User:Karmella Haynes/Notebook/miR Trigger/2011/07/18

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07/18/11

  • ✓ Transfection: miR-luc sensor "177/MV8" set



Transfections: miR-luc sensors, 177/MV8 set
> Lipofectamine LTX
> Follow Jason's protocol, 12-well plates

Plate 2

Wells Plasmid (MV8 vector) 3 μg DNA =
1-3 186-177 22 μL
4-6 184-177 22 μL
7-9 187-177 16 μL
10-12 188-177 18 μL
13-15 189-177 17 μL
16-18 190-177 15 μL
19-21 191-177 13 μL
22-24 183-177 11 μL
25-27 192-177 12 μL
28-30 no DNA n/a

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

> Flow cytometry tomorrow (7/19/11)