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Method for rapidly extracting DNA (Desoxvribose Nucleic Acid) of plant genome with high flux

A plant genome, high-throughput technology, applied in the basic field of molecular biology, can solve problems such as large differences in DNA results, difficult EP tube numbering, cross-contamination, etc., to achieve no protein and RNA contamination, and the extraction method is simple and fast. Extracted effects

Inactive Publication Date: 2014-06-25
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the most commonly used method for extracting plant genomic DNA is to use a large number of EP tubes (1.5-2ml and other specifications) for manual extraction. The process is time-consuming and labor-intensive, and the number of samples extracted per day is very limited, only about 200-400 samples / person; and there are a series of problems, such as cross-contamination caused by repeated use of mortar, difficult and error-prone numbering of a large number of EP tubes, large human errors in the sample extraction process, large differences in DNA results, high cost of single sample extraction, and inability to standardize the scale operations, etc.
These conventional DNA artificial extraction methods cannot meet the needs of molecular marker screening and seed purity identification of a large number of plant materials in a short period of time.

Method used

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  • Method for rapidly extracting DNA (Desoxvribose Nucleic Acid) of plant genome with high flux
  • Method for rapidly extracting DNA (Desoxvribose Nucleic Acid) of plant genome with high flux
  • Method for rapidly extracting DNA (Desoxvribose Nucleic Acid) of plant genome with high flux

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] This example uses the Zephyr workstation combined with the improved CTAB method to extract genomic DNA from cucumber leaves. For the basic process, see figure 1 .

[0049] A: Test materials:

[0050] Take fresh cucumber leaves or vacuum freeze-dried leaves that have been stored for a long time and place them at the bottom of a 96-well deep-well plate. The volume of the leaves accounts for about one-fifth of the well volume. The amount should be kept as consistent as possible, otherwise the extraction efficiency of DNA will be affected.

[0051] B: Pre-freezing and quick-freezing and crushing of cucumber leaves

[0052] Before pre-freezing, remove the cover of the 96-well deep-well plate, put 2 steel balls (3mm in diameter, or balls of other materials) into each well; then place the deep-well plate (without the cover) The cover) is put into liquid nitrogen for quick freezing, or placed in a low-temperature drying device for low-temperature vacuum drying of the sample ...

Embodiment 2

[0070] This example is the Zephyr workstation combined with the improved NaOH method for high-throughput extraction of cucumber seed genomic DNA.

[0071] A: Test materials:

[0072] Put the cucumber seeds soaked and accelerated germination (the germ length is about 1cm) in a 96-well plate, or directly place the cucumber seeds (embryo facing down) in a 96-well plate, add an appropriate amount of water and then germinate at 27°C, and observe carefully during the period. It is optimal when the germ length exceeds 1cm (see figure 2 ).

[0073] Note: During the germination process, pay attention to replenishing water. Lack of water will cause the germ to lose water, affect the growth of the germ, and then affect the quality of seed DNA extraction.

[0074] B: Pre-freezing, quick-freezing and crushing of cucumber seeds / germs

[0075] If germination is directly carried out in a 96-well plate, before pre-freezing, absorb the water added during the germination process, and then pu...

Embodiment 3

[0090] This example is the Zephyr workstation combined with the improved SDS method to extract genomic DNA from cucumber leaves. For the basic process, see figure 1 .

[0091] A: Test materials:

[0092] Take fresh cucumber leaves or vacuum freeze-dried leaves that have been stored for a long time and place them at the bottom of a 96-well deep-well plate. The volume of the leaves accounts for about one-fifth of the well volume. The amount should be kept as consistent as possible, otherwise the extraction efficiency of DNA will be affected.

[0093] B: Pre-freezing and quick-freezing and crushing of cucumber leaves

[0094] Before pre-freezing, remove the cover of the 96-well deep-well plate, put 2 steel balls (3mm in diameter, or balls of other materials) into each well; then place the deep-well plate (without the cover) The cover) is put into liquid nitrogen for quick freezing, or placed in a low-temperature drying device for low-temperature vacuum drying of the sample for...

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Abstract

The invention discloses a method for rapidly extracting DNA (Desoxvribose Nucleic Acid) of a plant genome with a high flux. The method comprises the following steps: respectively putting plant materials to be extracted in hole plates 96; then putting a steel ball in each hole and performing liquid nitrogen flash freezing on each hole; fully grinding the plant materials to be extracted in the hole plates 96 by virtue of a crusher; and finally, extracting the DNA from the grinding powder via an improved NaOH, CTAB (Cetyltrimethyl Ammonium Bromide) or SDS (Sodium Dodecyl Sulfate) method, wherein various reagents are all added by utilizing channels 96 of a Zephyr MBW nucleic acid workstation during the DNA extracting process. The method has the advantages of high flux, low cost, rapid speed and the like. In addition, the extraction method is simple; and the obtained DNA of the plant genome is high purity and good in stability.

Description

technical field [0001] The invention belongs to the basic field of molecular biology, and specifically relates to a method for extracting plant genome DNA rapidly and with high throughput. The method is fast, high-throughput, and low-cost. Genomic DNA was extracted from seeds. Background technique [0002] The acquisition of genomic DNA of plant materials is the basis for related research such as gene mapping research, molecular marker-assisted breeding, seed purity identification and transgenic material identification. Plant seed DNA extraction, purity identification, and disease resistance testing are necessary steps and important guarantees to ensure the high quality and safety of seeds on the market. At present, the most commonly used method for extracting plant genomic DNA is to use a large number of EP tubes (1.5-2ml and other specifications) for manual extraction. The process is time-consuming and labor-intensive, and the number of samples extracted per day is very l...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 温常龙许勇于拴仓赵泓董从娟
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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