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== IVT: == :'''2nd Strand''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Purified cDNA||||||||||7.7ul |- |FS Buffer||||||||||0.2ul |- |SS Buffer||||||||||1ul |- |dNTP||||||||||0.8ul |- |DNA Pol||||||||||0.2ul |- |RNase H||||||||||0.1ul |- |Total||||||||||10ul |- |} :::''Incubate @16C 2hours'' :'''Add T7 Promoter''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |dsDNA||||||||||10ul |- |KAPA qPCR MM||||||||||25ul |- |[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#IVT_3.27_primer_sequence RL.Add.T7] (10uM)||||||||||0.5ul |- |dH2O||||||||||14.5ul |- |Total||||||||||50ul |- |} :::''Incubate @94C 2min'' :::''Incubate @60C 2min'' :::''Incubate @72C 5min'' :'''Beads Purify''' ::*Add 50ul beads ::*Wash twice with 80% EtOH ::*Resuspend in 6.4ul :'''IVT RXN''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |dsDNA||||||||||6.4ul |- |ATP||||||||||1.6ul |- |GTP||||||||||1.6ul |- |CTP||||||||||1.6ul |- |UTP||||||||||1.6ul |- |10x T7 Buffer||||||||||1.6ul |- |T7 Polymerase||||||||||1.6ul |- |Total||||||||||16ul |- |} :::''Incubate @37C 10 hours; lid 70C'' :::''Incubate @4C until ready'' :'''RT RXN''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |synthesized RNA||||||||||5ul |- |10uM [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#IVT_5.27_primer_sequence RL.IVT.N9]||||||||||1ul |- |Water||||||||||1ul |- |} :::''Incubate @65C 5min'' :::''Incubate Ice 1min'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |10x RT Buffer||||||||||1ul |- |10mM dNTP||||||||||0.5ul |- |100mM DTT||||||||||0.5ul |- |RNase Inh||||||||||0.5ul |- |SSIII||||||||||0.5ul |- |Total||||||||||10ul |- |} :::''Incubate @25C 5min'' :::''Incubate @50C 20min'' :::''Incubate @70C 10min'' :'''Beads Purify''' ::*Add 10ul beads ::*Wash twice with 80% EtOH ::*Resuspend in 23ul dH2O :'''PCR (PhusionHF) and SYBR''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |SSIII Reaction||||||||||23ul |- |2x Buffer||||||||||25ul |- |10uM [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence PB_PCR_F]||||||||||1ul |- |10uM PCR_N2_idX||||||||||1ul |- |Total||||||||||50ul |- |} ::{| {{table}} |- |98C 30 sec|||||||||| |- |98C 15s -> 58C 20s -> 72C 30s||||||||||6x |- |98C 10s -> 72C 20s||||||||||10x (9x for Sybr) |- |72C, 5min |- |} :::*Ampure Beads Purify Phusion Amplified(0.8x) :::*Resuspend in 10ul :::*Run 1ul on gel [[File:08202013_IVT_PCR1_gel1.jpg]]
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