User:Karmella Haynes/Notebook/Polycomb project/2010/10/27

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10/27/10

  • ✓ Transfections: HEK293 Gal4EED + Pc-ATF's


Transfections
> Try transfecting with Pc-ATF first, then inducing H3K27me3
> HEK293 Gal4-EED 23;4;9 cells, no dox induction
> Use Fugene, 12-well format, 1.5 mL medium per well
> 2 plates total (1 control, 1 for dox induction time course)


Wells Plasmid DNA Volume Fugene Opti-MEM
1. KAH126/MV2 500 ng 1.0 μL 1.5 μL 48.5 μL
2. KAH154/MV2 " 2.0 μL " "
3. KAH128/MV2 " 1.0 μL " "
4. KAH157/MV2 " 2.0 μL " "
5. KAH129/MV2 " 1.0 μL " "
6. KAH156/MV2 " 2.0 μL " "
7. KAH130/MV2 " 1.5 μL " "
8. KAH158/MV2 " 1.5 μL " "
9. KAH132/MV2 " 1.0 μL " "
10. KAH60/pcVN " 1.0 μL " "
11. no DNA --- --- " "

> Master mix (2x, 1 per plate): 3 μL Fugene + 97 μL Opti-MEM --> R.T./ 5 min.
> Add 2x vol. DNA --> R.T./ 20 min.
> Remove 0.5 mL medium from each well (decrease volume to improve transfection efficiency)
> Add 50 μL DNA/Fugene mix to each well (1.5 ml medium/ well); Grow cells at 37°C
> Check RFP after 18-24 hours



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