User:Karmella Haynes/Notebook/Polycomb project/2009/12/21

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12/21/09

  • ✓ Transfections: Polycomb ATF's for stable lines

Transfections
> U2OS Flp-in T-REx cells + Polycomb-ATF plasmids (puro)
> Use Lipofectamine, 6-well format

Wells Plasmid DNA Volume Lipo Opti-MEM (total)
1 KAH126/MV2 2 μg 4.1 μL 4 μL 500 μL
2 KAH127/MV2 " 4.0 μL " "
3 KAH128/MV2 " 4.0 μL " "
4 KAH129/MV2 " 4.1 μL " "
5 KAH130/MV2 " 5.8 μL " "
6 KAH131/MV2 " 3.9 μL " "
7 KAH132/MV1 " 4.4 μL " "
8 KAH133/MV1 " 3.9 μL " "
9 no DNA --- --- " "
10 untreated --- --- --- "

> 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.
> Remove 500 μL medium from each well; Add 500 μL complexes to each well (4 ml med. each); Grow cells at 37°C
> Refresh medium after 5 hours (ab-free)
> Grow for two days, then plate dilution cultures in selection medium (puro)



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