Nuclear DNA- Microsatellites
Singleplexed PCR was performed in 16 µl reactions containing 2.0 µl of DNA template, 4.5 µl of ddH2O, 0.5 µl of each primer, and 8.5 µl of DreamTaq Green PCR Master Mix. When amplifying microsatellites with the GS-2 and GS-4 primers, 0.3 µl of ddH2O was replaced with bovine serum albumin (BSA, New England Biolabs, Inc, 12 mg) to promote reaction specificity. For all samples a touchdown PCR profile was used with the following conditions: 95°C for 1 min; 2 cycles of 95°C for 15 sec, 60°C for 30 sec, 72°C for 45 sec; 2 cycles changing 60°C to 58°C; 2 cycles changing 58°C to 54°C; 2 cycles changing 54°C to 52°C; 35 cycles of 95°C for 15 sec, 50°C for 30 sec, 72°C for 45 sec; 72°C for 5 mins. Successful PCR amplifications were replicated twice in tissue samples and three times in museum samples. Following amplification, 1.5% agarose gels were run with a 100 bp size standard (Invitrogen) and stained with GelRed (Biotium) or SYBR Green (Invitrogen). To remove residual primers, dNTPs and nontarget molecules, solid phase reversible immobilization (SPRI hereafter) cleaning via magnetic beads was performed (following Rohland & Reich, 2012) in a ratio of 1 part PCR product to 1.5X magnetic beads (KAPA beads, Roche). Cleaned PCR products were eluted in 20 ul ddH2O and quantified on a NanoDrop Lite Spectrophotometer (ThermoScientific).