User:Karmella Haynes/Notebook/Polycomb project/2010/06/03

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06/03/10

  • ✓ Transfection: histone methylation reporter



Lipo transfection
> KAH96 [5xGal4 : HSVtkTATA : K : YFP : YFP : NLSstop2 : polyA : HS4 : HS4]
> KAH142 [DPRE : 5xGal4 : HSVtkTATA : K : YFP : YFP : NLSstop2 : polyA : HS4 : HS4]

Wells Plasmid DNA Volume Lipo Opti-MEM (total)
1 (KAH126-1) KAH96/ FlpE 1.5, 0.5 μg 2.5, 1.0 μL 4 μL 500 μL
2 (KAH126-1) KAH142/ FlpE 1.5, 0.5 μg 2.5, 1.0 μL " "
3 (KAH126-1) none --- --- μL " "
4 (FTRx) KAH96/ FlpE 1.5, 0.5 μg 2.5, 1.0 μL 4 μL 500 μL
5 (FTRx) KAH142/ FlpE 1.5, 0.5 μg 2.5, 1.0 μL " "
6 (FTRx) none --- --- μL " "

> Add Lipo to 250 μL Opti-MEM --> R.T/ 5 min.
> Add DNA to 250 μL Opti-MEM
> Add DNA mix to Lipo mix --> R.T./ 20 min.
> Add 500 μL complexes to each well (4 ml med. total each); Grow cells at 37°C
> Refresh medium after 5 hours (ab-free)



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