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Method for extracting nucleuses of mature leaves of fruit trees suitable for flow cytometry analysis

A technology of flow cytometry and mature leaves, which is applied in the field of cell biology, can solve the problems of flow cytometry limitations and other problems, achieve good detection results, little impact, and maintain the integrity of the effect

Inactive Publication Date: 2012-12-26
CHANGLI INST OF POMOLOGY HEBEI ACADEMY OF AGRI & FORESTRY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The selection of fresh and young materials in fruit tree production and breeding is limited to leaves and test tube seedlings that have just germinated in early spring, so the analysis by flow cytometry is greatly limited

Method used

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  • Method for extracting nucleuses of mature leaves of fruit trees suitable for flow cytometry analysis
  • Method for extracting nucleuses of mature leaves of fruit trees suitable for flow cytometry analysis
  • Method for extracting nucleuses of mature leaves of fruit trees suitable for flow cytometry analysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Take 200mg of mature leaves of Fuji apple in the field, put them into a pre-cooled petri dish, first treat with 5% sodium hypochlorite for 10 minutes, then add 3ml of pre-cooled ether, pour off the ether immediately after 30 seconds, and quickly wash with cold extraction buffer 5 times, grind in a mortar with 2 mL of nucleus extraction buffer, filter through a 500-mesh screen, centrifuge the filtrate at 4°C (2000 rpm) for 5 minutes, rinse 3 times, and collect deposited nuclei. Suspended with 400 μl of staining buffer, placed in a dark place at room temperature, stained for 30 min, and then tested on the machine.

[0031] The components of the cell nucleus extracting solution are: 15mmol / L Tris, 40mmol / L KCl, 20mmol / L NaCl, 5mmol / L Na 2 EDTA, 10mmol / L MgSO 4 , 50mmol / L sucrose and 1.0% (v / v) Triton X-100, add ddH by volume 2 0 to 200ml, after adjusting the pH to 7.5, add 200 μl of 15mmol / L mercaptoethanol.

[0032] The staining buffer used was to add 20 mg / L RNase A a...

Embodiment 2

[0038] Mix 200 mg of mature leaves of Muscat (2X) and Kyoho (4X) grapes in the field, first treat with 8% sodium hypochlorite for 15 minutes, then put it into a pre-cooled petri dish, add 3ml of pre-cooled ether, pour it out immediately after 40 seconds Ethyl ether was quickly washed 3 times with cold extraction buffer, ground in a mortar with 1 mL of extraction buffer, filtered through a 600-mesh sieve, and the filtrate was centrifuged (2000 rpm) at 4 °C for 5 minutes, rinsed 3 times, and collected deposited nuclei. Suspended with 500 μl of staining buffer, placed in a dark place at room temperature, stained for 15 minutes, and then tested on the machine.

[0039] The components of the cell nucleus extracting solution are: 20mmol / L Tris, 60mmol / L KCl, 10mmol / L NaCl, 2mmol / L Na 2 EDTA, 20mmol / L MgSO 4 , 40mmol / L sucrose and 0.5% (v / v) Triton X-100, add ddH by volume 2 O (deionized water) to 200ml, after adjusting the pH to 7.8, add 200μl of 15mmol / L mercaptoethanol.

[004...

Embodiment 3

[0046] Take 200 mg of pineapple leaves in the field, first treat them with 10% sodium hypochlorite for 10 minutes, then put them into a pre-cooled petri dish, add 3ml of pre-cooled ether, pour off the ether immediately after 50 seconds, and quickly wash 3 times with cold extraction buffer , put into a mortar with 2ml of extraction buffer and grind, filter through a 400-mesh sieve, centrifuge the filtrate at 2°C (1500r / min) for 10 minutes, rinse 3 times, and collect the deposited nuclei. Suspended with 600 μl of staining buffer, placed in a dark place at room temperature, stained for 30 minutes, and then tested on the machine. Such as Figure 3a , Figure 3b shown.

[0047] The components of the cell nucleus extracting solution are: 20mmol / L Tris, 40mmol / L KCl, 10mmol / L NaCl, 5mmol / L Na 2 EDTA, 20mmol / L MgSO 4 , 50mmol / L sucrose and 1.0% (v / v) Triton X-100, add ddH by volume 2 O (deionized water) to 200ml, after adjusting the pH to 8.0, add 200μl of 15mmol / L mercaptoethano...

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Abstract

The invention provides a method for extracting nucleuses of mature leaves of fruit trees suitable for flow cytometry analysis. In the method, a nucleus extracting solution comprises the following components: 15 to 20mmol / L Tris, 40 to 60mmol / L KCl, 10 to 20mmol / L NaCl, 2 to 5mmol / L Na2EDTA (ethylene diamine tetraacetic acid), 10 to 20mmol / L MgSO4, 40 to 50mmol / L cane sugar, and 0.5 to 1.0 percent Triton X-100; ddH2O is added until the volume is 200ml; and after the pH is regulated to be 7.5 to 8, 200mu liters of 15mmol / L mercaptoethanol is added. The method has wide ranges of applicable varieties and material species, the prepared nucleuses have high purity, the extraction process needs short time, nucleus degradation is prevented, the flow cytometry analysis is facilitated, and the peak of an acquired flow map is thin and high and little influenced by impurity peaks, so that the DNA (deoxyribonucleic acid) ploidy of a plant to be detected can be easily judged.

Description

technical field [0001] The invention relates to a method for extracting cell nuclei, in particular to a method for extracting cell nuclei from mature leaves of fruit trees suitable for flow cytometric analysis. in the field of cell biology. Background technique [0002] Flow cytometry (flow cytometry, FCM) is a method that uses flow cytometry (flowcytometer) to analyze and measure the DNA content of the nucleus. Flow cytometer is an instrument for flow cytometric analysis. It integrates electronic technology, computer technology, laser technology, and fluid theory. It is a very advanced detection and analysis instrument and is known as the "CT" of the laboratory. This technology can perform multi-parameter, rapid quantitative analysis and sorting of tiny biological particles (cells, nuclei, chromosomes, etc.) in fast flow. In plant genetics and breeding research, it can be used for plant ploidy identification, nuclear DNA quantity (genome size) determination, reproductive ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N1/28G01N1/30G01N33/50
Inventor 吴雅琴程和禾赵艳华吴永杰李玉生
Owner CHANGLI INST OF POMOLOGY HEBEI ACADEMY OF AGRI & FORESTRY SCI
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