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Toad oil genome dna extraction method, kit and application thereof

An extraction method, the technology of toad oil, which is applied in the field of genomic DNA extraction of Chinese medicinal materials, can solve problems such as difficult extraction of toad oil dry material, inability to perform DNA extraction steps of toad oil medicinal materials, and inability to identify DNA barcodes, etc., to achieve expansion The effect of increasing the success rate

Active Publication Date: 2019-01-18
INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the Chinese Pharmacopoeia animal medicine toad oil sample is not a fresh dry material sample, rich in substances such as mucus, colloid and hemicellulose. The medicinal material of Houha toad oil is highly swollen, and the expansion degree is as high as more than 55 times. After centrifugation in a water bath, there is no supernatant or very little supernatant, and the subsequent steps of DNA extraction of the toad oil medicinal material cannot be performed.
In addition, the fragmentation and oxidative degradation of genomic DNA in the dry wood of Toad oil
Therefore, it is difficult to extract the genomic DNA of the dry wood samples of Hatoad oil with the existing commercial DNA extraction kits, so that subsequent work such as identification of DNA barcodes cannot be carried out

Method used

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  • Toad oil genome dna extraction method, kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Obtaining the Genomic DNA Extraction Method of Toad Oil Medicinal Material of High Purity Genomic DNA

[0031] Establish a standard method for the extraction of genomic DNA from the medicinal material of toad oil, the extraction method is:

[0032] 1. Take 5 mg of Herba Herb Oil, add 500 μl of buffer FB, grind until there are no obvious particles and mix well. Wherein the buffer FB is acid-containing active water, specifically water containing 0.1%-2% hydrochloric acid.

[0033] 2. Add 360 μl buffer ATL and 40 μl proteinase K (20 mg / mL), vortex and mix well to lyse the sample. It is preferred to use a vortex mixer to mix well, incubate at 56°C until the solution is clear, mix the sample by inversion 2-3 times per hour, or use a water bath shaker, usually 1 hour can completely lyse.

[0034] The buffer ATL contains 1%-2.5% sodium dodecylsulfonate, 10-20mM glycine and 10mM sodium edetate.

[0035] 3. Optionally, add 4 μl RNase A (100 mg / ml), vortex and mix we...

Embodiment 2

[0044] Example 2 Large sample verification

[0045] In order to verify the general effectiveness of the method for extracting the toad oil genome DNA, reagent composition and concentration determined in Example 1 to obtain high-purity genomic DNA, a variety of toad oil samples were used to verify, specifically:

[0046] 1) Realize the DNA extraction of toad oil medicinal materials from different origins (Heilongjiang Province, Jilin Province and Liaoning Province) and the standard control Toad oil medicinal materials of China Institute for the Control of Pharmaceutical and Biological Products to obtain high-purity genomic DNA.

[0047] 2) Realize the DNA extraction of toad oil medicinal materials with different commercial specifications (line oil and block oil) to obtain high-purity genomic DNA.

[0048]1. Take about 5 mg of Herba Haba oil, add 500 μl of buffer FB, and use a grinding pestle to grind repeatedly for about 30 seconds until there are no obvious particles and mix w...

Embodiment 3

[0068] Example 3 Kazakh oil high-purity genomic DNA extraction kit

[0069] The kit includes the following components:

[0070] 1. Adsorption column / collection tube;

[0071] 2. Buffer FB: water containing 0.1% to 2% hydrochloric acid

[0072] 3. Buffer ATL: 1% to 2.5% sodium dodecylsulfonate, 10 to 20mM glycine and 10mM sodium edetate;

[0073] 4. Buffer AL: 10-20 mM glycine and 10 mM sodium edetate.

[0074] 5. Buffer AW1: 36% to 50% guanidine hydrochloride and 20% to 50% isopropanol;

[0075] 6. Buffer AW2: 0.1% to 1% sodium azide and 20% to 50% isopropanol

[0076] 7. Buffer TE

[0077] 8. Proteinase K: 20mg / mL

[0078] 9. Grinding pestle

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Abstract

The invention discloses an extraction method of genome DNA of forest frog's oviduct, comprising the steps: taking a forest frog's oviduct medicinal material, adding acid containing active water to carry out renaturation on the sample, and adding a buffer solution ATL and a protease K cracking sample; adding a fat tissue removing solution phenol / chloroform / isoamyl alcohol to remove fat and protein; adding a washing solution to wash, absorbing DNA with an adsorption column, and finally eluting to obtain high-purity genome DNA. The invention also provides an extraction kit of the genome DNA of forest frog's oviduct and application thereof. The extraction method disclosed by the invention can effectively extract the genome DNA from the forest frog's oviduct medicinal material to obtain high-purity genome DNA, and provides a good base for wider application of the DNA bar code technology.

Description

technical field [0001] The invention relates to the field of genomic DNA extraction of traditional Chinese medicinal materials, in particular to a method for extracting genomic DNA from hazel oil, a kit and an application thereof. Background technique [0002] The identification of traditional Chinese medicine is the premise and basis for studying the variety and quality of traditional Chinese medicine, formulating the standard of traditional Chinese medicine, and finding and expanding the source of medicine. The four traditional identification methods of traditional Chinese medicine are base identification, character identification, microscopic identification and physical and chemical identification. The theoretical basis of traditional identification methods is based on the analysis of the traits of taxa. These traits are phenotypes closely related to the growth stage and environment of organisms. The identification results are easily affected by subjective and objective f...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12Q1/6806
CPCC12N15/101C12Q1/6806C12Q2521/537C12Q2523/113C12Q2523/308
Inventor 石林春刘建辉宋经元刘金欣唐先明刘景波姚辉周建国王福宾于海龙
Owner INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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