Ptc 0200 dna engine cycler

DNA was extracted from tissue samples using a DNeasy Blood and Tissue Kit (Qiagen), with a final elution volume of 50 μL. We amplified 19 dinucleotide microsatellite loci (Scv1, Scv3, Scv4, Scv6, Scv8, Scv9, Scv12, Scv13, Scv14, Scv15, Scv16, Scv18, Scv19, Scv20, Scv23, Scv24, Scv25, Scv27, Scv31, Scv32; Hale, Lurz, et al., 2001 (link)). Microsatellite loci were amplified individually in 15 μL reactions containing 0.5 μL template DNA, 1 × AmpliTaq buffer, 1.5 mM MgCl₂, 0.75 mM each dNTP, 0.5 mM each primer, and 0.2 U AmpliTaq DNA polymerase (Applied Biosystems) under the following cycling conditions: 95°C for 2 min; 35 cycles of 94°C for 15 s, annealing temperature (48, 52, or 54°C) for 15 s (see Hale, Lurz, et al., 2001 (link)), and 72°C for 15 s; followed by 72 °C for 10 min. PCR amplifications were performed in a PTC 0200 DNA Engine Cycler (Bio‐Rad). Forward primers were labeled with a fluorescent dye and PCR products were visualized and sized on an Applied Biosystems 3730S capillary DNA analyzer, under standard run conditions, with 500 LIZ as the internal size standard. Electropherograms generated by the DNA analyzer were scored using GeneMarker software (v.2.6.3) and all genotypes were checked manually (Hulce et al., 2011 ).