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Reagent for extracting total DNA of bamboo shoots and application

A technology of reagent and bamboo fungus, applied in the field of molecular biology of genetic engineering technology, can solve the problems of short DNA fragments, toxic reagents, long time consumption, etc., and achieves the effects of good integrity, avoiding pollution and less raw material consumption.

Inactive Publication Date: 2018-12-18
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 bamboo fungus

Method used

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  • Reagent for extracting total DNA of bamboo shoots and application
  • Reagent for extracting total DNA of bamboo shoots and application
  • Reagent for extracting total DNA of bamboo shoots and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Extraction of Dictyophora mycelium and fruiting body total DNA

[0030] Collect the mycelium and fruiting bodies of Dictyophora under the same culture conditions, the specific steps are as follows:

[0031] (1) Add 200 μL of equilibrium solution to the DNA adsorption column, centrifuge at 10,000 rpm for 1 min, discard the waste liquid, and pretreat the DNA adsorption column;

[0032] (2) Grind 2-50 mg of Dictyophora mycelium and fruiting body samples respectively, add 450 μL of lysate, mix well, and centrifuge at room temperature at 10,000 rpm for 30 s;

[0033] (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;

[0034] (4) Add 600 μL of DNA washing solution 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 liquid collection tube, and centrifuge at ...

Embodiment 2

[0038] Example 2 Different Kinds of Mushroom DNA Extraction Test

[0039] (1) Sample pretreatment: take 2-50 mg samples of Dictyophora fruiting bodies, Dictyophora mycelium, Flammulina velutipes, Agaricus blazei, and Lentinus edodes fruiting bodies for grinding;

[0040] (2) Add 200 μL of equilibrium solution to the DNA adsorption column, centrifuge at 10,000 rpm for 1 min, discard the waste liquid, and pretreat the DNA adsorption column;

[0041] (3) Put the ground sample into a centrifuge tube, add 450 μL of lysate, mix well, and centrifuge at 10,000 rpm at room temperature for 30 s;

[0042] (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;

[0043] (4) Add 600 μL of DNA washing solution 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 liquid collection tube, and...

Embodiment 3

[0046] Example 3: PCR amplification of Dictyophora ITS sequence

[0047] According to the ITS sequence of Dictyophora, the extracted Dictyophora fruiting bodies, Dictyophora hyphae, Dictyophora dried samples, Flammulina velutipes fruiting bodies, Agaricus blazei fruiting bodies, Lentinus edodes fruiting bodies PCR detection.

[0048] The primer sequences are:

[0049] ITS-4: 5’-TCCTCCGCTTATTGATATGC-3’

[0050] ITS-5: 5’- GGAAGTAAAAGTCGTAACAAGG-3’

[0051] The reagents used for PCR amplification were purchased from Beijing Quanshijin Biotechnology Co., Ltd., and the reaction system was:

[0052]

[0053] 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.

[0054] PCR amplification products were detected by 1% agarose gel electrophoresis, the loading buffer was 10×Loading Buffer, the electrophoresis buffer was 1×TAE, the molecular weight standard marker was Trans 15K, ethidium bromide staining, and UV gel im...

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Abstract

The invention discloses a reagent for extracting total DNA of bamboo shoots and an application. The method comprises the steps of column balancing, lysing crackinga sample, absorbing the DNA, cleaning, and eluting the DNA. Compared with the traditional DNA extraction method such as a the CTAB method, the a SDS method, the an alkali lysising method and a conventional kit, the method provided byof the invention has the advantages of less consumption of samples, high purity of the DAN samples, good completeness, simple operation and the like, the extraction time can be reduced from 1 to 2 hours to 15 minutes, in the whole extraction process, no toxic reagent such as chloroform, phenol, and beta-mercaptoethanol and the like is used, and the research personnel is ensured enabled not to be harmed by the toxic reagent. An experiment result shows that the reagent is suitable for extracting the total DNA of various fungi such as the bamboo shoots.

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 Dictyophora and its application. 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 analysis, molecular hybridization, genetic diversity analysis, and genomics. Therefore, it is extremely important to efficiently and rapidly prepare total DNA samples with high purity and good integrity. [0003] At present, traditional methods for preparing fungal total DNA include CTAB, SDS, and alkaline lysis. CTAB and SDS use ionic surfactants to lyse fungal cells to release the genome, and then phenol / chloroform / isoamyl Multiple extract...

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辉罗海凌林占熺
Owner FUJIAN AGRI & FORESTRY UNIV
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