User:Karmella Haynes/Notebook/Polycomb project/2010/11/27

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11/27/10

 * &#x2713; HEK293 Gal4-EED: luc repression assay
 * &#x2713; luc reactivation: Fugene transfection
 * &#x2713; HEK293 Gal4-EED stocks: change to puro medium

Transfections > HEK293 luc-repressed cells + Gal4/mCh/VP64 constructs

> Do luc assay to confirm silencing in 8, 6, 4, 2 and 0 day silenced cells --> 4, 6, and 8-day treatments show full luc repression

> Set up 24-well plates (5 ml from 10 ml plate; added to ; aliquot 1 ml cells per well):
 * 1) 8-day silenced (10 cm plate #1)
 * 2) 4-day silenced (#3)
 * 3) no silencing (#5)
 * Note: dox wash-out; cells were washed 1x w/ PBS before resuspending in complete medium

Set-up per plate > Use Fugene, 24-well format > Dilute each plasmid to 100 ng/ul in 1x PBS

> 25x master mix: 45 ul Fugene + 455 ul Opti-MEM --> R.T/ 5 min. > Add 8.0, 7.5, 7.0, 6.0, 4.0, 0.0 ul PBS to each row of tubes > Add 0.0, 0.5, 1.0, 2.0, 4.0, 8.0 ul 100 ng/ul DNA to each row > Add 20 ul Fugene master mix to each sample --> R.T./ 20 min. > Add complexes to each well (1 ml med. each); 37&deg;C > Assay RFP & luc after 48 hours


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