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Protocols and settings for the nucleofector.

Reusing Cuvettes

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

  1. 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.
  2. 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.
  3. Rinse once with 70% ethanol, flicking the cuvette strongly afterwards to remove as much ethanol as possible.
  4. Leave to dry in the hood, with the UV lamp on to limit contamination.
  5. Store in a sealed tip box; cuvettes should be marked in some fashion to track their use.

DIY Buffers

Buffer V

  1. 6 ml of ddH2O
  2. 1.8 ml of 0.5 M NaH2PO4, pH 7.2.
  3. 50 μl of 1 M KCL
  4. 100 μ of 1 M MgCl
  5. 1 ml of 0.2 M HEPES (do not adjust pH)
  6. 1 ml of 0.24 M sodium succinate

Final volume should be 10 ml, filter using a 0.2 μm syringe filter to sterilize.

THP-1 Cells

Use the THP1 cell line kit from Amaxa, or Buffer V, whichever is available.

Transfection Protocol:

  1. Subculture THP1 cells 2-3 days before nucleofection, aiming for a density of 3-4 x 105/ml on the day of transfection.
  2. Count the cells, and then transfer 1 x 106 cells to a centrifuge tube and pellet at 1,000x G, 10 min, room temperature.
  3. Remove the media and resuspend in 100 μl of Nucleofector Solution V.
  4. Add 0.5 μg DNA or 30 - 300 nM siRNA (3-30 pmol).
  5. Transfer into the cuvette and cap.
  6. Electroporate using program U-001 or V-001; the former gives higher viability but at the cost of lower transfection efficiency.
  7. Add 500 μl of pre-warmed media to the cuvette and pipette gently to mix.
  8. 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).
  9. Clean the cuvette for re-use (see protocol, above)
  10. Culture a minimum of 24 hours before use.