User:Karmella Haynes/Notebook/Polycomb project/2010/04/22

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04/22/10

 * &#x2713; Transfections: Polycomb-ATF negative controls for stable lines

Transfections > U2OS Flp-in T-REx cells + Polycomb-ATF plasmids (puro) > Use Lipofectamine, 6-well format > Make duplicate plates (one will be induced with dox); 12 wells total

> Add 8 μL (2x) Lipo to 500 μL (2x) Opti-MEM --> R.T/ 5 min. > Add 2x DNA to 500 μL (2x) 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. total each); Grow cells at 37&deg;C > Refresh medium after 5 hours (ab-free) --> Add 1μg/mL dox to one plate, check for RFP expression tomorrow > Grow for one/two days, then plate dilution cultures in selection medium (puro)

> 4/23/10 --> Dox+ plate results: ~50% of the cells are RFP+ (high expression and efficiency). For colony plates, use ~50x105 (1/20) and ~25x105 (1/40) cells in a large (~20 cm) gridded plate


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