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Simplified method for rapidly extracting DNA from plant leaves

A plant leaf and extraction method technology, applied in the field of plant leaf DNA extraction, can solve the problems of high cost and low efficiency, and achieve the effect of simple instrument, simple and fast operation process, and easy operation

Inactive Publication Date: 2017-05-31
NORTHEAST AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0002] DNA extraction is one of the most common molecular operations in the laboratory, especially in the identification of large-scale molecular markers in plant breeding, it is often necessary to extract the DNA of thousands of materials, and the traditional extraction method will have low efficiency. Problems; advanced instrument extraction requires the purchase of reagent kits and other supporting consumables and reagents, and the cost is relatively high; although ordinary rapid extraction technology greatly simplifies the extraction process, it basically relies on instruments for the early grinding process, which puts forward requirements for laboratory hardware conditions

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  • Simplified method for rapidly extracting DNA from plant leaves
  • Simplified method for rapidly extracting DNA from plant leaves
  • Simplified method for rapidly extracting DNA from plant leaves

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specific Embodiment approach 1

[0023] Embodiment 1: This embodiment provides a simplified and rapid extraction method for plant leaf DNA. The specific steps are as follows:

[0024] 1. Sampling

[0025] Put 0.1-0.2g of fresh tomato or cucumber leaves directly into a 1.5mL centrifuge tube, and mark the sample name or serial number. The extraction within 48 hours can be stored at 4°C, and the extraction after 48 hours should be stored at -20°C, and do not freeze and thaw repeatedly.

[0026] 2. Grinding

[0027] Take an appropriate amount of quartz sand (due to the different quality of different batches of samples, you can pre-grind 1 to 2 samples, whichever is easier to grind the sample, the amount is not strictly required, but it is necessary to ensure that the amount of each sample is basically the same to facilitate subsequent centrifugation ) into the centrifuge tube containing the leaf sample, add 300μl, 0.4M NaOH solution, grind it directly with a sterilized 1000μl pipette tip until there is no obvio...

specific Embodiment approach 2

[0038] Specific embodiment 2: This embodiment takes the identification marker amplification of tomato Ty-2 gene as an example, and compares the experimental effects of the present invention and the traditional CTAB extraction method.

[0039] CTAB method extraction process:

[0040] (1) Grind the quick-frozen leaves in liquid nitrogen into powder with a sterile pipette tip. After grinding, quickly add 700 μL of CTAB solution preheated in a 65°C water bath, cover the lid and mix well.

[0041] (2) Place the mixed sample in a 65°C water bath for 1 hour, and turn the sample up and down every few minutes during the water bath to make it evenly heated and fully contact the tissue sample with the solution.

[0042] (3) After the water bath, the sample was taken out and cooled at room temperature. After cooling, 700 μL of chloroform: isoamyl alcohol (24:1) mixture was added, mixed evenly and allowed to stand for 10 minutes.

[0043] (4) Place the sample after standing in a centrifug...

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Abstract

The invention discloses a simplified method for rapidly extracting DNA from plant leaves. The simplified method comprises the following steps: firstly, placing fresh plant leaves in a centrifuge tube; (2) taking and placing quartz sand in the centrifuge tube with leaf samples, adding an NaOH solution, using a sterilized tip of 1000-microliter pipettor to grind directly until no apparent leaf block exists, covering a tube cover and sealing with a sealing film to prevent the tube cover is jacked to open by steam during boiling water bath; thirdly, placing the grinded and sealed centrifuge tube in the boiling water bath; fourthly, placing the centrifuge tube in a centrifuge for centrifugation at room temperature; and fifthly, adsorbing supernatant, adding to a new centrifuge tube, and also adding a Tris-cl buffer solution. The method provided by the invention has low requirements on laboratory conditions, adopts extremely simple instruments and reagents, is simple to operate, good in effect, suitable for rapid extraction of leaves of tomatoes and cucumbers, and particularly suitable for large-scale extraction of different samples.

Description

technical field [0001] The invention relates to a method for extracting plant leaf DNA. Background technique [0002] DNA extraction is one of the most common molecular operations in the laboratory, especially in the identification of large-scale molecular markers in plant breeding, it is often necessary to extract the DNA of thousands of materials, and the traditional extraction method will have low efficiency. Problems; advanced instrument extraction requires the purchase of reagent kits and other supporting consumables and reagents, and the cost is relatively high; although ordinary rapid extraction technology greatly simplifies the extraction process, it basically relies on instruments for the early grinding process, which puts forward requirements for laboratory hardware conditions . Contents of the invention [0003] The purpose of the present invention is to provide an extremely simple and efficient method for extracting DNA from plant leaves by simplifying the gri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 许向阳赵婷婷姜景彬张贺陈秀玲李景富王傲雪
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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