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Safe and rapid method for extracting genomic DNA of tea tree old leaf

A genome and tea tree technology, applied in the field of safe and rapid extraction of genomic DNA from old leaves of tea trees, can solve the problems of being unsuitable for old leaves and taking a long time to achieve the effect of short time and protection of body safety.

Inactive Publication Date: 2012-11-28
SICHUAN AGRI UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to overcome the disadvantages and deficiencies of the prior art that are not suitable for old leaves, use toxic reagents and take a long time, aiming at the characteristics of polyphenols and polysaccharides in old leaves of tea trees, to provide a safe and fast method for extracting genomic DNA from old leaves of tea trees Methods

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  • Safe and rapid method for extracting genomic DNA of tea tree old leaf
  • Safe and rapid method for extracting genomic DNA of tea tree old leaf
  • Safe and rapid method for extracting genomic DNA of tea tree old leaf

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Embodiment Construction

[0025] The present invention will be described in detail below in conjunction with specific embodiments.

[0026] 1 Materials and methods

[0027] 1) Material

[0028] The fresh old leaves of tea trees are collected from 12 tea tree cultivars (lines) planted in Sichuan Agricultural University and the provincial famous mountain breeding field, namely Anji White Tea, Qingxin Oolong, Gulin Cowhide Tea, Wuniuzao, Shuyong 307 , Nanjiang No. 4, Fuding Dabai Tea, Qianmei 502, Longjing 43, Mengshan No. 11, Tieguanyin and Yinghong No. 2.

[0029] 2) DNA extraction method

[0030] Take 0.3g of old tea tree leaves, add liquid nitrogen to submerge the surface of the leaves, grind to powder, place in a 1.5mL centrifuge tube, then immediately add 800 μL of preheated DNA extraction buffer (DNA extraction buffer consists of 1.5% (W / V) SDS (pH5.5), 100mmol / L Tris-HCl (pH8.0), 40mmol / L EDTA (pH8.0), 1mol / L NaCl, 2.5% PVP, 10mmol / L β-mercaptoethanol composition), Shake well and place in 65°...

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Abstract

The present invention discloses a safe and rapid method for extracting genomic DNA of tea tree old leaves, comprising the following steps: grinding the tea tree leaves into powder in liquid nitrogen; then adding 800 [mu]L of preheated DNA extraction buffer; placing in a 65 DEG C water bath for 30 min after shaking; adding 125 [mu]L of KAC of 5mol / L; placing in an ice bath for 20 min after shaking; centrifuging for 3 min after reacting sufficiently; adding the supernatant to a adsorption column, loading in a collection tube and centrifuging for 1 min; adding the filtrate in the collection tube to the adsorption column again, loading in collection tubes, centrifuging for 1 min, and discarding the liquid waste; adding 800 [mu]L of ethanol of 70% to the adsorption column, centrifuging for 1min, and discarding the liquid waste; adding 800 [mu]L of ethanol of 70% again to the adsorption column, centrifuging for 1min, and discarding the liquid waste; re-loading the adsorption column into the collection tube, and centrifuging for 3 min to remove the remaining ethanol; removing the adsorption column and placing at 50 DEG C for 5min; and placing the adsorption column in a new centrifuge tube, adding 60 [mu]L of TE, placing at 40 DEG C for 5 min, and centrifuging for 3min. The liquid in the centrifuge tube is the extracted tea tree genomic DNA. The method is safe and rapid for the extraction of genomic DNA from tea tree leaves.

Description

technical field [0001] The invention relates to a method for safely and rapidly extracting genomic DNA from old leaves of tea trees. Background technique [0002] DNA molecular marker technology is an important tool for studying the origin, molecular evolution, genetic diversity and variety identification of tea trees. A prerequisite for these studies is the extraction of high-quality genomic DNA. Because tea tree leaves are rich in phenolic substances, the content of phenolic substances in fresh leaves can reach 18% to 36% of the total dry matter, and contain a large amount of alkaloids and other various secondary substances, which is very important for the extraction of DNA. It brings great difficulties, such as phenolic substances can cause DNA degradation, discoloration and affect subsequent analysis, and residual polysaccharides can inhibit PCR reactions. [0003] At present, the common extraction methods of tea tree genomic DNA include SDS method and CTAB method. Alt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 齐桂年陈盛相李成磊
Owner SICHUAN AGRI UNIV
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