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== totoRNASeq Library Preparation == :'''TSO Reaction''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Purified cDNA||||||||||3ul |- |5x RT buffer [MMLV]||||||||||2ul |- |10mM dNTP mix||||||||||1ul |- |100mM DTT||||||||||0.25ul |- |10uM TSO||||||||||1ul |- |Betaine (5M)||||||||||2ul |- |RNase Inhibitor||||||||||0.25ul |- |Smarter MMLV||||||||||0.5ul |- |Total||||||||||10ul |- |} :::''Incubate @42C 20min'' :::''Incubate @70C 10min'' :'''PCR (PhusionHF)''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |TSO Reaction||||||||||10ul |- |2x Buffer||||||||||25ul |- |10uM P1-STRT||||||||||1ul |- |10uM PCR_R_N2_id||||||||||1ul |- |H2O||||||||||13ul |- |Total||||||||||50ul |- |} ::{| {{table}} |- |98C 30 sec|||||||||| |- |98C 15s -> 58C 20s -> 72C 30s||||||||||6x |- |98C 10s -> 72C 20s||||||||||9x (1ng); 12x (100pg) |- |72C, 5min |- |} *Run 5ul on gel: [[File:03052014_PCR1_Gel1_toto.jpg|300px]] Results: Decent amplification for all conditions. Negative control (NC) which does have ERCC (10pg level) shows a distinct band at around 300bp for both Mg protocol (above) and the RNaseIII protocol (see STRT gel below). Mg protocol does show more high molecular weight fragments. * Beads purified (0.8x) twice and eluted in 10ul. '''Repeat RNaseIII Protocol using Sten TSO''' :*Repeated exactly the same way except with the following substitutions: :**TSO.r06 replaced with C1-P1-RNA-TSO :**P1-STRT replaced with C1-P1-PCR-2 :*Run 5ul on gel: [[File:03052014_PCR1_Gel1_toto_sten.jpg|200px]] Note: Possible mistake with the quantification of the input RNA obtained from Illumina - may be double that intended. Indicates that the efficiency of this TSO in the totoRNAseq protocol might be too low to be truly viable...
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