DNA (deoxyribonucleic acid) extracting solution and method for quickly extracting DNA from plant leaves by using DNA extracting solution

Abstract

The invention provides a DNA extracting solution and a method for rapidly extracting DNA from plant leaves by using the DNA extracting solution, and relates to the field of DNA extraction, 500 ml of the DNA extracting solution is formed by fully mixing 10 ml of 1 mol / L Tris-HCl, 2.5 ml of 5 mol / L NaCl, 2.5 ml of 0.5 mol / L EDTA (Ethylene Diamine Tetraacetic Acid), 0.25 g of SDS (Sodium Dodecyl Sulfate) and H2O. The method comprises the following steps: taking fresh and tender plant leaves of 3 mm < 2 >, putting the fresh and tender plant leaves into a centrifugal tube of 2 ml, and putting the centrifugal tube into an ice box for storage; adding a steel ball with the diameter of 3mm and 300 microliters of the DNA extracting solution as described in claim 1 into the centrifugal tube; placing the centrifugal tube in the step 2 on a grinding machine provided with a proper clamp, and grinding for 15s at the frequency of 50Hz until the blades are completely crushed; putting the ground centrifugal tube into a centrifugal machine, centrifuging for 1 minute at the speed of 10000 rpm, and then taking out supernate, namely an extracted DNA solution. The method provided by the invention solves the problems of complexity, low efficiency and high cost of leaf DNA extraction in the prior art, and can be used for conveniently and efficiently extracting DNA at low cost.

Description

Technical Field

[0001] The invention relates to the field of DNA extraction, in particular to a DNA extracting solution and a method for rapidly extracting DNA from plant leaves using the DNA extracting solution. Background Art

[0002] DNA (deoxyribonucleic acid) plays a vital role in life activities. It is an important molecule present in the cell nucleus and mitochondria. It has important functions such as storage and transmission of genetic information, regulation of genes, and synthesis of proteins. It is one of the main objects of molecular biology research.

[0003] In recent years, with the development and improvement of modern molecular biology, researchers have used molecular biology techniques to conduct a lot of in-depth research in various fields of plants, such as seed purity identification, molecular-assisted breeding, gene library construction, genetic diversity analysis, genetic map construction, gene localization, etc. However, the development of all these studies depends on an efficient, fast and low-cost method for extracting plant genomic DNA.

[0004] From the current development, the extraction of plant genomic DNA mainly includes CTAB method, SDS method, magnetic bead method, kit method, etc. Although the DNA obtained by CTAB method and SDS method is of high purity and good integrity, toxic reagents such as chloroform are required, and there are disadvantages such as many steps, complicated operation, long time consumption, environmental pollution, and high consumables. In addition, freezing plant tissues requires the use of liquid nitrogen and harmful reagents, which is dangerous during operation and cannot meet the needs of high-throughput analysis in modern molecular biology research. The magnetic bead method is currently a DNA extraction method with relatively high extraction quality and throughput, but the required reagents are expensive and are not suitable for large-scale commercial molecular marker-assisted selection breeding with fewer markers. Even if a relatively simple DNA extraction kit is used, there are problems of high cost and low extraction efficiency. Summary of the invention

[0005] In view of the above technical problems, the present invention provides a DNA extracting solution and a method for rapidly extracting DNA from plant leaves using the DNA extracting solution, providing a convenient, efficient and low-cost DNA extraction method.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] The DNA extract provided by the present invention comprises 500 ml of a DNA extract prepared by fully mixing 10 ml of 1 mol Tris-HCl, 2.5 ml of 5 mol NaCl, 2.5 ml of 0.5 mol EDTA, 0.25 g SDS and H2O.

[0008] The method for rapidly extracting DNA from plant leaves provided by the present invention comprises the following steps:

[0009] S101: Take 3mm 2 Place fresh and tender plant leaves in a 2 ml centrifuge tube and store the tube in an ice box to keep the leaf samples at low temperatures;

[0010] S102: Add a steel ball with a diameter of 3 mm and 300 μl of the DNA extraction solution as described in claim 1 into the centrifuge tube of step S101;

[0011] S103: placing the centrifuge tube in step S102 on a grinder equipped with an appropriate fixture, and grinding at a frequency of 50 Hz for 15 seconds until the leaves are completely broken;

[0012] S104: Place the ground centrifuge tube in step S103 into a centrifuge, centrifuge at 10,000 rpm for 1 minute, and then take out the supernatant, which is the extracted DNA solution.

