User:Karmella Haynes/Notebook/miR Trigger/2010/01/04

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01/04/10

 * RT-PCR: RFP reporter-transfected samples + controls

RT-PCR > Round 1: Controls KAH108 (RFP), KAH108 +Dox (RFP, YFP), mock (untransfected) --> Use dilutions of cDNA: 1. 1:10, 2. 1:100, 3. 1:1000, 4. 1:10000 --> 12 templates x 6 primer pairs YFP1, YFP2, RFP1, RFP2, MV2 (vector backbone), GAPDH --> Make 13x master mix for each primer pair

--> PCR: 95°C/ 3 min.; [95°C/ 30 sec, 57°C/ 30 sec, 72°C/ 30 sec] x 30; 72°C/ 3 min.; 4°C/ ∞

> Round 2: All samples --> Use 1:1000 dilution of cDNA --> 10 templates x 4 primer pairs: 1. YFP2, 2. RFP1, 3. GAPDH, 4. MV2 (vector backbone) --> Make 11x master mix for each primer pair

--> PCR: 95°C/ 3 min.; [95°C/ 30 sec, 57°C/ 30 sec, 72°C/ 30 sec] x 30; 72°C/ 3 min.; 4°C/ ∞


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