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Electrotransfection buffer solution used in cooperation with high-density matrix pin electrodes and preparing method thereof

A buffer and electrotransfection technology, which is applied in the field of electrotransfection buffer and its preparation, can solve the problems of low cell transfection efficiency and high cell death rate, and achieve improved transfection efficiency, permeability and high compatibility Effect

Active Publication Date: 2016-04-06
ETTA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] When trying to use the existing buffer solution and our self-developed high-density matrix needle electrode for electrotransfection, high cell death rate, low cell transfection efficiency, or both phenomena will appear

Method used

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  • Electrotransfection buffer solution used in cooperation with high-density matrix pin electrodes and preparing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Experimental equipment, Suzhou Yida cell electrotransfection instrument model EBXP-H1, Nunc 24-well plate;

[0034]K562, human chronic myelogenous leukemia cells, cultured in RPMI1640 medium (containing 10% GibcoFBS); HL60, human acute myeloid leukemia cells, cultured in RPMI1640 medium (containing 10% GibcoFBS); BHK21, suckling hamster kidney cells, DMEM high glucose Culture medium (containing 10% GibcoFBS);

[0035] UV sterilize the ultra-clean bench for 30 minutes, and at the same time put 1×PBS, DMEM high-glucose medium, 0.25% Trypsin-EDTA in a 37°C water bath for 30 minutes;

[0036] Discard the medium, add an appropriate amount of 1×PBS to wash the cells, add an appropriate amount of 0.25% Trypsin-EDTA to digest the cells after discarding, remove 0.25% Trypsin-EDTA, and digest in a 37°C incubator for 2 minutes; after the digestion is complete, take an appropriate amount of complete Medium was digested and pipetted down to single cells for counting by hemocytomete...

Embodiment 2

[0046] The experimental equipment, Bio-Rad electroporation cup, Nunc 24-well plate, and experimental cells are the same as those in Example 1.

[0047] Digestion: Take the cell culture dish to absorb the culture medium, wash it twice with PBS (3ml each time), add 1ml of trypsin, and digest; digestion stop: add 3ml of culture medium after the digestion to stop the digestion, and pipette several times, as far as possible May cause adherent cells to become suspended;

[0048] Centrifugation: transfer all the samples in step 2 into a centrifuge tube, count at 1000rpm for 5min;

[0049] Resuspension: Remove the original medium from the sample in step 3, replace it with Opti-MEM medium, dilute and resuspend;

[0050] Electroporation: Take 60 μl of the diluted cell culture solution in the Bio-Rad electrode cup, put it into the electrotransfer machine according to the instructions, and operate according to the instructions in the instructions; the electroporation conditions are: the ...

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Abstract

The invention discloses an electrotransfection buffer solution capable of being used in cooperation with high-density matrix pin electrodes. The content of potassium chloride, glucose, triphosadenine and cane sugar in the buffer solution is 10-20 mMol / l, 10-20 mMol / l, 5-6 mMol / l and 350-450 mMol / l respectively. It is indicated through experiments that when electroporation tests are carried out on cells K562, HL60 and BHK 21 by using the buffer solution in cooperation with the high-density matrix pin electrodes, the cell survival rate and electrotransfection efficiency are higher than those of the method of using a Bio-Rad electric rotating cup, and an unexpected technical effect is achieved.

Description

technical field [0001] The invention relates to an electrotransfection buffer and a preparation method thereof, in particular to a buffer for electrotransfection with high-density matrix needle electrodes and a preparation method thereof. Background technique [0002] Electrotransfection is a method of mechanically introducing polar molecules into host cells through the cell membrane. (such as DNA, etc.) into the cell and eventually into the nucleus of the technology. [0003] The process of electrotransfection is briefly described as follows: during the electroporation process, the cell membrane is perforated, and the plasmid contacts the cell membrane under the action of electrophoretic force, and forms a transferable complex with the electroporated area on the cell membrane. The transient voltage causes the plasmid to break away from the complex and diffuse into the cytoplasm, while a small part of the plasmid enters the nucleus and integrates with the chromosome, and th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/87
CPCC12N15/87
Inventor 汪莹戴晓兵
Owner ETTA BIOTECH