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Preparation method of thermomyces PCR template

A fungal and template technology, applied in microorganism-based methods, biochemical equipment and methods, and microbial assay/inspection, etc., can solve the problems of unfavorable high-throughput screening of transformants, cumbersome operations, and large amount of bacterial cells.

Inactive Publication Date: 2018-10-12
YUNNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the traditional methods for obtaining fungal DNA genome templates are based on CTAB and SDS methods. These two methods require grinding a large number of mycelial cells with liquid nitrogen, which not only requires a large amount of bacteria (more than 50 mg), but also is cumbersome and time-consuming (1- 2 hours); in addition, organic solvents such as phenol and chloroform used in the extraction process have certain toxicity, and these factors have seriously affected the progress of the experiment, which is not conducive to large-scale high-throughput screening of transformants. no exception

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  • Preparation method of thermomyces PCR template
  • Preparation method of thermomyces PCR template
  • Preparation method of thermomyces PCR template

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Construction of knockout plasmids

[0042] Inoculate Dupont thermophiles NRRL 2155 wild bacteria from slant medium onto PDA medium, place them in a 45°C incubator and cultivate them for 7 days, then scrape about 100 mg of mycelia, and use the CTAB method to extract genomic PCR templates. The steps are as follows:

[0043] (1) Add 10mL of CTAB separation buffer (CTAB 4g / L, NaCl 16.364g / L, 1M Tris-HCl 20ml) into a 50mL centrifuge tube and preheat in a 60°C water bath.

[0044] (2) Weigh 100 mg of mycelium, place it in a pre-cooled mortar, pour liquid nitrogen into it, and grind the mycelia as soon as possible.

[0045] (3) Add the ground powder directly into the preheated CTAB separation buffer, and gently swirl to make it evenly mixed.

[0046] (4) The sample was incubated at 65°C for 30 minutes.

[0047] (5) Add an equal volume (10 mL) of chloroform-isoamyl alcohol, and gently invert to mix.

[0048] (6) Centrifuge at 4000 rpm for 10 minutes at room temperature.

[...

Embodiment 2

[0058] Preparation, transformation and validation of protoplasts from Dupontella thermophilica NRRL 2155

[0059] Preparation of TE buffer: 10mM Tris-HCl and 0.1mM EDTA, pH 7.5.

[0060] (1) Inoculate the mycelium block of Dupont thermophilic bacteria NRRL 2155 in the center of the PDA medium, and place it in an incubator at 45°C for 7 days. Use 1mL pipette tip and sterile water (add 0.05% Tween 20) to scrape the culture from the above plate, filter with four layers of lens paper, and divide the liquid containing spores into 1.5mL centrifuge tubes, 10000rpm , centrifuge at room temperature for 5 min to enrich the spores, discard the supernatant, enrich to 1×10 8 cells / mL and washed twice with sterile water.

[0061] (2) Transfer 200 μL of the spore suspension to 100 mL of YPS liquid medium, culture at 45° C. and shake at 180 rpm for 20 h.

[0062] (3) Pour the mycelium cultivated in step (2) into a sterile funnel (containing four layers of lens-cleaning paper) and filter to...

Embodiment 3

[0081] The thermophilic Dupont bacteria NRRL 2155 wild bacteria were inoculated on the PDA medium from the slant medium, and placed in a 45°C incubator for 5 days.

[0082] The PCR template preparation was the same as step (17) in Example 2. The difference is that the mycelium selected is the hyphae of the wild fungus cultured in this example; and the formula of the TE buffer is: 8mM Tris-HCl and 0.05mM EDTA, pH 7.

[0083]The PCR verification operation is the same as step (18) in Example 2. The difference is that the primers used in PCR are ER5F (SEQ ID No.1) / ER5R (SEQ ID No.2), and the PCR template is the template prepared in this example.

[0084] see results image 3 Lane 3 in the figure, it can be judged from the figure that a clear target band can be effectively amplified, but there are fuzzy non-specific bands below the target band. Therefore, although the quality of the lysed bacterial cell suspension obtained under the conditions of this example is not the best, it...

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Abstract

The invention provides a preparation method of a thermomyces PCR template. The method comprises the following steps that 1, hyphae of thermomyces is suspended in a TE buffer solution, and a thallus suspension is obtained; 2, the thallus suspension is heated under the temperature of 90 DEG C or above for at least 10 min, and an obtained cracked thallus suspension is adopted as the PCR template. Themethod for obtaining a thermomyces genome can be used for PCR identification of thermomyces genetic converters, the method adopts a small number of thalli, the cultivation time of the converters canbe shortened, converter detection can be performed at the early cultivation period, operation is easy, convenient and rapid, time and labor are saved, the method is economical, free of toxin and environmentally friendly, the follow-up PCR sensitivity is high, specificity is high, it becomes possible that converters can be screened out in a large-scale and high-throughput mode, and the method for rapidly identifying the thermomyces converter has important scientific significance and value.

Description

technical field [0001] The application provides a method for preparing a PCR template of a fungus of the genus Thermomyces. Background technique [0002] Thermomyces is a kind of temperature-specific fungi suitable for growth at 45-50°C. It is widely distributed and found in tropical, subtropical, temperate, frigid, humid and arid regions of the earth. Due to a series of unique enzymes and metabolites in the cells of thermophilic fungi, they have unique survival adaptability under high temperature conditions. And because many of the enzymes and metabolites, such as thermostable enzymes and biologically active small molecules, have important economic value in production and life, thermophilic fungi have become a current research hotspot. [0003] Genetic transformation is an important means of studying biomolecular biology. In genetic transformation, the most commonly used method is mainly to perform PCR screening using the genome of the transformant as a template. Most of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/04C12Q1/686C12R1/645
CPCC12Q1/6806C12Q1/686C12Q2527/125
Inventor 牛雪梅黄为平张克勤
Owner YUNNAN UNIV
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