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Method for extracting nucleic acid of endangered semi-mangrove plant Hernandia nymphaeifolia

A technology of tung nucleic acid and lotus leaf, which is applied in the field of molecular biology, can solve the problems of viscous supernatant, unclean polysaccharide treatment, and wire drawing of pipette tips, etc., and achieve the effect of reducing the loss of nucleic acid

Active Publication Date: 2021-07-20
HAINAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The technical problem to be solved in the present invention is: how to provide a kind of method that is applicable to the extraction of the chrysanthemum nucleic acid, solve the supernatant that occurs when adopting the traditional SDS method to extract Viscous, the polysaccharides are not handled cleanly during cracking, and the tip of the pipette is drawn when the sample is run.

Method used

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  • Method for extracting nucleic acid of endangered semi-mangrove plant Hernandia nymphaeifolia
  • Method for extracting nucleic acid of endangered semi-mangrove plant Hernandia nymphaeifolia
  • Method for extracting nucleic acid of endangered semi-mangrove plant Hernandia nymphaeifolia

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Experimental program
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Effect test

Embodiment 1

[0031] (1) Take an appropriate amount of fresh plant leaves, put them in a pre-cooled mortar, add liquid nitrogen and grind them thoroughly. Transfer the powder to a 2mL centrifuge tube, add 5% SDS (m / v) lysate at a preheated temperature of 65°C, mix upside down, add RNase, and vortex for 1 min;

[0032] (2) After mixing, bathe in water at 65°C for 10 minutes; add an equal volume of chloroform: isoamyl alcohol = 24: 1, shake and mix well, at a speed of 11000-135000 rpm / min Centrifuge at room temperature for 10-15min.

[0033] (3) Take the supernatant and transfer it to a new centrifuge tube, add an equal volume of Tris-balanced phenol (pH>7.8) and shake to mix evenly. rpm Centrifuge at 4°C for 15-20 minutes.

[0034] (4) Take the supernatant and transfer it to a new centrifuge tube, add 1 / 10 times the volume of 3mol / L NaAc (pH5.6) and an equal volume of isopropanol, mix well, and put it at -20°C overnight. When the speed is 11000~135000 rpm Centrifuge at 4°C for 15-20 min...

Embodiment 2

[0037] (1) Take an appropriate amount of fresh plant leaves, put them in a pre-cooled mortar, add liquid nitrogen and grind them thoroughly. Transfer the powder to a 2mL centrifuge tube, add lysate preheated at 65°C, mix up and down, add RNase, and vortex for 1 min. The composition of the lysate is: 100mmol / L Tris-HClpH8.0, 50mmol / L EDTA, 500mmol / L NaCl, 2% (V / V) β-mercaptoethanol, 5% (m / v) SDS, 2% (m / v) PVP. The preparation method of improving lysate is: with 5g, the SDS that pH value is 8.0, 2g polyvinylpyrrolidone, 2mlβ mercaptoethanol, 2.925g sodium chloride, 1.461g ethylenediaminetetraacetic acid, the Tris-HCl solution of 10ml, mix Mix well, then add distilled water to make up to 100ml.

[0038] (2) After mixing in a water bath at 65°C for 10 minutes, add an equal volume of extract and shake to mix. The extract consists of Tris balanced phenol: chloroform: isoamyl alcohol = 25: 24: 1, centrifuged at room temperature at a speed of 11,000 to 135,000 rpm 10~15min.

[003...

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Abstract

The invention belongs to the aspect of molecular biology, and particularly relates to a method for extracting nucleic acid of endangered semi-mangrove plant Hernandia nymphaeifolia. The method comprises the following steps: cracking cells by using an improved lysis solution, performing extraction by using a mixed solution of Tris balanced phenol-chloroform-isoamyl alcohol in a certain proportion, and performing extraction by using Tris balanced phenol with the same volume in order to more thoroughly remove glycoprotein; and precipitating and purifying the DNA by using sodium acetate-isopropanol with improved volume, so as to obtain the high-quality genome DNA. The DNA extracted by the method provided by the invention is high in concentration and purity, is not easy to degrade, the damage of superoxide anion free radicals or active oxygen to the DNA is prevented, and a foundation is laid for subsequent molecular biology experiments such as PCR amplification, molecular marker technology development, genome library construction and the like.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a method for extracting nucleic acid of the endangered semi-mangrove plant Palismanthus japonicus. Background technique [0002] Hernandia nymphaeifolia (J.Presl) Kubitzki is an evergreen tree belonging to the nymphaeifolia family and the nymphaeifolia genus. Hernandia nymphaeifolia is rich in polysaccharides and polyphenols and has special ingredients. The extract is extremely viscous and easy to extract. Browning, so nucleic acid extraction is more difficult. [0003] Traditional SDS method [0004] (1) Weigh 0.2g sample and grind it into powder with liquid nitrogen, put it in a 1.5ml centrifuge tube, add 800μL SDS lysate (100mmol / L Tris-HCl, pH8.0, 50mmol / L EDTA , 500mmol / L NaCl, 2% β-mercaptoethanol (v / v), 5% SDS (m / v)), put it in a 65°C water bath for 20min. [0005] (2) Centrifuge at 4°C, 12 000rpm for 10min. Transfer the supernatant into a new 1.5ml centrifu...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003C12Q2521/327C12Q2527/125Y02A50/30
Inventor 王勇倪靓谭佐莉张修含于靖张世杰
Owner HAINAN NORMAL UNIV
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