User:Karmella Haynes/Notebook/Polycomb project/2010/04/20

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04/20/10

  • ✓ Transfection: Test H3me reporter constructs



Transfections
> U2OS cells + (H3me reporter + Gal-VP64) constructs
> Use Fugene, 12-well format


Wells Plasmid DNA Volume Fugene Opti-MEM
1. KAH96/V0201 500 ng 0.8 μL 1.5 μL 48.5 μL
2. KAH96/V0201 + KAH60/VNpc 250 ng + 250 ng 0.4 μL + 0.6 μL " "
3. KAH142/V0201 500 ng 0.8 μL 1.5 μL 48.5 μL
4. KAH142/V0201 + KAH60/VNpc 250 ng + 250 ng 0.4 μL + 0.6 μL " "
5. KAH146/V0201 500 ng 2.0 μL 1.5 μL 48.5 μL
6. KAH146/V0201 + KAH60/VNpc 250 ng + 250 ng 1.0 μL + 0.6 μL " "
7. KAH147/V0201 500 ng 3.2 μL 1.5 μL 48.5 μL
8. KAH147/V0201 + KAH60/VNpc 250 ng + 250 ng 1.6 μL + 0.6 μL " "

> Master mix (10x): 15 μL Fugene + 485 μL Opti-MEM --> R.T./ 5 min.
> Add 50 μL Fugene mix to DNA --> R.T./ 20 min.
> Add DNA/Fugene mix to each well (2 ml medium/ well); Grow cells at 37°C
> Refresh ab-free medium after 5 hours
> Check RFP/ YFP after 18-24 hours


> 4/21/10

Microscopy 4/21/10

--> Looks like DPRE might confer some leaky expression (KAH142, 147), but overall, looks like Gal4-VP64 is needed to induce expression. Constructs look functional.



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