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Method for extracting genome DNA of jellyfish

An extraction method, genome technology, applied in the field of genetic engineering

Inactive Publication Date: 2011-03-23
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Kit method has high cost and low yield, and it is not practical for large-scale population analysis; CTAB method samples are ground with liquid nitrogen and extracted with phenolic form, which is cumbersome and toxic, and it is not suitable for jellyfish disc genome Extraction, can not guarantee the later experiment
At the same time, the existing technology mostly uses kit extraction or phenol-chloroform extraction to extract the genome of marine animals. These two methods for the extraction of jellyfish genome DNA cannot guarantee the quality and quantity to meet the needs of the experiment, and cannot achieve satisfactory results.

Method used

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  • Method for extracting genome DNA of jellyfish
  • Method for extracting genome DNA of jellyfish

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The jellyfish polyp was picked up from the corrugated plate (or its attachment) with a toothpick, and collected into a 1.5ml centrifuge tube. Carefully shred with surgical scissors until there is no obvious granular tissue, add 400ul CTABbuffer, and set aside. Add 5ul proteinase K, digest at 55°C for 24 hours until the solution is clear and ready for use.

[0024] Then add the mixed solution of phenol, chloroform and isoamyl alcohol equal to the volume of the clarified solution, mix evenly and centrifuge at 12000rpm for 30min to suck the supernatant, then add the mixed solution of chloroform and isoamyl alcohol equal to the volume of the gained supernatant, and mix upside down Evenly centrifuge at 12000rpm for 30min to aspirate the supernatant. Add 0.7 times the volume of isopropanol to the obtained supernatant to precipitate at -20°C for 10 minutes, centrifuge at 12,000 rpm for 20 minutes, and discard the liquid. Wash twice with 500 ul of 70% ethanol at 12000 rpm for...

Embodiment 2

[0028] Collect the jellyfish sphenoids preserved in 95% alcohol into a 1.5ml centrifuge tube. Add 400ul homogenate buffer, 40ul 10% SDS, 2ul proteinase K, seal and digest at 55°C for 1 hour. After digestion, take out and add 400ul 6M NaCl, shake for 30 seconds and then centrifuge at 12000rpm for 30 minutes. Aspirate 800ul of the supernatant and add 560ul of isopropanol, precipitate at -20°C for 10 minutes, then centrifuge at 12000rpm for 20min, and remove the supernatant. Wash twice by centrifugation with 70% ethanol at 12,000 rpm for 5 minutes, and dry at room temperature. That is, the DNA of the jellyfish disc is obtained; the DNA of the jellyfish disc is stored in sterilized pure water at -20°C.

[0029] The DNA that obtains the jellyfish polyp is dissolved in 40ul sterilized pure water, and can be used for agarose gel electrophoresis detection after 30min (see figure 2 ).

[0030] The buffer solution contains 0.01M Tris-Hcl and 0.1M EDTA; the isopropanol and ethanol w...

Embodiment 3

[0032] The difference from Example 1 is:

[0033] Shred the jellyfish polyp with surgical scissors until there is no obvious granular tissue, add CTAB buffer 300ul, and set aside;

[0034] Then add 3ul of proteinase K with a concentration of 20mg / ml, digest at 56°C for 26 hours until the solution is clear, and set aside;

[0035] Add 1 times the volume of isopropanol to the supernatant after centrifugation with chloroform and isoamyl alcohol, precipitate at -18°C for 15 minutes, take out after precipitation, centrifuge, discard the liquid, and use a volume concentration of 70 % ethanol, centrifuged and washed once, and the excess liquid was poured out at room temperature and dried naturally to obtain the genomic DNA of the jellyfish polyp.

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Abstract

The invention relates to the genetic engineering technique, in particular to a method for extracting the genome DNA of a jellyfish. The method specifically comprises the steps of: shearing the jellyfish into tissues without obvious particles by using a pair of surgical scissors, adding CTAB buffer and protease K, and digesting at 55-56 DEG C for 24-26 h until the solution is clear; or dehydratingthe jellyfish in 95% of alcohol, adding buffer, SDS and the protease K, and sealing and digesting at 55-56 DEG C for 0.8-1.0 h; mixing phenol, chloroform and isoamyl alcohol, centrifuging and absorbing the supernatant, then adding a chloroform and isoamyl alcohol mixture for mixing, centrifuging and absorbing the supernatant; adding isopropyl alcohol in the supernatant, precipitating for 10-15 min, taking out after the precipitation, centrifuging, discarding the liquid, centrifugally cleaning with ethanol, pouring the surplus liquid and naturally airing at the room temperature to obtain the genome DNA of the jellyfish. Through the application of the invention, high-quality genome DNA of the jellyfish can be extracted; and the method is simple, economic, easy to operate and suitable for the common biological laboratory.

Description

technical field [0001] The invention relates to genetic engineering technology, in particular to a method for extracting jellyfish genome DNA. Background technique [0002] Due to the biological characteristics of jellyfish, such as high water content, easy decomposition and autolysis, the extraction of its genomic DNA is limited. The life history of jellyfish includes sexual and asexual generations. Polyps live sessilely and belong to the asexual generation of jellyfish. Discs live planktonic and belong to the sexual generation of jellyfish. The two are very different in external shape and biological function, which leads to the extraction of genomic DNA in two stages. The method is different. At present, the research on jellyfish at home and abroad has not separated different life generations to study the extraction of genomic DNA. The reported jellyfish genomic DNA extraction methods are mostly Kit method and CTAB method. Kit method has high cost and low yield, and it...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C07H21/04C07H1/08
Inventor 崔朝霞张姝栾维莎刘媛
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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