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Fungus DNA (Deoxyribose Nucleic Acid) extracting method suitable for PCR (Polymerase Chain Reaction) amplification

An extraction method, fungal technology, applied in the field of extraction, separation, and purification of DNA, can solve the problems of liquid nitrogen easy to frostbite the skin, time-consuming, and low efficiency, and achieve the ideal effect of DNA concentration and purity

Inactive Publication Date: 2012-07-11
SHANDONG HOMEY AQUATIC DEV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Through these methods, a large amount of DNA can be extracted at one time and the quality is good, but the above methods have certain disadvantages: first, liquid nitrogen is easy to frostbite the skin, and liquid nitrogen and dry ice are not easy to obtain in some places and are limited; second, it is time-consuming and inefficient. ; Third, relatively more equipment and medicines are used
The above-mentioned wall-breaking method either has the disadvantages of slow extraction speed, cumbersome steps, or high technical requirements.

Method used

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  • Fungus DNA (Deoxyribose Nucleic Acid) extracting method suitable for PCR (Polymerase Chain Reaction) amplification
  • Fungus DNA (Deoxyribose Nucleic Acid) extracting method suitable for PCR (Polymerase Chain Reaction) amplification
  • Fungus DNA (Deoxyribose Nucleic Acid) extracting method suitable for PCR (Polymerase Chain Reaction) amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] 1. DNA extraction

[0029] a. Inoculate Aspergillus sydowii in liquid culture medium, cultivate at 37°C for 36 hours, then in a 1.5ml centrifuge tube, control the rotation speed to 12000r / min, centrifuge for 10min, and collect mycelium;

[0030] b. Weigh 0.2g of mycelium with a wet weight, place it in a clean mortar, add 1ml of TRIS saturated phenol extract to the mortar, and grind it to a paste; wherein, the TRIS saturated phenol extract The pH value is 8.0;

[0031] c. Transfer all the paste to a 1.5ml centrifuge tube with a blue tip that cuts off 0.5cm from the tip, shake fully for about 1min, then add 0.5ml of chloroform-isoamyl alcohol extract, in which chloroform-isoamyl The volume ratio of the alcohol extraction solution: 25:1, gently oscillate and mix to extract the protein; control the speed at 12000r / min, centrifuge for 5min, and take the supernatant;

[0032] d. Transfer the supernatant to a new centrifuge tube, add 2 times the volume of pre-cooled absolute...

Embodiment 2

[0037] 1. DNA extraction

[0038] a. Inoculate Epicoccum spp. in liquid culture medium, culture at 28°C for 72 hours, then in a 1.5ml centrifuge tube, control the rotation speed to 15000r / min, centrifuge for 5min, and collect mycelia;

[0039] b. Weigh 0.3g of mycelium with a wet weight, put it in a clean mortar, add 1ml of TRIS saturated phenol extract to the mortar, and grind it fully until it is paste; wherein, the TRIS saturated phenol extract The pH value is 8.0;

[0040] c. Transfer all the paste to a 1.5ml centrifuge tube with a blue tip that cuts off 0.5cm from the tip, shake fully for about 1min, then add 0.5ml of chloroform-isoamyl alcohol extract, in which chloroform-isoamyl The volume ratio of the alcohol extract: 25:1, gently oscillate and mix to extract the protein; control the rotation speed at 15000r / min, centrifuge for 8min, and take the supernatant;

[0041] d. Transfer the supernatant to a new centrifuge tube, add 2 times the volume of pre-cooled absolute ...

Embodiment 3

[0046] 1. DNA extraction

[0047] a. Inoculate Alternaria sp. in liquid culture medium, cultivate at 28°C for 60 hours, then in a 1.5ml centrifuge tube, control the rotation speed at 13000r / min, centrifuge for 8min, and collect mycelium;

[0048] b. Weigh 0.5g of mycelium with a wet weight, place it in a clean mortar, add 1ml of TRIS saturated phenol extract to the mortar, and grind it thoroughly until it becomes a paste; wherein, the TRIS saturated phenol extract The pH value is 8.0;

[0049] c. Transfer all the paste to a 1.5ml centrifuge tube with a blue tip that cuts off 0.5cm from the tip, shake fully for about 1min, then add 0.5ml of chloroform-isoamyl alcohol extract, in which chloroform-isoamyl The volume ratio of the alcohol extraction solution: 25:1, gently oscillate and mix to extract the protein; control the rotation speed at 18000r / min, centrifuge for 8min, and take the supernatant;

[0050] d. Transfer the supernatant into a new centrifuge tube, add 2 times the...

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Abstract

The invention relates to a fungus DNA (Deoxyribose Nucleic Acid) extracting method suitable for PCR (Polymerase Chain Reaction) amplification. The method comprises the following steps of: performing liquid culturing on thalli, centrifuging and collecting mycelia; adding a TRIS (tris(hydroxymethyl)aminomethane) saturated phenol extracting solution into grinding equipment for grinding into paste; transferring the paste into a centrifuge tube, adding a chloroform-isopentyl alcohol extracting solution, mixing uniformly, extracting proteins, centrifuging and taking a supernatant; adding absolute ethyl alcohol into the supernatant for precipitating, centrifuging and collecting a precipitate; and washing the precipitate with ethanol, drying and dissolving to obtain a fungus genome DNA. According to the method, the use of liquid ammonia is avoided, the use of reagents is reduced, and a large quantity of samples can be extracted within a short period of time; and the method has the characteristics of easiness, convenience, high efficiency, rapidness and low price. The concentration and purity of the DNA extracted with the method are ideal, and conventional molecular biology researches such as subsequent PCR amplification and the like can be met.

Description

technical field [0001] The invention relates to a method for separating, extracting and purifying DNA, in particular to a method for extracting fungal DNA suitable for PCR amplification. Background technique [0002] With the development of molecular biology, it is of great significance to quickly and efficiently extract high-quality DNA in the molecular biology research of fungi. Fungi have a special cell wall structure. Compared with other microorganisms, their cell walls are thicker and their polysaccharide content is higher, so the extraction of fungal DNA is more difficult. At present, the commonly used fungal DNA extraction methods are CTAB method, SDS method and kit method. The main technical routes of the first two methods are: use liquid or solid medium to cultivate fungi, collect mycelium, grind and break the cell wall with liquid nitrogen, and then use each extract to extract DNA, and then obtain DNA after purification. Through these methods, a large amount of D...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 贾爱荣刘昌衡孙永军张永刚孟秀梅张绵松胡炜刘新袁文鹏夏雪奎
Owner SHANDONG HOMEY AQUATIC DEV
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