Difference between revisions of "Electroporation of 3T3-L1 Adipocytes"

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(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% ...')
 
Line 16: Line 16:
 
#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

Revision as of 17:41, 7 May 2009

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

  1. Warm media and PBS but not trypsin in water bath
  2. Wash cells twice with PBS (-/-), then trypsinize and with 2 mL 0.25% trypsin in the incubate
  3. When cells have fallen off the plate, add 9 mL Media and pipet into a 15 mL falcon tube
  4. Spin at 2000 RPM for 5 min.
  5. Wash cells with PBS +/+
  6. Reuspend cells to a final volume of 0.5 mL per electroporation in PBS +/+
  7. 500 uL of cells is mixed with 50-200 ug of DNA (or 10 uL siRNA) in a 0.4 cm cuvette
  8. Electroporate at 260V and 950 uF
  9. Add 1 mL media to cuvette and transfer to a 15 mL tube with media for 10 min.
  10. Aspirate floating debris before replating
  11. Bring up tube to final required volume, mix and plate