Jun 29, 2023

Public workspaceCells electroporation for cell transfection with NEON system

DOI
dx.doi.org/10.17504/protocols.io.e6nvwd9jwlmk/v1
  • 1University of Glasgow
Juan.Gonzalez
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.e6nvwd9jwlmk/v1
Protocol Citation: Juan.Gonzalez 2023. Cells electroporation for cell transfection with NEON system. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwd9jwlmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 29, 2023
Last Modified: June 29, 2023
Protocol Integer ID: 84238
Abstract
Protocol for cell electroporation with the NEON transfection system
Warm the resuspension buffer and the E2 buffer in the water bath out of the water at Temperature37 °C

Put the electrode part inside the left hand side hood in 419
Put a NEON glass tube matching the electrode position
Add Amount3 mL of E2 buffer in the glass tube.
Note
The buffer should cover the electrode level

Switch on the NEON equipment
Load the saved settings.
Note
The settings depend on the cells used. I.e., we used 3 pulses of 10 ms at 1650 mV for C2C12 myoblasts


Add Amount5 µg of the plasmid in a sterile Eppendorf tube
Add Amount3 mL 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 Amount150 µL of sterile DPBS and discard the supernatant carefully
Wash the cell pellet with Amount150 µL of resuspension buffer and discard the supernatant carefully
Resuspend the cell pellet with Amount130 µL of resuspension buffer and discard the buffer

Transfer Amount130 µL of the cell solution to the Eppendorf tube containing the plasmid

Mix without making any bubble
Take a Amount100 µL tip with the NEON pipette

Take Amount100 µL 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

AfterDuration24:00:00 , check the cells under the microscope and perform the required experiment

1d