User:Karmella Haynes/Notebook/Polycomb project/2010/11/01

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11/01/10

  • ✓ HEKGal4EED + PcATF's: split cells and do luc assay
  • ✓ ChIP: H3K27me3 IP for expt. samples (overnight)
  • ✓ Transfection: HepG2's (Pc-ATF set + pcDNA6/TetR)
  • ✓ Frozen stocks: transfer HepG2's and HEK293's to liq. nitrogen tank.



Lipo transfection
> Co-transfect Pc-ATF with pcDNA6/TetR tet repressor plasmid (Invitrogen)


Wells Plasmid DNA Volume 0.5 μg/μL pcDNA6 Lipo Opti-MEM (total)
1 KAH126/MV2 1 μg 2.0 μL 2.0 μL 4 μL 500 μL
2 KAH154/MV2 " 3.7 μL " " "
3 KAH128/MV2 " 2.0 μL " " "
4 KAH157/MV2 " 3.7 μL " " "
5 KAH129/MV2 " 2.0 μL " " "
6 KAH156/MV2 " 4.1 μL " " "
7 KAH130/MV2 " 2.8 μL " " "
8 KAH158/MV2 " 3.0 μL " " "
9 KAH131/MV2 " 2.0 μL " " "
10 KAH159/MV2 " 3.7 μL " " "
11 KAH132/MV2 " 2.2 μL " " "
12 (Tet R only) --- --- " " "

> Add Lipo to 250 μL Opti-MEM --> R.T/ 5 min.
> Add Pc-ATF and pcDNA6 plasmid 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



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