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Method for extracting nuclear DNA of lotus

A cell nucleus and weighing technology, which is applied in the field of DNA extraction, can solve the problems of low purity, many impurities, affecting the uniformity of lotus genome sequencing, and the usefulness of unbiased sequences, so as to achieve less pigment and protein, high quality and purity Effect

Inactive Publication Date: 2013-03-20
WUHAN BOTANICAL GARDEN CHINESE ACAD OF SCI
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Problems solved by technology

[0004] The CTAB (cetyltrimethylammonium bromide) method is a quick and easy routine method for extracting total plant DNA (including grinding, cracking, extracting, precipitating DNA, removing RNA, etc.), but the CTAB method The obtained lotus DNA sample contains more chloroplast DNA and mitochondrial DNA, and has many impurities and low purity, which greatly affects the uniformity, unbiasedness and usefulness of the sequence of the lotus genome, thereby ultimately affecting the sequence. Assembly quality, not suitable for lotus genome sequencing

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  • Method for extracting nuclear DNA of lotus

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Embodiment Construction

[0026] 1. Two buffers

[0027] 1. Nucleus Extraction Buffer Solution Ⅰ

[0028] (1) Components of Nucleus Extraction Buffer Solution Ⅰ

[0029] Tris-hydroxymethylaminomethane (Tris-base) 10 mmol / L;

[0030] Ethylenediaminetetraacetic acid (EDTA) 10 mmol / L;

[0031] Potassium chloride (KCl) 80 mmol / L;

[0032] Sucrose (Sucrose) 0.5 mol / L;

[0033] Polyethylene glycol octylphenyl ether (TritonX-100) 0.5%;

[0034] Polyvinylpyrrolidone-30 (PVP-30) 2%;

[0035] Spermidine 1 mmol / L;

[0036] Spermine (Spermine) 1 mmol / L;

[0037] β-Mercaptoethanol 0.15%.

[0038] (2) Preparation method of cell nucleus extraction buffer Solution Ⅰ

[0039] ① After the first 5 components are dissolved and mixed, adjust the pH to 9.4-9.5, and then put it in a sterilized bottle to sterilize at 121°C and 0.1Mpa for 12-16 minutes;

[0040] ② After adding, the 4 components are mixed;

[0041] ③Seal and reserve.

[0042] 2. Cell nucleus lysis buffer solution II

[0043] (1) Components of Nucle...

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Abstract

The invention discloses a method for extracting the nuclear DNA of a lotus, relating to a method for extracting DNA in the field of molecular biology. The method mainly comprises the following steps of: adding a nucleus extraction buffer solution to etiolated seedling leaves of the lotus, which are grinded into powder, and magnetically stirring, filtering, centrifugalizing and rinsing to obtain apurified nucleus; adding a nucleus lysis buffer solution for water bath at 65 DEG C; extracting through chloroform: isoamylol and precipitating through isopropanol so as to obtain floccus DNA precipitates; and washing through 70% ethanol and drying at room temperature, and then adding a Tris-EDTA (Ethylene Diamine Tetraacetic Acid) buffer solution to dissolve the DNA, and preserving at -20 DEG C for later use. The DNA obtained by the invention is the high-quality nuclear DNA of the lotus, has low content of chlorophyll DNA and mitochondrion DNA and contains few polysaccharides, polyphenols, pigments and proteins; and in addition, the invention is suitable for the extraction of the high-quality nuclear DNA needed by the aspects of establishing a BAC (Binary-Analog Conversion) library, sequencing genomes, and the like.

Description

technical field [0001] The invention relates to a method for extracting DNA in the field of molecular biology, in particular to a method for extracting lotus cell nucleus DNA; in particular, it relates to a method for effectively extracting high-quality lotus cell nucleus DNA using leaves of yellow lotus seedlings as materials. Background technique [0002] Lotus, commonly known as lotus, is one of the earliest plants among angiosperms, known as "living fossil". It has a long history of cultivation in our country and is an important aquatic economic crop, which is edible, medicinal and ornamental. Because lotus has unique economic, cultural and ornamental value, it has attracted widespread attention. However, the application of molecular biotechnology in lotus research is currently mainly focused on species identification, evolutionary relationship analysis, and genetic diversity evaluation among germplasms. In other aspects, in-depth research on lotus genome has not yet be...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
Inventor 杨美刘艳玲徐立铭
Owner WUHAN BOTANICAL GARDEN CHINESE ACAD OF SCI
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