Fugene Transfection of 293T/COS Cells - Revision history

Davebrid: added catalog number

2011-08-29T13:22:01Z

added catalog number

← Older revision		Revision as of 13:22, 29 August 2011
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	==Materials==		==Materials==
	* Fugene-6(Roche cat#)		* Fugene-6 (Roche cat# 11815091001)
	* OptiMEM or DMEM without serum		* OptiMEM or DMEM without serum
	* Cells (log-phase growing)		* Cells (log-phase growing)

Davebrid: updated protocol

2011-08-29T13:21:31Z

updated protocol

← Older revision		Revision as of 13:21, 29 August 2011
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			==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 -/-.
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	#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.
	#~~Incubat~~ 5-20 min.		#Incubate 5-20 min. Split cells while waiting
	#Split confluent cells 2~~-3X into fresh dishes as follows~~:		#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 25 mL COS/FBS		##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 ]]

Davebrid at 15:28, 15 July 2009

2009-07-15T15:28:36Z

← Older revision		Revision as of 15:28, 15 July 2009
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	#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 ]]

Jpecherer: /* Protocol */

2009-06-10T14:07:43Z

Protocol

← Older revision		Revision as of 14:07, 10 June 2009
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	#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.		#Add 51 uL OptiMEM/Fugene per ug DNA to each DNA tube.
	#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:

Davebrid at 14:35, 8 June 2009

2009-06-08T14:35:34Z

← Older revision		Revision as of 14:35, 8 June 2009
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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 ug of DNA need 3 uL Fugene.
	*Per uL of Fugene need 16 uL of OptiMEM.		##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

Davebrid: wrote initial page

2009-06-08T14:34:18Z

wrote initial page

New page

==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