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. | ||
| Line 9: | Line 9: | ||
#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 | ||
Revision as of 14:35, 8 June 2009
Protocol
- Warm OptiMEM, COS-FBS Media and PBS -/-.
- 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 required amount of DNA to eppendorf tubes.
- Add 51 uL/ug OptiMEM/Fugene to each DNA tube.
- Incubat 5-20 min.
- Split confluent cells 2-3X into fresh dishes as follows:
- 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 25 mL COS/FBS
- Aliquot cells into wells as required (1 mL per 12well, 2 mL per 6 well)
- Add DNA/Fugene/DMEM to cells
- Leave mixture on Cells for 24-48h to allow protein to accumulate