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Method for breaking wall for filamentous fungus genomic DNA extraction

A filamentous fungus and genome technology is applied in the necessary wall-breaking field in the filamentous fungal genome extraction process, which can solve the problems of high cost, long time, and hidden dangers of chemical reagents for environmental protection, and achieves reduction of dependence, convenient operation, and reduction of bacterial contamination. Effect

Inactive Publication Date: 2014-04-02
CHINA THREE GORGES UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Chemical reagents have hidden dangers in environmental protection
[0003]The methods developed to obtain high-quality DNA are generally time-consuming, expensive, and require some special equipment, which is difficult for ordinary biological laboratories to meet

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] A simple wall-breaking method for extracting filamentous fungal genome DNA, the method comprising the following steps:

[0016] 1) Filamentous fungus culture: Filamentous fungus A was cultured in NBRIP [National Botanical Research Institute’s phosphate medium (pH 7.0), containing: 10 g glucose, 5 g Ca per liter 3 (PO 4 ) 2 , 5 g MgCl 2 ·6H 2 O, 0.25 g MgSO 4 7H 2 O, 0.2 g KCl, 0.1 g (NH 4 ) 2 SO 4 ] medium, a 250 ml Erlenmeyer flask was loaded with 50 ml liquid medium, cultured with shaking at 28°C for 2 days, and then cultured statically for 4 days;

[0017] 2) Collect fungal mycelium: Use a sterilized toothpick to pick the fungus A cultured in step 1) and place it in a 1.5 ml centrifuge tube for centrifugation (12000 rpm, 10 min), pour off the supernatant, and then use sterilized double Centrifuge (12000 rpm, 10 min) after mixing mycelia with distilled water, discard the supernatant, and keep the fungal mycelia;

[0018] 3) Add extraction solution: add 500 u...

Embodiment 2

[0025] A simple wall-breaking method for extracting filamentous fungal genome DNA, the method comprising the following steps:

[0026] 1) Filamentous fungus culture: Filamentous fungus B was cultured in NBRIP [National Botanical Research Institute’s phosphate medium (pH 7.0), containing: 10 g glucose, 5 g Ca per liter 3 (PO 4 ) 2 , 5 g MgCl 2 ·6H 2 O, 0.25 g MgSO 4 7H 2 O, 0.2 g KCl, 0.1 g (NH 4 ) 2 SO 4 ] medium, a 250 ml Erlenmeyer flask was loaded with 50 ml liquid medium, cultured with shaking at 28°C for 2 days, and then cultured statically for 6 days;

[0027] 2) Collect fungal mycelium: pick fungus B cultured in step 1) with a sterilized toothpick, put it in a 1.5 ml centrifuge tube (12000 rpm, 10 min), pour off the supernatant, and then use sterilized double Centrifuge (12000 rpm, 10 min) after mixing mycelia with distilled water, discard the supernatant, and keep the fungal mycelia;

[0028] 3) Add extraction solution: add 500 ul of DNA extraction buffer [2%...

Embodiment 3

[0036] A simple wall-breaking method for extracting filamentous fungal genome DNA, the method comprising the following steps:

[0037] 1) Filamentous fungus culture: Filamentous fungus C was cultured in potato medium (20% potatoes, 2% sucrose), 250 ml Erlenmeyer flask was filled with 50 ml liquid medium, shake culture at 28 ℃ for 1 day, and then static culture 6 days;

[0038] 2) Collect fungal mycelium: Use a sterilized toothpick to pick the fungus C cultured in step 1) and place it in a 1.5 ml centrifuge tube for centrifugation (12000 rpm, 10 min), pour off the supernatant, and then use sterilized double Centrifuge (12000 rpm, 10 min) after mixing mycelia with distilled water, discard the supernatant, and keep the fungal mycelia;

[0039] 3) Add extraction solution: add 500 ul of DNA extraction buffer [2% SDS, 1.4 M NaCl, 0.2 M Tris-HCl (pH8.0), 0.02 M EDTA (pH8.0)], and mix the bacteria with a sterile toothpick and extraction buffer;

[0040] 4) Freezing: Immediately put...

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Abstract

The invention discloses a method for breaking walls for filamentous fungus genomic DNA extraction. The walls are broken in a multi-step centrifugation and glass rod extrusion mode; the method specifically comprises the following steps in sequence: culturing fungi, collecting the thallus, adding an extraction liquid in multiple steps, freezing, crushing the filamentous thallus, collecting a genomic DNA solution, and precipitating the genomic DNA. According to the method, an ordinary refrigerator of -20 DEG C and a self-made glass rod are adopted, the experimental equipment is easily available, the dependence on liquid nitrogen in the conventional method is reduced, living contaminants in grinding by using a mortar are reduced, the method is convenient to operate, economic and practical, filamentous fungus genomic DNA can be conveniently and rapidly obtained, and thus the method is suitable for popularization and application.

Description

technical field [0001] The invention relates to a wall-breaking method for genome extraction, in particular to a wall-breaking method necessary for the genome extraction process of filamentous fungi. Background technique [0002] Filamentous fungi widely exist in nature and have a variety of functions. Therefore, the study of filamentous fungi has not declined for a long time, but has attracted more and more attention from researchers. The first step in microbial research is species identification. The traditional classification and identification of fungi is mainly based on the morphological characteristics during culture, and the differences in culture characteristics are very significant, thus affecting the accuracy of classification. The modern taxonomic identification of fungi is a comprehensive identification combining morphology, physiology, and DNA molecular level. DNA extraction is the first step in identification at the molecular level, and there are many methods ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 陈国华吴广陈汉臣王凤玲
Owner CHINA THREE GORGES UNIV
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