Electroporation of 3T3-L1 Adipocytes - Revision history

Davebrid: changed default siRNA to 5 uL (500 pmoles) and made some extra notes

2010-02-17T18:45:23Z

changed default siRNA to 5 uL (500 pmoles) and made some extra notes

← Older revision		Revision as of 18:45, 17 February 2010
Line 1:		Line 1:
	==Materials==		==Materials==
	*Differentiated cells FBS day 3 or less.		*Differentiated cells FBS day 3 or less. If cells are difficult to trypsinize, they will not recover well.
			**To calculate the number of cells, use 1 - 15cm dish per final dish (10cm/6well/12well).
			**For example for 2 knockdowns, each being seeded in 6 - 12 well sized wells, use 1 dish, and 2 electroporations.
	*Gene Pulser cuvette		*Gene Pulser cuvette
	*DNA: CsCl Purified or siRNA (resuspended to 0.1 nmol/uL with 200 uL water)		*DNA: CsCl Purified or siRNA (resuspended to 0.1 nmol/uL with 200 uL water)
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	==Protocol==		==Protocol==
	#Warm media and PBS but not trypsin in water bath		#Warm media and PBS but not trypsin in water bath
	#Wash cells twice with PBS (-/-), then trypsinize and with 2 mL 0.25% trypsin in the ~~incubate~~		#Wash cells twice with PBS (-/-), then trypsinize and with 2 mL 0.25% trypsin in the incubator
	#When cells have fallen off the plate, add 9 mL Media and pipet into a 15 mL falcon tube		#When cells have fallen off the plate, add 9 mL Media and pipet into a 15 mL falcon tube
	#Spin at 2000 RPM for 5 min.		#Spin at 2000 RPM for 5 min.
	#Wash cells with PBS +/+		#Wash cells with PBS +/+
	#Reuspend cells to a final volume of 0.5 mL per electroporation in PBS +/+		#Reuspend cells to a final volume of 0.5 mL per electroporation in PBS +/+
	#500 uL of cells is mixed with 50-200 ug of DNA (or 10 uL siRNA) in a 0.4 cm cuvette		#500 uL of cells is mixed with 50-200 ug of DNA (or 5 uL siRNA) in a 0.4 cm cuvette. These volumes are for half of a final plate of cells. Scale up or down accordingly.
	#Electroporate at 160V and 950 uF		#Electroporate at 160V and 950 uF
	#Add 1 mL media to cuvette and transfer to a 15 mL tube with media for 10 min.		#Add 1 mL media to cuvette and transfer to a 15 mL tube with media for 10 min. The amount of media should correspond to the final volume of plated cells (ie one electroporation of half a plate of cells would go into 6 mL final volume of media).
	#Aspirate floating debris before replating		#Aspirate floating debris before replating
	#Bring up tube to final required volume, mix and plate		#Bring up tube to final required volume, mix and plate

Davebrid: changed voltage to 160V

2009-06-07T21:32:21Z

changed voltage to 160V

← Older revision		Revision as of 21:32, 7 June 2009
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	#Reuspend cells to a final volume of 0.5 mL per electroporation in PBS +/+		#Reuspend cells to a final volume of 0.5 mL per electroporation in PBS +/+
	#500 uL of cells is mixed with 50-200 ug of DNA (or 10 uL siRNA) in a 0.4 cm cuvette		#500 uL of cells is mixed with 50-200 ug of DNA (or 10 uL siRNA) in a 0.4 cm cuvette
	#Electroporate at ~~260V~~ and 950 uF		#Electroporate at 160V and 950 uF
	#Add 1 mL media to cuvette and transfer to a 15 mL tube with media for 10 min.		#Add 1 mL media to cuvette and transfer to a 15 mL tube with media for 10 min.
	#Aspirate floating debris before replating		#Aspirate floating debris before replating
	#Bring up tube to final required volume, mix and plate		#Bring up tube to final required volume, mix and plate

Davebrid at 17:41, 7 May 2009

2009-05-07T17:41:45Z

← Older revision		Revision as of 17:41, 7 May 2009
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	#Wash cells with PBS +/+		#Wash cells with PBS +/+
	#Reuspend cells to a final volume of 0.5 mL per electroporation in PBS +/+		#Reuspend cells to a final volume of 0.5 mL per electroporation in PBS +/+
	#500 uL of cells is mixed with 50-200 ug of DNA in a 0.4 cm cuvette		#500 uL of cells is mixed with 50-200 ug of DNA (or 10 uL siRNA) in a 0.4 cm cuvette
	#Electroporate at 260V and 950 uF		#Electroporate at 260V and 950 uF
	#Add 1 mL media to cuvette and transfer to a 15 mL tube with media for 10 min.		#Add 1 mL media to cuvette and transfer to a 15 mL tube with media for 10 min.
	#Aspirate floating debris before replating		#Aspirate floating debris before replating
	#Bring up tube to final required volume, mix and plate		#Bring up tube to final required volume, mix and plate

Davebrid: Created page with '==Materials== Differentiated cells FBS day 3 or less. Gene Pulser cuvette DNA: CsCl Purified or siRNA (resuspended to 0.1 nmol/uL with 200 uL water) PBS +/+ PBS -/- 0.25% ...'

2009-05-07T17:39:05Z

Created page with '==Materials== *Differentiated cells FBS day 3 or less. *Gene Pulser cuvette *DNA: CsCl Purified or siRNA (resuspended to 0.1 nmol/uL with 200 uL water) *PBS +/+ *PBS -/- *0.25% ...'

New page

==Materials==
*Differentiated cells FBS day 3 or less.
*Gene Pulser cuvette
*DNA: CsCl Purified or siRNA (resuspended to 0.1 nmol/uL with 200 uL water)
*PBS +/+
*PBS -/-
*0.25% Trypsin
*L1 FBS Media

==Protocol==
#Warm media and PBS but not trypsin in water bath
#Wash cells twice with PBS (-/-), then trypsinize and with 2 mL 0.25% trypsin in the incubate
#When cells have fallen off the plate, add 9 mL Media and pipet into a 15 mL falcon tube
#Spin at 2000 RPM for 5 min.
#Wash cells with PBS +/+
#Reuspend cells to a final volume of 0.5 mL per electroporation in PBS +/+
#500 uL of cells is mixed with 50-200 ug of DNA in a 0.4 cm cuvette
#Electroporate at 260V and 950 uF
#Add 1 mL media to cuvette and transfer to a 15 mL tube with media for 10 min.
#Aspirate floating debris before replating
#Bring up tube to final required volume, mix and plate

Electroporation of 3T3-L1 Adipocytes - Revision history

Davebrid: changed default siRNA to 5 uL (500 pmoles) and made some extra notes

Davebrid: changed voltage to 160V

Davebrid at 17:41, 7 May 2009

Davebrid: Created page with '==Materials== *Differentiated cells FBS day 3 or less. *Gene Pulser cuvette *DNA: CsCl Purified or siRNA (resuspended to 0.1 nmol/uL with 200 uL water) *PBS +/+ *PBS -/- *0.25% ...'

Davebrid: Created page with '==Materials== Differentiated cells FBS day 3 or less. Gene Pulser cuvette DNA: CsCl Purified or siRNA (resuspended to 0.1 nmol/uL with 200 uL water) PBS +/+ PBS -/- 0.25% ...'