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

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02/21/11

 * &#x2713; HepG2 transfection: pcDNA6/TR electroporation (Amaxa protocol)
 * &#x2713; 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.


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