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

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07/07/11

  • ✓ Daniel - Transfections: Lipo LTX miR-34 sensors in U2OS plain



Transfections: miR-34 sensors
> Lipofectamine LTX
> Follow Jason's protocol, 12-well plates


Wells Plasmid (MV8 vector) </u>
1-3 183-DF4
4-6 183-DF5
7-9 183-DF6
10-12 184-DF6 (no Gal4 b.s.)
13-15 184-DF6 (no Gal4 b.s.)
16-18 184-DF6 (no Gal4 b.s.)
19-21 177 (RFP, no miR-34 t.s.)
22-24 no DNA


Lipo LTX
Note: set up 3x vol reactions
> Dilute 1 μg DNA in dH2O (10 μL final); 3x = 3 μg DNA in dH2O (30 μL final)
> Add 190 μL Opti-MEM to 10 μL DNA; 3x = 570 μL Opti-MEM, 30 μL DNA
> Add 1 μL PLUS reagent --> R.T/ 5 min.; 3x = 3 μL PLUS
> Add 3 μL Lipo LTX --> R.T/ 30 min.; 3x = 9 μL Lipo LTX
> Add ~200 μL complexes (drop-wise) to each well (1 ml med. each); Grow cells at 37°C

> Microscopy and flow cytometry tomorrow (7/08/11)