User:Karmella Haynes/Notebook/miR Trigger/2009/12/24

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12/24/09

 * &#x2713; RNA prep & cDNA for KAH93, 94, 95, 108/MV2 transfected Dox+/- JDS33-47 cells
 * &#x2713; Split U2OS plain: 1x 1:10 flask; 2x 6-well, 2.5x105/well, ab-free
 * &#x2713; Split JDS33-47: 1x 1:10 flask

RNA prep & cDNA > 6-well plates: use 1 mL TRIZol per well > Dissolve pellet in 15 μL THE RNA Storage Solution

> Measure RNA concentrations

> oligo(dT) Primer annealing --> Discard low yeild samples (2, 4); retry later --> 2 rxns for KAH108, 108/Dox, no DNA, no DNA/ Dox (12 total)

> cDNA synthesis mix

--> Add 10 μL mix to each annealing rxn. --> 50°C/ 50 min., 80°C/ 5 min., ice --> Add 1.0 μL RNase H, 37°C/ 20 min. --> Store at -20°C


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