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Chris:LabNotes/sci-Methyl Seq/Calendar/2017/2017-7-3
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==Barcode 2 Adapter Design (Adpt2_v4)== *As described above, the following is how we would eventually create dsDNA Adpt2_v4: Adpt2_v4: (second strand synthesis; cut by DraIII) <span style="color:blue">3' / / /</span> 5' -----<u>CACNNN|GTG</u><span style="color:blue">-----[Barcode2]DDDDDDDD</span>----- 3' ----- <span style="color:blue">\ \ \ 5'</span> | Second strand synthesis V <span style="color:blue">3' / / /</span> 5' -----<u>CACNNN|GTG</u><span style="color:blue">-----[Barcode2]DDDDDDDD</span>----- 3' -----<u>GTG|''NNN''CAC</u><span style="color:red">-----[''Barcode2'']HHHHHHHH</span>----- <span style="color:blue">\ \ \ 5'</span> | Cut by DraIII V <span style="color:blue">3' / / /</span> 5' /5Phos/<u>GTG</u><span style="color:blue">-----[Barcode2]DDDDDDDD</span>----- 3' <u>''NNN''CAC</u><span style="color:red">-----[''Barcode2'']HHHHHHHH</span>----- <span style="color:blue">\ \ \ 5'</span> *Below are the two oligos I'll be ordering today to perform second strand synthesis and restriction enzyme digestion: (using Barcode2 = GATAGG, DDDDDDDD = TGGAGAGG, NNN=CAG [such that won't anneal to the A-tailed sticky end of the original template]) <u>Adpt2_v4 Barcode Seq</u> (This is top strand containing Barcode2 Sequence) 5' AATGA<u>CACCAGGTG</u><span style="color:blue">GATGTGGTGGTGAAGGTGATGGGTAGTGAGGGA[GATAGG]TGGAGAGG</span>TGAAACGCGGCAACACGGGAAA<span style="color:blue">''GAGGTGGAGGGAGAAGTGAAAGTTG''</span> 3' (italicized portion is part of Y-tail) <u>Adpt2_v4 Universal Primer</u> (Primer to start second strand synthesis and complete Y-adapter formation) 5' <span style="color:blue">''GGTTTTGGGTGGGATTAGTGATTGG''</span>TTTCCCGTGTTGCCGCGTTTCA 3' (italicized portion is part of Y-tail) *If we wanted to order the primers fully formed, then we would order the following: (remove the sequences that would otherwise be digested away) Adpt2_v4: 5' /5Phos/<u>GTG</u><span style="color:blue">GATGTGGTGGTGAAGGTGATGGGTAGTGAGGGA[GATAGG]TGGAGAGG</span>TGAAACGCGGCAACACGGGAAA<span style="color:blue">''GAGGTGGAGGGAGAAGTGAAAGTTG''</span> 3' (italicized portion is part of Y-tail) Adpt2_v4_comp: 5' <span style="color:blue">''GGTTTTGGGTGGGATTAGTGATTGG''</span>TTTCCCGTGTTGCCGCGTTTCACCTCTCCACCTATCTCCCTCACTACCCATCACCTTCACCACCACATC<u>CACCTG</u> 3'
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