[0013] The above technical solution has the following advantages or beneficial effects:

[0014] (1) The CTAB method and the SDS method usually involve multiple steps, including cell disruption, protein removal, DNA precipitation, washing and drying, etc., which are complicated and time-consuming. The method provided by the present invention simplifies the cell disruption step by adding steel beads and using a grinder for rapid grinding, and reduces the number of centrifugation and precipitation, thereby shortening the extraction time.

[0015] (2) The CTAB method and the SDS method often require the use of toxic reagents such as chloroform for protein removal, which not only pollutes the environment but also may cause harm to operators. The method provided by the present invention adopts a milder DNA extraction solution formula, avoids the use of toxic reagents such as chloroform, and reduces environmental pollution and operation risks.

[0016] (3) The magnetic bead method and the kit method are usually expensive, especially when large-scale DNA extraction is required. The method provided by the present invention reduces the cost of reagents and equipment by optimizing the extracting solution formula and using equipment such as a grinder, making the method more economical for large-scale extraction.

[0017] (4) The CTAB method, SDS method, magnetic bead method and kit method have different extraction efficiencies, but generally have problems such as long extraction time and complicated steps. The method provided by the present invention significantly improves the DNA extraction efficiency through rapid grinding and simplified operation steps, making this method have greater application potential in high-throughput analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the polypropylene gel electrophoresis result of the rapid extraction of cabbage leaf genomic DNA provided in Example 1 of the present invention.

[0019] Figure 2 This is the polypropylene gel electrophoresis result of the rapid extraction of genomic DNA from pepper leaves provided in Example 2 of the present invention.

[0020] Figure 3 This is the polypropylene gel electrophoresis result of the rapid extraction of genomic DNA from spinach leaves provided in Example 3 of the present invention.

[0021] Figure 4 This is the polypropylene gel electrophoresis result of the rapid extraction of genomic DNA from green onion leaves provided in Example 4 of the present invention.

[0022] Figure 5 It is a wireframe diagram of the steps of rapidly extracting genomic DNA provided by the present invention. DETAILED DESCRIPTION

[0023] Embodiment 1:

[0024] Take 3mm 2 Place the cabbage cotyledons in a 2 ml centrifuge tube, place in an ice box and bring back to the laboratory.

[0025] A steel bead with a diameter of 3 mm and 300 μl of DNA extract were added into a 2 ml centrifuge tube to obtain an A1 solution, wherein 500 ml of DNA extract was composed of 10 ml of 1 mol Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride), 2.5 ml of 5 mol NaCl, 2.5 ml of 0.5 mol EDTA (ethylenediaminetetraacetic acid), 0.25 g SDS (sodium dodecyl sulfate) and H2O mixed thoroughly.

[0026] Place the centrifuge tube containing A1 solution on the grinder and grind at a frequency of 50 Hz for 15 seconds until the leaves are completely broken;

[0027] The centrifuge tube of the ground A1 solution was placed in a centrifuge and centrifuged at 10,000 rpm for 1 minute, and then the supernatant was taken out to complete the rapid DNA extraction solution of the cabbage leaves.

[0028] The A1 solution is subjected to electrophoresis (existing technology) to obtain an electrophoresis pattern. Figure 1 .

[0029] Among them, Tris-HCl (provides a buffer environment), NaCl (helps dissolve and precipitate DNA), EDTA (chelates metal ions to prevent DNA degradation), and SDS (acts as a detergent to destroy cell membranes and nuclear membranes). Compared with the prior art, no further purification steps (such as chloroform / isoamyl alcohol extraction, ethanol precipitation, etc.) are required. This is because the method of this embodiment is intended to quickly extract DNA for preliminary analysis or screening.

[0030] Embodiment 2:

[0031] Take 3mm 2 Place the fresh pepper leaves in a 2 ml centrifuge tube, place in an ice box and bring back to the laboratory.

[0032] A steel bead with a diameter of 3 mm and 300 μl of DNA extract were added into a 2 ml centrifuge tube to obtain A2 solution, wherein 500 ml of DNA extract was composed of 10 ml of 1 mol Tris-HCl, 2.5 ml of 5 mol NaCl, 2.5 ml of 0.5 mol EDTA, 0.25 g SDS and H2O fully mixed.

