Reagent for extracting total DNAs of Grifola frondosa and application thereof

A technology of Grifola frondosa and reagents, applied in the field of genus, can solve the problems of short DNA fragments, toxic reagents, and long time consumption, and achieve the effects of increased binding rate, high integrity, and simple operation

Inactive Publication Date: 2018-12-14
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This solves the shortcomings of the existing extraction technology, such as large sample consumption, long time consumption, toxic reagents, and short extracted DNA fragments, so as to meet the needs of molecular biology experiments on precious materials such as Grifola frondosa

Method used

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  • Reagent for extracting total DNAs of Grifola frondosa and application thereof
  • Reagent for extracting total DNAs of Grifola frondosa and application thereof
  • Reagent for extracting total DNAs of Grifola frondosa and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1, adopt this method to extract the total DNA of Grifola frondosa mycelium and fruiting body

[0032] Collect the mycelium and fruiting bodies of Grifola frondosa respectively, and the specific steps are as follows:

[0033] (1) Add 200 μL of BB to the DNA adsorption column, centrifuge at 10,000 rpm for 1 min, discard the waste liquid, and pretreat the DNA adsorption silica gel column;

[0034] (2) Grind 2-100 mg of the sample with dry ice, add 600 µL of LB, mix well, and centrifuge at 10,000 rpm for 30 s at room temperature;

[0035](3) Transfer the supernatant to a treated DNA adsorption column, centrifuge at 10,000 rpm for 1 min at room temperature, and discard the waste liquid;

[0036] (4) Add 600 µL of WB to the DNA adsorption silica gel column, centrifuge at 10,000 rpm at room temperature for 1 min, discard the waste liquid, put the DNA adsorption silica gel column back into the waste liquid collection tube, and centrifuge at 10,000 rpm at room temper...

Embodiment 2

[0046] Collect the total DNA of Grifola frondosa fruiting bodies, mycelium, and the total DNA of mushrooms, oyster mushrooms, and Cordyceps militaris respectively. The specific steps are as follows:

[0047] (1) Add 200 μL of BB to the DNA adsorption column, centrifuge at 10,000 rpm for 1 min, discard the waste liquid, and pretreat the DNA adsorption silica gel column;

[0048] (2) Grind 2-100 mg of the sample with dry ice, add 600 µL of LB, mix well, and centrifuge at 10,000 rpm for 30 s at room temperature;

[0049] (3) Transfer the supernatant to a treated DNA adsorption column, centrifuge at 10,000 rpm for 1 min at room temperature, and discard the waste liquid;

[0050] (4) Add 600 µL of WB to the DNA adsorption silica gel column, centrifuge at 10,000 rpm at room temperature for 1 min, discard the waste liquid, put the DNA adsorption silica gel column back into the waste liquid collection tube, and centrifuge at 10,000 rpm at room temperature for 1 min;

[0051] (5) Move...

Embodiment 3

[0059] Embodiment 3, Grifola frondosa ITS sequence PCR amplification

[0060] ITS-4 / 5, a common primer for ITS fragments of edible fungi, was used to detect the genomes of Grifola frondosa fruiting body, mycelium, dry sample DNA and total DNA of shiitake mushroom, ganoderma lucidum and Flammulina velutipes.

[0061] The primer sequences are:

[0062] ITS-4: 5’-TCCTCCGCTTATTGATATGC-3’

[0063] ITS-5: 5’- GGAAGTAAAAGTCGTAACAAGG-3’

[0064] The reagents used for PCR amplification were purchased from Beijing Quanshijin Biotechnology Co., Ltd., and the reaction system was:

[0065]

[0066] The conditions of the PCR reaction are: 94°C 7min → [94°C 30s → 56°C 30s → 72°C 30s] ×34cicles → 72°C 10min → 4°C.

[0067] Agarose gel electrophoresis analysis

[0068] The total DNA and PCR amplification products were detected by 1% agarose gel electrophoresis, the loading buffer was 10×LoadingBuffer, the electrophoresis buffer was 1×TAE, the molecular weight standard marker was Trans 1...

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Abstract

The invention provides a reagent for extracting total DNAs of Grifola frondosa and an application thereof. Total DNAs are fast and efficiently extracted from about 50mg of mycelium and sporocarp samples of Grifola frondosa through crushing and cleavage, DNA adsorption, rinsing and elution. The extraction time of a single sample is shortened to 15min, and the extracted DNAs have good integrity andhigh purity and can be directly used for downstream molecular biology experiments such as PCR, Real-Time PCR and molecular labeling. The toxic reagents such as chloroform, phenol and beta-mercaptoethanol are not used in the whole extraction process so that the researchers are protected from toxic reagents.

Description

technical field [0001] The invention belongs to the field of molecular biology of genetic engineering technology, and in particular relates to a reagent for extracting total DNA of Grifola frondosa and its application. The method develops a method suitable for downstream molecular biology tests such as PCR, Real-Time PCR, and molecular markers. The rapid total DNA extraction method of fungi such as Grifola frondosa, can quickly extract the total DNA of fruiting bodies, mycelia and other fermentation products of fungi such as Grifola frondosa, and the extracted products can be used in molecular biology and genetics Research and Molecular Identification. Background technique [0002] DNA is the carrier of genetic information in living organisms, including the genetic traits of organisms. High-quality, high-purity total DNA is the primary condition to ensure the development of molecular biology research such as PCR amplification, restriction endonuclease digestion, genetic map...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1006
Inventor 李晶张煜隆曹秀明胡应平张双双刘艳玲刘朋虎林辉罗海凌童金华王泽辉林占熺
Owner FUJIAN AGRI & FORESTRY UNIV
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