User:Karmella Haynes/Notebook/Polycomb project/2010/02/09

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02/09/10

 * &#x2713; U2OS Flp-in T-REx: add dox to Dox+ wells (6-well plate from 2/08)
 * &#x2713; Transfection: Gal4-EED constructs in HEK293 luc reporter line
 * &#x2713; Polycomb-ATF lines: frozen stocks for 90% confluent plates: 127-4, 128-8, 132-8; 1x 1:10 plate (per line)

Transfection > HEK293 cells + Gal4-EED constructs (KAH139, 140, 141/V0201) > Use Fugene, 24-well format > Serial dilutions of plasmids: 800 - 25 ng in 5 μL --> Start with 1600 ng in 10 μL OptiMEM --> Add 5 μL plasmid to 5 μL OptiMEM for each dilution step

> Master mix: 90 μL Fugene + 910 μL Opti-MEM --> R.T/ 5 min. > Add 20 μL Fugene mix to DNA --> R.T./ 20 min. > Add 25 μL complexes to each well (1 ml med. each); Grow cells at 37&deg;C > Assay Luc after 18-24 hours

> Day 2 --> Refresh medium. Do luc assay next day (2/11).


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