User:Karmella Haynes/Notebook/miR Trigger/2009/12/15

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12/15/09

  • ✓ Transfection: RFP reporters into U2OS JDS37-43 (luc miRNA-YFP)
  • ✓ Split cells: U2OS plain (1x 1:10)

Transfections
> U2OS JDS37-43 (luc miRNA-YFP) cells + RFP-luc target reporters
> Use Lipofectamine, 6-well format

Wells Plasmid DNA Volume Lipo Opti-MEM (total)
1 KAH93/MV2 2 μg 4.4 μL 4 μL 500 μL
2 KAH93/MV2(Dox) " " " "
3 KAH94/MV2 " 4.8 μL " "
4 KAH94/MV2(Dox) " " " "
5 KAH95/MV2 " 8.7 μL " "
6 KAH95/MV2(Dox) " " " "
7 KAH14/MV1 " 6.1 μL " "
8 KAH14/MV1(Dox) " " " "
9 no DNA --- --- " "
10 no DNA(Dox) --- --- " "
11 untreated --- --- --- "
12 no DNA(Dox) --- --- --- "

> 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
> After 5 hours, change medium (ab-free) and add 1 μg/mL doxycycline to "Dox" samples > Check RFP & YFP expression (microscopy) after 18-24 hours



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