User:Karmella Haynes/Notebook/Polycomb project/2010/04/20

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04/20/10

 * &#x2713; Transfection: Test H3me reporter constructs

Transfections > U2OS cells + (H3me reporter + Gal-VP64) constructs > Use Fugene, 12-well format

> Master mix (10x): 15 μL Fugene + 485 μL Opti-MEM --> R.T./ 5 min. > Add 50 μL Fugene mix to DNA --> R.T./ 20 min. > Add DNA/Fugene mix to each well (2 ml medium/ well); Grow cells at 37&deg;C > Refresh ab-free medium after 5 hours > Check RFP/ YFP after 18-24 hours

> 4/21/10



--> Looks like DPRE might confer some leaky expression (KAH142, 147), but overall, looks like Gal4-VP64 is needed to induce expression. Constructs look functional.


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