User:Karmella Haynes/Notebook/Polycomb project/2010/11/29

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11/29/10

 * &#x2713; Microscopy: take photos of RFP expression
 * &#x2713; Luc assay: HEK293 luc-repressed cells; reactivation by Gal4-ATF plus controls

Luc assay > Take photos of RFP expression for plate 1 (8-day repression set), 400 ng DNA wells: KAH60(#5), 36(#17), 59 (#23); KAH54 omitted b/c it has no mCherry > Resuspend cells in ~500 uL medium (aspirate off 500); tansfer to 1.5 mL tube > For each sample, aliquot 100 ul to 3 wells in 96-well solid white plate --> Use 1 row of medium for background measurements > Incubate for 3 min.; use standard plate reader "luciferase atp" protocol (2 reads per plate)

Cell counts > Cell counts: dilute 25 ul cells in PBS (100 ul final vol.) > Read with Millipore Scepter counter > Total cells per 100 ul luc assay sample = x cells/mL * 4 dln. factor / 10 > Calculate luc per cell

Results: Success! KAH60 activator de-represses luc in a Gal4 and VP64 dependent manner (neg ctrl deletions do not activate luc)


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