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Method for extracting filamentous fungi genome DNA and rapid screening method for kit and genetic transformant

A filamentous fungus and kit technology, applied in the field of molecular biology, can solve the problems of long time, time-consuming and laborious, and many impurities, and achieve the effect of short time-consuming

Active Publication Date: 2013-10-23
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] One of the purposes of the present invention is to provide a method and kit for rapidly extracting genome from filamentous fungi, thereby solving the problems of long time consumption, use of toxic organic reagents, and many impurities in traditional filamentous fungal genomic DNA extraction methods
[0005] The second object of the present invention is to provide a method for rapid screening of filamentous fungal genetic transformants, thereby solving the problems of time-consuming and labor-intensive screening methods for filamentous fungal transformants and low screening efficiency.

Method used

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  • Method for extracting filamentous fungi genome DNA and rapid screening method for kit and genetic transformant
  • Method for extracting filamentous fungi genome DNA and rapid screening method for kit and genetic transformant
  • Method for extracting filamentous fungi genome DNA and rapid screening method for kit and genetic transformant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Aspergillus oryzae genome extraction kit, made according to the following formula:

[0044] Electric grinder: speed 1800rpm, equipped with disposable 70mm plastic conical grinding pestle;

[0045]Solution A: 50mM Tris-HCl, 50mM EDTA-2Na, 3%SDS, 2M NaCl, 1% β-mercaptoethanol, pH8.0;

[0046] Solution B: 4M guanidine hydrochloride, 20% isopropanol, 10mM EDTA-2Na, 0.2M acetic acid-sodium acetate buffer at pH 5.0;

[0047] Solution C: 70% absolute ethanol, 0.2M acetic acid-sodium acetate buffer at pH 5.0;

[0048] Solution D: 20 μg / mL RNase aqueous solution, adjust the pH to 8.0 with 1M NaOH solution.

[0049] DNA adsorption column: silica gel column.

[0050] Among them, the electric grinder was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

Embodiment 2

[0052] Utilize the method for the kit of embodiment 1 to extract Aspergillus oryzae genome, the steps are as follows:

[0053] (1) Take a 1.5mL conical bottom centrifuge tube and add 100μL of solution A;

[0054] (2) Add 0.1 mg of Aspergillus oryzae nia D300 (purchased from NRIB Research Institute, Japan) to the centrifuge tube of (1);

[0055] (3) Grind the bacteria with an electric grinder for 2 minutes;

[0056] (4) Add solution A to the ground mycelium to make the final volume about 150 μL;

[0057] (5) Shake in a vortex shaker for 30s to disperse the cells as much as possible;

[0058] (6) Centrifuge at a speed of 12000g and centrifuge for 5 minutes;

[0059] (7) Carefully take 100 μL of the supernatant into a new 1.5 mL centrifuge tube, add 300 μL of solution B, and mix well by inverting;

[0060] (8) Add the mixture of (7) into the adsorption column, centrifuge at 12000g for 1min;

[0061] (9) Pour off the liquid in the collection tube, add 500 μL solution B to the...

Embodiment 3

[0069] The rapid screening method of aspergillus oryzae genetic transformants, the steps are as follows:

[0070] 1. Transform Aspergillus oryzae niaD300 (purchased from NRIB Research Institute, Japan) into plates (0.3% sodium nitrite, 0.2% KCl, 0.05% MgSO 4 .7H 2 O, 0.001% FeSO 4 .7H 2 O, 0.1%K 2 PHO 4 , 2% glucose, 0.8M NaCl, 0.1μg / mL pyrithione (PT), 15mM CsCl, pH 5.5), the transformants grown in, such as figure 1 As shown, use a sterile toothpick to pick onto the screening plate (0.3% sodium nitrite, 0.2% KCl, 0.05% MgSO 4 .7H 2 O, 0.001% FeSO 4 .7H 2 O, 0.1% K 2 PHO 4 , 2% glucose, 0.1 μg / mL pyrithione (PT), pH 5.5);

[0071] 2. When the transformant is cultured on the screening plate to a mycelium amount of 0.1mg, pick the mycelium of the transformant into a 1.5mL centrifuge tube with a toothpick and extract the genome by the method in Example 2, such as figure 2 shown, and measure the concentration.

[0072] 3. Design two pairs of amplification primers to ...

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Abstract

The invention discloses a method for extracting filamentous fungi genome DNA and a rapid screening method for a kit and a genetic transformant. The kit comprises an electric grinder, a solution A (lysate), a solution B (DNA combination solution), a solution C (DNA washing solution), a solution D (DNA eluent) and a DNA adsorption column. The method for extracting filamentous fungi genome DNA is short in time consumption (within 30 min) and high in quality of extracted genome, has no use of toxic reagents such as phenol and chloroform, and can well meet subsequent experiments. The method for rapidly screening the filamentous fungi genetic transformant overcomes the disadvantages of time consumption, labor consumption, low screening efficiency and the like in a conventional screening method for the transformant, and is particularly suitable for large-scale screening of the genetic transformant.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a filamentous fungus DNA extraction kit and an extraction method, and a rapid screening method for filamentous fungal genetic transformants. Background technique [0002] Filamentous fungi widely exist in nature, play an important role in industry, agriculture, medicine and basic biological research, and have been widely studied for many years. With the development of molecular technology, the genetic transformation of filamentous fungi has made great progress in theory and application (Yan Peisheng et al., 1999). The main problem encountered in the current transformation system of filamentous fungi is how to screen genetic transformants with high throughput. Ordinary colony PCR cannot be applied to filamentous fungi with complex cell wall structures (Pan Li et al., 2009). Although Pan Li et al. have used some improved methods in the PCR of filamentous fun...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68C12Q1/04C12R1/69
Inventor 潘力周斌何攀吕扬勇
Owner SOUTH CHINA UNIV OF TECH
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