Day 1- 2018.01.23:
- Prepare 7 plates of 60mm of diameter with 12 coverslips of 12mm.
- Treatment with 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.
- Manual counting: 7.05×10^6 cells/ml.
- For every plate of 60mm: 5ml of HEK medium + the volume that correspond to 1.000.000 of cells: 141.84microL.
- Incubate at 37ºC for 24h.
Day 2- 2018.01.24:
Based on the results of the previous experiment ICC GDAP1L1 with colonies 2 and 15 and in permeated and non-permeated conditions, we select the col. number 15 and we are going to perform this experiment with permeated cells.
CONCENTRATION OF PLASMID GDAP1L1 col. 15 [ ]=0.2995 microg/microL.
*For every plate of 60mm of diameter –> 8 microg of DNA + 12 microL of Lipofectamine.
- GDAP1L1: 7 plates
- 187 microL of DNA in 1.75 mL of Optimem.
- 84microL of Lipo in 1.75mL of 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: 5ml of HEK medium and 500microL of every mix of DNA and Lipo.
– Incubate O/N at 37ºC.
Day 3- 2018.01.25:
- Fix with PFA 4%. Incubation at RT during 10min.
- Wash with PBS 1x.
- PERMEATION with triton 0.3% (5min)
- Wash with PBS 1x.
- Blocking solution -> 5% goat serum in PBS for 1h at RT.
- Remove the blocking solution and freeze at -80ºC.
Day 4 – 2018.01.26: Check the GDAP1L1 transfection – ICC:
- Blocking solution: 5% goat serum in PBS
- Primary antibody: Mouse GDAP1L1 diluted 1/200
- Secondary antibody: GAM488 diluted 1/500
- Vectashield mounting medium
Results: The samples were well transfected.
