NSO J558L electroporation

• • 1*10⁷ cells

  • 2 washes PBS

  • Resuspend the cells in 200l of PBS and transfer them in a BIORAD cuvette 0,4 cm

  • Add 10 g of plasmidic linearized DNA (precipitated with AcNa/Ethanol – washed in 70% ethanol – resuspended in 20 l PBS)

  • Incubate 10 min ice

  • Add 600l of PBS without mixing the cells

  • Elettroporation with BIORAD apparatus:NSO 5 shots/400VS/25F

  • Incubate 20 min ice

  • Check mortality: 30% mortality is to be considered good for the transfection

  • Dilute the cells in 24 ml DMEM/10%FCS and put them into a 24 well plate (1ml/w)

  • After 24 hours remove the medium and add selection medium

It is advisable always add a mock control of the transfection (use PBS instead of plasmind)