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Separation and purification method of crosslinked tetrahymena micronucleus

A technology of Tetrahymena and thermophilic Tetrahymena, applied to animal cells, etc., can solve the problems of unsuitable cells, difficult to carry out effectively, unsuitable for separation, etc.

Inactive Publication Date: 2015-12-30
UNIV OF SCI & TECH OF CHINA
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Problems solved by technology

It is mainly operated on living cells, the operation time is long and it is not suitable for the separation of crescent nuclei, because the crescent nuclei are extremely extended from the original round nuclei to become linear nuclei, which are easy to break when mechanically broken. broken
In addition, because the cross-linked cells such as formaldehyde are difficult to break mechanically, the current experimental method is not suitable for cross-linked cells.
[0003] Tetrahymena thermophila is a good research model, but it cannot effectively lyse the cross-linked cells and separate the small nuclei, and it is difficult to obtain better crescent small nuclei
It makes it difficult to effectively carry out related experiments (ChIP, Hi-C) targeting small nuclei

Method used

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  • Separation and purification method of crosslinked tetrahymena micronucleus

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Embodiment Construction

[0024] The present invention is achieved by the following means.

[0025] I. Sample collection and storage

[0026] 1. Take Tetrahymena in the crescent stage 3 hours after conjugation, the density is about 2.5x10 5 cells / ml, add 37% formaldehyde to a final concentration of 1%, mix well, and leave at room temperature for 10 minutes.

[0027] 2. Add 2.5M glycine to a final concentration of 125mM to neutralize formaldehyde, mix well, place at room temperature for 5 minutes, and place on ice for 15 minutes.

[0028] 3. Centrifuge at 1500g for 2 minutes at 4°C, remove the supernatant, and wash once with 10mM Tris-HCl. Centrifuge again to remove supernatant.

[0029] 4. Transfer to a 1.5ml centrifuge tube, about 2x10 per tube 7 Cells were centrifuged several times at 2000g at 4°C to remove residual liquid as much as possible. Samples can be stored at -80°C or used directly for subsequent experiments.

[0030] II. Cell Lysis and Isolation of Crescent Nuclei

[0031] 1. Take th...

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Abstract

The invention relates to a separation and purification method of crosslinked tetrahymena micronucleus. The separation and purification method of the crosslinked tetrahymena micronucleus comprises the following specific steps of performing crosslinking treatment on tetrahymena cells by using crosslinking agents such as formaldehyde; splitting the cells by using chemical cleavage; and finally separating micronucleus of the tetrahymena in a centrifugal mode. Owing to the separation and purification method of the crosslinked tetrahymena micronucleus, Hi-C and correlational studies in tetrahmena thermophila are feasible.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a separation method for purifying cross-linked Tetrahymena, especially the small nucleus of the thermophilic Tetrahymena, especially the small nucleus of the crescent stage. Background technique [0002] The existing method for separating the large and small nuclei of Tetrahymena is generally to separate the large and small nuclei by means of differential centrifugation or density gradient sedimentation after mechanical crushing. It is mainly operated on living cells, the operation time is long and it is not suitable for the separation of crescent nuclei, because the crescent nuclei are extremely extended from the original round nuclei to become linear nuclei, which are easy to break when mechanically broken. broken. In addition, because the cross-linked cells such as formaldehyde are difficult to mechanically disrupt, the current experimental method is not suita...

Claims

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Application Information

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IPC IPC(8): C12N5/07
Inventor 宋晓元罗正誉
Owner UNIV OF SCI & TECH OF CHINA
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