User:Karmella Haynes/Notebook/miR Trigger/2011/06/28

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06/28/11

  • ✓ Transfection: miR sensor test set #1 (no Gal4 binding sites), U2OS plain



Transfections: miR sensor set 1, 0 Gal4 BS
Plate 1: Lipo LTX
> Lipofectamine LTX
> Follow Jason's protocol, 12-well plates


Wells Plasmid (MV8 vector) DNA Volume PLUS reagent Lipo Opti-MEM (total)
1-3 KAH186 1 μg 5 μL 1 uL 3 μL 200 μL
4-6 KAH184 " 5 μL " " "
7-9 KAH187 " 5 μL " " "
10-12 KAH186-196 " 5 μL " " "
13-15 KAH184-196 " 5 μL " " "
16-18 KAH187-196 " 5 μL " " "
19-21 KAH186-177 " 5 μL " " "
22-24 KAH184-177 " 5 μL " " "
25-27 KAH187-177 " 5 μL " " "
28-30 KAH186-198 " 5 μL " " "
31-33 KAH184-198 " 5 μL " " "
34-36 KAH187-198 " 5 μL " " "

--> 37-39. no transfection

Lipo LTX
Note: set up 3x vol reactions
> Add 180 μL Opti-MEM to 5 μL DNA; 3x = 540 μL Opti-MEM, 15 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


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