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Method for purifying small fragment DNA in gel by grinding method and use thereof

A grinding method and small fragment technology, applied in the field of molecular biology, can solve the problems of low recovery efficiency, failure to obtain, and failure to obtain target fragments, etc., and achieve the effect of improving recovery efficiency

Inactive Publication Date: 2009-09-09
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is very effective for separating molecular weight DNA. However, the recovery rate of DNA is related to the molecular weight of DNA. When the molecular weight of DNA is in an appropriate range, a high recovery rate can be obtained; when the molecular weight is greater than 20kb, the recovery The recovery rate is only about 20%. Similarly, for small molecular weight DNA, its recovery efficiency is also very low, especially for fragments with a molecular weight less than 100bp, and even the target fragment cannot be obtained, which cannot meet the requirements of routine experiments.
[0003] At present, many biological companies have developed kits that can be used to recover DNA. These kits are effective for DNA with a general molecular weight, but due to their own defects, the recovery efficiency for small molecular weight DNA is low, and even the target fragment cannot be obtained.

Method used

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  • Method for purifying small fragment DNA in gel by grinding method and use thereof
  • Method for purifying small fragment DNA in gel by grinding method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] 1) Add 400 μl of 0.1 times TE buffer or sterilized water to the recovered high-concentration gel containing 70 bp DNA PCR products, grind and break with a homogenizer, centrifuge at 10,000 rpm for 10 minutes, and take the supernatant;

[0022] 2) Add 1 / 3 volume of 3M NaAc with a pH of 5.2 and 2 volumes of absolute ethanol, or 0.6 volumes of isopropanol to the supernatant to precipitate DNA; centrifuge at 10,000 rpm for 10 minutes, discard the supernatant;

[0023] 3) Wash the DNA pellet with 600 μl of 70% cold ethanol, centrifuge at 10,000 rpm for 5 minutes, and discard the supernatant;

[0024] 4) Vacuum-dry the DNA precipitate to evaporate the residual ethanol, dissolve the DNA precipitate in 0.1 times TE buffer or sterilized water, and store at -20°C. Take 5ul electrophoresis results as figure 1 shown.

Embodiment 2

[0026] 1) Add 800 μl of 0.1 times TE buffer or sterilized water to the recovered high-concentration gel containing 98bp DNA PCR products, grind and break with a homogenizer, centrifuge at 12,000 rpm for 5 minutes, and take the supernatant;

[0027] 2) Add 1 / 3 volume of 3M NaAc with a pH of 5.2 and 2 volumes of absolute ethanol, or 0.6 volumes of isopropanol to the supernatant to precipitate DNA; centrifuge at 12,000 rpm for 5 minutes, discard the supernatant;

[0028] 3) Wash the DNA pellet with 1000 μl of 70% cold ethanol, centrifuge at 12000 rpm for 3 minutes, and discard the supernatant;

[0029] 4) Vacuum-dry the DNA precipitate to evaporate the residual ethanol, dissolve the DNA precipitate in 0.1 times TE buffer or sterilized water, and store at -20°C. Take 5ul electrophoresis results as figure 1 shown.

Embodiment 3

[0031] 1) Add 600 μl of 0.1 times TE buffer or sterilized water to the recovered high-concentration gel containing the 225bp DNA fragment generated by enzyme digestion, grind and break with a homogenizer, centrifuge at 11,000 rpm for 6 minutes, and take the supernatant;

[0032] 2) Add 1 / 3 volume of 3M NaAc with a pH of 5.2 and 2 volumes of absolute ethanol, or 0.6 volumes of isopropanol to the supernatant to precipitate DNA; centrifuge at 11,000 rpm for 6 minutes, discard the supernatant;

[0033] 3) Wash the DNA pellet with 800 μl of 70% cold ethanol, centrifuge at 11,000 rpm for 6 minutes, and discard the supernatant;

[0034] 4) Vacuum-dry the DNA precipitate to evaporate the residual ethanol, dissolve the DNA precipitate in 0.1 times TE buffer or sterilized water, and store at -20°C. Take 5ul electrophoresis results as figure 1 shown.

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Abstract

The invention discloses a method for quickly and efficiently purifying and recovering small fragment DNA by using a homogenizer to grind gel. Grind the gel containing small fragments of DNA in TE buffer directly with a homogenizer, collect the supernatant by centrifugation, precipitate the DNA under the action of salt ions and absolute ethanol, and redissolve the precipitated DNA in TE buffer. So as to achieve efficient purification and recovery of small fragments of DNA. The method has simple operation steps and improves recovery efficiency. At the same time, the method provides a new means for the purification and recovery of small and medium molecular weight DNA for research and application such as gene cloning, mutation detection, gene diagnosis and therapy.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a method and application of purifying small fragment DNA from gel by grinding method. Background technique: [0002] Separation and purification of DNA is a basic technology in the field of genetic engineering. At present, the main method of DNA recovery is to separate DNA fragments by gel electrophoresis, then cut the gel containing the target fragment, dissolve the gel under the action of NaI, and then pass Column or glass strain to recover DNA. This method is very effective for separating molecular weight DNA. However, the recovery rate of DNA is related to the molecular weight of DNA. When the molecular weight of DNA is in an appropriate range, a high recovery rate can be obtained; when the molecular weight is greater than 20kb, the recovery The recovery rate is only about 20%. Similarly, for small molecular weight DNA, the recovery efficiency is very low, especially for fragm...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07H21/04C07H1/06
Inventor 林旭瑷陈寅严杰
Owner ZHEJIANG UNIV
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