High-flux plant tissue genome DNA extraction method based on paramagnetic particle method

A plant tissue and extract technology, applied in the field of molecular biology, can solve the problems of low scale standardization, many manual operation steps, pollution of the environment, etc., and achieve the effects of stable and reliable DNA quality, reduced quality impact, and high safety.

Pending Publication Date: 2022-03-25
INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to provide a high-throughput method for extracting plant tissue genomic DNA based on the magnetic bead method to solve the problems in the above-mentioned prior art. Low standardization, organic reagents are harmful to the human body and pollute the environment, and the DNA extraction efficiency is increased by 10 times. The quality of the prepared DNA is stable and reliable, which can meet the needs of large-scale molecular marker-assisted breeding for high-throughput nucleic acid extraction.

Method used

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  • High-flux plant tissue genome DNA extraction method based on paramagnetic particle method
  • High-flux plant tissue genome DNA extraction method based on paramagnetic particle method
  • High-flux plant tissue genome DNA extraction method based on paramagnetic particle method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Establishment of a high-throughput genomic DNA extraction method for plants based on the magnetic bead method

[0037] (1) Take about 0.10 g of the plant leaves to be extracted (one seed is one), put them in a 2.00 mL 96 deep-well plate and put steel balls with a diameter of 0.4 cm. The 96-deep well plate was placed in a -80°C ultra-low temperature freezer for one hour.

[0038] (2) Place the 96-well plate in a high-throughput grinder and grind at a frequency of 1500 rpm for 30s (the seeds are ground twice using this condition).

[0039] (3) Add 400 μL of 2×CTAB extract to the deep-well plate, mix well, and bathe in water at 65°C for 30 minutes; the formula of the 2×CTAB extract is: 1.40mol / L NaCl, 0.10mol / L Tris-HCl, 0.02 mol / L EDTA, 20.00g CTAB, 5.00g sorbitol, 10.00g PVP. (Seeds use SDS extract, the SDS extract formula is: 0.70mol / L NaCl, 0.05mol / L Tris-HCl, 0.01mol / L EDTA, 10.00g SDS, 5.00g sorbitol, 10.00g PVP).

[0040] (4) Put the sample after the wa...

Embodiment 2

[0073] About 0.10 g of cotton leaves to be extracted was placed in a 2 mL 96 deep-well plate and steel balls with a diameter of 0.40 cm were placed. The 96 deep-well plate was frozen in a -80°C ultra-low temperature refrigerator for one hour, then taken out, and ground in a high-throughput grinder with a frequency of 1500r for 30s.

[0074] Add 400 μL of 2×CTAB extract solution to the deep-well plate, mix well, and heat in a water bath at 65°C for 30 min; put the sample after the water bath into a centrifuge, and centrifuge at 4500 rpm for 1 min. Add 200 μL of deproteinized solution to the deep-well plate, set the oscillator frequency to 1000 rpm for 10 seconds, then centrifuge at 4500 rpm for 10 min; put the deep-well plate into the centrifuge and centrifuge at 4500 rpm for 1 min. Place the centrifuged deep-well plate in the liquid transfer workstation to open the nucleic acid extraction program, and place the magnetic frame, absolute ethanol, 70% ethanol, magnetic beads, ddH...

Embodiment 3

[0082] Take one cotton seed to be extracted, place it in a 2mL 96 deep-well plate and put a steel ball with a diameter of 0.40cm. The 96 deep-well plate was frozen in a -80°C ultra-low temperature refrigerator for one hour, then taken out, and ground in a high-throughput grinder with a frequency of 1500r for 1 min.

[0083] Add 400 μL of SDS extract solution to the deep-well plate, mix well, and heat in a water bath at 65°C for 30 min; put the sample after the water bath into a centrifuge, and centrifuge at 4500 rpm for 1 min. Add 200 μL of deproteinized solution to the deep-well plate, set the oscillator frequency to 1000 rpm for 10 seconds, then centrifuge at 4500 rpm for 10 min; put the deep-well plate into the centrifuge and centrifuge at 4500 rpm for 1 min. Place the centrifuged deep-well plate in the liquid transfer workstation to open the nucleic acid extraction program, and place the magnetic frame, absolute ethanol, 70% ethanol, magnetic beads, ddH in sequence accordi...

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Abstract

The invention discloses a high-flux plant tissue genome DNA extraction method based on a paramagnetic particle method, and belongs to the field of molecular biology. The invention specifically discloses a DNA extracting solution. The DNA extracting solution comprises the following components: (a) the DNA extracting solution comprises the following components: NaCl, Tris-HCl, EDTA (Ethylene Diamine Tetraacetic Acid), CTAB (Cetyltrimethyl Ammonium Bromide), sorbitol and PVP (Polyvinyl Pyrrolidone); (b) the DNA extracting solution is prepared from the following components: NaCl, Tris-HCl, EDTA (Ethylene Diamine Tetraacetic Acid), SDS (Sodium Dodecyl Sulfate), sorbitol and PVP (Polyvinyl On the basis of a magnetic bead method, the DNA extracting solution is combined with a pipetting workbench, the defects that time and labor are consumed, manual operation steps are many, the scale standardization degree is low, organic reagents are harmful to human bodies and pollute the environment and the like in a traditional method are overcome, the DNA extracting efficiency is improved by 10 times, and the prepared DNA is stable and reliable in quality and high in practicability. The requirements of large-scale molecular marker-assisted breeding on high-throughput nucleic acid extraction can be met.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a high-throughput method for extracting genomic DNA from plant tissues based on a magnetic bead method. Background technique [0002] In recent years, with the continuous development of molecular biology, studies such as cotton genome sequencing, variety identification and population genetic analysis have also been widely used and promoted. However, the realization of these technologies often needs to be based on high-purity and high-integrity whole-genome DNA, which puts forward higher requirements for the throughput of whole-genome DNA extraction. [0003] At present, relatively mature DNA extraction methods include SDS method, CTAB method and kit method. SDS method and CTAB method are cheap to use, and the extracted DNA can meet the requirements of most molecular experiments, but the operation process is too cumbersome and time-consuming. It is time-consuming and laborious, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1013
Inventor 陈丽婷匡猛黄龙雨吴立强黄义文彭军周大云吴玉珍
Owner INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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