Method for extracting DNA from single eggs of copepods

An extraction method and technology for copepods, which are applied in the field of DNA extraction and can solve the problems of damage, straight elution efficiency, and no work report on genome DNA extraction from a single egg of copepods.

Inactive Publication Date: 2010-04-21
XIAMEN UNIV
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  • Abstract
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AI Technical Summary

Problems solved by technology

It is worth mentioning that there are many problems in the two commonly used methods for extracting DNA from single zooplankton eggs and the commercial kits that have emerged in an endless stream. In summary, there are mainly the following points: 1. The two methods that are currently popular Both use physical means and tools to destroy the structure of the egg. This operation will make the amount of DNA extracted less than the theory. These are man-made losses caused by the operation, and the DNA content is not high. For a single egg, this loss is huge; 2. In the method of Chelex-100, the resin added by itself virtually increases the chance of contamination, and practice has proved that the addition of resin has a great impact on downstream PCR experiments. Inhibition; 3. The commercial kit mainly completes the collection and purification of DNA through the process of membrane adsorption and elution. While the quality of DNA is improved, the elution efficiency directly affects the concentration of DNA, which will also directly affect the downstream. PCR experiment
[0003] The above are the research progress in this field abroad, so far, there is no domestic report on the extraction of genome DNA from a single egg of copepods

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  • Method for extracting DNA from single eggs of copepods

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Embodiment 1

[0017] Place a single copepod egg in 5-10 μL of 1×TE buffer solution (pH 8); add proteinase K to make the final concentration of proteinase K reach 4 mg / mL (the purpose of adding proteinase K is mainly through the action of proteinase K to destroy the external structure of the egg so that the internal DNA can be released smoothly, and at the same time avoid the loss of DNA caused by physical damage); after adding proteinase K, a mixed extract can be obtained, and the mixed extract obtained will be passed through A heat incubation program, the specific program is as follows: place at 55°C for 30 minutes, then place at 95°C for 5 minutes. After the above procedures are completed, the crude extract of single egg DNA can be obtained, and then precipitated by the nucleic acid co-precipitation agent (DNA Mate) produced by Takara Biotechnology Co., Ltd. (Takara). The specific steps of precipitation are as follows: Add 1 / 10 volume of 3M sodium acetate solution and 4 μL DNA Mate to the...

Embodiment 2

[0020]Similar to Example 1, a single copepod egg was placed in 5-10 μL of 1×TE buffer (pH 7.5); proteinase K was added to make the final concentration of proteinase K reach 2.5 mg / mL (the purpose of adding proteinase K Mainly through the action of proteinase K to destroy the external structure of the egg so that the internal DNA can be released smoothly, and at the same time avoid the loss of DNA caused by physical damage); after adding proteinase K, a mixed extract can be obtained, and then The obtained mixed extract was subjected to a heat incubation procedure, and the specific procedure was as follows: standing at 60° C. for 25 minutes, and then standing at 95° C. for 5 minutes. After the above procedures are completed, the crude extract of single egg DNA can be obtained, and then precipitated by the nucleic acid co-precipitation agent (DNA Mate) produced by Takara Biotechnology Co., Ltd. (Takara). The specific steps of precipitation are as follows: Add 1 / 10 volume of 3M so...

Embodiment 3

[0022] Similar to Example 1, a single egg of the copepod was placed in 5-10 μL of 1×TE buffer (pH 8.5); proteinase K was added to make the final concentration of proteinase K reach 5.5 mg / mL (the purpose of adding proteinase K Mainly through the action of proteinase K to destroy the external structure of the egg so that the internal DNA can be released smoothly, and at the same time avoid the loss of DNA caused by physical damage); after adding proteinase K, a mixed extract can be obtained, and then The obtained mixed extract was subjected to a heat incubation procedure, the specific procedure was as follows: 120 minutes at 37°C, and then 5 minutes at 95°C. After the above procedures are completed, the crude extract of single egg DNA can be obtained, and then precipitated by the nucleic acid co-precipitation agent (DNA Mate) produced by Takara Biotechnology Co., Ltd. (Takara). The specific steps of precipitation are as follows: Add 1 / 10 volume of 3M sodium acetate solution and...

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Abstract

The invention discloses a method for extracting DNA from single eggs of copepods, relating to a DNA extraction technology. The single eggs have different egg diameters and are generated by different egg producing ways. The method can ensure the extraction efficiency of DNA, and the extracted DNA can be directly applied to downstream PCR experiments. The method comprises the following steps of placing the single eggs of copepods in a 1*TE buffer solution, adding protease K until the final concentration of the protease K reaches 2.5-5.5mg/mLto obtain a mixed extraction solution; carrying out a thermal incubation program on the mixed extraction solution obtained to obtain a crude extract of DNA of the single eggs; and depositing the crude extract of DNA of the single eggs with a nucleic acid coprecipitator to obtain DNA precipitate. The precipitate can be dissolved in sterilized water and stored in a refrigerator for later use.

Description

technical field [0001] The invention relates to a DNA extraction technology, especially a novel DNA extraction method that is practical, rapid and effective for single eggs of copepods, including different egg-laying modes, different physiological states and different egg diameters. Background technique [0002] At present, there are mainly two methods for extracting single eggs of zooplankton in the world, one is Montero-Pau et al. Application of an inexpensive and high-throughput genomic DNA extraction method for the molecular ecology of zooplanktonic diapausing eggs .Limnology and Oceanography: Methods, 2008, 6: 218-222) proposed the HotSHOT method, this method is mainly aimed at the extraction of a single egg genome DNA of zooplankton diapause eggs, and its operation steps mainly include the following steps: adding certain A large amount of lysis buffer was used; diapause eggs were broken by physical means and tools; finally, genomic DNA was released through a process of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C07H21/04
Inventor 吴荔生徐智焕王桂忠蒋洁兰王超陈丹
Owner XIAMEN UNIV
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