(A) Comparison between linear and circular primer extension using the CHES reaction buffer system (similar to main text Figure 1D that is in the Tris buffer system). Extensions performed at −7°C for 2 weeks. Template sc12GAA-p and primer P9 were used. 100 pmol triplets were used. (B) The periodic oscillations were observed with various repeat sequence templates (CGG, GAC, and GAA, termed sc12CGG-p, sc12GAC-p, and sc12GAA-p, respectively) in both CHES and Tris buffer systems. Primers P9 (CGG), P9 (GAC), or P9 were used respectively. Extensions performed at −7°C for 4 weeks. 200 pmol triplet was used in the CHES buffer system samples, 100 pmol triplets was used in the Tris buffer system samples. (C) Dilution of samples increased the efficiency. The plot in (C) shows the difference in extension efficiency (Δ-extension efficiency) between the undiluted (Un.) and the 2–50-fold diluted (×2–×50) samples. The Δ-extension efficiency of the ligations before invasion (mean of bands 1–9) were unaffected by the dilution (giving a Δ-extension efficient of ~1). However, the Δ-extension efficiency of band 10 (full length +1 triplet, invasion) increased strongly with dilution. Extensions performed at −7°C for 1 week. (D) The same effect of dilution (improving invasion) was seen over a range of MgCl2 concentrations (50–200 mM). Extensions performed at −7°C for 1 week. All extension reactions presented here were run at standard reaction conditions described in main text Materials and methods except when specified otherwise for dilution, salt, or buffer system. In (C, D), template sc12GAA-p and primer P9 were used. Original gels and numeric values are supplied in Figure 2—figure supplement 1—source data 1.