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Method for extracting Cryptococcus humicola mitochondrion

A technology of Cryptococcus terrestris and mitochondria, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of low cost, time-consuming and laborious, and difficulty in cell wall breaking, so as to prolong the incubation temperature and increase the yield. Efficient, easy-to-operate effects

Inactive Publication Date: 2014-12-03
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The sucrose density gradient centrifugation method is a traditional method, which is low in cost, but time-consuming and laborious, and requires an ultra-fast low-temperature centrifuge, and the kit for extracting mitochondria is relatively expensive
The differential centrifugation method is only suitable for the extraction of Saccharomyces cerevisiae mitochondria. When it is used for the extraction of Cryptococcus terrestris, it is difficult to break the cell wall, and the yield of protoplasts is low, which further leads to the inability to obtain mitochondria

Method used

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  • Method for extracting Cryptococcus humicola mitochondrion
  • Method for extracting Cryptococcus humicola mitochondrion
  • Method for extracting Cryptococcus humicola mitochondrion

Examples

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Embodiment 1

[0029] This embodiment utilizes a strain of Cryptococcus terrestris ( Cryptococcus humicolus ) BSLL1-1 strain, through the method of the present invention, high-quality active mitochondria are obtained, and the specific operations are as follows:

[0030] (1) Centrifuge the Cryptococcus terrestris cultured in the GM liquid medium to the logarithmic growth phase to collect the bacteria, wash with 0.05mol / L Tris buffer at pH 7.5 three times, and then add the pretreatment buffer (pH8.0 0.01mol / L PBS solution containing 0.1mol / L EDTA and 1% β-mercaptoethanol), shake well, and shake at 100 rpm for 90 min at 28°C;

[0031] (2) Centrifuge the above solution at 5000 rpm for 5 min, discard the supernatant, wash the precipitate twice with 0.01mol / L PBS, then centrifuge at 5000 rpm for 5 min, and collect the precipitate;

[0032] (3) Add enzymatic hydrolysis buffer (pH7.4 containing 40 mg / mL helicase, 4 mg / mL cellulase, 0.01mol / L PBS solution of 0.9mol / L sorbitol, helicase and cellulos...

Embodiment 2

[0039] Embodiment 2: Utilize a strain Cryptococcus terrestris ( Cryptococcus humicolus ) BSLL1-1 strain, through the method of the present invention, high-quality active mitochondria are obtained, and the specific operations are as follows:

[0040] (1) Centrifuge the Cryptococcus terrestris cultured in the GM liquid medium to the logarithmic growth phase to collect the bacteria, wash with 0.05mol / L Tris buffer at pH 7.5 three times, and then add the pretreatment buffer (pH8.0 0.01mol / L PBS solution containing 10mmol / L EDTA, 0.5% β-mercaptoethanol), shake well, shake at 150rpm for 120min at 25℃;

[0041] (2) Centrifuge the above solution at 10000rpm for 4min, discard the supernatant, wash the precipitate twice with 0.01mol / L PBS, then centrifuge at 10000rpm for 4min, and collect the precipitate;

[0042] (3) Add enzymatic hydrolysis buffer (pH7.4 containing 60 mg / mL helicase, 2 mg / mL cellulase, 0.35mol / L sorbitol in 0.01mol / L PBS solution, helicase and cellulose All enzyme...

Embodiment 3

[0049] Embodiment 3: Utilize a strain Cryptococcus terrestris ( Cryptococcus humicolus ) BSLL1-1 strain, through the method of the present invention, high-quality active mitochondria are obtained, and the specific operations are as follows:

[0050] (1) Centrifuge the Cryptococcus terrestris cultured in the YPD liquid medium to the logarithmic growth phase, wash three times with 0.05mol / L Tris buffer at pH 7.5, and then add pretreatment buffer (pH8.0 0.01mol / L PBS solution containing 50mmol / L EDTA, 0.8% β-mercaptoethanol), shake well, shake at 200rpm for 60min at 30℃;

[0051] (2) Centrifuge the above solution at 8000rpm for 3min, discard the supernatant, wash the precipitate twice with 0.01mol / L PBS, then centrifuge at 8000rpm for 3min, and collect the precipitate;

[0052] (3) Add enzymatic hydrolysis buffer (pH7.4 containing 20 mg / mL helicase, 5 mg / mL cellulase, 0.01mol / L PBS solution of 0.5mol / L sorbitol, helicase and cellulose All enzymes were purchased from Shanghai ...

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Abstract

The invention discloses a method for extracting a Cryptococcus humicola mitochondrion. According to the method, the operation steps of the existing differential centrifugation method, the extraction of the components of a buffer and the preparation method are improved, the pretreatment time of a thallus is prolonged, and the shaking is continuously carried out; in the enzymatic and wall-breaking steps, the components of the enzymatic buffer are improved, the amount of the snailase is increased and meanwhile the cellulose is added, the enzymolysis and wall-breaking time and the incubation temperature are prolonged, the wall of the thallus is completely broken, and the yield of a protoplast is increased; in order to mildly lyse the protoplast so as not to break the mitochondrion, the protoplast lysate and a mitochondrion washing buffer solution are both prepared from PBS. Due to adoption of the method, the high-quality mitochondrion can be extracted, the mitochondrion has a complete structure and activity and the cytochrome oxidase system is not destroyed. The mitochondrion extracted by the method can be used for physiology and biochemistry studies with the mitochondrion as an object of the study. The method is simple, economical, easy in operation and the like and is suitable for being operated and popularized in an ordinary biological laboratory.

Description

technical field [0001] The invention relates to a method for extracting mitochondria, in particular to a method for extracting mitochondria of Cryptococcus terrestris. Background technique [0002] Mitochondria are an important organelle in cells and exist in most living cells. Mitochondria play an important role in cell metabolism, and their main function is to provide the energy required for the metabolism of various substances in cells. Therefore, the study of the structure, function and physical and chemical properties of mitochondrial inner and outer membranes, respiratory chain enzymes and mitochondrial DNA has become an important topic in cell biology research, and the extraction of active mitochondria is the basis of these studies. [0003] At present, there are many methods for extracting mitochondria, including sucrose density gradient centrifugation, differential centrifugation and kit extraction methods. The sucrose density gradient centrifugation method is a t...

Claims

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Application Information

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IPC IPC(8): C12N1/16C12R1/645
Inventor 年洪娟李金金张晶晶陈丽梅
Owner KUNMING UNIV OF SCI & TECH
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