High throughput method for rapidly extracting genomic DNA of fungus

A high-throughput, genomic technology, applied in recombinant DNA technology, DNA preparation, etc., can solve the problems of large consumption of chemical reagents, human health hazards, cumbersome experimental steps, etc., to shorten the extraction time, avoid toxic carcinogenic reagents, and speed up experiments. effect of speed

Inactive Publication Date: 2012-03-28
CHINA JILIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For the removal of proteins released by cell rupture, most of them use phenol and β-mercaptoethanol, etc., and these drugs are very toxic and have a greater harmful effect on human health
At present, these methods reported at home and abroad consume a large amount of chemical reagents, the experimental steps are cumbersome, the extraction time is long, and the most important thing is that they are more exposed to toxic reagents.
None of these can meet the needs of current high-throughput fungal genome research

Method used

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  • High throughput method for rapidly extracting genomic DNA of fungus
  • High throughput method for rapidly extracting genomic DNA of fungus
  • High throughput method for rapidly extracting genomic DNA of fungus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] (1) Take a 1.5ml centrifuge tube, add 100μl improved and optimized CTAB buffer (1.5% CTAB, NaCl 1.4mol / L, TrisCl 100mmol / L, EDTA 20mmol / L, 1% SDS, 1% PVP, solvent for deionized water);

[0027] (2) Pick out rice koji bacteria [Ustilaginoidea virens (Cooke) Tak] with a diameter of 1 to 2 cm from the culture medium, put them into a centrifuge tube, and perform the following operations:

[0028] (3) Fully grind the bacterial block with a grinding rod sterilized by 75% alcohol;

[0029] (4) After grinding sufficiently, add 400 μl CTAB buffer and 4 μl RNase A (10 μg / ml, produced by sigma);

[0030] (5) Place the ground sample in a water bath at 60-70°C, shake gently every 8-10mins, and take it out after 20-30mins;

[0031] (6) Add 700ul of CIA solution (chloroform:isoamyl alcohol=24:1, v / v), shake and mix for 1min;

[0032] (7) 12000rpm, centrifuge for 10mins;

[0033] (8) Carefully pipette the upper aqueous phase into another centrifuge tube, add 0.7 times the volume of i...

Embodiment 2

[0038] Take Pichia pastoris (Pichia pastoris) mycelium, black mold (Aspergillus niger) mycelium, Oyster mushroom (Oyster mushroom) mycelium and black fungus (Auricularia auricular) mycelium with a diameter of about 1-2 cm. Genomic DNA was extracted according to the method in Example 1. Extract gained DNA and embodiment 1 gained DNA and carry out agarose gel electrophoresis and ultraviolet spectrophotometer method detection respectively by following method:

[0039] Agarose gel electrophoresis detection: first prepare 0.8% agarose gel, and use 1×TAE electrophoresis buffer to carry out electrophoresis detection on the extracted fungal genome total DNA, and the composition of the 1×TAE electrophoresis buffer is as follows: 0.04mol / L Tris-acetic acid, 0.001mol / L EDTA. Pipette 2-4 μL of fungal genomic DNA and mix it with 1 ul of 6× loading buffer (manufactured by Quanshijin Biological Co., Ltd.). At the same time, 15K Marker (manufactured by Quanshijin Biological Co., Ltd.) was u...

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Abstract

The invention provides a high throughput method for rapidly extracting genomic DNA of a fungus. According to the method, a fungal bacterium block or a mycelium undergoes a simple physical grinding process and is subjected to impurity removal by an improved CTAB solution to obtain the purified DNA. The method of the invention mainly has the following beneficial effects: 1, a complex process that aspecial agent liquid nitrogen must be used to grind in present methods is avoided; 2, the usage of toxic carcinogenic regents of phenol and the like in the present methods is avoided; 3, the usage ofa toxic odorous chemical reagent beta-mercaptoethanol is avoided; and 4, the method of the invention only needs 1h, the extraction time is shortened by 2h, the experiment speed is greatly accelerated, and the high throughput rapid extraction can be realized. The method of the invention is also suitable for the rapid extraction of the genomic DNA of other various fungi.

Description

(1) Technical field [0001] The invention relates to a method for rapidly extracting genomic DNA of fungi with high throughput, through which the genomic DNA of various fungi, such as yeast, mold, fungus, and animal and plant pathogenic fungi, can be rapidly extracted. (2) Background technology [0002] Fungi (fungus) are widely distributed in nature, and there are many species. The morphology of fungi varies greatly. Some are very small and can only be seen under a microscope, such as yeast-type colonies; some are very large, such as the fruiting bodies of mushrooms such as Ganoderma lucidum. The nutritional mode of fungi is heterotrophic absorption type, that is, absorbs soluble nutrients from the surrounding environment through the cell surface, and it can produce a large number of asexual and sexual spores for reproduction. The vegetative body of most fungi is a mycelium with many hyphae together. [0003] Fungi are generally divided into three groups, namely yeasts, mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 邹克琴胡东维李素芳王为民
Owner CHINA JILIANG UNIV
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