User:Karmella Haynes/Notebook/miR Trigger/2011/07/25

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07/25/11

  • ✓ Transfection: for real-time microscopy



Transfections: miR-luc sensors, 196/MV8 set
> Lipofectamine LTX
> Follow Jason's protocol, 12-well plates

Plate 2

Wells Cells Plasmid / treatment 3 μg DNA =
1 U2OS 183-DF4/MV8 5 μL
2 U2OS 183-DF5/MV8 5 μL
3 U2OS 183-DF6/MV8 4 μL
4 U2OS 183-177/MV8 4 μL
5 JDS33-47 176/MV8 1.3 μL
6 JDS33-47 176/MV8 +dox 1.3 μL
7 JDS33-47 177/MV8 1.8 μL
8 JDS33-47 177/MV8 +dox 1.8 μL
9 JDS33-47 183/MV8 5 μL
10 JDS33-47 183/MV8 +dox 5 μL
11 JDS33-47 183-177 4 μL
12 JDS33-47 183-177 +dox 4 μL

> 2 hours prior to microscopy (10 am)
Lipo LTX
Note: set up 3x vol reactions
> Dilute 1 μg DNA in dH2O (10 μL final)
> Add 190 μL Opti-MEM to 10 μL DNA
> Add 1 μL PLUS reagent --> R.T/ 5 min.
> Add 3 μL Lipo LTX --> R.T/ 30 min.
> Add ~200 μL complexes (drop-wise) to each well (1 ml med. each)

> Added transfection complexes at 11 am.
> Real-time microscopy started at 1 pm (48 hrs, 2 hr time points)



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