Electroporation using Neon Electroporation from Invitrogen
Overview
This protocol is for transfecting plasmid DNA into mammalian cells using the Neon machine from Invitrogen to perform electroporation.
Protocol 1) Electroporation of 1e6 adherent cells in 100 µl
Preparation steps
Perform all steps using sterile tissue culture ingredients and technique
Move Neon machine into the tissue culture hood, spray down with 70% ethanol.
Place media, PBS, and trypsin in 37ºC water bath
Bring "R" buffer to room temp.
Thaw DNA and measure concentration. Ideally DNA should be at 1-5 µg/µl in TE or H2O. Invitrogen recommends using 5 to 30 µg DNA per transfection. Volume of DNA should not exceed 10% of total volume (10 µl). Calculate amount of DNA that will be added to 100 µl of cells.
Prepare 60 mm plates with 5 ml DMEM + 10% FBS (No antibiotic), keep at 37ºC.
Fill plastic neon tube with 3 ml of Invitrogen Electrolyte buffer and insert tube into the Neon pipette station. Note: Invitrogen Instructions say to use buffer "E" if electroporating in 10 µl and "E2" if electroporating in 100 µl.
Set the desired Pulse parameters on the Neon machine. For HEK293T cells in 100 µl use the following:
Pulse voltage = 1,100
Pulse width (ms) = 20
Pulse Number = 2
Electroporation steps
Pipette the appropriate amount of plasmid into a 1.5 ml microfuge tube . The total amount of plasmid should be between 5 and 30 µg per 100 µl. For two transfections add 15 to 75 µg DNA to the tube, which will eventually be mixed with 250 µl of cells at 1e7 cells/ml.
Trypsinize cells, resuspend in 10 ml DMEM, count cells.
Centrifuge 100-400g (1200 rpm with 15 ml falcon tubes) for 5 min, remove DMEM supernatant, resuspend in 10 ml PBS (No Ca/Mg).
Centrifuge 1200 rpm 5 min, remove PBS completely.
Resuspend in Invitrogen Buffer "R" at 1e7 cells/ml at R.T. If preparing to do 2 transfections, resuspend 2.5e6 cells in 250 µl buffer "R". Do not let sit more than 15-30 minutes in this buffer. Buffer "R" is appropriate for all established adherent and suspension cell lines. Buffer "T" is for primary blood-derived suspension cells.
Gently transfer the 250 µl of cells in Buffer "R" to the microfuge tube containing the DNA and mix gently.
Press the push-button on the Neon Pipette down to the second stop to open the pipette clamp, then insert the 100 µl Neon Tip onto the Neon Pipette pushing down firmly to make sure the tip snaps all the way onto the pipette. Gently release the push-button while continuing to press down firmly on the pipette tip in order to ensure a good seal between the pipette and the tip. Any gaps between the pipette tip and the pipette will cause poor transfection.
Press the push-button on the Neon Pipette down to the first stop, immerse in the cell/DNA mixture and slowly release the push-button to suck up 100 µl of cell/DNA mixture. Very important to make sure that there are no bubbles in the tip.
Insert pipette and tip into the tube filled with electrolyte buffer on the Neon Pipette Station until you hear a click sound. Note, the handle protrusion should be facing towards the rear.
Press "Start" on neon machine. Monitor the needle to make sure there are no sparks arcing, which will ruin the transfection.
Remove the pipette from the station and gently expel the cell/DNA mixture by pressing the push-button down to the first stop into a tissue culture plate containing pre-warmed media without antibiotics.