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==Exp. 1 Making ds-cDNA for the total RNA from the hybrid lines== Templates: *Hybrid 1 total RNA: 1.9ug/ul *Hybrid 2 total RNA: 0.44ug/ul *BJ fibroblast total RNA: 110ng/ul First strand synthesis: H1 H2 BJ x 2 RNA 2.5ul 8ul 8ul 10mM dNTP mix 1.0ul 1ul 1ul 50uM Oligo(dT)20 1.0ul 1ul 1ul H2O 5.5ul - - 65C 5min -> place on ice for 1min Add 10x RT buffer 2ul 2ul 2ul 25mM MgCl2 4ul 4ul 4ul 0.1M DTT 2ul 2ul 2ul RNase OUT(40U/ul) 1ul 1ul 1ul SS III RT(200U/ul) 1ul 1ul 1ul 50C 50min -> 85c 5min -> place on ice -> add 1ul RNase H, 37C 20min Purify with the Qiaquick column (combine the two BJ reactions), eluted with 30ul ddH2O. Second strand synthesis: DNA 28ul 10X NEBuffer 4ul 50ng/ul random hexamers 2ul 10mM dNTP 0.4ul H2O 3.4ul 65C 5min -> place on ice for 1min -> add 2ul 10U/ul DNA Pol I -> 16C 2h -> 75C 20min Purify with Qiaquick column.
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