AMSBIO LLC
GenePORTER® Transfection Reagent
T201015
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Aim of the present study was to over express dominant negative protein kinase in rat pancreatic beta cells by transecting pCMV vector based over expression DNA plasmid.
Transfection of pCMV vector based protein over expression plasmid in rat cells.
Rat pancreatic beta cell culture, GenePORTER® Transfection Reagent, Serum free media, FBS, CO2 incubator etc.
The day before transfection, cells were seeded to 60 mm culture dish so that next day it will be up 70% confluent. On the day of transfection 0.5 micro gram of plasmid DNA was mixed with serum free medium using half of the transfection volume. After that GenePORTER® transfection reagent of was added to diluted DNA, mixed rapidly and incubated at room temperature for 30 minutes. Next, cell culture medium from 60 mm culture dish was aspirated and DNA/GenePORTER reagent mix was gently added to the cells and incubated at 37 degree Celsius for 4 hours. After incubation, transfection medium was replaced with complete medium and kept in incubator for 36 hours.
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After 36 hours of incubation, cells were examined in fluorescent microscope for the presence of green fluorescence as a result of over expression of GFP labeled kinase dead protein. Almost, 98% of examined cells showed green fluorescence as a result of efficient transfection of expogenous DNA plasmid by GenePORTER® Transfection Reagent.
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