User:Karmella Haynes/Notebook/Polycomb project/2010/08/30

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


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08/30/10

 * &#x2713; Transfection: Pc-ATF's into H3me3 reporter cells
 * &#x2713; Freeze-down: KAH142-3
 * &#x2713; ChIP/ Co-IP: x-link KAH130-4, 131-4 (formaldehyde x-link only, no DMA)

Fugene transfection > Transfect the following cell lines (in 12-well plates, 6 wells per line): with six different Pc-ATF constructs and neg. ctrls. (dox inducible). Test to see if Pc-ATF can activate YFP. > Pc-ATF test constructs (human only for this trial):
 * KAH96-11
 * KAH142-3
 * KAH142-5
 * KAH146-1
 * KAH126: human PCD-ATF
 * KAH154: human PCD-RFP (no act. domain)
 * KAH130: human CBX8-ATF
 * KAH158: human CBX8-RFP (no act. domain)
 * KAH132: ATF (no Polycomb)

Use the same set-up (above) for: 1-6: KAH96-11 cells 7-12: KAH142-3 cells 13-18: KAH142-5 cells 19-24: KAH146-1 cells

> Master mixes (4, 4x mixes): 7.2 μL Fugene + 72.8 μL Opti-MEM --> R.T/ 5 min. > Add 4x vol DNA --> R.T./ 20 min. > Add 20 μL complexes to each well (2 ml med. each); Grow cells at 37&deg;C overnight > Tomorrow, photograph RFP/YFP without dox, then add dox and check for RFP/YFP in the evening


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