Day 1- 2018.01.22:
- Prepare 7 plates of 60mm of diameter with 12 coverslips of 12mm.
- Poly-D-Lysina (diluted 1/40 with PBS 1x)
- Incubate for 1h (37ºC, 5% CO2).
- Wash with PBS 1x.
- Trypsin-EDTA a plate of HEK293 (previously defreezed).
- Manual counting: 4.475×10^6 cells/ml.
- For every plate of 60mm: 5ml of HEK medium + the volume that correspond to 1.000.000 of cells: 223microL.
- For every maintenance plate (x2): 10ml of HEK medium + the volume that correspond to 1.500.000 of cells: 335microL.
- Incubate at 37ºC for 24h.
Day 2- 2018.01.23:
CONCENTRATION OF PLASMIDS:
- CNTN2 (2016/06/09): [ ]=500.6ng/ml
- CASPR2: [ ]=500ng/ml
- GDAP1L1 col. 2: [ ]=298.5ng/ml
- GDAP1L1 col. 15: [ ]=299.5ng/ml
For every plate of 60mm of diameter –> 8 microg of DNA + 12 microL of Lipofectamine.
- CNTN2: 1 plate
- 15.98microL pf DNA in 250microL of Optimem.
- 12microL of Lipo in 250microL of Optimem.
- CASPR2: 1 plate
- 16microL of DNA in 250microL of Optimem.
- 12microL of Lipo in 250microL of Optimem.
- GDAP1L1 col. 2: 2 plates
- 53.6microL of DNA in 500microL of Optimem.
- 24microL of Lipo in 500microL of Optimem.
- GDAP1L1 col. 15: 2 plates
- 53.42microL of DNA in 500microL of Optimem.
- 24microL of Lipo in 500microL of Optimem.
*We are going to leave 1 plate with HEK293 cells without any transfection to perform another experiment.
– Incubate at RT for 5 min.
– Unify every DNA with the aproppriate 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, 5% CO2.
Day 3- 2018.01.24:
- Fix with PFA 4%. Incubation at RT during 10min.
- Wash with PBS 1x.
- PERMEATION with triton 0.3% (5min): Only for CASPR2, 1 plate of GDAP1L1 col. 2 and 1 plate of GDAP1L1 col. 15.
- Wash with PBS.
- Blocking solution -> 5% goat serum in PBS for 1h at RT.
- Remove the blocking solution and freeze at -80ºC.
Check the transfection – ICC:
- For CNTN2:
- HEKs 293 Cells Transfection: 2018.01.23
- Blocking solution: 5% goat serum in PBS
- Primary antibody: Rabbit CNTN2 diluted 1/200
- Secondary antibody: GAR488 diluted 1/500
- Vectashield mounting medium
- For CASPR2:
- HEKs 293 Cells Transfection: 2018.01.23
- Blocking solution: 5% goat serum in PBS
- Primary antibody: Rabbit CASPR2 diluted 1/500
- Secondary antibody: GAR488 diluted 1/500
- Vectashield mounting medium
- For GDAP1L1: 2 conditions – permeated HEK293 cells and non-permeated HEK293 cells.
- HEKs 293 Cells Transfection: 2018.01.23
- Blocking solution: 5% goat serum in PBS
- Primary antibody: Mouse GDAP1L1 diluted 1/100
- Secondary antibody: GAM488 diluted 1/500
- Vectashield mounting medium
Results: All samples were well transfected. We found the plasmid GDAP1L1 in both colonies (number 2 and 15) in permeated and non-permeated conditions. We visualized the cells better in permeated conditions.
