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Griffin:siRNA transfection: Difference between revisions
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Verify the transfection efficiency with the use of a fluorescent dye-labeled siRNA; [http://datasheets.scbt.com/sc-36869.pdf Control siRNA (FITC Conjugate)-A: sc-36869] (FITC, CY5, etc.). A flow cytometer can be used to measure cell transfection efficiency. The transfection efficiency must be measured 5-7 hours after cells are first exposed to the transfection reagent. This is important as the siRNA reagent complex with the siRNA will maximal enter the cell during this time frame. After 5-7 hours, the cells could begin to degrade or exotise the control siRNA-FITC conjugate and this will. When transfecting adherent cells, 5-7 hours after transfection, wash the slide then fix and mount the cells prior to visualization under a fluorescent scope. | Verify the transfection efficiency with the use of a fluorescent dye-labeled siRNA; [http://datasheets.scbt.com/sc-36869.pdf Control siRNA (FITC Conjugate)-A: sc-36869] (FITC, CY5, etc.). A flow cytometer can be used to measure cell transfection efficiency. The transfection efficiency must be measured 5-7 hours after cells are first exposed to the transfection reagent. This is important as the siRNA reagent complex with the siRNA will maximal enter the cell during this time frame. After 5-7 hours, the cells could begin to degrade or exotise the control siRNA-FITC conjugate and this will. When transfecting adherent cells, 5-7 hours after transfection, wash the slide then fix and mount the cells prior to visualization under a fluorescent scope. | ||
'''1)''' In a six well tissue culture plate, seed | '''1)''' In a six well tissue culture plate, seed 2e5 cells per well in 2 ml antibiotic-free normal growth medium supplemented with FBS. | ||
'''2)''' Incubate the cells at 37° C in a CO2 incubator until the cells are 50 - 80% confluent. This will usually take 18-24 hours. | '''2)''' Incubate the cells at 37° C in a CO2 incubator until the cells are 50 - 80% confluent. This will usually take 18-24 hours. | ||
