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

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  • ✓ Microscopy: take photos of RFP expression
  • ✓ 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)