2.5.2. PCR Amplification

To reliably assign samples to individual reindeers, we used eight dinucleotide microsatellite markers that were sorted into two multiplex assays for e fficient genotyping (multiplex 1 and 2); six of the markers were taken from Wilson et al. [19] (RT1, RT6, RT13, RT20, RT27, RT30) and two from Røed and Midthjell [20] (NVHRT22, NVHRT46). The markers can be found in GeneBank using the accession numbers found in Table 1. The forward-primers were labeled with one of three fluorescent dyes (6FAM, NED or PET, Table 1). In addition, the pigtale sequence [21] was added to the 5 end of one of the reverse primers to facilitate accurate genotyping.

The PCR reactions were carried out in 10 μL reaction volume: 5 μL 2× multiplex PCR master mix (Qiagen Multiplex kit), 0.05 μg/μ<sup>L</sup> BSA (NEB) and adjusted primer set concentrations (Table 1). The PCR conditions for both multiplexes were 10 min at 95 ◦C, 35 cycles of 30 s at 94 ◦C, 30 s at 58 ◦C, 1 min 72 ◦C and a final extension for 45 min at 72 ◦C.

The PCR products (1 μL) were then mixed with Genescan 500 LIZ (Applied Biosystems) size standard (0.24 μL) and Hi-Di formamide (10.00 μL), following a 2 min denaturation at 95 ◦C on a 2720 Thermal cycler. Capillary electrophoresis was carried out on an ABI 3730 DNA Analyzer (Applied Biosystems). The POP-7 ™ Polymer was used as a separation matrix and the sample injection times were set to 4 s/2 kv. The PCR fragments were analyzed in GeneMapper 4.1 (Applied Biosystems). The alleles were automatically scored and then manually checked.

To check for possible contamination, every eighth sample added was a negative control. Negative controls contained all of the PCR master-mix components except the DNA template (water was added instead of DNA). Four samples (two tissue- and two scat samples) from previously known reindeer were added as positive controls. Two samples were excluded from the analysis due to an error in registration, leaving 303 samples for the genetic analysis. The combination of the eight markers comprised the genetic profile of an individual reindeer. The samples with the same genetic profile were grouped together and linked to the same individual reindeer.



"Pigtail" sequence added to the 5 end of the primer according to Brownstein et al. [21].
