2018.11.05
- Prepare 6 plates of 60mm of diameter with 26 coverslips of 9mm.
- 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.
2018.11.06
- Prepare the DNA and lipofectamine: [FLOT1] = 0,48 µg/µL. [FLOT2]= 0,43 µg/µL. Per cada placa –> 6 µg FLOT1 + 3 µg FLOT2
- 36 µg FLOT1 (75 µL) + 18 µg FLOT2 (42 µL) + 1,5 ml Optimem
- 72 µL of Lipofectamine + 1,5 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.
2018.11.08 (48 hours later)
- 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-FLOT1: diluted1/50
- GAR 488 IgM diluted 1/1000
- Ac anti-FLOT2 biotinilated: diluted 1/25
- Streptavidine 594 diluted 1/400
- Vectashield mounting medium
RESULT: both FLOT1 and FLOT2 are transfected, but there are some dead cells. Maybe it’s better doing the transfection during 36 hours instead of 48 hours.
