Preparation method of flow cytometry water lily sample and cell lysis buffer solution

A technology of flow cytometry and lysis buffer, applied in the field of cell lysis buffer, it can solve the problems of complex genetic background and inability to obtain ideal results, and achieve the effect of maintaining stability, protecting DNA from degradation and improving accuracy

Inactive Publication Date: 2018-01-19
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Due to the rich variety of water lilies, wide distribution, various shapes, and complex genetic background, some varieties of water lilies cannot get ideal results when treated with conventional cell lysis buffe

Method used

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  • Preparation method of flow cytometry water lily sample and cell lysis buffer solution
  • Preparation method of flow cytometry water lily sample and cell lysis buffer solution
  • Preparation method of flow cytometry water lily sample and cell lysis buffer solution

Examples

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Embodiment 1

[0059] In this embodiment, Indian red water lily (Nymphaea rubra Roxb.ex Andrews) is used as a processing sample, and the preparation method of the flow cytometry sample suitable for water lily provided in this embodiment is described. The preparation method includes the following steps:

[0060] 1 Take 1 g of Indian red water lily leaves, put them into a petri dish dripped with 1 ml of pre-cooled cell lysis buffer, and cut the water lily leaves into pieces within 1 min with a blade;

[0061] Wherein, the component of cell lysis buffer is:

[0062] 0.2mol / L Tris-HCl, 10mmol / L MgCl 2 , 2mmol / L EDTA-2Na·2H 2 O, 80mmol / L NaCl, 10mmol / L Na 2 S 2 o 5 , 2% (w / v) PVP-10, 1% (V / V) Triton X-100, 5mmol / L DTT; pH value is 7.5.

[0063] 2 Add 1ml of pre-cooled cell lysis buffer to the fragments to release the nuclei and obtain a nuclei suspension;

[0064] 3 Filter the cell nucleus suspension with a 400-mesh nylon mesh to obtain the cell nucleus filtrate into a disposable 2ml plasti...

Embodiment 2

[0070] This embodiment uses Egyptian blue water lily (Nymphaea caerulea Savigny) as a processing sample, and the preparation method of the flow cytometry sample suitable for water lily provided by this embodiment is described, and the preparation method includes the following steps:

[0071] 1 Take 1 g of Egyptian blue water lily leaves, put them into a petri dish dripped with 1 ml of pre-cooled cell lysis buffer, and cut the water lily leaves into pieces within 1 min with a blade;

[0072] Wherein, the component of cell lysis buffer is:

[0073] 0.13mol / L Tris-HCl, 7mmol / L MgCl 2 , 1mmol / L EDTA-2Na·2H 2 O, 72mmol / L NaCl, 15mmol / L Na 2 S 2 o 5 , 3% (w / v) PVP-10, 0.5% (V / V) Triton X-100, 4mmol / L DTT; pH value is 7.5.

[0074] 2 Add 1ml of pre-cooled cell lysis buffer to the fragments to release the nuclei and obtain a nuclei suspension;

[0075] 3 Filter the cell nucleus suspension with a 500-mesh nylon mesh to obtain the cell nucleus filtrate into a disposable 2ml plastic ...

Embodiment 3

[0081] The present embodiment uses fragrant water lily (Nymphaea odorata Ait) as the processing sample, and the preparation method of the flow cytometry sample applicable to the water lily provided by the present embodiment is described, and the preparation method includes the following steps:

[0082] 1 Take 1g of water lily leaves, put them into a petri dish dripped with 1ml of pre-cooled cell lysis buffer, cut the water lily leaves into pieces within 1min with a blade;

[0083] Wherein, the component of cell lysis buffer is:

[0084] 0.16mol / L Tris-HCl, 13mmol / L MgCl 2 , 2mmol / L EDTA-2Na·2H 2 O, 86mmol / L NaCl, 18mmol / L NaCl 2 S 2 o 5 , 2.6% (w / v) PVP-10, 0.8% (V / V) Triton X-100, 6mmol / L DTT; pH value is 7.5.

[0085] 2 Add 1ml of pre-cooled cell lysis buffer to the fragments to release the nuclei and obtain a nuclei suspension;

[0086] 3 Filter the cell nucleus suspension with a 400-mesh nylon mesh to obtain the cell nucleus filtrate into a disposable 2ml plastic cen...

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Abstract

The invention discloses a preparation method of a flow cytometry water lily sample and a cell lysis buffer solution, and relates to the field of flow cytometry detection. The preparation method of theflow cytometry water lily sample, disclosed by the invention, comprises a lysing step and a staining step, wherein in the lysing step, a water lily tissue sample is lysed, filtered and centrifuged toobtain cell nuclei to be dyed, and in the staining step, the cell nuclei to be dyed are stained to obtain dyed cell nuclei. By the preparation method, the improved cell lysis buffer solution is usedfor treating the water lily tissue sample, and the cell lysis buffer solution can remove residual cytoplasmic fragments from the complete cell nuclei of water lily, maintain the stability of the cellnuclei in a suspension, prevent agglutination, protect DNA from degradation and provide a suitable environment for specific nuclear DNA chemical staining, so that negative effects of cytoplasmic components on staining are effectively reduced.

Description

technical field [0001] The invention relates to the field of flow cytometry detection, in particular to a method for preparing a flow cytometry sample suitable for water lilies and a cell lysis buffer. Background technique [0002] Nymphaeaceae (Nymphaeaceae) Nymphaea plants are perennial herbaceous plants. Its flowers and leaves float on the water surface. The flowers and leaves are gorgeous and graceful. It has high ornamental value. It plays a very important role in water purification. There are more than 50 species of water lily plants in the world, and 5 species are native to China. After long-term artificial hybridization and selection, there have been thousands of water lily species. Determining the genome size of some water lily species with important research value and economic traits can provide valuable reference for future water lily genome sequencing, research on its origin and evolution, and cross-breeding work. Understanding the ploidy of water lily cells is...

Claims

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Application Information

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IPC IPC(8): C12N1/06G01N1/30G01N15/14
Inventor 蒋玮唐雪明吕贝贝吴潇
Owner SHANGHAI ACAD OF AGRI SCI
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