Additional file 2: of Comparing species tree estimation with large anchored phylogenomic and small Sanger-sequenced molecular datasets: an empirical study on Malagasy pseudoxyrhophiine snakes

PCR and Sequencing Protocols. All PCR reactions were 10 μL reactions consisting of 5 μL of GoTaq® Green Master Mix, 3 μL of H2O, 0.5 μL each of forward and reverse primers at a 10 μM concentration, and 1 μL of DNA extract. Samples were incubated at 96 °C for 15 min initially, 96 °C for 45 s, followed by 45 s at the appropriate temperature for the primer pair (see below), with a 72 °C extension period for 1 min. This procedure, minus the initial 15 min incubation period, was repeated for 35 cycles. Reactions were cleaned with 2 μL of ExoSap-IT® following the Exosap-IT® protocol. Sequencing reactions used the same primers as for the PCR reactions. We used the BigDye® Terminator v3.1 Cycle Sequencing Kit; each sequencing reaction consisted of 0.2 μL of ABI BigDye® Terminator Ready Reaction Mix, 1.5 μL of ABI BigDye® 5X Sequencing Buffer, 4.3 μL of H2O, 1 μL of the cleaned PCR reaction template, and 0.5 μL of the 10 μM primer for each direction. Sequencing reactions were incubated initially at 96 °C for 1 min, followed by 96 °C for 10 s, 50 °C for 5 s, and 60 °C for 1 min 15 s and repeated for 15 cycles, minus the initial 1 min incubation period; the reaction was then incubated at 96 °C for 10 s, 50 °C for 5 s, and 60 °C for 1 min 30 s and repeated for 6 cycles; the reaction was then incubated at 96 °C for 10 s, 50 °C for 5 s, and 60 °C for 2 min and repeated for 5 cycles. Sequencing reactions were cleaned prior to sequencing using ethanol precipitation. (PDF 155 kb