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A kind of fungal hyphae total dna extraction liquid and the method for extracting fungal mycelia total dna

A technology for extracting liquid and fungus, which is applied in the field of extracting liquid of total DNA of fungal hyphae and extracting total DNA of fungal mycelium, which can solve the problems of long operation time, many steps, time-consuming and laborious, etc., and achieve the goal of reducing operation time and simplifying operation steps Effect

Active Publication Date: 2018-03-20
BEIJING UNIV OF AGRI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the fungal cell wall is rich in polysaccharides and other substances, even after liquid nitrogen freeze-drying and grinding, it is difficult to be broken by ordinary extracts, and direct digestion with enzymes often does not get a good wall-removing effect
In fact, the wall-breaking methods currently used in most laboratories are liquid nitrogen freeze-drying and grinding, but most of these methods use toxic organic solvents, phenol, etc., and the operation time is long and there are many steps
In addition, grinding with liquid nitrogen is easy to frostbite the skin, requires strong grinding, time-consuming and labor-intensive, cross-contamination is likely to occur due to the limitation of the number of mortars, fungal hyphae are also easily lost during the grinding process, and the efficiency is not high, especially for some units and institutions. Severely limited due to lack of availability of liquid nitrogen and dry ice

Method used

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  • A kind of fungal hyphae total dna extraction liquid and the method for extracting fungal mycelia total dna

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The extract I: including 200mM Tris-HCl, 10mM C 12 The aqueous solution of fatty alcohol ether ammonium sulfate and 200mM sodium chloride has a pH value of 8.0;

[0033] The extract II: including 200mM Tris-HCl and 1ppm cationic polyacrylamide, its pH value is 8.0.

[0034] (1) Collect the fungal mycelium of powdery mildew;

[0035] (2) Put 1 mg of fungal mycelium in 500 microliters of extract I, and mix it upside down;

[0036] (3) Freeze and thaw twice at minus 196°C and 60°C respectively, that is, freeze at minus 196°C and thaw at 60°C;

[0037] (4) Add 50 microliters of extract solution II, invert and mix well, and let stand for 2 minutes;

[0038] (5) Centrifuge at 10,000 rpm for 5 minutes to remove insoluble matter and obtain a supernatant containing fungal DNA.

Embodiment 2

[0040] Extraction solution I: including 150mM sodium phosphate, 5mM C 15 The aqueous solution of fatty alcohol ether ammonium sulfate and 100mM sodium chloride has a pH value of 7.8;

[0041] Extract solution II: including 150mM sodium phosphate and 3ppm cationic polyacrylamide, its pH value is 7.8.

[0042] (1) Collect the fungal mycelium of powdery mildew;

[0043] (2) Put 1 mg of fungal mycelium in 500 microliters of extract I, and mix it upside down;

[0044] (3) Freeze and thaw three times at minus 80°C and 50°C respectively, that is, freeze at minus 80°C and thaw at 50°C;

[0045] (4) Add 75 microliters of extract solution II, invert and mix well, and let stand for 2 minutes;

[0046] (5) Centrifuge at 12,000 rpm for 3 minutes to remove insoluble matter and obtain a supernatant containing fungal DNA.

[0047] (6) Add an appropriate amount of isopropanol, place at minus 30°C for 30 minutes, centrifuge at 12,000 rpm for 10 minutes, and then dissolve in an appropriate a...

Embodiment 3

[0049] Extraction solution I: including 100mM disodium hydrogen phosphate, 5mM C 15 The aqueous solution of fatty alcohol ether ammonium sulfate and 100mM sodium chloride has a pH value of 8.2;

[0050] Extract solution II: including 100 mM disodium hydrogen phosphate and 3 ppm cationic polyacrylamide, and its pH value is 8.2.

[0051] (1) Collect the fungal mycelium of powdery mildew;

[0052] (2) Put 1 mg of fungal mycelium in 500 microliters of extract I, and mix it upside down;

[0053] (3) Freeze and thaw twice at minus 50°C and 50°C respectively, that is, freeze at minus 50°C and thaw at 50°C;

[0054] (4) Add 125 microliters of extract solution II, invert and mix well, and let stand for 2 minutes;

[0055] (5) Centrifuge at 12,000 rpm for 5 minutes to remove insoluble matter and obtain a supernatant containing fungal DNA.

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Abstract

The invention belongs to the technical field of total DNA extraction of fungal hyphae, and in particular relates to a solution for extracting total DNA of fungal mycelium and a method for extracting total DNA of fungal mycelia. The total DNA extract of fungal hyphae includes extract I and extract II, wherein the extract I includes buffer salt, non-ionic surfactant, and an aqueous solution of neutral salt, and its pH value is 7.5-8.5; The extract II includes buffer salt and cationic surfactant, and its pH value is 7.5-8.5. The following beneficial effects can be obtained by using the total DNA extraction solution of fungal hyphae of the present invention: 1) the total DNA extraction steps are simplified; 2) it is suitable for micro-extraction of total DNA, and provides effective experimental means for experimental materials that are difficult to be scarce; 3 ) avoids the use of toxic organic solvents; 4) can simplify the operation steps and time; 5) also avoids the possibility of frostbite skin easily caused during the liquid nitrogen grinding process.

Description

technical field [0001] The invention belongs to the technical field of total DNA extraction of fungal hyphae, and in particular relates to a solution for extracting total DNA of fungal mycelium and a method for extracting total DNA of fungal mycelia. Background technique [0002] In the study of molecular biology of fungi, the extraction of total fungal DNA is crucial. At present, the commonly used extraction methods mainly include CTAB method, SDS extraction method, urea extraction method and enzymatic hydrolysis method. The extraction steps are generally similar, and the key lies in how to effectively break the cell wall, some of which are mechanically broken, and some are digested and removed by enzymes and other substances. Because the fungal cell wall is rich in polysaccharides and other substances, even after liquid nitrogen freeze-drying and grinding, it is difficult to be broken by ordinary extracts, and direct digestion with enzymes often does not get a good wall-r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003C12Q2527/125
Inventor 郭巍毕扬张艳杰李亚宁赵丹张汀刘大群杨宝东陈艳
Owner BEIJING UNIV OF AGRI
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