Electroporation of LNCaP Cells
Dominik Haudenschild June 24, 2002

Procedure

1. Grow cells to approximately 80% confluency in complete media
   
2. Harvest cells by trypsinization, centrifuge 1500g for 5 minutes
   
3. Wash cells by resuspending in 10 ml media without serum
   
4. Remove 50µl cells to count, meanwhile centrifuge 1500g for 5 minutes
   
5. Resuspend cells at 8,000,000 cells/ml (8e6) in media without serum
   
6. Pipet 500µl cells to 0.4cm cuvette
   
7. Add 20µg DNA in 20µl, tap gently to mix
   
8. Incubate on ice for 10 minutes
   
9. Electroporate (325 Volts, 1000µF, infinite resistance)
   
10. Incubate at RT for 10 minutes
    
11. Add 1ml complete media (10%FBS) to cells and gently aspirate
    
12. Pipet cells to 100mm dish with 9ml complete media
    
13. 48 hours later, begin selection with 3µg/ml Blasticidin in complete media

Materials

1. 20µg DNA at 1mg/ml per electroporation, in TE, 10mM Tris, or Water
   
2. 4 million healthy and growing cells per electroporation
   
3. Complete media: RPMI-1640 with Pen/Strep/Glutamine and 10% FBS
   
4. RPMI-1640 Media without Pen/Strep/Glutamine, NO FBS
   
5. 0.4cm electroporation cuvettes