User:Karmella Haynes/Notebook/Polycomb project/2011/02/21

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  • ✓ HepG2 transfection: pcDNA6/TR electroporation (Amaxa protocol)
  • ✓ HepG2: split 1:10

HepG2 transfection, Amaxa protocol
> Previous Lipofectamine attempt was unsuccessful. Try electrporation.
> pcDNA6/TR, tet repressor plasmid (Invitrogen), blasticidin resistance
> Plasmids (2 μg each)

  1. pcDNA6/TR (4 μL)
  2. pmaxGFP positive control (4 μL)

> Prepare DNA: add 2 μg plasmid DNA (10 μl max.) to one labeled sterile 0.5 ml tube per transfection.
> Set up the amaxa machine in the flow hood. Select the HepG2 high viability (H-022) Nucleofector program.
> Fill wells in 6-well plate with 2.0 ml DMEM plain culture medium.
> Cell count (1/4 dilution): 9.54 x104/ mL x 4 = 3.8 x105/mL; use 2.6 mL/ sample
> Pellet 106 cells per sample, 90x g/ 10 min.
> Resuspend cells in 100 μl of Nucleofector solution per sample.
> Mix 100 μl cells with 2 μg DNA.
> Transfer the cells to an Amaxa cuvette (avoid bubbles); close w/ the blue cap.
> Insert the cuvette into the holder and press “X” to start the program.
> After display shows “OK” immediately remove the cuvette, add ~500 μl of culture medium to the cells and transfer to the 6-well plate (using an Amaxa plastic pipette).
> Press “X” to reset the Nucleofector.
> Grow cells at 37°C. Control pmaxGFP expression should be visible in 4 – 24 hours.