User:Karmella Haynes/Notebook/miR Trigger/2009/12/29

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12/29/09

 * &#x2713; Transfection 1: RFP-reporters KAH93-95 for time-lapse microscopy
 * &#x2713; Transfection 2: RFP-reporters KAH93-95 for RNA(RT-PCR)

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); Grow cells at 37&deg;C overnight

Day 2 > Change to CO2 independent medium (L-15, no phenol red, 10% T.S. FBS, 1% P/S) > 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 > Add 1 μg/mL Dox; record images every 2-4 hours

Transfection 2 > KAH93-95/MV2 + JDS33-47 U2OS > 6-well format, Lipofectamine 2000

> 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. > Culture vol. = 3.5, no need to remove 500 mL. Add 500 μL complexes to each well (4 ml med. each); Grow cells at 37&deg;C > Refresh medium after 5 hours (ab-free)

Day 2 > Add 1μg/mL Dox to "Dox" wells.

Day 3 > RNA preps/ cDNA synthesis


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