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Method for extracting and purifying columnar high-purity animal mtDNA

A purification method and high-purity technology, which is applied in the field of nucleic acid extraction and purification in molecular biology, can solve the problems of harmfulness and residues in environmental operators, and achieve high-purity results

Inactive Publication Date: 2010-06-02
LIAOCHENG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Because the mitochondrial DNA extracted by this technique often has problems such as residual nuclear DNA, RNA and phenol, it cannot meet the requirements of molecular biology research, such as PGR amplification, enzyme digestion and sequencing, etc.
In addition, phenol and the like are corrosive chemicals, and long-term use is harmful to the environment and operators

Method used

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  • Method for extracting and purifying columnar high-purity animal mtDNA
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  • Method for extracting and purifying columnar high-purity animal mtDNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Extraction of frog mitochondrial DNA

[0024] (1) Take 2-15g of liver, gonad or muscle of black-spotted frog, chop it into a small amount of SE homogenization buffer, add about 20-30mL of SE solution, homogenize up and down with an electric homogenizer at 1500r / min for 30 seconds, and filter , pipette the homogenate into a centrifuge tube.

[0025] (2) Centrifuge at 1500r / min for 15min to absorb the supernatant. Centrifuge at 12000r / min for 20min to take the precipitate, add 10ml SE solution to suspend the sediment, repeat centrifugation once, add 10ml STM solution to suspend mitochondria, add solution IV to make the final concentration 100μg / ml, warm bath at 37℃, 30min, centrifuge at 12000r / min for 10min, discard the supernatant solution, add 4ml SE solution to suspend mitochondria, and then transfer to four 1.5ml Eppendorf tubes, 1ml in each tube.

[0026] (3) Centrifuge at 12000r / min for 8min to discard the supernatant, add 150μl solution I to each tube,...

Embodiment 2

[0031] Example 2: Extraction of fish mitochondrial DNA.

[0032] (1) Take 5-6g fish liver or gonad tissues, rinse them in a small amount of SE solution, cut them into pieces, add about 50ml SE, and homogenize them up and down with an electric homogenizer at 1500r / min for 30s.

[0033] (2) The homogenate was centrifuged at 1000r / min for 15min to absorb the supernatant, and then centrifuged at 12000r / min for 20min, and the precipitate was mitochondria. Add 4ml of STM solution to suspend the mitochondria, add solution IV to make the final concentration 100μg / ml, incubate at 37°C for 30min, centrifuge at 12000r / min for 10min, discard the supernatant; add 4ml of SE solution to suspend the mitochondria, transfer to four 1.5ml Eppendorf tubes.

[0034] (3) Centrifuge at 12000r / min for 10min, discard the supernatant, add 150μl of solution I to each tube, suspend the precipitate, add 300μl of newly prepared solution II, mix well, and ice-bath for 10min, then add 225μl of cold solution ...

Embodiment 3

[0038] Example 3: Extraction and purification of honeybee mitochondrial DNA

[0039] (1) Take fresh honey bee or alcohol soaked specimens, add liquid nitrogen to grind, add about 5-10mL SE solution, mix well, filter, and suck the homogenate into a centrifuge tube.

[0040] (2) Centrifuge at 1500r / min for 15min to absorb the supernatant. Centrifuge at 12000r / min for 20min to take the precipitate, add 2ml of STM solution to suspend, add solution IV to make the final concentration 100μg / ml, warm bath at 37°C for 30min, centrifuge at 12000r / min for 10min, discard the supernatant, add 2mL of SE solution to suspend, and then transfer to 2 1.5ml Eppendorf tubes, 1ml per tube.

[0041] (3) Centrifuge at 12000r / min for 8min to discard the supernatant, add 150μL of solution I to each tube, pipette the precipitate evenly, add 300μL of freshly prepared solution II, mix well, and ice-bath for 10min, add 225μL of cold solution III each, mix well, and ice-bath 60min, centrifuged at 12000r / ...

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Abstract

The invention discloses a method for extracting and purifying a columnar high-purity animal mtDNA. In the method, an alkaline denaturation method is taken as a basis and improved by adding a DNaseI digestion step to eliminate the residue of nuclear DNA, and by adding a RNase digestion step to eliminate the residue of RNA; a column chromatography is adopted to replace a phenol extraction method for purifying the mtDNA to solve the problems of phenol toxicity and residue thereof. The mitochondrial DNA obtained by the method has high purity; and electrophoresis detection displays that an electrophoresis band is clear, neat and even with clear background and without the situations of macromolecular DNAs near sample application holes, front-end DNA trailing, and the like.

Description

technical field [0001] The invention belongs to the technical field of molecular biology nucleic acid extraction and purification, and in particular relates to a high-purity animal mitochondrial DNA extraction and purification technology. Background technique [0002] The existing animal mitochondrial DNA extraction technology is mainly alkaline denaturation method. The technology uses differential centrifugation to extract mitochondria; SDS alkaline denaturation method to lyse mitochondria; phenol extraction to purify mtDNA. The prominent feature of this technology is its simplicity and speed, so it has been widely used in the fields of biology, medicine and forensic identification. [0003] Because the mitochondrial DNA extracted by this technique often has problems such as residual nuclear DNA, RNA, and phenol, it cannot meet the requirements of high-demand molecular biology research, such as PGR amplification, enzyme digestion, and sequencing. In addition, phenol and t...

Claims

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Application Information

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IPC IPC(8): C12N15/10C07H21/04C07H1/08
Inventor 闫华超杜廷广
Owner LIAOCHENG UNIV
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