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Reagent for extracting total DNA of lucid ganoderma and application of reagent

A reagent, the technology of Ganoderma lucidum, applied to the reagent for extracting the total DNA of Ganoderma lucidum and its application field, can solve the problems of short DNA fragments, toxic reagents, long time-consuming, etc., and achieve the effects of increasing the binding rate, high integrity, and simple operation.

Inactive Publication Date: 2018-12-21
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 Ganoderma lucidum

Method used

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  • Reagent for extracting total DNA of lucid ganoderma and application of reagent
  • Reagent for extracting total DNA of lucid ganoderma and application of reagent
  • Reagent for extracting total DNA of lucid ganoderma and application of reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1, adopt this method to extract the total DNA of Ganoderma lucidum mycelium and fruiting body

[0033] The mycelium and fruiting bodies of Ganoderma lucidum were collected under the same culture conditions, and the specific steps were as follows:

[0034] (1) Add 200 μL of Balance Buffer to the DNA adsorption column SC, centrifuge at 10,000 rpm for 1 min, discard the waste liquid, and pretreat the DNA adsorption column;

[0035](2) Add 400 µl of Lysis Buffer after grinding 2-50 mg of the sample, mix well, and centrifuge at 10,000 rpm at room temperature for 30 s;

[0036] (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;

[0037] (4) Add 600 µl of Wash Buffer to the DNA adsorption column, centrifuge at 10,000 rpm at room temperature for 1 min, discard the waste liquid, put the DNA adsorption column back into the waste collection tube, and centrifuge at 10,000...

Embodiment 2

[0047] Collect ganoderma lucidum fruiting bodies, mycelium, and Cordyceps militaris, Agaricus blazei, and Grifola frondosa fruiting bodies respectively. The specific steps are as follows:

[0048] (1) Add 200 μL of Balance Buffer to the DNA adsorption column SC, centrifuge at 10,000 rpm for 1 min, discard the waste liquid, and pretreat the DNA adsorption column;

[0049] (2) Add 400 µl of Lysis Buffer after grinding 2-50 mg of the sample, mix well, and centrifuge at 10,000 rpm at room temperature for 30 s;

[0050] (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;

[0051] (4) Add 600 µl of Wash Buffer to the DNA adsorption column, centrifuge at 10,000 rpm at room temperature for 1 min, discard the waste liquid, put the DNA adsorption column back into the waste collection tube, and centrifuge at 10,000 rpm at room temperature for 1 min.

[0052] (5) Move the DNA adsorption col...

Embodiment 3

[0060] Embodiment 3, Ganoderma lucidum ITS sequence PCR amplification

[0061] According to the Ganoderma lucidum ITS sequence, the edible fungus ITS fragment general primer ITS-4 / 5 was used to detect the extracted Ganoderma lucidum fruiting body, mycelium, dry sample DNA and the total DNA genome of Cordyceps militaris, Agaricus blazei and Grifola frondosa.

[0062] The primer sequences are:

[0063] ITS-4: 5’-TCCTCCGCTTATTGATATGC-3’

[0064] ITS-5: 5’- GGAAGTAAAAGTCGTAACAAGG-3’

[0065] The reagents used for PCR amplification were purchased from Beijing Quanshijin Biotechnology Co., Ltd., and the reaction system was:

[0066]

[0067] 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.

[0068] Agarose gel electrophoresis analysis

[0069] 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×TA...

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Abstract

The invention relates provides a reagent for extracting total DNA of lucid ganoderma and the application of the reagent. The total DNA is rapidly and efficiently extracted from about 50 mg of myceliumand sporocarp sample by adopting the steps of crushing, cracking, DNA adsorption, rinsing, eluting and the like; for fresh dry samples, and total DNA suitable for ITS identification and other molecular biology experiments also can be extracted. Further experiment proves that the method also can be applied to edible-medicinal fungi such as cordyceps militaris, agaricus blazei murill and grifola frondosa. According to the method, the extraction time of single sample is shortened to 15 minutes; in addition, the extracted DNA has the advantages of good integrity and high purity, and can be directly used for downstream molecular biology experiments such as PCR, Real-Time PCR and molecular markers.

Description

technical field [0001] The invention belongs to the field of molecular biology of genetic engineering technology, and relates to a reagent for extracting total DNA of Ganoderma lucidum and its application. The method develops a rapid Ganoderma lucidum suitable for downstream molecular biology tests such as PCR, Real-Time PCR, and molecular markers. The total DNA extraction method of fungi such as Ganoderma lucidum can quickly extract the total DNA of fruiting bodies, mycelia and other fermentation products of fungi such as Ganoderma lucidum. The extracted products can be used for 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 anal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1006
Inventor 刘艳玲张煜隆王安南李晶王泽辉林兴生罗海凌林占熺
Owner FUJIAN AGRI & FORESTRY UNIV
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