Fugene Transfection of 293T/COS Cells: Difference between revisions
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#Warm OptiMEM, COS-FBS Media and PBS -/-. | #Warm OptiMEM, COS-FBS Media and PBS -/-. | ||
#Calculate amount of Fugene needed. | #Calculate amount of Fugene needed. | ||
##Per ug of DNA need 3 uL Fugene. | |||
##Per uL of Fugene need 16 uL of OptiMEM. | |||
#Add Fugene to OptiMEM, incubate ~5 min. | #Add Fugene to OptiMEM, incubate ~5 min. | ||
#Add required amount of DNA to eppendorf tubes. | #Add required amount of DNA to eppendorf tubes. | ||
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#Incubat 5-20 min. | #Incubat 5-20 min. | ||
#Split confluent cells 2-3X into fresh dishes as follows: | #Split confluent cells 2-3X into fresh dishes as follows: | ||
#Wash confluent COS cell plate(s) twice with 10 mL D-PBS -/- | ##Wash confluent COS cell plate(s) twice with 10 mL D-PBS -/- | ||
#Add 1 mL Trypsin solution (0.05%) and incubate at 37C until cells are detached | ##Add 1 mL Trypsin solution (0.05%) and incubate at 37C until cells are detached | ||
#Add 25 mL COS/FBS | ##Add 25 mL COS/FBS | ||
#Aliquot cells into wells as required (1 mL per 12well, 2 mL per 6 well) | ##Aliquot cells into wells as required (1 mL per 12well, 2 mL per 6 well) | ||
#Add DNA/Fugene/DMEM to cells | #Add DNA/Fugene/DMEM to cells | ||
#Leave mixture on Cells for 24-48h to allow protein to accumulate | #Leave mixture on Cells for 24-48h to allow protein to accumulate | ||