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Method for extracting whole genome DNA of atractylodes macrocephala koidz leaves

A whole-genome, leaf-based technology, applied in the field of molecular biology, can solve the problems of low quality DNA extraction, small amount of extracted DNA, and low extraction volume, and achieve the effects of common reagents, shortened experiment time, and improved quality

Inactive Publication Date: 2021-03-23
湖北省农业科学院中药材研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Taking the leaves of Atractylodes macrocephala as the research object, SDS method, conventional CTAB method and kit method can all be used to extract genomic DNA of medicinal plants such as Atractylodes macrocephala, but the conventional CTAB method and SDS method have low quality of extracted DNA, less extraction amount, The procedures are cumbersome and time-consuming, etc.
Although the kit method can effectively remove polysaccharides and polyphenols in DNA, the cost is high, the amount of extracted DNA is small, and it is accompanied by certain degradation.

Method used

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  • Method for extracting whole genome DNA of atractylodes macrocephala koidz leaves
  • Method for extracting whole genome DNA of atractylodes macrocephala koidz leaves
  • Method for extracting whole genome DNA of atractylodes macrocephala koidz leaves

Examples

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Effect test

Embodiment 1

[0043] This instance has studied the influence of pyrolysis temperature on the whole genome DNA extraction method of Atractylodes macrocephala leaf of the present invention, and concrete process is as follows:

[0044] First, weigh Atractylodes macrocephala leaves in a pre-cooled mortar at 4°C, and quickly grind them into fine powder in liquid nitrogen, and divide them into five parts: A, B, C, D, and E; then, after grinding each 200mg Add 1.5mL of nuclear separation liquid to the fine powder, add the nuclear separation liquid to A, B, C, D, E, mix well and then centrifuge, discard the supernatant to collect the precipitate; Add 800 μL of CTAB nuclear lysate and 5 μL of proteinase K, add CTAB nuclear lysate and 20 mg / mL proteinase K to the resulting pellet, and incubate A, B, C, D, and E at 45°C, 55°C, and 65°C, respectively. , 70°C, 75°C in a water bath for 40min; to the mixed solution after the water bath, add a volume of chloroform-isoamyl alcohol mixed solution that is 2 / 3...

Embodiment 2

[0053] This instance has studied the impact of cracking time on the whole genome DNA extraction method of Atractylodes Rhizoma Atractylodes Rhizoma Atractylodes Rhizome Leaf of the present invention, and concrete process is as follows:

[0054] First, weigh Atractylodes macrocephala leaves in a pre-cooled mortar at 4°C, and quickly grind them into fine powder in liquid nitrogen, and divide them into five parts: A, B, C, D, and E; then, after grinding each 200mg Add 1.5mL of nuclear separation liquid to the fine powder, add the nuclear separation liquid to A, B, C, D, E, mix well and then centrifuge, discard the supernatant to collect the precipitate; Add 800 μL of CTAB nuclear lysate and 5 μL of proteinase K, add CTAB nuclear lysate and 20 mg / mL of proteinase K to the obtained precipitate, and place A, B, C, D, and E in water bath at 65°C for 5 min, 10 min, 20min, 30min, 40min; add the chloroform-isoamyl alcohol mixed solution whose volume is 2 / 3 of the above-mentioned CTAB nu...

Embodiment 3

[0063] This example compares the DNA extraction method of Atractylodes macrocephala leaf whole genome of the present invention, and the improved SDS method and the kit method for extracting Atractylodes macrocephala DNA.

[0064] Using the whole genome DNA extraction method of Atractylodes macrocephala leaves of the present invention, the specific steps are as follows:

[0065] Weigh Atractylodes macrocephala leaves in a pre-cooled mortar at 4°C, and quickly grind them into fine powder in liquid nitrogen, then add nuclear separation liquid at a ratio of 1.5 mL of nuclear separation liquid per 200 mg of ground fine powder, and fully After mixing, centrifuge, discard the supernatant to collect the precipitate; then add 800 μL CTAB nuclear lysate and 5 μL proteinase K for every 200 mg of ground fine powder, add CTAB nuclear lysate and 20 mg / mL proteinase K to the obtained precipitate, Water bath at 65°C for 20 min; add a chloroform-isoamyl alcohol mixture whose volume is 2 / 3 of t...

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Abstract

The invention belongs to the technical field of molecular biology, and particularly provides a method for extracting whole genome DNA of atractylodes macrocephala koidz leaves, which mainly comprisesthe following steps: sufficiently grinding an atractylodes macrocephala koidz leaf sample by using liquid nitrogen, dissociating by using nuclear separation liquid, carrying out CTAB lysate water bathcracking, protease digestion, chloroform / isoamyl alcohol extraction, isopropanol precipitation, ethanol washing and RNA enzyme treatment, thus the whole genome DNA of the atractylodes macrocephala koidz is rapidly extracted. Compared with an improved SDS method, the consumed time is shortened, the DNA concentration is increased, and the quality is obviously improved; compared with a DNA extraction kit method, the method has the advantages that the consumed time is equivalent, the DNA concentration is increased by 1 / 2, the DNA quality is higher, and the cost is greatly reduced. The problems that in the prior art, DNA extraction quality is low, the extraction amount is small, procedures are tedious, and time is long are solved, high-quality atractylodes macrocephala genome DNA can be extracted, and important technical support is provided for molecular biology research such as atractylodes macrocephala germplasm resource genetic diversity analysis, germplasm identification and classification.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, in particular to a method for extracting whole genome DNA from Atractylodes macrocephala leaves. Background technique [0002] Atractylodes macrocephala Koidz. belongs to the perennial herbaceous plant of Atractylodes genus in Compositae. Its rhizome is used as medicine, which has the effects of invigorating the spleen and replenishing qi, drying dampness and promoting diuresis, antiperspirant and antiabortive. Due to the long history of Atractylodes atractylodes cultivation, the long-term introduction and hybridization of different production areas have caused the problems of mixed germplasm and uneven quality of Atractylodes rhizome, so studying the genetic diversity of Atractylodes rhizome at the molecular level is very important for the identification and classification of Atractylodes rhizome . DNA extraction is the premise of genetic diversity analysis based on PCR technology. Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 蒋小刚张美德王华
Owner 湖北省农业科学院中药材研究所