User:Karmella Haynes/Notebook/Polycomb project/2010/07/01

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07/01/10

  • ✓ Transfection: FTRx H3me reporter clones



Transfections
> U2OS cells + (H3me reporter + Gal-VP64) constructs
> Use Fugene, 12-well format
> Samples (plated in duplicate, transfect just one):
--> KAH96-2, 3, 5, 6
--> KAH142-1, 2, 3, 4, 6, 7, 8


Wells Plasmid DNA Volume Fugene Opti-MEM
11 total KAH60/VNpc 500 ng 1.2 μL 1.5 μL 48.5 μL

> Master mix (12x): 18 μL Fugene + 582 μL Opti-MEM --> R.T./ 5 min.
> Add 14.4 μL DNA --> R.T./ 20 min.
> Add 51.2 μL DNA/Fugene mix to each well (2 ml medium/ well); Grow cells at 37°C
> Check RFP/ YFP after 18-24 hours


7/02/10
> Good RFP (Gal4-VP64 activator) signal in all Fugene transfected samples, but no YFP induction. The co-transfection experiment showed that Gal4-VP64 does activate H3me reporter, so my conclusion is that these cells are not stably transfected w/ the H3me sensor genes. Repeat transfection of H3me sensors.