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Method for purifying genomic deoxyribonucleic acid (DNA) of plant tissues by using goldmag particles

A technology of gold magnetic particles and plant tissue, which is applied in the field of plant tissue genomic DNA, can solve the problems of lack of versatility, low purification rate, and complex operation of the background technology, and achieve shortened DNA extraction time, high purification rate, and purification process. fast effect

Active Publication Date: 2012-02-08
XIAN GOLDMAG NANOBIOTECH
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AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a simple and fast method for purifying plant tissue genomic DNA using magnetic nanocomposite materials, so as to solve the problems of complex operation, low purification rate and lack of versatility in the background technology; it can be applied to the purification of different plants Tissues and different tissues of the same plant

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  • Method for purifying genomic deoxyribonucleic acid (DNA) of plant tissues by using goldmag particles
  • Method for purifying genomic deoxyribonucleic acid (DNA) of plant tissues by using goldmag particles

Examples

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Effect test

Embodiment 1

[0039] Embodiment 1: the method for purifying genomic DNA from tobacco leaves with gold magnetic particles

[0040] 1) Lysis of plant samples

[0041] 1.1) Take 100 mg of fresh plant leaf tissue, wash it with distilled water, dry the surface moisture, cut it into pieces, put it in a 2ml centrifuge tube, and add liquid nitrogen to grind. Before tissue thawing, add 100 μl Lysis Solution 1 [2%-5% polyvinylpyrrolidone (PVP), 2%-5% cetyltrimethylammonium bromide (CTAB), 0.5%-2% β-mercaptoethanol , 0.005~0.01M ethylenediaminetetraacetic acid (EDTA), 0.5~2M NaCl, 0.05~0.2M Tris-HCl] and 100μl Lysis Solution 2 (3~6M GuHCl, 2%~5% Triton X-100), mixed After homogenization, place in a 65°C water bath for 25 minutes (during which the centrifuge tube is inverted once every 5 minutes).

[0042] 1.2) Add 10 μl of 10 mg / ml RNase solution into the centrifuge tube, blow and aspirate to mix, put in a water bath at 37° C. for 5 min, and take out the centrifuge tube.

[0043] 2) Chloroform extr...

Embodiment 2

[0055] Embodiment 2: the method for purifying genomic DNA from tomato seedlings with gold magnetic particle

[0056] 1) Lysis of plant samples

[0057] 1.1) Take 100 mg of fresh tomato seedling tissue, wash it with distilled water, dry the surface moisture, cut it into pieces, put it in a 2ml centrifuge tube, and add liquid nitrogen to grind. Before tissue thawing, add 200 μl Lysis Solution 1 [2%-5% polyvinylpyrrolidone (PVP), 2%-5% cetyltrimethylammonium bromide (CTAB), 0.5%-2% β-mercaptoethanol , 0.005~0.01M ethylenediaminetetraacetic acid (EDTA), 0.5~2M NaCl, 0.05~0.2M Tris-HCl] and 200μl lysate 2 (3~6M GuHCl, 2%~5% Triton X-100), mixed After homogenization, place in a 65°C water bath for 25 minutes (during which the centrifuge tube is inverted once every 5 minutes).

[0058] 1.2) Add 10 μl of 10 mg / ml RNase solution into the centrifuge tube, blow and aspirate to mix, put in a water bath at 37° C. for 5 min, and take out the centrifuge tube.

[0059] 2) Chloroform extrac...

Embodiment 3

[0071] Embodiment 3: the method for purifying genomic DNA from tobacco root with gold magnetic particle

[0072] 1) Lysis of plant samples

[0073] 1.1) Take 100 mg of fresh tobacco root, wash it with distilled water, dry the surface moisture, cut it into pieces, put it in a 2ml centrifuge tube, and add liquid nitrogen to grind it. Before tissue thawing, add 400 μl Lysis Solution 1 [2%-5% polyvinylpyrrolidone (PVP), 2%-5% cetyltrimethylammonium bromide (CTAB), 0.5%-2% β-mercaptoethanol , 0.005~0.01M ethylenediaminetetraacetic acid (EDTA), 0.5~2M NaCl, 0.05~0.2M Tris-HCl] and 400μl lysate 2 (3~6M GuHCl, 2%~5% Triton X-100), mixed After homogenization, place in a 65°C water bath for 25 minutes (during which the centrifuge tube is inverted once every 5 minutes).

[0074] 1.2) Add 10 μl of 10 mg / ml RNase solution into the centrifuge tube, blow and aspirate to mix, put in a water bath at 37° C. for 5 min, and take out the centrifuge tube.

[0075] 2) Chloroform extraction

[00...

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Abstract

The invention aims to provide a method for simply and quickly purifying genomic deoxyribonucleic acid (DNA) of plant tissues by using a magnetic nano composite material. The method comprises the following steps of: performing sample lysis by using two kinds of lysis solution, wherein the first lysis solution comprises PVP (poly vinyl pyrrolidone) with mass-to-volume ratio of 2 to 5 percent, 2 to 5 percent of CTAB (cetyl trimethyl ammonium bromide), and beta-mercaptoethanol with volume fraction of 0.5 to 2 percent, and the second lysis solution comprises 3 to 6M of guanidine hydrochloride and Triton X-100 with volume fraction of 2 to 5 percent; blending, and performing full lysis for 5 to 30 min in a water bath; and adding 10 to 50 microliters of 10 mg / ml RNase solution, and thus obtainingthe sample lysis solution with the genomic DNA. The purification method is easy to operate, has quick purification process, and has the advantages of high purification rate, high DNA integrity, high purity and the like. According to the method, fractional tissues lysis is not required, and the requirement of extracting the genomic DNA of various plants and different places is met to the great extent.

Description

technical field [0001] The invention relates to a method for purifying genome DNA of various types of plants and different parts of plants by using gold magnetic particles. Background technique [0002] It is the practical application of plant genetic engineering and molecular biotechnology in agriculture to directly transform the recipient with the donor plant DNA with the target gene, and to screen the offspring with the target trait variation. It is a key link to extract high-quality genomic DNA from different species of plants and their different parts for downstream experiments such as subsequent PCR reactions. The established method has high requirements for the purity and concentration of the extracted genomic DNA. At present, the extraction methods of plant genome DNA are highly targeted, and most of them can only be extracted from fresh plant leaves, which will bring great limitations to downstream applications. [0003] Nucleic acid purification methods can be rou...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 崔亚丽魏旭旭晁旭张景阁赵稳操李谭杰
Owner XIAN GOLDMAG NANOBIOTECH
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