Objective: to test the doble transfection of FLOT1 AND FLOT2.
Day 1- 2018.08.20:
- Prepare 4 plates of 60mm of diameter with 14 coverslips of 12mm.
- Treatment with Poly-D-Lysina (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.
Day 2- 2018.08.21:
[FLOT1] = 0,48 µg/µL. [FLOT2= 0,43 µg/µL.
4 conditions are made (1 plate/condition):
- A) 4 µg FLOT1 (8,3 µL) + 4 µg FLOT2 (9,2 µL)
- B) 5 µg FLOT1 (10,4 µL) + 5 µg FLOT2 (11,5 µL)
- C) 3 µg FLOT1 (6,3 µL) + 5 µg FLOT2 (11,5 µL)
- D) 5 µg FLOT1 (10,4 µL) + 3 µg FLOT2 (6,9 µL)
*For every plate of 60mm of diameter –> 12 microL of Lipofectamine.
- 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: 5ml of HEK medium and 500microL of every mix of DNA and Lipo.
- Incubate O/N at 37ºC.
Day 3- 2018.08.22:
- 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: 2 coverslips of every plate.
- Blocking solution: 5% goat serum in PBS
- Primary antibody:
- FLOT1: diluted 1/100
- FLOT2: diluted 1/200
- Secondary antibody: GAR488 diluted 1/1000
- Vectashield mounting medium
RESULT: in the 4 conditions the FLOT1 is less transfected than the FLOT2 (there are few FLOT1 transfected cells). Maybe it’s necessary to put more FLOT1 DNA in the following transfections.
