5 ng) except that cycling was performed on a Mastercycler Nexus PCR Cycler with aluminium block (Eppendorf, Hamburg, Germany). The genotypes obtained were compared to those previously generated using the Investigator® buy R428 ESSplex Plus Kit [24]. For ChargeSwitch® purified samples, a standard 25 μL

Investigator® ESSplex Plus reaction volume with maximum of 15 μL of template DNA was used. Maxwell-extracted samples were amplified using a reduced 16.7 μL reaction volume with maximum of 10 μL of template DNA. Investigator® ESSplex Plus amplification reactions were performed with a standard 30 cycle protocol on a Mastercycler Nexus PCR Cycler with aluminium block except for an additional 3 min final extension step at 68 °C. One microliter of amplification product or allelic ladder was combined with 11.5 μL Hi-Di™ formamide and 0.5 μL of BTO Size Standard (Qiagen N.V., Venlo, Netherlands). Electrophoresis was done on an Applied Biosystems 3500xL Genetic Analyzer (injected at 3.0 kV for 8 s). The PowerPlex® ESI 17 Fast and ESX 17 Fast Systems were used to genotype

DNA from anonymous liquid blood samples from 656 unrelated individuals and 720 father and son pairs that were previously typed with the PowerPlex® ESX 17, ESI 17, and ESI 17 Pro Systems [5] and [25] along with six samples from the Standard Reference Materials 2391c, PCR Based DNA Profiling Standard and 10 samples from the Standard Reference Materials 2391b, PCR Based DNA Profiling Standard. Amplification products http://www.selleckchem.com/products/PD-98059.html were analyzed on an Applied Biosystems 3130xl Genetic Analyzer. All genotyping was performed with GeneMapper ID-X v1.4 software. Data tables were exported into Excel (Microsoft, Redmond, WA) and compared to data generated previously with the PowerPlex® ESX 17 and ESI17 Systems [25], and the Powerplex® ESI 17 Pro System [5]. N − 4 and N + 4 (N − 3 and N + 3 for D22S1045) stutter percentages

were calculated for all loci based on peak height from the data generated from unrelated individuals with the STR_StutterFreq Excel based software developed at NIST [26]. To ensure that data was not used from main allele peaks that were saturating, or where the main allele peak was too low and potentially in the stochastic range, BCKDHB stutter percentages were only calculated where the major allele was between 200 and 4000 RFU. In addition, to exclude contributions from N + 4 stutter that could artificially raise the height of the N − 4 stutter peak, N − 4 stutter was not calculated for alleles at heterozygous loci where the larger allele was two repeats away from the smaller allele at that locus. N − 2 stutter was calculated for D1S1656 and SE33. Full profiles were obtained in the presence of 0.5 mM EDTA for both the PowerPlex® ESI Fast and ESX Fast configurations (Supplemental Fig. 1). Signal decreased at all loci with increasing EDTA concentration for both configurations, except at vWA.