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Eriocheir sinensis blood DNA extraction method for Southern hybridization analysis

A technology of Chinese mitten crab and extraction method, applied in the field of DNA extraction of animals, can solve problems such as differences in DNA extraction methods

Inactive Publication Date: 2010-11-17
FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This is obviously different from the composition formula of most fish blood cells, and there are also obvious differences in the DNA extraction methods

Method used

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  • Eriocheir sinensis blood DNA extraction method for Southern hybridization analysis
  • Eriocheir sinensis blood DNA extraction method for Southern hybridization analysis
  • Eriocheir sinensis blood DNA extraction method for Southern hybridization analysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] A kind of Chinese mitten crab blood DNA extraction method for Southern hybridization analysis of the present invention adopts the following process steps:

[0024] Chinese mitten crabs were purchased from Wuxi Zhongqiao Market. Take 0.5ml of ACD anticoagulant from a disposable 3ml syringe, insert the needle point directly from the pia mater of the base joint of Eriocheir sinensis rubbed with alcohol, the depth is 0.35cm, suck 1ml of blood, then take out the syringe and inhale Mix the ACD anticoagulant thoroughly with the blood by inverting it upside down with a small amount of air. Remove the needle, slowly inject the mixture into a 1.5ml centrifuge tube, centrifuge speed: 2000 rpm, centrifuge for 10 minutes, when the centrifugation is over, remove the supernatant and leave the blood cell pellet. Quickly add 0.6 ml of lysis buffer to suspend the pellet. The lysis solution is composed of sodium lauryl sarcosinate, proteinase K, disodium ethylenediaminetetraacetic acid ...

Embodiment 2

[0026] A kind of Chinese mitten crab blood DNA extraction method for Southern hybridization analysis of the present invention adopts the following process steps:

[0027] Chinese mitten crabs were purchased from Wuxi Zhongqiao Market. Take 0.5ml of ACD anticoagulant from a disposable 3ml syringe, insert the needle point directly from the pia mater of the basal joint of Eriocheir sinensis that has been wiped with alcohol, the depth is 0.4cm, absorb 1ml of blood, then take out the syringe and inhale Apply a small amount of air and invert up and down to mix the anticoagulant thoroughly with the blood. Remove the needle, slowly inject the mixture into a 1.5ml centrifuge tube, centrifuge speed: 3000 rpm, centrifuge for 5 minutes, after the centrifugation is over, remove the supernatant and leave the blood cell pellet. Quickly add 0.6 ml of lysis buffer to suspend the pellet. 0.6 ml of lysate contains 3 mg of sodium lauryl sarcosinate; 0.12 mg of proteinase K; 11.17 mg of disodium...

Embodiment 3

[0029] A kind of Chinese mitten crab blood DNA extraction method for Southern hybridization analysis of the present invention adopts the following process steps:

[0030] Chinese mitten crabs were purchased from Wuxi Zhongqiao Market. Take 0.5ml of ACD anticoagulant into a disposable 3ml syringe, insert the needle tip directly from the pia mater of the base joint of Eriocheir sinensis pedicure cleaned with alcohol, the depth is 0.45cm, suck 1ml of blood, then take out the syringe and inhale Apply a small amount of air and invert up and down to mix the anticoagulant thoroughly with the blood. Remove the needle, slowly pour the mixture into a 1.5ml centrifuge tube, centrifuge speed: 2500 rpm, centrifuge for 10 minutes, when the centrifugation is over, remove the supernatant and leave the blood cell pellet. Quickly add 0.6 ml of lysis buffer to suspend the pellet. 0.6 ml of lysis solution contains 6 mg of sodium lauryl sarcosinate; 0.12 mg of proteinase K; 11.17 mg of disodium ...

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Abstract

The invention relates to an eriocheir sinensis blood DNA extraction method for Southern hybridization analysis, which is characterized by comprising the following steps of: sucking ACD anticoagulant by using a syringe, inserting the syringe into a soft membrane of a walking leg base joint of eriocheir sinensis to suck blood, and fully mixing the ACD anticoagulant and the blood uniformly to obtain mixed solution; centrifuging the mixed solution, removing supernate and leaving blood cell sediment; suspending the blood cell sediment into lysing solution to obtain mixed solution; adding Tris saturated phenol into the mixed solution, shaking the solution uniformly and centrifuging the solution; adding chloroform into the supernate, shaking the mixture uniformly and then centrifuging the mixture, and taking the supernate; adding ammonium acetate and anhydrous ethanol into the supernate, shaking the mixture uniformly and then centrifuging the mixture, and collecting DNA; and washing the DNA by using 70 percent ethanol, and airing and dissolving the DNA. The DNA extracted by the method has high concentration, and can meet a Southern hybridization method.

Description

technical field [0001] The invention relates to a method for extracting DNA from the blood of Eriocheir sinensis for Southern hybridization analysis, which is the DNA extraction of animals. Background technique [0002] Southern blotting is a classical experimental method for molecular biology. The basic principle is to immobilize the DNA sample to be detected on a solid-phase carrier, hybridize it with a labeled nucleic acid probe, and display a hybridization signal at the position of the solid-phase DNA that has a homologous sequence with the probe. Whether there is a fragment homologous to the probe in the detected DNA sample and the length of the fragment can be determined by Southern hybridization. [0003] Chinese mitten crab is a unique aquatic economic animal in my country. In the long-term evolution, different geographical populations have been formed, such as the Liaohe population, the Yellow River population, the Yangtze River population, the Oujiang population a...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
Inventor 唐永凯俞菊华徐跑李建林李红霞尤洋
Owner FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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