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Method for rapid extraction of genomic DNA of melons

A genome and melon technology, applied in the field of DNA extraction, can solve the problems of long extraction time, cumbersome extraction steps, and strong toxicity of related chemicals, and achieve the effect of improving quality and simple process steps

Inactive Publication Date: 2013-11-27
HEFEI FENGLE SEED
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  • Abstract
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  • Application Information

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Problems solved by technology

[0003] Because the leaves of melon plants contain a large amount of polyphenols, they are easily oxidized with oxygen in the air during the extraction process, thus affecting the quality of DNA extraction. At the same time, polyphenols will combine with Taq enzyme in the PCR reaction, affecting Enzymatic reaction efficiency, thereby reducing the success rate of PCR reaction
Traditionally, the CTAB method is generally used for the extraction of genomic DNA of melon plants. Although it can avoid the influence of polyphenols, it has disadvantages such as long extraction time, cumbersome extraction steps, and strong toxicity of related chemicals. Alkaline boiling method avoids the CTAB method Related problems, but there is a shortcoming that the polyphenols in the leaves cannot be removed, resulting in subsequent related experiments such as melon crop breeding improvement, variety purity molecular marker identification, etc., the DNA quality cannot meet the relevant requirements

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  • Method for rapid extraction of genomic DNA of melons

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1. A method for rapidly extracting the genomic DNA of the watermelon variety Seedless No. 1, which operates in sequence as follows:

[0017] (1) Plant tissue selection:

[0018] Choose 2-2.5mm 3 Seedless watermelon No. 1 copper coin-sized young leaf tissue sown in the sand bed indoors;

[0019] (2) Extraction of genomic DNA:

[0020] Place the selected leaf tissue at the bottom of a 96-well PCR plate, add 40 μL of 0.70 mol / L NaOH solution to each well, place in a boiling water bath for 1 min, then add 30 μL of 0.70 mol / L Tris-HCl, and place in a boiling water bath at 100 °C for 1 min;

[0021] (3) Precipitation of genomic DNA:

[0022] Place the 96-well PCR plate boiled with alkali above in a centrifuge at 3500 rpm and centrifuge for 20 minutes, then extract the supernatant in each well and transfer to each well of another 96-well PCR plate, and add 80 μL of Isopropanol, stored in a freezer at -20°C;

[0023] (4) Redissolution of genomic DNA:

[0024] Pla...

Embodiment 2

[0035] Example 2. A method for rapidly extracting the genomic DNA of the muskmelon variety Jinlu No. 3, which operates in sequence according to the following steps:

[0036] (1) Plant tissue selection:

[0037] Choose 2-2.5mm 3 Young leaf tissues of the size of copper coins of melon Jinlu No. 3 sown in sand beds indoors;

[0038] (2) Extraction of genomic DNA:

[0039] Place the selected leaf tissue at the bottom of a 96-well PCR plate, add 40 μL of 0.70 mol / L NaOH solution to each well, place in a boiling water bath for 1 min, then add 30 μL of 0.70 mol / L Tris-HCl, and place in a boiling water bath at 100 °C for 1 min;

[0040] (3) Precipitation of genomic DNA:

[0041] Place the 96-well PCR plate boiled with alkali above in a centrifuge at 3500rpm and centrifuge for 20min, then extract the supernatant in each well and transfer to each well of another 96-well PCR plate, and add 100 μL of Isopropanol, stored in a freezer at -20°C;

[0042] (4) Redissolution of genomic DN...

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Abstract

The invention discloses a method for rapid extraction of genomic DNA of melons, especially a method for fast, convenient and large-scale extraction of genomic DNA of melon plants by NaOH and isopropanol. According to the method, a 96-hole PCR amplification board is employed for extraction by a traditional soda boiling process, isopropanol is adopted to conduct precipitation purification on the extracted product, so that the extraction quality, extraction speed and extraction scale of the melon genomic DNA are greatly improved, and the needs of subsequent melon crop breeding improvement, variety purity molecular marker identification and other related items for melon genomic DNA can be satisfied. At the same time, the method has the advantages of simple process steps, easy operation and small tissue need, thus being especially suitable for mass extraction of melon genomic DNA.

Description

technical field [0001] The invention relates to a method for extracting genomic DNA of melon plant tissue, belonging to the technical field of DNA extraction. Background technique [0002] The extraction of plant tissue genomic DNA is a basic operation that is often involved in laboratory operations, especially in plant-related molecular biology research, it is often necessary to extract relevant plant tissue genomic DNA for follow-up experiments. The extraction of genomic DNA from traditional plant tissues generally uses the CTAB method or SDS method, which is cumbersome, difficult, and slow, and cannot meet the needs of laboratories for large-scale extraction of genomic DNA from plant tissues. The genomic DNA alkaline cooking method developed in recent years is derived from the small preparation of bacterial plasmid DNA. In order to meet the extraction requirements of plasmid DNA, the bacterial cell membrane is broken. Under the alkaline condition with a pH value of 12.6, ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 周贤达王兆贤陈会中吴义兵周桂林林茂朱先飞徐丽媛王丽梅程国旺
Owner HEFEI FENGLE SEED