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Method for extracting whole genome DNA from Apocymum venetum L. leaves

A technology for complete genome and plant leaves, which is applied in the field of extracting complete genome DNA, which can solve the problems of difficult DNA extraction and inability to extract genomic DNA

Inactive Publication Date: 2007-05-30
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Since plants such as Apocynum apocynum have been growing in drought or saline-alkaline and other water-deficient areas for a long time, the leaves of the plants contain a large number of secondary metabolites, and the treated leaves have undergone serious cross-linking between histones and genomic DNA due to long-term drying. , which makes it very difficult to extract the DNA of plants such as apocynum growing in drought or saline-alkali and other water-deficient areas, as well as various processed Chinese medicinal materials and beverages.
The current commercially available kits and common laboratory methods are unable to extract complete genomic DNA from the leaves of Apocynum and other plants except for etiolated seedlings

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The leaves of Apocynum apocynum were collected in Zhajing area, Tongliao, Inner Mongolia in October 2005, and were preserved by natural drying. Weigh 15 mg of the leaf and grind it thoroughly with liquid nitrogen, transfer the material together with the liquid nitrogen to a 50 mL plastic centrifuge tube with a cover before the liquid nitrogen evaporates, add 0.5-1 mL of lysate (2% CTAB , and add 1.4mol / LNaCl, 1%PVP, 0.02mol / L EDTA, pH8.0 0.1mol / L TrisHCl and 2%SDS), then add DTT to it to a final concentration of 40mg / mL, 4~6μL RNase A (10mg / mL), papain (to a final concentration of 40-60mg / mL), shaking and warming at 35-45°C for 12-24h;

[0024] Transfer the solution into a dolph tube, add 0.6-1 mL of phenol-chlorophenol-isoamyl alcohol (volume ratio: 25 / 24 / 1), extract for 5-10 minutes, and centrifuge at 7000 g for 5 minutes; the supernatant is passed through chloroform-isoamyl alcohol again Multiple extractions of amyl alcohol (volume ratio: 25 / 1) until there is no whi...

Embodiment 2

[0030] Collected in 1988 in Zhongba County, Ningxia, Apocynum purpurea was conventionally pressed into dry leaf specimens. In December 2005, 15 mg of the leaves on the specimen were weighed and fully ground by liquid nitrogen. Before the liquid nitrogen evaporated to dryness, the material was transferred together with the liquid nitrogen to a 50 mL plastic centrifuge tube with a cover. After the liquid nitrogen evaporated, 0.5 ~1mL lysate (2%CTAB, containing 1.4mol / L NaCl, 1%PVP, 0.02mol / L EDTA, pH8.0 0.1mol / L Tris HCl and 2%SDS), then add DTT to it to the final concentration 40mg / mL, 4-6μL RNaseA (10mg / mL), papain (to a final concentration of 40-60mg / mL), shaking incubation at 35-45°C for 12-24h;

[0031]Transfer the solution into a dolph tube, add 0.6-1 mL of phenol-chlorophenol-isoamyl alcohol (volume ratio: 25 / 24 / 1), extract for 5-10 minutes, and centrifuge at 7000 g for 5 minutes; the supernatant is passed through chloroform-isoamyl alcohol again Multiple extractions of ...

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Abstract

The invention discloses an extracting method of entire genome DNA from apocynum leaf, which comprises the following steps: grinding apocynum leaf; adding cracking liquid to crack; adding reducer and protein hydrolase to dispose for a while; removing protein through chloroform / isoamyl alcohol solution; removing polysaccharide through sodium acetate solution; sedimenting DNA through anhydrous alcohol or isopropyl alcohol; dissolving DNA through TE solution.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a method for extracting complete genome DNA from leaves of Apocynum genus plants. Background technique [0002] Apocynum, also known as wild hemp, tea flower, Zeqima, etc., is a perennial herbaceous plant of Apocynaceae (Apocynaceae) genus Apocynum. Although there is a small amount of distribution in the eastern coast, it is mostly distributed in Inner Mongolia, Xinjiang and Gansu are areas with dry climate and high salt content. It is not only a traditional Chinese medicinal material listed in the Pharmacopoeia of the People's Republic of China, but also a typical psammophyte and halophyte, which has great research and application value in pharmacy and ecology. With the deepening of research and application, the research on Apocynum and related species has gradually penetrated to the molecular level. However, because the leaves of Apocynum genus contain many substances such ...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N15/29
Inventor 陆长梅张卫明顾龚平彭雪梅
Owner NANJING NORMAL UNIVERSITY
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