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Fungal hypha total DNA (deoxyribonucleic acid) extracting solution and method for extracting fungal hypha total DNA

A technology for extracting liquid and fungi, applied in the field of extracting the total DNA of fungal mycelium and extracting the total DNA of fungal mycelium, can solve the problems of long operation time, low efficiency, no wall removal effect, etc., and simplify the operation steps. , the effect of reducing operation time

Active Publication Date: 2016-05-25
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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  • Fungal hypha total DNA (deoxyribonucleic acid) extracting solution and method for extracting fungal hypha total DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The extract I: including 200mM Tris-HCl, 10mMC 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] Extract I: including 150mM sodium phosphate, 5mMC 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 12000rpm for 3 minutes to remove insoluble matter and obtain the 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 amount of TE b...

Embodiment 3

[0049] Extraction solution I: including 100mM disodium hydrogen phosphate, 5mMC 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 fungal hypha total DNA extraction, and particularly relates to a fungal hypha total DNA extracting solution and a method for extracting fungal hypha total DNA. The fungal hypha total DNA extracting solution comprises an extracting solution I and an extracting solution II, wherein the extracting solution I comprises a buffer salt, a nonionic surfactant and a neutral salt water solution, and the pH value of the extracting solution I is 7.5-8.5; and the extracting solution II comprises a buffer salt and a cationic surfactant, and the pH value is 7.5-8.5. The fungal hypha total DNA extracting solution simplifies the total DNA extraction steps, is suitable for extracting trace amounts of total DNA, provides an effective experimental means for the scarce experimental materials, avoids the use of the toxic organic solvents, can simplify the operation steps and shorten the time, and avoids the possibility of high tendency to cause frost damage to skin in 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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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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