User:Karmella Haynes/Notebook/Polycomb project/2010/04/23

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04/23/10

 * &#x2713; Transfections: H3me reporters into cell lines KAH126-1, 130-2, 132-8
 * &#x2713; Polycomb-ATF negative control lines: colony plates

Transfections > U2OS Flp-in T-REx lines (puro/zeo/blast) + Polycomb-ATF plasmids (hygro) --> Note: co-transfect with FlpE (1:3); hygro resistance replaces zeo resistance after "Flp" in > Use Lipofectamine, 6-well format > Plates:
 * 1) KAH126-1 Flp-in T-REx
 * 2) KAH130-2 Flp-in T-REx
 * 3) KAH132-8 Flp-in T-REx

> Add 16 μL (4x) Lipo to 1000 μL (4x) Opti-MEM --> R.T/ 5 min. > Add DNA to 250 μL Opti-MEM > Add 250 Lipo mix into each 250 DNA mix --> R.T./ 20 min. > Add 500 μL complexes to each 3.5 mL well (4 ml med. total each); Grow cells at 37&deg;C > Refresh medium after 5 hours (ab-free) > Grow for one/two days, then plate dilution cultures in selection medium (+hygro)

Polycomb-ATF negative control lines: colony plates > For each line make 2 plates: ~1x105 and 5x104 cells > 25 ml 1 μg/mL puro, 15 μg/mL blast, 200 μg/mL zeo > Also make 6-well back-up plate for all transfections (~3x105 cells in selection medium)


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