2018.11.28
- Prepare 2 plates of 60mm of diameter with 14 coverslips of 12mm.
- Treatment with Poly-D-Lysine (diluted 1/40 with boratte buffer)
- Incubate for 1h (37ºC, 5% CO2).
- Wash with PBS
- Trypsin-EDTA a plate of HEK293.
- For every plate of 60mm: 5ml of HEK medium + the volume that correspond to 1.000.000 of cells.
- Incubate at 37ºC for 24h.
2018.11.29
- Prepare the DNA and lipofectamine:
- [AXL] = 0,79 µg/µL. 8 µg = 10,2 µL + 250 µL Optimem
- [ALCAM] = 0,31 µg/µL. 8 µg = 25,8 µL + 250 µL Optimem
- 24 µL of Lipofectamine + 0,5 ml Optimem
- Incubate at RT for 5 min.
- Unify every DNA with the appropriate amount of Lipo and incubate at RT for 30 min.
- For every plate: 500microL of every mix of DNA and Lipo.
- Incubate O/N at 37ºC.
2018.11.30
- Fix with PFA 4%. Incubation at RT during 10min.
- Wash with PBS 1x.
- Blocking solution: 5% goat serum in PBS for 1h at RT.
- Remove the blocking solution and freeze at -80ºC.
Check the transfection – ICC:
- Blocking solution: 5% goat serum in PBS
- Ac anti-cmyc 1/100
- GAM488 1/500
- Vectashield mounting medium
RESULT: both AXL and ALCAM are well transfected.
