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Method for high flux rapid extraction of vegetable single seed DNA

An extraction method and high-throughput technology, applied in the field of molecular biology, can solve the problems of time-consuming, many extraction steps, and complexity, and achieve the effect of saving time, simple process and fast process.

Inactive Publication Date: 2015-08-19
TIANJIN INSTITUE OF QUALITY STANDARD & TESTING OF AGRICULTUAL PRODS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The purpose of the present invention is to overcome the shortcomings of traditional sample DNA extraction steps, such as many, complicated, and time-consuming, and discloses a method for rapidly extracting DNA from vegetable single seeds with high throughput

Method used

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  • Method for high flux rapid extraction of vegetable single seed DNA
  • Method for high flux rapid extraction of vegetable single seed DNA
  • Method for high flux rapid extraction of vegetable single seed DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Taking the cauliflower variety "Jinpin 70" hybrid as a sample, the method of the present invention was used to rapidly extract DNA. The preparation method is as follows:

[0066] (1) Take about 100 mg of a single cauliflower hybrid seedling and add it to a 1.5 mL sterilized centrifuge tube, and use a hand-held electric crusher to grind it to powder;

[0067] (2) Add 100 μL of 10% Chelex-100 solution, vortex and mix thoroughly, and place in a 65°C water bath for 20 minutes at a constant temperature;

[0068] (3) After the water bath, quickly freeze on ice for 10 minutes, centrifuge at 13,200 rpm / min for 3 minutes, take the supernatant, and save it for subsequent PCR amplification.

[0069] PCR detection:

[0070] 1. Amplify the cauliflower EST-SSR purity identification primer HYC33, the primer sequence is:

[0071] HYC33-F: 5'-AGACTTCGTCGATCCACCTG-3'

[0072] HYC33-R: 5'-GACCTCGTCTCCTCTTCCT-3'

[0073] 2. Reaction system:

[0074] 2× Master Mixes 5 μL, HYC33 upst...

Embodiment 2

[0082] Using the Chinese cabbage variety "Qiulu 75" hybrid as a sample, the method of the present invention was used to rapidly extract DNA. The preparation method is as follows:

[0083] (1) Take about 100 mg of a single Chinese cabbage hybrid seedling and add it to a 1.5 mL sterilized centrifuge tube, and use a hand-held electric crusher to grind it to powder;

[0084] (2) Add 100 μL of 10% Chelex-100 solution, vortex and mix thoroughly, and place in a 65°C water bath for 20 minutes at a constant temperature;

[0085] (3) After the water bath, quickly freeze on ice for 10 minutes, centrifuge at 13,200 rpm / min for 3 minutes, take the supernatant, and save it for subsequent PCR amplification.

[0086] PCR detection:

[0087] 1. Amplify the Chinese cabbage EST-SSR purity identification primer BC1, the primer sequence is:

[0088] BC1-F: 5'-TTTGTCCCTATTGCTCAGGG-3'

[0089] BC1-R: 5'-CCGAGAACGTCTTTCTCTTG-3'

[0090] 2. Reaction system:

[0091] 2× Master Mixes 5 μL, upstr...

Embodiment 3

[0099] Using the Chinese cabbage variety "Jinbai 56" hybrid as a sample, the method of the present invention was used to rapidly extract DNA. The preparation method is as follows:

[0100] (1) Take about 100 mg of a single Chinese cabbage hybrid seedling and add it to a 1.5 mL sterilized centrifuge tube, and use a hand-held electric crusher to grind it to powder;

[0101] (2) Add 100 μL of 10% Chelex-100 solution, vortex and mix thoroughly, and place in a 65°C water bath for 20 minutes at a constant temperature;

[0102] (3) After the water bath, quickly freeze on ice for 10 minutes, centrifuge at 13,200 rpm / min for 3 minutes, take the supernatant, and save it for subsequent PCR amplification.

[0103] PCR detection:

[0104] 1. Amplify the Chinese cabbage EST-SSR purity identification primer BC9, the primer sequence is:

[0105] BC9-F: 5'-ACCTTCCGTCTCTGGGTCTT-3'

[0106] BC9-R: 5'-TTTTGAACGAGGTGGAGGAC-3'

[0107] 2. Reaction system:

[0108] 2× Master Mixes 5 μL, upst...

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Abstract

The invention discloses a method for high flux rapid extraction of vegetable single seed DNA, an early seedling single plant which is developed from a seed by breaking of hull, a to-be-tested sample single plant is ground into powder in a 1.5 mL pointed bottom centrifugal tube by a handheld electric disruptor, then100 mu L of 10 % Chelex-100 solution is added, the mixture is in water bath at 65 DEG C for 20 min, the mixture is quickly frozen for 10 min, and centrifuged for 3 min ate the speed of 13200 RPM / min, the supernatant is sucked to obtain sample DNA, and the sample DNA can be directly used for PCR amplification. According to the DNA extraction method, the whole course of sample DNA extraction can be finished in 35 minutes. The extracted DNA quality standards meet the requirements of the subsequent PCR amplification, the method is more suitable for the purpose of breeding industry variety breeding, high flux rapid extraction of DNA in the sample, and has the advantages of being efficient, fast, low in cost, simple in operation and the like.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, relates to a template DNA preparation method, and is an efficient, fast, low-cost and easy-to-operate high-throughput vegetable single seed DNA extraction method. Background technique [0002] Molecular biology techniques have penetrated into various aspects of biology, medicine, botany, genetics and zoology. The extraction of plant DNA is the most basic step in molecular analysis, and plays an important role in plant genetic engineering and plant molecular biology research. The quality of the extracted DNA will directly affect the success or failure of later molecular biology research. Molecular markers based on PCR technology have been widely used in seed purity identification, genetic relationship analysis, molecular marker-assisted breeding, gene location and transgenic plant detection. Among them, markers based on PCR technology, such as simple repeat sequence markers (SSR markers...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 赵新刘莉莉王永王超楠兰青阔李梅陈锐朱珠郭永泽景海春
Owner TIANJIN INSTITUE OF QUALITY STANDARD & TESTING OF AGRICULTUAL PRODS
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