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Method for extracting larva DNA (Deoxyribonucleic Acid) before metamorphosis of echinoderm by freezing method

A technology of echinoderm and freezing method, which is applied in the field of extracting echinoderm metamorphic prelarvae DNA by freezing method, can solve the problems of large damage and trouble, and achieve the effect of easy control, simple operation, and no toxic side effects

Inactive Publication Date: 2012-04-11
DALIAN OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The DNA extraction of aquatic animals is mainly found in adults, and the extraction of DNA from larvae is less. Currently, the DNA extraction method of marine aquatic animal larvae is the DMSO fixation method. In the experiment, the larvae were taken out from the fixative solution, and the fixative solution was rinsed with pure water. This step is more troublesome, and DMSO is a highly toxic substance, which will cause great harm to the experimenter.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0019] Taking sea urchin as an example, the steps are as follows:

[0020] a. Use a micropipette to take a sea urchin larvae under a dissecting microscope, put it into a 0.2ml centrifuge tube, and store it in a freezer at -80°C;

[0021] b. Take out the frozen centrifuge tube containing sea urchin larvae and thaw it, and add 15ul of lysate and 1ul of 0.3mg / ml proteinase K to the centrifuge tube within 10 minutes after thawing, and then put it in a 55°C water bath for 3 Hours, shake once every half hour during this period; the components and final concentration of the lysate are 10mmol / L Tris-Cl, 50mmol / L KCL, 0.5% Tween-20, pH8.0;

[0022] c. After lysing, raise the temperature of the water bath to 85°C and bathe in water for 15 minutes to inactivate proteinase K; the lysed sample is directly used for PCR amplification to obtain the amplified product. The composition of the microsatellite amplification PCR system is as follows:

[0023] Reaction system needs to add volume

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Abstract

The invention discloses a method for extracting larva DNA (Deoxyribonucleic Acid) before metamorphosis of echinoderm by a freezing method. The method comprises the following steps of: filling echinoderm larva in a centrifugal tube and preserving in a freezing mode under the condition of -80 DEG C; unfreezing and then adding 15 ul of cracking solution and 1 ul of 0.3 mg / ml protease K into the centrifugal tube in 10 minutes; cracking in a water bath for 3 hours and oscillating and shaking up once every half an hour, wherein the cracking solution comprises the following components with the following final concentration: 10 mmol / L Tris-Cl, 50 mmol / L of KCL and 0.5 percent of Tween-20, and the pH is 8.0; heating to 85 DEG C and processing in water bath for 15 minutes; and directly performing PCR (Polymerase Chain Reaction) amplification on a sample after cracking to obtain an amplified product, wherein a micro-satellite amplified PCR system consists of the following substances: 1 ul of DNA template, 2.5 ul of 10*buffer, 1.0 ul / each of primer, 2.2 ul of d NTP, 1.5 ul of MgCl2, 0.35 ul of Taq and 15.45 ul of dd H2O, 25 ul in total. The method is easy to operate and does not cause any harm to operators.

Description

technical field [0001] The invention relates to a method for extracting DNA, in particular to a method for extracting DNA from echinoderm metamorphosis prelarvae by freezing method. Background technique [0002] Sea urchin belongs to Echinodermata Echinacea, is a relatively common marine invertebrate, and is also the earliest model organism used in the history of biological science. Its eggs and embryos play a pivotal role in the development of early developmental biology. The fertilized eggs of sea urchins develop into immature sea urchins through three main developmental stages: early embryonic development, planktonic larvae, and prostrate metamorphosis. Sea cucumbers belong to the class Echinodermata and are aquatic animals with high economic value. Sea cucumbers also develop from planktonic larvae to immature sea cucumbers. The larvae of sea cucumbers and sea urchins are small in shape, generally no more than 1mm. In addition, during the metamorphosis process, their mor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
Inventor 常亚青赵新亚丁君
Owner DALIAN OCEAN UNIV
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