Powerplex esi kit

Referent samples (12 buccal swabs) from potential living relatives were collected, recorded, and preliminarily labeled by local DNA experts. Living relatives were directly contacted by a local person (Mr Jure Lauc), who was present during the collection of each sample. Collection was performed with an Internal GENOS collection kit, and dried and labeled samples were transported to the laboratory. Upon arrival, the samples were relabeled and the relevant information entered into the Chain of Custody forms. The samples were stored at -80°C until DNA extraction was performed using the QIAmp DNA Mini Kit (14 ).
PowerPlex^® ESI kit (Promega Corp.) was used for further analysis. Similar amounts of DNA were used in all PCR reactions. Amplification was carried out as described previously (12 ). The total reaction volume was 10 μL. Additionally, PowerPlex^®Y23 kit (Promega Corp.) was used to simultaneously amplify 23 Y-STR loci according to manufacturer’s recommendations (13 ). PCR amplification was carried out by the Applied Biosystems® GeneAmp® PCR System 9700 (Life Technologies) according to manufacturer’s recommendations. Electrophoresis of the amplification products was performed on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The raw data were compiled and analyzed using 310 Data Collection Software and GeneMapper^TM 3.2.

The DNA concentration was determined using Quantifiler Human DNA Quantification Kit (Applied Biosystems Foster City, CA, USA) (11 ). The reaction was carried out in the AB 7300 Real-Time PCR System (Applied Biosystems) according to manufacturer’s recommendations.
The PowerPlex^®ESI kit (Promega Corp., Madison, WI, USA) was used to simultaneously amplify 15 STR loci: D3S1358, D8S1179, D18S51, D21S11, FGA, TH01, vWA, D2S441, D10S1248, D22S1045, D1S1656, D12S391, D2S1338, D16S539, D19S433 as well as the gender determination locus, Amelogenin. Amplification was carried out as described previously (12 ). The total volume of each reaction was 25 μL. Additionally, the PowerPlex^®Y23 kit (Promega Corp.) was used to simultaneously amplify 23 Y-STR loci according to manufacturer’s recommendations (13 ). PCR amplification was carried out using the Applied Biosystems® GeneAmp® PCR System 9700 (Life Technologies) according to manufacturer’s recommendations. Electrophoresis of the amplification products was performed on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The raw data were compiled and analyzed using 310 Data Collection Software and GeneMapper^TM 3.2.