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).