User:Karmella Haynes/Notebook/Polycomb project/2010/10/13

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10/13/10

 * &#x2713; Transfection: HepG2 cells w/ Pc-ATF constructs
 * &#x2713; HEK Gal4EED: induce 4-day wells with dox

Lipo transfection > Note: HepG2 can be difficult to transfect. Try all constructs at first, optimize as needed.

> Add Lipo to 250 μL Opti-MEM --> R.T/ 5 min. > Add DNA to 250 μL Opti-MEM > Add DNA mix to Lipo mix --> R.T./ 20 min. > Add 500 μL complexes to each 3.5 mL well (4 ml med. each); Grow cells at 37&deg;C

> Results: All 11 constructs are expressed in about 1% of the cells. Cells are very clumpy, so this might prevent better transfection. Proceed with puromycin kill curve for HepG2 and stables.


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