User:Karmella Haynes/Notebook/miR Trigger/2011/07/10

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07/10/11

 * Transfections: miR-34 sensor

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


 * 1) Plate 1: 12-well glass-bottom for time course microscopy
 * 2) Plate 2: 12-well plastic for KAH183-177/MV8 only control (no miR-34 target sites) for flow cytometry

Plate 1

Lipo LTX Note: set up 2x vol reactions > Dilute 1 μg DNA in dH2O (10 μL final); 3x = 2 μg DNA in dH2O (20 μL final) > Add 190 μL Opti-MEM to 10 μL DNA; 2x = 380 μL Opti-MEM, 20 μL DNA > Add 1 μL PLUS reagent --> R.T/ 5 min.; 2x = 2 μL PLUS > Add 3 μL Lipo LTX --> R.T/ 30 min.; 2x = 6 μL Lipo LTX > Add ~200 μL complexes (drop-wise) to each well (1 ml med. each); Grow cells at 37&deg;C

> Microscopy tomorrow (7/11/11)

Plate 2

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&deg;C

> Flow cytometry tomorrow (7/11/11)


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