube with a pipette. Magnetically separate until the supernatant is clear and discard the supernatant. Take out the centrifuge tube from the magnetic separator, add 3ml of binding solution (the binding solution is a mixed solution of polyethylene glycol and salt, wherein the concentration of polyethylene glycol is 10%, and the salt is NaCl with a concentration of 1M), blow and mix ,spare.

[0032] (2) Sample cracking: Add 200 μl proteinase K solution to the bottom of a 10ml centrifuge tube, add 1ml blood sample (or 100mg tissue sample) and 2ml guanidine hydrochloride lysis solution (the concentration of guanidine hydrochloride lysis solution is 4M, and the composition is: 3 ~4M GuHCl, 0.01~0.02M EDTA, 0.01~0.02M NaCl and 4~6% TX-100), pipetting and mixing, fully cracking in 56°C water bath for 5min.

[0033] (3) Add the pretreated magnetic particles in step (1) into the...

Embodiment 2

[0037] (1) Pretreatment of magnetic particles: transfer 6 ml of magnetic particles into a 50 ml centrifuge tube with a pipette. Magnetically separate until the supernatant is clear and discard the supernatant. Take out the centrifuge tube from the magnetic separator, add 18ml of binding solution (the binding solution is a mixed solution of polyethylene glycol and salt, wherein the concentration of polyethylene glycol is 10%, and the salt is NaCl with a concentration of 3M), blow and mix ,spare.

[0038] (2) Sample lysis: Add 1.2ml proteinase K solution to the bottom of a 50ml centrifuge tube, add 6ml blood sample (or 600mg tissue sample), 12ml guanidine hydrochloride lysate with a concentration of 5M, and 6μl RNase with a concentration of 10mg / ml Solution A, blow and mix evenly, take a water bath at 20-70°C for 10 minutes, and the effect is best in a water bath at 56°C.

[0039](3) Add the pretreated magnetic particles in step (1) into the lysed cell solution in step (2), mi...

Embodiment 3

[0043] (1) Pretreatment of magnetic particles: transfer 10 ml of magnetic particles into a 50 ml centrifuge tube with a pipette. Magnetically separate until the supernatant is clear and discard the supernatant. Take out the centrifuge tube from the magnetic separator, add 20ml of binding solution (the binding solution is a mixed solution of polyethylene glycol and salt, wherein the concentration of polyethylene glycol is 15%, and the salt is NaCl with a concentration of 5M), blow and mix ,spare.

[0044] (2) Sample cracking: Add 2ml proteinase K solution to the bottom of a 50ml centrifuge tube, add 10ml blood sample (or 1000mg tissue sample), 20ml guanidine hydrochloride lysis solution (the concentration of guanidine hydrochloride lysis solution is 6M, and the composition is: 3 ~4M GuHCl, 0.01~0.02M EDTA, 0.01~0.02M NaCl and 4~6% TX-100), pipetting and mixing, fully cracking in 56°C water bath for 20min.

[0045] (3) Add the pretreated magnetic particles in step (1) into the...

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Abstract

The invention relates to the application field of nanometer and micrometer materials, in particular to a method for quickly extracting large numbers of genome DNA from biological samples with magnetic particulates. The method comprises the following steps of: (1) cracking biological samples: taking a certain amount of biological samples such as blood, tissue and like, adding proteinase K solution and guanidine hydrochloride cracking liquid with the concentration of 4 to 6 M, evenly mixing, and fully cracking in 56 DEG C water bath for 5 to 20 min; (2) adding combined solution into sample cracking liquid to form mixed solution containing magnetic particulates (DNA compound); (3) separating the magnetic particulates (DNA compound) from the mixed solution; (4) washing; (5) eluting: adding eluting liquid to enable DNA to be eluted from the magnetic particulates, separating the magnetic particulates and the eluted DNA under the action of an exterior magnetic field, and collecting supernatant liquid to obtain rarefied DNA. By using the invention, high purity DNA can be successfully extracted from large numbers of samples, large-scale instruments such as a centrifuge and like are not needed, the operation steps are simple and convenient and need short time.

Description

technical field [0001] The invention relates to the application field of nano-micron materials, in particular to the rapid extraction and purification of a large amount of genome DNA from biological samples by using magnetic particles in molecular biology. Background technique [0002] Nucleic acid extraction is a key step in many molecular biology techniques, and its extraction method is particularly important due to the requirements for purity and integrity. Currently, there are various methods for extracting DNA. The traditional phenol-chloroform liquid-phase extraction method is cumbersome and time-consuming to operate, and the solution used is toxic; while the new solid-phase extraction method is easier to operate, non-toxic and environmentally friendly. [0003] Regarding the solid-phase extraction of nucleic acids, reported methods include the use of silicon surfaces, glass, ion-exchange resins, or modified magnetic beads to specifically adsorb DNA. Among them, Chin...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 崔亚丽南婧安德鲁·G·陈周瑞民李芳
Owner SHANXI LIFEGEN
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