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Anoectochilus roxburghii DNA extracting method suitable for RAPD analysis

An extraction method and technology for taking supernatant, applied in the field of molecular biology, can solve problems such as affecting experimental results and RAPD instability, and achieve the effect of clear bands and high repeatability

Inactive Publication Date: 2015-11-04
FUJIAN AGRI & FORESTRY UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because RAPD is very unstable, changes in external conditions can easily affect the experimental results

Method used

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  • Anoectochilus roxburghii DNA extracting method suitable for RAPD analysis
  • Anoectochilus roxburghii DNA extracting method suitable for RAPD analysis
  • Anoectochilus roxburghii DNA extracting method suitable for RAPD analysis

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Experimental program
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Embodiment 1

[0026] (1) An appropriate amount of sample (0.5g), ground with liquid nitrogen, poured into a 2mL centrifuge tube, and added CTAB solution (3%CTAB; 100mmol / L Tris-HC1, pH 8.0; 20mmol / LEDTA, pH 8.0; 2.0 mol / L NaCl; 4% PVP (K-30); before use, add 2% β-mercaptoethanol, shake, put in a 65°C water bath for 45 minutes, slightly invert several times every 5 minutes in the middle, at 20°C at 12000 rpm Centrifuge for 15 minutes.

[0027] (2) Take the supernatant, add 2 / 3 volume of chloroform: isoamyl alcohol (volume ratio 24:1), lie flat for 5 minutes, and then centrifuge at 12000 rpm for 10 minutes at 20°C.

[0028] (3) Take the supernatant, add 1 / 3 volume of chloroform, lie flat for 5 minutes, and centrifuge at 12,000 rpm for 10 minutes at 20°C.

[0029] (4) Take the supernatant, add an equal volume of -20°C pre-cooled isopropanol, add 1 / 5 of the supernatant volume 3mol / L NaAc (pH 5.2), place it in the -20°C refrigerator for 30min, Centrifuge at 12,000 rpm for 15 min to precipitate...

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Abstract

The invention belongs to the field of molecular biology and particularly relates to an anoectochilus roxburghii DNA extracting method suitable for RAPD analysis. By the method, high-purity anoectochilus roxburghii DNA can be obtained at high yield according to the plant features of anoectochilus roxburghii. The method has the advantages that in traditional CTAB extract, PVP use amount is increased to 4%, and salinity is increased to 2.0mol / L; chloroform and isoamyl alcohol with the volume ratio of 24:1 are used; after isopropanol precipitates DNA, 500 microliter of high salt TE (100mmol / L Tris-HC1, pH8.0; 20mmol / L EDTA, pH8.0; 1.4mol / L NaCl) is added for reprecipitation; RNaseA is added finally. In subsequent RAPD experiments, the anoectochilus roxburghii DNA extracted by the method is clear in band, bright, stable and high in repeatability.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a method for extracting DNA from Clematis aureus which is suitable for RAPD analysis. Background technique [0002] The quality of DNA extraction is the key to molecular biology experiments, which directly affects the accuracy and repeatability of subsequent experiments. At present, there are many DNA extraction methods in China, among which the most common and used methods are CTAB method and SDS method. Since the components and metabolites of different plants are quite different, it is very important to choose the most suitable extraction method according to the characteristics of different plants. Clematis is because it contains a large amount of polysaccharides and polyphenols, but its plant protein content is not high. The characteristic of the SDS method lies in its ability to remove proteins, but it is not as good as the CTAB method in terms of removal of poly...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 张重义王剑锴李明杰王建明冯法节张宝
Owner FUJIAN AGRI & FORESTRY UNIV
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