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

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10/13/10

  • ✓ Transfection: HepG2 cells w/ Pc-ATF constructs
  • ✓ 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.


Wells Plasmid DNA Volume Lipo Opti-MEM (total)
1 KAH126/MV2 2 μg 4.1 μL 4 μL 500 μL
2 KAH154/MV2 " 7.5 μL " "
3 KAH128/MV2 " 4.0 μL " "
4 KAH157/MV2 " 7.4 μL " "
5 KAH129/MV2 " 4.1 μL " "
6 KAH156/MV2 " 8.2 μL " "
7 KAH130/MV2 " 5.7 μL " "
8 KAH158/MV2 " 6.0 μL " "
9 KAH131/MV2 " 3.9 μL " "
10 KAH159/MV2 " 7.4 μL " "
11 KAH132/MV2 " 4.4 μL " "
12 none --- --- " "

> 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°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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