Method for quickly extracting fungal DNA for PCR amplification

An extraction method and a fungal technology, applied in the field of fungal molecular biology, can solve the problems of difficulty in applying batches of fungal DNA extraction, a large number of mycelium steps, time-consuming and labor-intensive, etc., and achieve improved molecular identification and detection efficiency, good integrity, cost reduction effect

Inactive Publication Date: 2010-04-21
INST OF FOREST ECOLOGY ENVIRONMENT & PROTECTION CHINESE ACAD OF FORESTRY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The DNA extracted by the CTAB method has a high purity, but a large amount of mycelium is required and the steps are cumbersome. First, the fungal mycelium is ground and broken, and incubated at high temperature, which is time-consuming and laborious.
In addition, there are also some reports on fungal DNA extraction methods, but they are all inseparable from the steps of grinding and breaking the wall and extracting proteins with phenol and chloroform.
The above methods are difficult to apply to batch fungal DNA extraction

Method used

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  • Method for quickly extracting fungal DNA for PCR amplification
  • Method for quickly extracting fungal DNA for PCR amplification
  • Method for quickly extracting fungal DNA for PCR amplification

Examples

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Embodiment 1

[0021] Inoculate Pleurotus eryngii (DC.) Gillet, Pleurotus eryngii (DC.) Gillet), Pleurotus eryngii (DC.) Gillet, Pleurotus eryngii (DC.) Gillet), Pleurotus tuber-regium (Rumph.ex Fr. ) Singer), Pleurotus ferulae (Lanzi) X.L.Mao), Pleurotus spodeleucus (Fr.) Quél.), Pleurotus otreatus (Jacq.) P.Kumm.), abalone Pleurotus ( Pleurotus abalonus Y.H.Han, K.M.Chen & S.Cheng) 8 kinds of 3 strains were grown in PDA medium at 30°C for one week, and a small amount of fungal mycelium was picked up by the inoculation needle and put into 200μl lysate (10mM Tris-HCl , pH8.0, 1mM EDTA, 250mM NaCl, 1% SDS; freshly prepared) in a microcentrifuge tube; quick-frozen in liquid nitrogen for 1min, quickly boiled in boiling water for 5min, and repeated once; 4°C, 12000rpm centrifuged for 2min, took Transfer the clear to a sterile centrifuge tube, add twice the volume of 95% ethanol, mix upside down, freeze in liquid nitrogen for 1 min, centrifuge at 4°C, 12000rpm for 10min, discard the supernatant; ...

Embodiment 2

[0024]Pleurotus otreatus (Jacq.) P.Kumm.), Alternaria tenuissima, Botryosphaeria dothidea (Moug.) Ces.&De Not.), Fusarium oxysporum ), Glomerella cingulata, Monilinia fructigena Honey, Colletotrichum mgloeosporioides, Paecilomyces lilacinus (Thom.) Samson, 3 each The strain was grown in PDA medium at 30°C for one week, and a small amount of fungal mycelium was picked up by an inoculation needle and placed in 200 μl of lysate (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 250 mM NaCl, 1% SDS; fresh prepared) microcentrifuge tubes, in which different lysate components were quick-frozen in liquid nitrogen for 1 min, quickly put into boiling water for 5 min, and repeated once; Double the volume of 95% ethanol, mix it upside down, freeze in liquid nitrogen for 1min; centrifuge at 12000rpm at 4°C for 10min, discard the supernatant; add 1ml of 75% ethanol to wash the precipitate; centrifuge at 12000rpm at 4°C for 1min, discard the supernatant, and precipitate Air-dry; add 50 μl 1×TE to dissolv...

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Abstract

The invention discloses a method for quickly extracting fungal DNA for PCR amplification, which comprises the steps of: (1) culturing fungi to obtain fungal mycelia; (2) placing the fungal myceliua into a centrifugal tube filled with a lysate; (3) alternately freezing and thawing the centrifugal tube; and (4) centrifuging the thawed fungal mycelia, taking a supernatant, adding 95 percent ethanol into the supernatant, mixing the mixture evenly, quickly freezing the mixture by liquid nitrogen, centrifuging the mixture, removing a supernatant, and air-drying a precipitate to obtain the fungal DNA. The method has simple operation, is time-saving and labor-saving, reduces the loss of the fungal DNA during the grinding, does not need phenol and chloroform for extraction, and avoids the contact of a toxic reagent. The DNA extracted by the method has good integrity, high purity and high yield, can be directly used for a PCR amplification reaction, and can efficiently amplify a target product for cloning and sequencing. The method improves the extraction efficiency of the fungal DNA, reduces the cost for the extraction of the fungal DNA, and can effectively improve the molecular identification and detection efficiency of the fungi.

Description

technical field [0001] The invention relates to a DNA extraction method of microorganisms, in particular to a rapid extraction method of fungal DNA for PCR amplification, belonging to the field of fungal molecular biology. Background technique [0002] With the in-depth study of fungal molecular biology, a small amount of fungal genomic DNA is needed as a template for PCR amplification when carrying out molecular identification and classification of fungi and cloning of their genes. Fungi have thick cell walls and high polysaccharide content. The commonly used fungal DNA extraction method is the CTAB method. The DNA extracted by the CTAB method has a high purity, but it requires a large amount of mycelium and the steps are cumbersome. First, the fungal mycelium is ground to break the wall and incubated at high temperature, which is time-consuming and laborious. In addition, there are also some reports on fungal DNA extraction methods, but they are all inseparable from steps...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12R1/645C12R1/79C12R1/77
Inventor 林彩丽王曦茁朴春根田国忠李永汪来法郭民伟
Owner INST OF FOREST ECOLOGY ENVIRONMENT & PROTECTION CHINESE ACAD OF FORESTRY
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