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Rapid extraction method for plant mitochondrion DNA

An extraction method and mitochondrial technology, applied in the biological field, can solve the problems of impurity pollution, low yield, complicated operation, etc., achieve the effect of improving yield and quality, maintaining stable pH value and osmotic pressure

Inactive Publication Date: 2015-12-02
SHANDONG CROP GERMPLASM CENT
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The mitochondrial DNA obtained by density gradient centrifugation has high purity, but the operation is complex, time-consuming, requires a large amount of materials, and the yield is low; ordinary differential centrifugation is easy to operate, but the extracted mtDNA is low in purity and easy to degrade, nuclear DNA, RNA Serious contamination with impurities such as protein
Due to the constraints of these unfavorable factors, the existing mitochondrial extraction methods cannot meet the needs of follow-up research work

Method used

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  • Rapid extraction method for plant mitochondrion DNA

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Embodiment 1

[0038] (1) Solution preparation

[0039] BufferA: 0.4M sucrose, 5mMEDTA (ethylenediaminetetraacetic acid), 50mM Tris-HCl, 10mM trimethylglycine, 8mM cysteine, 0.1% BSA (bovine serum albumin) and 1% PVP (polyvinylpyrrolidone), pH7.8;

[0040] BufferB: 0.4M sucrose, 1mM EDTA, 10mM Tris-HCl, 0.1% BSA, pH7.2;

[0041] Solution W1: 1% glucose, 50mM EDTA, 25mM Tris-HCl, pH8.0;

[0042] Solution W2: 0.2mM NaOH, 1% SDS (sodium dodecyl sulfate);

[0043] Solution W3: 5mol / L KAC (potassium acetate), pH4.8;

[0044] Rinsing solution WT: 70% alcohol;

[0045] Elution buffer TB: 10mM Tris-HCl, 1mM EDTA, PH=8.0;

[0046] (2) Tissue pretreatment:

[0047] Take 1g of fresh and young radish tissue and treat it in the dark at 4°C for 24h, and wash the material in pre-cooled (pre-cooled to 2-4°C) distilled water (if disinfection is required, soak it in a diluted solution of sodium hypochlorite with a final concentration of 0.7%) 5min);

[0048] (3) Grinding:

[0049] Once the material i...

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Abstract

The invention discloses a rapid extraction method for plant mitochondrion DNA. The method includes the steps of firstly, preparing Buffer A (containing betaine and cysteine), Buffer B (containing BSA), a solution W1, a solution W2, a solution W3, buffer WT and buffer TB; secondly, adding Buffer A for grinding after preprocessing fresh and tender tissue, collecting filtered fluid, keeping the pH value at 7.0 or above, and conducting 12000g centrifugation on the supermatant after conducting 1000g centrifugation on collected fluid; thirdly, taking sediment, and adding Buffer B to sediment for resuspension, and conducting 1000g centrifugation; fourthly, taking supermatant, adding DNase I and EDTA for reaction, and then conducting 12000g centrifugation; fifthly, adding the solution W1 to the sediment for suspending sediment; sixthly, adding the solution W2 and the solution W3 for gentle perversion, taking supermatant after centrifugation, and extracting the mitochondrion DNA through an adsorption column. By means of the method, the high-quality plant mitochondrion DNA can be efficiently and rapidly obtained.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for rapidly extracting plant mitochondrial DNA. Background technique [0002] Mitochondria is an important organelle of plant cells. It is a genome with a certain genetic functional structure, capable of self-replication, encoding some important enzyme components in the process of respiratory metabolism, and providing energy for cell physiological activities. Mitochondria, as the center of cellular energy metabolism and material conversion, play an important role in cell synthesis, material transport and information transmission. In addition, studies on cytoplasmic male sterility in many higher plants have shown that mitochondria are closely related to cytoplasmic male sterility and are important carriers of male sterility factors. The study of the structure and function of mitochondrial DNA (mtDNA) is of great significance for crop improvement and the exploration of the or...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 白静王俊峰谢坤王效睦余华马玉敏
Owner SHANDONG CROP GERMPLASM CENT
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