Survivors of Cycle XIV were divided in two aliquots and polymerase chain reaction (PCR) amplified under conditions that transliterated (10 (link),13 (link)) the Z:P pairs to C:G or T:A pairs in approximately equal amount. Translitation from Z:P to C:G used the following conditions: 0.4 μM each primer, 0.01 mM dZTP, 0.4 mM each dC/dGTP, 0.04 mM each dA/dTTP, 20 mM Tris–HCl pH 8.8, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton® X-100, 0.1 U/μl JumpStart Taq Polymerase (Sigma-Aldrich). Conversion from Z:P to T:A had the following conditions: 0.4 μM each primer, 0.4 mM dPTP, 0.04 mM each dC/dGTP, 0.4 mM each dA/dTTP, 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.001% (w/v) gelatin, 0.1 U/μl JumpStart Taq Polymerase.
Tags and barcodes were then added by 12 cycles of PCR with tagged primers. The amplified products were purified by native PAGE, recovered by gel-extraction and the collection of amplicons was submitted for Ion Torrent deep sequencing, according to the sequencing center guidelines.
Tags and barcodes were then added by 12 cycles of PCR with tagged primers. The amplified products were purified by native PAGE, recovered by gel-extraction and the collection of amplicons was submitted for Ion Torrent deep sequencing, according to the sequencing center guidelines.
