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===Making cDNA === 64 65 72 73 75 74 RNA 3ul 3ul 3ul 3ul 4ul 3ul 10X DNase I buffer 5ul 5ul 5ul 5ul 5ul 5ul RNase-Free DNase I 2.5ul 2.5ul 2.5ul 2.5 2.5 2.5 H2O 39.5ul 39.5ul 32.5ul 32.5 31.5 32.5 37C 15min Clean up and concentrate all RNAs with Zymo DNA-Free RNA kit * Add 4 volumes of RNA binding buffer; * Transfer to Zymo-spin columns in collection tubes, spin @15k rpm for 30s; * Add 200ul RNA Wash buffer, spin @15k rpm for 30s; * Add 10ul DNase/RNase-free water, spin @15k rpm for 30s; Reverse transcription 64~75 RNA 8ul 10mM dNTP 1ul 50uM dT12-18 1ul 65C 5min -> chill on ice for 1min Add 10X RT buffer 2ul 25mM MgCl2 4ul 0.1M DTT 2ul RNaseOUT 1ul Superscript III 1ul Incubate all tube at 50C for 50min. Cleanup the first-strand reactions with qiaquick columns. quantify: GM12864: 74.8ng/ul; 2.17/1.16; GM12865: 72.3ng/ul; 2.16/1.09; GM12872: 79.1ng/ul; 2.08/0.78; GM12873: 76.4ng/ul; 2.16/1.17; GM12875: 66.4ng/ul; 2.17/1.14; GM12874: 120.6ng/ul;2.14/1.70.
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