20181219-21: Coating and transfection for ANO2 (for ICC and for WB)

2018.12.19:

• Prepare:
  • 1 60mm-plate with 26 coverslips of 9mm.
  • 2 100mm-plates (without coverslips)
• 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 million cells.
  • 100mm: 8 ml of HEK medium + the volume that corespond to 3 million cells.
• Incubate at 37ºC for 24h.

2018.12.20

• Prepare the DNA and lipofectamine:
  • 60mm plate:
    • DNA: 8 µg = 6,9 µL + 250 µL Optimem
    • Lipofectamine: 12 µL + 250 µL Optimem
  • 100mm plates:
    • DNA: 12 µg/plate = 20,7 µL + 750 µL Optimem
    • Lipofectamine: 32 µL + 750 µL 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.

2018.12.21

• 60mm plate:
  • 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.
• 100mm plates:
  • Wash with PBS1x
  • Collect cells in 2 eppendorfs with a scrapper
  • Centrifuge the tubes and remove the supernatant.
  • Freeze the pellets at -20ºC