User:Karmella Haynes/Notebook/miR Trigger/2011/05/30

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05/30/11

  • ✓ Transfection: miR sensor constructs into U2OS plain



Lipo LTX transfection
> Follow Jason's protocol, 12-well plates
> Set up each in triplicate, 48 samples (4 plates) total
> Keep 5th plate as "blank" control for FACS

Wells Plasmid DNA Volume PLUS reagent Lipo Opti-MEM (total)
1-3 FC1* 1 μg ### μL 1 uL 3 μL 200 μL
4-6 FC2 " ### μL " " "
7-9 FC3 " ### μL " " "
10-12 FC4 " ### μL " " "
13-15 FC31 " ### μL " " "
16-18 FC32 " ### μL " " "
19-21 FC33 " ### μL " " "
22-24 FC34 " ### μL " " "
25-27 KAH100 " ### μL " " "
28-30 KAH101 " ### μL " " "
31-33 KAH118* " ### μL " " "
34-36 KAH119* " ### μL " " "
37-39 KAH121 " ### μL " " "
40-42 KAH122 " ### μL " " "
43-45 KAH124 " ### μL " " "
46-48 KAH125 " ### μL " " "
  • Poor quality DNA (see BioBrick Cloning entry 5/30/11)


> Dilute 1 ug DNA in 20 μL (total) st dH2O; set up 3x and aliquot 20 μL to other 2 tubes
> Add 180 μL Opti-MEM
> 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); Grow cells at 37°C



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