[0033] Place the centrifuge tube containing A2 solution on the grinder and grind at a frequency of 50 Hz for 15 seconds until the leaves are completely broken;

[0034] The centrifuge tube of the ground A2 solution was placed in a centrifuge and centrifuged at 10,000 rpm for 1 minute, and then the supernatant was taken out to complete the rapid DNA extraction solution of pepper leaves.

[0035] The A2 solution is subjected to electrophoresis (existing technology) to obtain an electrophoresis pattern. Figure 2 .

[0036] Embodiment 3:

[0037] Take 3mm 2 Place the fresh spinach leaves in a 2 ml centrifuge tube, place in an ice box and bring back to the laboratory.

[0038] A steel bead with a diameter of 3 mm and 300 μl of DNA extract were added into a 2 ml centrifuge tube to obtain A3 solution, wherein 500 ml of DNA extract was composed of 10 ml of 1 mol Tris-HCl, 2.5 ml of 5 mol NaCl, 2.5 ml of 0.5 mol EDTA, 0.25 g SDS and H2O fully mixed.

[0039] Place the centrifuge tube containing A3 solution on the grinder and grind at a frequency of 50 Hz for 15 seconds until the leaves are completely broken;

[0040] The centrifuge tube of the ground A3 solution was placed in a centrifuge and centrifuged at 10,000 rpm for 1 minute, and then the supernatant was taken out to complete the rapid DNA extraction solution of spinach leaves.

[0041] The A3 solution is subjected to electrophoresis (existing technology) to obtain an electrophoresis pattern. Figure 3 .

[0042] Embodiment 4:

[0043] Take 3mm 2 Place the fresh and tender green onion leaves in a 2 ml centrifuge tube, place it in an ice box and bring it back to the laboratory.

[0044] A steel bead with a diameter of 3 mm and 300 μl of DNA extract were added into a 2 ml centrifuge tube to obtain an A4 solution, wherein 500 ml of DNA extract was composed of 10 ml of 1 mol Tris-HCl, 2.5 ml of 5 mol NaCl, 2.5 ml of 0.5 mol EDTA, 0.25 g SDS and H2O, which were fully mixed.

[0045] Place the centrifuge tube containing A4 solution on the grinder and grind at a frequency of 50 Hz for 15 seconds until the leaves are completely broken;

[0046] The centrifuge tube of the ground A4 solution was placed in a centrifuge and centrifuged at 10,000 rpm for 1 minute, and then the supernatant was taken out to complete the rapid DNA extraction solution of the green onion leaves.

[0047] The A4 solution is subjected to electrophoresis (existing technology) to obtain an electrophoresis pattern. Figure 4 .

[0048] The DNA extracting solution and the method for rapidly extracting DNA from plant leaves using the DNA extracting solution provided in Example 1 of the present invention use a grinder to rapidly grind leaves, thereby simplifying the cell disruption step and reducing subsequent processing steps, thereby reducing the complexity of the operation; the DNA extracting solution formula used does not contain toxic reagents such as chloroform, thereby reducing environmental pollution and operational risks; the overall cost is reduced by optimizing the reagent formula and using equipment such as grinders with low costs; and the extraction time is significantly shortened and the efficiency is improved through rapid grinding and simplified operating steps.

Claims

1. A DNA extract, characterized in that: 500 ml of the DNA extraction solution is composed of 10 ml of 1 mol Tris-HCl, 2.5 ml of 5 mol NaCl, 2.5 ml of 0.5 mol EDTA, 0.25 g SDS and H2O mixed thoroughly.

2. A method for rapidly extracting DNA from plant leaves, characterized in that: Includes steps: S101: Take 3mm 2 Place fresh and tender plant leaves in a 2 ml centrifuge tube and store the tube in an ice box to keep the leaf samples at low temperatures; S102: Add a steel ball with a diameter of 3 mm and 300 μl of the DNA extraction solution as described in claim 1 into the centrifuge tube of step S101; S103: placing the centrifuge tube in step S102 on a grinder equipped with an appropriate fixture, and grinding at a frequency of 50 Hz for 15 seconds until the leaves are completely broken; S104: Place the ground centrifuge tube in step S103 into a centrifuge, centrifuge at 10,000 rpm for 1 minute, and then take out the supernatant, which is the extracted DNA solution.

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