20180416- 20180418: Transfection of NF155 in HEK293 cells + freeze (for WB Native PAGE)

Day 1- 2018.04.16:

• Trypsin-EDTA a plate of HEK293, count cells, and prepare 3 prepare 3 plates of 100mm of diameter (one plate for non-transfected HEK293 cells, and 2 plates for transfected HEK293 cells)
• For every plate: 10 ml of HEK medium + the volume that correspond to 3×10⁶ cells
• Incubate at 37ºC for 24h.

Day 2- 2018.04.17:

[NF155] = 504 ng/microL

• For every plate (100 mm diameter)  –> 13,4 microg of DNA + 20 microL of Lipofectamine. In this case (x2 plates):
  • 54 mcl of DNA in 834 ml of Optimem
  • 40 mcl Lipo in 834 ml of Optimem.
• Incubate at RT for 5 min.
• Unify the DNA solution with the Lipo solution and incubate at RT for 30 min.
• For every plate: 834 microL of the mix DNA-lipo
• Incubate O/N at 37ºC.

Day 3- 2018.04.18:

• Collect cells with a scrapper into a tube (in this case, 2 tubes: transfected and non-transfected cells).
• Centrifuge to pellet de cells. Discart the supernatant and freeze the cells at -80ºC.