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Method for extracting and purifying animal mitochondria DNA

A technology of animal mitochondria and purification methods, which is applied in the field of extraction and purification of animal mitochondrial DNA to achieve high purity

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 electrophoresis of mitochondrial DNA extracted by this technology often has macromolecular DNA (residual nuclear DNA) near the sample hole and RNA tailing (residual RNA) at the front end, it cannot meet the requirements of molecular biology research, such as PCR amplification, digestion and sequencing, etc.

Method used

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  • Method for extracting and purifying animal mitochondria DNA
  • Method for extracting and purifying animal mitochondria DNA
  • Method for extracting and purifying animal mitochondria DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Extraction of fish mitochondrial DNA.

[0030] (1) Take 5-6g fish liver tissue, rinse it in a small amount of SE solution, cut it into pieces, add about 50mL SE, and homogenize it up and down 12 times with an electric homogenizer at 1500r / min.

[0031] (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 mitochondria, add solution D to make the final concentration 100μg / mL (to remove nuclear DNA), incubate at 37°C for 30min, centrifuge at 12000r / min for 10min, discard supernatant; add 4mL of SE solution to suspend mitochondria, and then transfer to 4 in a 1.5mL Eppendorf tube.

[0032] (3) Centrifuge at 12000r / min for 10min, discard the supernatant, add 150μl solution A to each tube, suspend the precipitate, add 300μl freshly prepared solution B and mix well, after ice bath for 10min, add 225μl cold solution C...

Embodiment 2

[0038] Example 2: Extraction and purification of mitochondrial DNA from frogs.

[0039] (1) Homogenization: take 2-15g liver or muscle of black-spotted frog, chop it into a small amount of SE homogenization buffer, add about 20-30mL SE solution, and homogenize it up and down 10 times with an electric homogenizer at 1500r / min, each time After 5 seconds, filter the homogenate into a centrifuge tube.

[0040] (2) Purify mitochondria: centrifuge at 1500r / min for 15min for the first time to get the supernatant. Centrifuge at 12000r / min for 20min for the second time to collect the precipitate, add 2mL of STM solution to suspend the mitochondria, add solution D 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, Then transfer to four 1.5 mL Eppendorf tubes, 1 mL each.

[0041] (3) Centrifuge at 12000r / min for 8min, discard the supernatant, add 150μL of solution A to eac...

Embodiment 3

[0046] Example 3: Extraction and purification of honeybee mitochondrial DNA.

[0047] (1) Take fresh bees or alcohol-soaked specimens, cut them into small pieces of SE homogenization buffer, add about 2-5mL of SE liquid, and homogenize them up and down with an electric homogenizer at 1500r / min for 10 times, each time for 5 seconds, after filtering, Pipette the homogenate into a centrifuge tube.

[0048] (2) Centrifuge at 1500r / min for 15min for the first time to get the supernatant. Centrifuge at 12000r / min for 20min for the second time to take the precipitate, add 4mL SE solution to suspend mitochondria, add solution D to make the final concentration 100μg / mL (to remove nuclear DNA), warm bath at 37℃, 30min, centrifuge at 12000r / min for 10min, discard the supernatant , add 2mL of STM solution to suspend mitochondria, and then transfer to two 1.5mL Eppendorf tubes, 1mL in each tube.

[0049] (3) Centrifuge at 12000r / min for 8min, discard the supernatant, add 150μL of solutio...

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Abstract

The invention discloses a method for extracting animal mitochondria DNA, which has the innovativeness that a DNasel digestion step is added on the basis of alkaline denaturation to eliminate residual nuclear DNA; and an RNase digestion step is added to remove residual RNA. The mitochondria DNA extracted by the method has high purity. The electrophoretic detection shows that an electrophoretic band is a clear, regular and uniform band; and the background is clear, and the conditions such as the trailing of macromolecular DNA and front-end RNA and the like do not occur near a sample application pore.

Description

technical field [0001] The invention belongs to the technical field of molecular biology nucleic acid extraction and purification, in particular to a method for extracting and purifying animal mitochondrial DNA. 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 electrophoresis of mitochondrial DNA extracted by this technology often has macromolecular DNA (residual nuclear DNA) near the sample hole and RNA tailing (residual RNA) at the front end, it cannot meet the requirements of molecular biology research, such as PCR amplification, digestion and sequenci...

Claims

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Application Information

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IPC IPC(8): C12N15/10C07H21/04C07H1/08
Inventor 闫华超贾少波王春明
Owner LIAOCHENG UNIV
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