Electroporation of 3T3-L1 Adipocytes

Materials

• 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)
• 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 incubator
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 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.
8. Electroporate at 160V and 950 uF
9. 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).
10. Aspirate floating debris before replating
11. Bring up tube to final required volume, mix and plate