User:Karmella Haynes/Notebook/miR Trigger/2010/01/14

{| width="800"
 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Project name
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

01/14/10

 * Outline of experiments/ tasks

Transfection 1 > KAH93-95, 108/MV2 + JDS33-47 U2OS > 12-well format, Fugene

> Master mixes (4, x3): 10.8 μL Fugene + 139.2 μL Opti-MEM --> R.T/ 5 min. > Add 3x vol DNA --> R.T./ 20 min. > Add 50 μL complexes to each well (2 ml med. each) > Refresh medium after ~8 hours; Grow cells at 37&deg;C overnight

Day 2 > Add 1 μg/mL doxycycline @ 8 am; Incubate at 37&deg;C 10 hours (until 6pm) > Change to CO2 independent medium (L-15, no phenol red, 10% T.S. FBS, 1% P/S) + 1 μg/mL doxycycline > Mark anchor point on well 1 > Set up plate under scope; secure plate to ensure no shifting; set to 34°C (Note: heater is +3-4°C off) > Find RFP+ cells and set staging > Record images every 4 hours for 24 hours


 * }