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A method for transfecting exogenous nucleic acid using atmospheric pressure cooled plasma jet

A technology of cold plasma and exogenous nucleic acid is applied in the field of plasma jet transfection of exogenous nucleic acid and exogenous nucleic acid, which can solve the problems of low transfection efficiency, high cell death rate, unstable transfection, etc. To achieve the effect of convenient transfection, short transfection time and convenient operation

Inactive Publication Date: 2017-04-26
XI AN JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the transfection is unstable and the transfection efficiency is not high
2. The calcium phosphate co-precipitation method is widely used in the research of cell transfection because the reagents are easy to obtain and cheap. First, DNA and CaCl 2 Mix, then add to PBS to slowly form a DNA calcium phosphate precipitate, and finally add the suspension containing the precipitate to the cultured cells, and absorb DNA through the endocytosis of the cell membrane. The disadvantage of this method is also transfection low efficiency
The efficiency of introduction is related to the intensity and duration of the pulse. The disadvantage of this method is that the cell death rate is high, the transfection instrument is not easy to operate, the solution preparation and setting parameters are complicated, etc.

Method used

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  • A method for transfecting exogenous nucleic acid using atmospheric pressure cooled plasma jet
  • A method for transfecting exogenous nucleic acid using atmospheric pressure cooled plasma jet
  • A method for transfecting exogenous nucleic acid using atmospheric pressure cooled plasma jet

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Experimental program
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Effect test

Embodiment 1

[0031] After the myeloma cells LP cells were changed the day before, the cells were counted and planted in a 24-well plate at a density of 5×10 5 For each well, the culture medium was RPMI1640, and 300uL of culture medium was added to each well as cells to be transfected.

[0032] Set up the plasma jet source, connect the positive and negative electrodes, and connect the gas flow meter. The bottom of the quartz tube is 1.5cm away from the cell liquid surface. Turn on the Ar gas, select the gas flow rate as 2SLM, and wait for the gas flow to stabilize. Turn on the oscilloscope and high voltage power supply. Adjust the power supply frequency to 10kHz, slowly turn the high voltage knob, observe the power supply voltage display on the oscilloscope, stop the knob until the voltage stabilizes at 10kv, and fix the voltage. At this time, a blue-purple plasma flow will blow out from the bottom of the quartz tube.

[0033] After waiting for one minute for the plasma to stabilize, you ...

Embodiment 2

[0035] Other operations of this embodiment are the same as those in Embodiment 1, the difference is that other auxiliary gases can also be added when the basic gas is introduced, and when the Ar gas is open, oxygen, nitrogen or water vapor and other gases can be added as Auxiliary gas, adjust the respective auxiliary gas flow rate 20sslm.

[0036] Leave it for 1 minute until the gas is fully mixed and stabilized, turn on the high voltage power supply to 10kv in the original way, wait for 1 minute and then the plasma jet is stable, place the cells to be transfected under the jet source, and after 20-60 seconds, add After the exogenous nucleic acid is transfected, mix it gently and put it into the incubator to continue culturing to complete the transfection process.

[0037] Specific experimental verification:

[0038] 1. Detection of transfection effect by fluorescence microscope

[0039] After the LP cells were changed the day before, the cells were counted and planted in a ...

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Abstract

The invention discloses a method for transfecting an exogenous nucleic acid by use of atmospheric pressure cold plasma jet and belongs to the technical field of biology. The method comprises the following steps: 1) counting to-be-transfected cells, planting the cells in a porous cell culture plate, and adding a culture solution to obtain a to-be-transfected cell solution; 2) adjusting a plasma jet source device, introducing a basic gas, applying sinusoidal high voltage after the gas flow is stable, and keeping the voltage constant until bluish violet plasma jet is blown out from the bottom of a quartz tube; 3) treating the to-be-transfected cells for 5-60 seconds directly under the plasma jet source, moving away the cells and adding the to-be-transfected exogenous nucleic acid, mixing evenly and then putting the mixed solution back into an incubator for continuous culture. The method for transfecting the exogenous nucleic acid by use of the atmospheric pressure cold plasma jet is capable of conveniently transfecting the exogenous nucleic acid; after transfection, the cell mortality rate is relatively lower than those of methods by use of lipidosome, electrotransfection and the like. Besides, the transfection time is very short and the transfection can be completed within dozens of seconds.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for transfecting exogenous nucleic acid, in particular to a method for transfecting exogenous nucleic acid by using atmospheric pressure cooling plasma jet. Background technique [0002] At present, there are several methods for transfecting nucleic acids such as exogenous DNA into cells: [0003] 1. DEAE-dextran is one of the earliest transfection reagents used in mammalian cells. DEAE-dextran is a cationic polymer, which is absorbed by the cell membrane after binding to negatively charged nucleic acids. However, the transfection is unstable and the transfection efficiency is not high. 2. The calcium phosphate co-precipitation method is widely used in the research of cell transfection because the reagents are easy to obtain and cheap. First, DNA and CaCl 2 Mix, then add to PBS to slowly form a DNA calcium phosphate precipitate, and finally add the suspension containing the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/87
Inventor 许德晖杨延洁王碧菁孔刚玉
Owner XI AN JIAOTONG UNIV
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