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A technology of germ cells and chromosomes, applied in the preparation of test samples, etc., to achieve the effects of easy observation, simple and efficient operation, and reduced damage
Inactive Publication Date: 2013-05-08
SHANGHAI OCEAN UNIV
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However, there is no report about a method for preparing shrimp germ cell chro
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Embodiment 1
[0035] Example 1 Preparation of Testicular Germ Cell Chromosomes
[0036] 1. Select a lively individual male Procambarus clarkii, weighing about 35~40g, carefully cut the carapace along the center of the rear edge of the carapace, avoiding the heart, pick the white kidney-shaped testis between the two livers under the heart, and put it in a petri dish.
[0037] 2. Incubate the testis with 500ug / ml colchicine at 16°C in a biochemical incubator for 4 hours, then remove the colchicine.
[0038] 3. Hypotonic treatment with 0.1mol / L KCl at room temperature for 30 minutes, carefully remove the hypotonic solution, and then pre-mix with Carnoy’s fixative (absolute ethanol: glacial acetic acid = 3:1) pre-cooled to 4 degrees. fixed.
[0039] 4. Carnot’s fixative (absolute ethanol: glacial acetic acid = 3:1) was changed at 4°C at fixed intervals of 1 hour for a total of 2 times, and then pre-cooled to 4°C postfixate (absolute ethanol: Glacial acetic acid = 1:1) fixed once, 20min.
...
Embodiment 2
[0044] Example 2 Preparation of Ovarian Germ Cell Chromosomes
[0045] 1. Select a lively female Procambarus clarkii, weighing about 35-40g, carefully cut the carapace along the center of the rear edge of the carapace, avoiding the heart, pick the yellow Y-shaped ovary under the heart, and put it in a petri dish Inside.
[0046]2. Incubate the ovaries with 500ug / ml colchicine at 16°C in a biochemical incubator for 4 hours, then remove the colchicine.
[0047] 3. Hypotonic treatment with 0.1mol / L KCl at room temperature for 30 minutes, carefully remove the hypotonic solution, and then pre-mix with Carnoy’s fixative (absolute ethanol: glacial acetic acid = 3:1) pre-cooled to 4 degrees. fixed.
[0048] 4. Carnot’s fixative (absolute ethanol: glacial acetic acid = 3:1) was changed at 4°C at fixed intervals of 1 hour for a total of 2 times, and then pre-cooled to 4°C postfixate (absolute ethanol: Glacial acetic acid = 1:1) fixed once, 20min.
[0049] 5. Aspirate and discard ...
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Abstract
The invention relates to a preparation method of a shrimp germ cell chromosome. The preparation method comprises the following steps: culturing testis/ovary by colchicine; performing KCl hypotonic treatment and pre-fixing by a Carnoy fixing solution; fixing by an after-fixing solution, wherein the after-fixing solution comprises absolute ethyl alcohol and glacial acetic acid according to the volume ratio of 1:1; dispersing spermatocytes/oocytes to prepare a cell suspension; dyeing by a Giemsa phosphate buffered solution; and performing microscopical examination under a Motic microscope. The preparation method disclosed by the invention has the following advantages that the operation is simple and efficient and chromosome slides can be mass-produced; sufficient chromosome samples can be fast, simply and conveniently obtained, the obtained samples are uniform in dispersion, shallow in background and easy to observe, and the obtained chromosome number is high in accuracy; the simple and easy-to-operate method is provided for preparing shrimp chromosomes; and the non-toxic fixing solutions are adopted, and the harm to a slide making operator can be reduced.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing shrimp germ cell chromosomes. Background technique [0002] It is a development direction in aquaculture to apply the new technology of bioengineering to the genetic breeding and variety improvement of shrimp. [0003] According to the literature search of the prior art, it is found that the chromosome specimens made by the traditional chromosome pressing technology destroy the structure of the chromosomes, making it impossible to carry out higher-level chromosome experiments, and a large amount of cleavage phase will be lost during the initial stage of the experiment. Chromosomes are not conducive to the development of subsequent experiments. Qian Guoying, Wang Caisheng, etc. compared the hypoosmotic method and tube infiltration method in the article "Comparative Study on Macrobrachium Chromosome Preparation" published in "Journal of Experimental Biology", Vo...
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