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

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01/14/10

  • Outline of experiments/ tasks

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

Wells Plasmid DNA Volume Fugene Opti-MEM
Col.1 (1-3) KAH93/MV2 400 ng 1.9 μL 3.6 μL 46.4 μL
Col.2 (4-6) KAH94/MV2 " 2.4 μL " "
Col.3 (7-9) KAH95/MV2 " 2.3 μL " "
Col.4 (10-12) KAH108/MV2 " 0.9 μL " "

> 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°C overnight

Day 2
> Add 1 μg/mL doxycycline @ 8 am; Incubate at 37°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