Cells electroporation for cell transfection with NEON system

Warm the resuspension buffer and the E2 buffer in the water bath out of the water at 37°C

Put the electrode part inside the left hand side hood in 419

Put a NEON glass tube matching the electrode position

Add 3mL of E2 buffer in the glass tube.

Switch on the NEON equipment

Load the saved settings.

Add 5µg of the plasmid in a sterile Eppendorf tube

Add 3mL of fresh cell media without antibiotics in a T25 flask

Trypsinise the cells as usual

Once you have the cell pellet, discard the supernatant carefully

Wash the pellet with 150µL of sterile DPBS and discard the supernatant carefully

Wash the cell pellet with 150µL of resuspension buffer and discard the supernatant carefully

Resuspend the cell pellet with 130µL of resuspension buffer and discard the buffer

Transfer 130µL of the cell solution to the Eppendorf tube containing the plasmid

Mix without making any bubble

Take a 100µL tip with the NEON pipette

Take 100µL of the mixture with the NEON pipette and place it in the glass tube matching the electrode position

Press "START" in the NEON display

Once the "COMPLETE" message appears in the NEON display, put the cells with the NEON pipette in the T25 flask

Put the T25 flask in the “Antibiotics free” incubator

After24h 0m 0s , check the cells under the microscope and perform the required experiment