Difference between revisions of "Fugene Transfection of 293T/COS Cells"

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==Materials==
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* Fugene-6 (Roche cat# 11815091001)
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* OptiMEM or DMEM without serum
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* Cells (log-phase growing)
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* DNA - typically transfect 50-1000 ng DNA per well
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==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.
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##Per ug of DNA need 3 uL Fugene.
*Per uL of Fugene need 16 uL of OptiMEM.
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##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/ug OptiMEM/Fugene to each DNA tube.
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#Add 51 uL OptiMEM/Fugene per ug DNA to each DNA tube.
#Incubat 5-20 min.
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#Incubate 5-20 min. Split cells while waiting
#Split confluent cells 2-3X into fresh dishes as follows:
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#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 -/-
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##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
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##Add 1 mL Trypsin solution (0.05%) and incubate at 37C until cells are detached
#Add 25 mL COS/FBS
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##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)
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##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
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#Add DNA/Fugene/DMEM to cells.
#Leave mixture on Cells for 24-48h to allow protein to accumulate
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#Leave mixture on Cells for 24-48h to allow protein to accumulate.
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[[ Category: Cell Culture ]]
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[[ 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

  1. Warm OptiMEM, COS-FBS Media and PBS -/-.
  2. Calculate amount of Fugene needed.
    1. Per ug of DNA need 3 uL Fugene.
    2. Per uL of Fugene need 16 uL of OptiMEM.
  3. Add Fugene to OptiMEM, incubate ~5 min.
  4. Add required amount of DNA to eppendorf tubes.
  5. Add 51 uL OptiMEM/Fugene per ug DNA to each DNA tube.
  6. Incubate 5-20 min. Split cells while waiting
  7. 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:
    1. Wash confluent COS cell plate(s) twice with 10 mL D-PBS -/-
    2. Add 1 mL Trypsin solution (0.05%) and incubate at 37C until cells are detached
    3. Add 24-48 mL COS/FBS depending on density (24 mL to split 1:2, 48 mL to split 1:4)
    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.
  8. Add DNA/Fugene/DMEM to cells.
  9. Leave mixture on Cells for 24-48h to allow protein to accumulate.