Method for extracting mitochondrial DNA of cotton

A technology for mitochondria and cotton is applied in the field of extracting mitochondrial DNA from cotton, which can solve the problems of difficulty in grasping, difficult to purify mtDNA, and complicated operation process.

Inactive Publication Date: 2011-07-13
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the operation process of these methods is relatively complicated, the cost is high, and the purity of the extracted mtDNA is not high, which affects subsequent experiments.
In the process of extracting mtDNA from different plants, various influencing factors are extremely complex and difficult to grasp, and the require

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  • Method for extracting mitochondrial DNA of cotton
  • Method for extracting mitochondrial DNA of cotton
  • Method for extracting mitochondrial DNA of cotton

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, the extraction of cotton mitochondrial DNA

[0038] 1. Solution preparation

[0039] Buffer A was prepared as follows: Dilute 0.05 mol Tris-HCl, 1.2 mol NaCl, 2 mmol EDTA, 8 g polyvinylpyrrolidone (PVP), 4 mmol β-mercaptoethanol and 0.5 g bovine serum albumin (BSA) to 1 L with water , adjust the pH value to 8.0.

[0040] The preparation method of buffer B is as follows: 0.05mol Tris-HCl, 0.3mol sucrose, 0.03mol MgCl 2 and 3 g of polyvinylpyrrolidone (PVP) were adjusted to 1 liter with water, and the pH value was adjusted to 8.0.

[0041] The preparation method of buffer C is as follows: mix 0.05mol Tris-HCl, 0.02mol EDTA-Na 2 and 0.3mol sucrose with water to make up to 1 liter, adjust the pH value to 7.5.

[0042] The preparation method of buffer D is as follows: mix 0.05mol Tris-HCl, 0.02mol EDTA-Na 2 and 0.6mol sucrose with water to make up to 1 liter, and adjust the pH value to 8.0.

[0043] The preparation method of lysate is as follows: 0.01mol ...

Embodiment 2

[0058] Embodiment 2, the extraction of cotton mitochondrial DNA

[0059] 1. Solution preparation

[0060] The preparation method of buffer A is as follows: dilute 0.05mol Tris-HCl, 1.2mol NaCl, 2mmol EDTA, 6g polyvinylpyrrolidone (PVP), 2mmolβ-mercaptoethanol and 0.1g BSA to 1 liter with water, adjust the pH value to 7.5.

[0061] The preparation method of buffer B is as follows: 0.05mol Tris-HCl, 0.1mol sucrose, 0.01mol MgCl 2 and 1 g of polyvinylpyrrolidone (PVP) were adjusted to 1 liter with water, and the pH value was adjusted to 7.5.

[0062] The preparation method of buffer C is as follows: mix 0.05mol Tris-HCl, 0.02mol EDTA-Na 2 and 0.1mol sucrose with water to make up to 1 liter, adjust the pH value to 7.5.

[0063] The preparation method of buffer D is as follows: mix 0.05mol Tris-HCl, 0.02mol EDTA-Na 2 and 0.2mol sucrose with water to make up to 1 liter, and adjust the pH value to 7.5.

[0064] The preparation method of lysate is as follows: 0.01mol Tris-HCl, 0...

Embodiment 3

[0079] Embodiment 3, the extraction of cotton mitochondrial DNA

[0080] 1. Solution preparation

[0081] The preparation method of buffer A is as follows: dilute 0.05mol Tris-HCl, 1.2mol NaCl, 2mmol EDTA, 10g polyvinylpyrrolidone (PVP), 5mmolβ-mercaptoethanol and 0.8g BSA to 1 liter with water, adjust the pH value to 8.5.

[0082]The preparation method of buffer B is as follows: 0.05mol Tris-HCl, 0.5mol sucrose, 0.05mol MgCl 2 and 5 g of polyvinylpyrrolidone (PVP) were adjusted to 1 liter with water, and the pH value was adjusted to 8.5.

[0083] The preparation method of buffer C is as follows: mix 0.05mol Tris-HCl, 0.02mol EDTA-Na 2 and 0.5mol sucrose with water to make up to 1 liter, adjust the pH value to 7.5.

[0084] The preparation method of buffer D is as follows: mix 0.05mol Tris-HCl, 0.02mol EDTA-Na 2 and 1.0mol sucrose with water to make up to 1 liter, and adjust the pH value to 8.5.

[0085] The preparation method of lysate is as follows: 0.01mol Tris-HCl, 0...

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Abstract

The invention discloses a method for extracting mitochondrial deoxyribonucleic acid (DNA) of cotton. The method comprises the following steps of: (1) homogenizing cotton, and centrifuging and collecting precipitate; (2) removing nuclear DNA, adding ethylene diamine tetraacetic acid (EDTA) solution, and centrifuging to obtain the precipitate; (3) adding buffer solution, and centrifuging to obtain mitochondria; (4) adding lysis solution and acetate solution, centrifuging, collecting supernatant, and extracting by using chloroform-isoamylol; (5) adding acetate solution and absolute ethanol, centrifuging to obtain the precipitate, and washing by using ethanol; (6) dissolving in triisopropylphenylsulfonyl (Tris)-EDTA (TE); (7) removing ribonucleic acid (RNA) and protein, extracting by using Tris saturated phenol-chloroform-isoamylol, and centrifuging and collecting the supernatant; and (8) adding the acetate solution and the ethanol, and centrifuging to obtain the precipitate; and washing by using the ethanol to obtain the mitochondrial DNA. Aiming at defects of the conventional extraction of the mitochondrial DNA, the conventional cetyltrimethylammonium bromide (CTAB) method is improved, and the extraction conditions are optimized by adjusting the time and times of centrifugation, changing reagents, increasing extraction and washing times, and the like. On the basis of ensuring that the mechanical damage degree of the mitochondria is minimum, the nuclear DNA, phenols and polysaccharides outside the mitochondria are effectively removed, pollution rate is reduced, the purity and the quality of the mitochondrial DNA are improved, and the mitochondrial DNA can meet the requirement of subsequent experiments of the gene engineering of the cotton mitochondria.

Description

technical field [0001] The invention relates to a method for extracting mitochondrial DNA of cotton. Background technique [0002] Mitochondria is the most important organelle of higher organisms and one of the cytoplasmic genetic systems. Mitochondrial genomic DNA (mtDNA) is relatively independent outside the nucleus, capable of self-replication, and controls many genetic traits. Its configuration mainly includes open ring, closed ring, superhelical, linear and multimer. Compared with higher animals, the mitochondrial genomes of higher plants are large and complex. [0003] Plant cytoplasmic male sterility is a relatively common phenomenon in nature, and the study of plant cytoplasmic male sterility is an important topic in the study of genetics. A large number of studies in the fields of genetics and molecular biology have shown that the genes that cause plant cytoplasmic male sterility are mainly distributed in the mitochondrial genome, that is, the mitochondrial genom...

Claims

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Application Information

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IPC IPC(8): C12N15/10C07H21/04C07H1/08
Inventor 华金平李双双薛龙飞熊敏张曦苏爱国王玉美
Owner CHINA AGRI UNIV
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