Fugene Transfection of 293T/COS Cells: Difference between revisions
Jump to navigation
Jump to search
Davebridges (talk | contribs) mNo edit summary |
Davebridges (talk | contribs) updated protocol |
||
| Line 1: | Line 1: | ||
==Materials== | |||
* Fugene-6(Roche cat#) | |||
* OptiMEM or DMEM without serum | |||
* Cells (log-phase growing) | |||
* DNA - typically transfect 50-1000 ng DNA per well | |||
==Protocol== | ==Protocol== | ||
#Warm OptiMEM, COS-FBS Media and PBS -/-. | #Warm OptiMEM, COS-FBS Media and PBS -/-. | ||
| Line 7: | Line 13: | ||
#Add required amount of DNA to eppendorf tubes. | #Add required amount of DNA to eppendorf tubes. | ||
#Add 51 uL OptiMEM/Fugene per ug DNA to each DNA tube. | #Add 51 uL OptiMEM/Fugene per ug DNA to each DNA tube. | ||
# | #Incubate 5-20 min. Split cells while waiting | ||
#Split confluent cells 2 | #Split cells to desired density. Cells double about in ~24h so for confluent cells the next days split 1:2. For immunofluoresence split 1:4 from a confluent dish. If cells are subconfluent split to a higher density: | ||
##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 | ##Add 24-48 mL COS/FBS depending on density (24 mL to split 1:2, 48 mL to split 1:4) | ||
##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). Place sterile coverslips in wells if immunofluoresence is required. | ||
#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. | ||
[[ Category:Cell Culture ]] | [[ Category: Cell Culture ]] | ||
[[ Category: Transfection ]] | |||
Revision as of 13:21, 29 August 2011
Materials
- Fugene-6(Roche cat#)
- OptiMEM or DMEM without serum
- Cells (log-phase growing)
- DNA - typically transfect 50-1000 ng DNA per well
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 OptiMEM/Fugene per ug DNA to each DNA tube.
- Incubate 5-20 min. Split cells while waiting
- Split cells to desired density. Cells double about in ~24h so for confluent cells the next days split 1:2. For immunofluoresence split 1:4 from a confluent dish. If cells are subconfluent split to a higher density:
- 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 24-48 mL COS/FBS depending on density (24 mL to split 1:2, 48 mL to split 1:4)
- Aliquot cells into wells as required (1 mL per 12well, 2 mL per 6 well). Place sterile coverslips in wells if immunofluoresence is required.
- Add DNA/Fugene/DMEM to cells.
- Leave mixture on Cells for 24-48h to allow protein to accumulate.