User:Karmella Haynes/Notebook/Polycomb project/2010/07/06

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07/06/10

  • ✓ Transfection: KAH130, 131 into FTRx



Lipo transfection
> Details

Wells Plasmid DNA Volume Lipo Opti-MEM (total)
1 KAH130/MV2 2 μg 5.8 μL 4 μL 500 μL
2 " + dox " 5.8 μL " "
3 KAH131/MV2 " 4.0 μL " "
4 " + dox " 4.0 μL " "
5 no DNA --- --- " "
6 untreated --- --- --- "

> 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. each); Grow cells at 37°C
> Refresh medium after 5 hours (ab-free); add 1 μg/mL dox to dox+ samples
> Next day: check transfection efficiency in dox+ samples


7/07/10
> ~50% RFP+ cells in dox+ samples
> ~1% leaky expression in dox- cells



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