User:Karmella Haynes/Notebook/miR Trigger/2009/12/17

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12/17/09

 * &#x2713; Transfection: RFP reporter into JDS37-43 (luc miRNA-YFP)

Transfections > U2OS JDS37-43 (luc miRNA-YFP) cells + RFP-luc target reporters > Use Lipofectamine, 6-well format

Day 1 (today) > 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. > '''Remove 0.5 mL growth medium from each well before adding transf. mix.''' Add 500 μL complexes to each well (total vol./well = 4 mL); Grow cells at 37&deg;C > After 5 hours, change medium (ab-free)

Day 2 (12/18/09) > Transfection (DNA+Lipo) was slightly toxic, no need to split cells; change medium to 15μg/mL puro(morning) > Do microscopy to find RFP+ cells (record positions using Metamorph multi-position staging) > Add 1 μg/mL doxycycline

Day 3 > Microscopy: record RFP vs. YFP expression at set stage positions

Day 4 > Repeat Day 3


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