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A method that enables ctab method to extract high-quality genomic dna from fungi

A high-quality, genomic technology, used in recombinant DNA technology, DNA preparation, etc., can solve the problems of small DNA extraction, limited success rate, and difficulty in meeting molecular experiments.

Inactive Publication Date: 2018-08-10
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the effects of CTAB and SDS methods for extracting DNA are quite satisfactory, they also have major shortcomings. Among them, although the CTAB method can effectively remove impurities such as phenols in fungal materials, the steps are cumbersome and time-consuming. The amount is not large, so that the concentration and purity of the obtained DNA are not high enough, which limits the success rate of subsequent experiments
Although the SDS method can quickly extract genomic DNA for molecular markers, the extracted DNA is limited to molecular markers and other experiments that do not require high DNA quality, and it is difficult to meet molecular experiments that require high DNA quality, such as gene cloning and molecular hybridization. Wait

Method used

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  • A method that enables ctab method to extract high-quality genomic dna from fungi
  • A method that enables ctab method to extract high-quality genomic dna from fungi
  • A method that enables ctab method to extract high-quality genomic dna from fungi

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Prepare 1×10 with sterilized 0.02% Tween water 7 spore suspension per ml, take 100 µL of the suspension and spread it evenly on the glass paper on the surface of the SDAY plate, and incubate at 25°C for 2.5 days. Then scrape the mycelium on the cellophane and put it into a mortar pre-cooled in a -70°C refrigerator, add 0.3 g of sterilized fine sand of 60 mesh and add liquid nitrogen for rapid grinding for 3 minutes until the mycelium finally becomes a silky ultrafine powder (600-1000 mesh), and then follow the steps of CTAB method to minimize exposure to the air during the extraction process, and use a refrigerated centrifuge to centrifuge at 4°C for all centrifugation processes.

Embodiment 2

[0030] Prepare 1×10 with sterilized 0.02% Tween water7 spore suspension per ml, take 150 µL of the suspension and inoculate it on the cellophane on the surface of the SDAY plate, add 0.1 g of 70-mesh sterilized fine sand, and incubate at 25°C for 2.5 days. Then scrape the mycelium on the cellophane and put it into a mortar pre-cooled in a -70°C refrigerator, add liquid nitrogen for rapid grinding for 4 minutes until the mycelium finally presents a silky ultrafine powder (600-1000 mesh), and then Follow the steps of the CTAB method, minimize exposure to air during the extraction process, and use a refrigerated centrifuge to centrifuge at 4°C for all centrifugation processes.

Embodiment 3

[0032] Spread a layer of sterilized cellophane on the SDAY plate, add 120 μL of 0.02% Tween water, and then add an appropriate amount (0.2 g) of 80 mesh fine sand containing the target strain to the SDAY cellophane and coat the plate evenly with a coating stick; 25 Cultivate for 3 days. Then scrape the mycelium on the cellophane into a mortar pre-cooled in a -70°C refrigerator, add liquid nitrogen for rapid grinding for 5 minutes until the mycelium finally presents a silky ultrafine powder (600-1000 mesh), and then follow the The CTAB method is operated step by step, and the exposure to the air is minimized during the extraction process. All centrifugation processes are centrifuged at 4°C in a refrigerated centrifuge.

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Abstract

The invention relates to a method capable of extracting high-quality genome DNA from fungus by adopting a CTAB process and belongs to the technical field of molecular biology. In inoculation of DNA extraction by adopting the CTAB process, fine sand containing strains is selected for inoculating directly, and / or spore suspension is adopted for inoculation, then the fine sand is added for culturing, and / or the cultured hyphae are added with the fine sand for grinding samples during sample grinding and DNA extraction until the hyphae are silky superfine powder finally. The method capable of extracting the high-quality genome DNA from the fungus by adopting the CTAB process has the advantages that content of the extracted DNA can be obviously increased, and quality and yield of the DNA are obviously improved; meanwhile, the method provided by the invention is easy to operate, strong in experimental stability, high in repeatability, accurate and prompt, so that DNA extraction quality is met while labour intensity can be greatly reduced in a sample grinding link, high sample grinding speed is still maintained even if a large amount of DNA samples are extracted, sample grinding time is greatly shortened, fatigue of a person during the sample grinding and extraction failure probability are greatly reduced.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to a method for extracting high-quality genomic DNA of fungi by CTAB method. Background technique [0002] DNA is the carrier of genetic information and important genetic material. As a basic and important technical means in molecular experiments, genomic DNA extraction is used in various research fields. Many studies require a large amount of experimental materials, and a certain amount and high-quality DNA samples are the guarantee for PCR amplification, restriction enzyme digestion, Southern hybridization, sequencing and other molecular biology research. The workload of DNA extraction is heavy, and the purity requirement is high. The quality and efficiency of DNA extraction seriously affect the experimental results. Therefore, it is extremely important to obtain high-quality and high-quantity DNA with appropriate methods. There are many DNA extraction meth...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
Inventor 童森淼冯明光
Owner ZHEJIANG UNIV
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