User:Karmella Haynes/Notebook/Polycomb project/2009/12/18

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12/18/09

 * Transfection: transfection and neo-induction of K-stop constructs

Transfection > Test of Neo-inducible translation in U2OS (plain) cells

> Master mixes (4, x6): 10.8 μL Fugene + 109.2 μL Opti-MEM --> R.T/ 5 min. > Add 6x vol DNA --> R.T./ 20 min. > Add 20 μL complexes to each well (1 ml med. each); Grow cells at 37&deg;C overnight

Day 2 > Check RFP expression --> Much less RFP in K-stop samples than K controls; some leaky K-stop expression (recorded images) > Refresh medium and add varying amounts of neomycin (G418) --> Make neo dilutions in 50 μL Opti-MEM (see table)

> Assay RFP after 18-24 hours --> Checked after 48 hours on 12/21/09(snowstorm on Sunday); indication of induction in some cells --> Move forward with neo-inducible Gal4-EED; quantify by Western blot


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