User:Karmella Haynes/Notebook/Polycomb project/2010/06/25

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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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06/25/10

 * &#x2713; Minipreps: KAH96, 142, 146, 147/V0201 DNAme-
 * &#x2713; Transfection: KAH96, 142, 146, 147/V0201 DNAme-
 * &#x2713; ChIP: cross-link FTRx and 154-2 cells

Minipreps > DNA methylation-free plasmid preps for transfection (grown in C2925) > Check KAH96, 142, 146, 147/V0201 with XbaI digest (add 641 to expected insert size)

Lipo transfection > KAH96 [5xGal4 : HSVtkTATA : K : YFP : YFP : NLSstop2 : polyA : HS4 : HS4] > KAH142 [DPRE : 5xGal4 : HSVtkTATA : K : YFP : YFP : NLSstop2 : polyA : HS4 : HS4] > KAH146 [[KAH96] + EED expression cassette] > KAH147 [[KAH142] + EED expression cassette]

> Add Lipo to 250 μL Opti-MEM --> R.T/ 5 min. > Add DNA to 250 μL Opti-MEM > Add DNA mix to Lipo mix --> R.T./ 20 min. > Add 500 μL complexes to each well (4 ml med. total each); Grow cells at 37&deg;C > Refresh medium after 5 hours (ab-free)


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