User:Karmella Haynes/Notebook/miR Trigger/2010/02/17

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02/17/10

 * &#x2713; Transfection: for RFP-repressor knock-down experiment

Lipo transfection > Wells 1-9: JDS33-47 cells; Wells 10-12: U2OS (Flp-in T-REx) cells

> Add Lipo to 250 μL Opti-MEM --> R.T/ 5 min. > Add DNA to 250 μL Opti-MEM > Add DNA mix to Lipo mix --> R.T./ 20 min. > Add 500 μL complexes to each well (4 ml final vol.); Grow cells at 37&deg;C > Refresh medium after 5 hours (ab-free) > Wells 3, 6, 9 (48hr dox): Add dox, final = 1 μg/mL

Day 2 > Wells 2, 5, 8 (24hr dox): Add dox, final = 1 μg/mL

Day 3 > Flow cytometry analysis of RFP and YFP expression


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