Protocols and settings for the nucleofector.
Nucleofector cuvettes can be reused several times, allowing for far more transfections to be conducted than the kits claim. Reuse is limited to 5-8 transfections, and it is a good idea to try and use the same cuvette for the same plasmids to limit the risk of cross-contamination. Decreasing transfection efficiency, or sparking during electroporation, are signs that the cuvette is no longer usable.
Cleaning the Cuvette
- Prepare your transfection and transfect the cells as quickly as possible. Remove the transfected cells as quickly as possible from the cuvette - the longer media is in the cuvette, the more likely the electrodes are to corrode.
- As quickly as possible, rinse the cuvette 5 times with sterile distilled water. Perform this in the tissue culture hood to keep the cuvette sterile. "Flick" the cuvette after each wash to remove as much water as possible.
- Rinse once with 70% ethanol, flicking the cuvette strongly afterwards to remove as much ethanol as possible.
- Leave to dry in the hood, with the UV lamp on to limit contamination.
- Store in a sealed tip box; cuvettes should be marked in some fashion to track their use.
- 6 ml of ddH2O
- 1.8 ml of 0.5 M NaH2PO4, pH 7.2.
- 50 μl of 1 M KCL
- 100 μ of 1 M MgCl
- 1 ml of 0.2 M HEPES (do not adjust pH)
- 1 ml of 0.24 M sodium succinate
Final volume should be 10 ml, filter using a 0.2 μm syringe filter to sterilize.
Use the THP1 cell line kit from Amaxa, or Buffer V, whichever is available.
- Subculture THP1 cells 2-3 days before nucleofection, aiming for a density of 3-4 x 105/ml on the day of transfection.
- Count the cells, and then transfer 1 x 106 cells to a centrifuge tube and pellet at 1,000x G, 10 min, room temperature.
- Remove the media and resuspend in 100 μl of Nucleofector Solution V.
- Add 0.5 μg DNA or 30 - 300 nM siRNA (3-30 pmol).
- Transfer into the cuvette and cap.
- Electroporate using program U-001 or V-001; the former gives higher viability but at the cost of lower transfection efficiency.
- Add 500 μl of pre-warmed media to the cuvette and pipette gently to mix.
- Transfer the electroporated cells to a well of a 12-well plate along with 900 μl of pre-warmed media (final volume of 1.5 ml).
- Clean the cuvette for re-use (see protocol, above)
- Culture a minimum of 24 hours before use.