Method for rapidly extracting fungal genome DNA

A genome and fungal technology, applied in the field of rapid extraction of fungal genomic DNA, can solve the problems of unstable DNA quality, low DNA purity, and low DNA content, and achieve the effects of good integrity and stability, low extraction cost, and simple operation

Inactive Publication Date: 2015-09-30
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The content of DNA extracted by CTAB method and SDS method is relatively high, but the purity of DNA is relatively low; the content of impurities such as phenols and sugars in DNA extracted by urea method is relatively high, which has a certain impact on subsequent research; benzyl chloride Although the extraction steps are simple and easy, the DNA quality is not stable enough. For materials with poor dispersion in the extraction buffer, the DNA extraction effect is not

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  • Method for rapidly extracting fungal genome DNA
  • Method for rapidly extracting fungal genome DNA

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Effect test

Embodiment 1

[0038] 1. Materials

[0039] Shiitake mushrooms (Lentinulaedodes, variety: Qiuzai No. 7) and poria cocos (Poria cocos variety: Northeast) are preserved strains of the Institute of Applied Fungi, Huazhong Agricultural University.

[0040] 2. Reagents

[0041] Lysis solution: 100mM Tris-HCl (pH=7.8), 20mM EDTA, 1.4M NaAc, 2% (w / v) CTAB, 0.5% (v / v) SDS;

[0042] PCA: chloroform: phenol: isoamyl alcohol = 25:24:1, volume ratio;

[0043] 3M NaAc;

[0044] Anhydrous ethanol;

[0045] 75% ethanol;

[0046] sterile ddH 2 O;

[0047] 10 mg / ml RNase.

Embodiment 2

[0049] A method for rapidly extracting fungal genome DNA, the steps are as follows:

[0050] Extraction of Genomic DNA from Lentinus edodes Mycelium and Fruiting Body

[0051] (1) Take the mycelium or fruiting bodies kept at -70°C and put them in a pre-cooled (-70°C) mortar.

[0052] (2) Add liquid nitrogen to grind to powder, and put the powder into a sterilized 1.5ml centrifuge tube (1 / 3 of the centrifuge tube).

[0053] (3) Add 400μl of the mixed lysate, mix well, and place for 10-15s.

[0054] (4) Add 500μLPCA, mix well, and let stand for 30s.

[0055] (5) Centrifuge at 12000rpm for 2min.

[0056](6) Take the upper aqueous phase to another centrifuge tube, add 1 / 10 volume of 3M sodium acetate, then add twice the volume of pre-cooled absolute ethanol, and place at -20°C for 5-10min.

[0057] (7) Centrifuge at 12000rpm for 2min.

[0058] (8) Discard the supernatant and rinse the pellet twice with 75% ethanol. Pour the ethanol out of the centrifuge tube, centrifuge at 1...

Embodiment 3

[0068] Embodiment 3: the extraction of Poria cocos mycelium genome DNA

[0069] (1) Take the Poria cocos mycelium kept at -70°C and put it in a pre-cooled (-70°C) mortar.

[0070] (2) Add liquid nitrogen to grind to powder, and put the powder into a sterilized 1.5ml centrifuge tube (1 / 3 of the centrifuge tube).

[0071] (3) Add the mixed lysate, mix well, and place for 10-15s. (400μl)

[0072] (4) Add 500μLPCA, mix well, and let stand for 30s.

[0073] (5) Centrifuge at 12000 rpm for 2 minutes.

[0074] (6) Take the upper aqueous phase to another centrifuge tube, add 1 / 10 volume of 3M sodium acetate, then add twice the volume of pre-cooled absolute ethanol, and place at -20°C for 5-10min.

[0075] (7) Centrifuge at 12000rpm for 2min.

[0076] (8) Discard the supernatant and rinse the pellet twice with 75% ethanol. Pour the ethanol out of the centrifuge tube, centrifuge at 12000rpm for 30s, and suck out the remaining ethanol with a pipette.

[0077] (9) Add sterile 100 μ...

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Abstract

The invention discloses a method for rapidly extracting fungal genome DNA. The method comprises the following steps: adding lysate into hyphae or sporocarp which is grinded with liquid nitrogen, adding PCA, and centrifuging; adding 3M of sodium acetate into the upper-layer water phase, further adding precooled absolute ethyl alcohol, and leaving to stand for 5-10 minutes at -20 DEG C; centrifuging, abandoning the supernate, bleaching and precipitating with 75% alcohol, centrifuging, and abandoning the supernate; adding ddH2O and RNase to dissolve the components, and preserving at -20 DEG C. Compared with other methods, the method for rapidly extracting fungal genome DNA is simple to operate, high in feasibility, small in DNA extraction material, high in DNA extraction efficiency, good in integrity and stability, relatively low in impurity content, and can be directly applied to further research such as ITS sequencing. DNA of sporocarp can be also extracted, so that the hypha activation and culture time can be shortened, and an efficient and feasible molecular technical method for macro fungus molecular biological research is provided.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for rapidly extracting fungal genome DNA. technical background [0002] Macrofungi are extremely important microorganisms in the world and play important roles in people's lives. The control effect of Trichoderma on plant diseases; the metabolites of Penicillium have a good effect on the prevention and treatment of human diseases; edible fungi have important nutritional value and health care function, which can meet people's demand for healthy food, and the edible fungus industry can be transformed and utilized in large quantities Crop stalks, edible fungi are increasingly closely related to human life. For example, Poria cocos is one of the four major adjuvants of traditional Chinese medicine. Ganoderma lucidum can improve people’s immunity. Shiitake mushrooms are rich in dietary fiber and polysaccharides that can regulate human immunity. Cordyceps sinensis has...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 边银丙王刚正樊晓琳
Owner HUAZHONG AGRI UNIV
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