User:Karmella Haynes/Notebook/miR Trigger/2011/07/27

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07/27/11

  • Transfection: miR-luc sensors "198/MV8" set (9) and CFP-only controls (9)



'Transfections: miR-luc sensors, 198/MV8 set', CFP-only conrols
> Lipofectamine LTX
> Follow Jason's protocol, 12-well plates

Plate 2

Wells Plasmid (MV8 vector) 3 μg DNA =
1-3 186-198 19 μL
4-6 184-198 26 μL
7-9 187-198 27 μL
10-12 188-198 17 μL
13-15 189-198 26 μL
16-18 190-198 26 μL
19-21 191-198 22 μL
22-24 183-198 22 μL
25-27 192-198 22 μL
28-30 186 12 μL
31-33 184 17 μL
34-36 187 12 μL
37-39 188 14 μL
40-42 189 11 μL
43-45 190 14 μL
46-48 191 12 μL
49-51 183 14 μL
52-54 192 12 μL
55-57 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 (7/28/11)



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