Electroporation of 3T3-L1 Adipocytes: Difference between revisions

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==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

*DNA: CsCl Purified or siRNA (resuspended to 0.1 nmol/uL with 200 uL water)

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==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~~

#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

#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

#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 ~~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. 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

#Bring up tube to final required volume, mix and plate

@@ Line 1: / Line 1: @@
 ==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
 *DNA:  CsCl Purified or siRNA (resuspended to 0.1 nmol/uL with 200 uL water)
@@ Line 11: / Line 13: @@
 ==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
+#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
 #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
+#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 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.  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
 #Bring up tube to final required volume, mix and plate