Difference between revisions of "Fugene Transfection of 293T/COS Cells"
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| + | ==Materials== | ||
| + | * Fugene-6 (Roche cat# 11815091001) | ||
| + | * 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 -/-. | ||
#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. | ||
| − | #Add 51 uL | + | #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: Transfection ]] | ||
Latest revision as of 13:22, 29 August 2011
Materials
- Fugene-6 (Roche cat# 11815091001)
- 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.
