User:Karmella Haynes/Notebook/Polycomb project/2010/02/08

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02/08/10

 * &#x2713; RNA preps/ cDNA synth.: Polycomb-ATF lines (dox-induced, ~2 days)
 * &#x2713; HEK293 #4 Luc: 1x 1:10 flask, 1x 24-well plate (~10,000 cells per well) for Gal4-EED transfection/ luc assay
 * &#x2713; U20S Flp-in T-REx: 1x 1:10 flask, 1x 6-well (for Pc-ATF gene expression control)

RNA Preps/ cDNA synth.

> RNA Preps --> Use 1 mL TriZol/ well (6-well dish) --> Combine duplicate after resuspending RNA pellet in 6 μL THE RNA Storage Solution

> oligo(dT) Primer annealing

> cDNA synthesis mix --> 8 reactions total

--> 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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