Electroporation of 3T3-L1 Adipocytes: Difference between revisions
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Davebridges (talk | contribs) 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% ...' |
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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 | ||
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
- 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 (or 10 uL siRNA) 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