Figure 7.

Homopolymeric tract formation due to 3′ unblocked dNTP contamination during TiEOS. Here, n represents the initiator strand; n + 1, n+2, etc., represent single-thymidine nucleotide additions, where anything greater than n+1 is considered a failure. This sample was generated using 60 units of TdT (M0315, NEB), 2 μL of buffer, 2 μL of CoCl₂, 1 μL of MgCl₂ (50 mM), 500 μM dTTP (3′ unblocked), and 50 pmol of T₂₀ initiator (water added to a final volume of 20 μL), for 10 min at 37 °C. The reaction was terminated using 5% 0.4 M EDTA. The chromatogram for this figure was generated using reverse-phase high-performance liquid chromatography (RP-HPLC); conditions included a DNASep column (C-18) using reverse-phase buffers (ADS Biotec). Samples were processed at 80 °C with ultraviolet detection at 260 nm.