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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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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 comp

Method used

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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

Experimental program
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Example Embodiment

[0039] Example 1: Method for purifying genomic DNA from tobacco leaves using gold magnetic particles

[0040] 1) Plant sample lysis

[0041] 1.1) Take 100mg of fresh plant leaf tissue and wash it with distilled water, wipe off the surface water, cut it and place it in a 2ml centrifuge tube, add liquid nitrogen for grinding. Before thawing the tissue, add 100μl 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 lysate 2 (3~6M GuHCl, 2%~5% Triton X-100), mixed After homogenization, water bath at 65°C for 25 min (invert the centrifuge tube once every 5 min during this period).

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

[0043] 2) Chloroform extraction

[0044] Add 0.7ml of chloroform to the ...

Example Embodiment

[0055] Example 2: Method for purifying genomic DNA from tomato seedlings using gold magnetic particles

[0056] 1) Plant sample lysis

[0057] 1.1) Take 100mg of fresh tomato seedling tissue and wash it with distilled water, wipe off the surface water, cut it and place it in a 2ml centrifuge tube, add liquid nitrogen for grinding. Before thawing the tissue, add 200μl of 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 lysis buffer 2 (3~6M GuHCl, 2%~5% Triton X-100), mixed After homogenization, water bath at 65°C for 25 min (invert the centrifuge tube every 5 min during this period).

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

[0059] 2) Chloroform extraction

[0060] Add 0.8ml of chlorof...

Example Embodiment

[0071] Example 3: Method for purifying genomic DNA from tobacco roots using gold magnetic particles

[0072] 1) Plant sample lysis

[0073] 1.1) Take 100 mg of fresh tobacco roots and wash them with distilled water, wipe off the surface moisture, cut them and place them in a 2ml centrifuge tube, add liquid nitrogen for grinding. Before thawing the tissue, add 400μl 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, water bath at 65°C for 25 min (invert the centrifuge tube every 5 min during this period).

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

[0075] 2) Chloroform extraction

[0076] Add 0.6ml of chloroform to the ...

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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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