The orange dye channel is reserved for the CC5-labelled Internal Lane Standard 500 Pro (CC5 ILS 500 Pro) size standard. Unless otherwise specified, amplification reactions were performed in triplicate. Each 25 μL amplification PD-1 inhibiton reaction contained 5 μL of PowerPlex® ESI/ESX Fast 5× Master Mix and 2.5 μL of the respective 10× Primer Pair Mix, with 17.5 μL available for purified DNA sample and amplification grade water. Direct amplification reactions were set up in the same way except that 5 μL of 5× AmpSolution™ Reagent and 12.5 μL of amplification grade water

(10.5 μL if performing an amplification with 2 μL of SwabSolution™ extract) were used to bring the volume to 25 μL. AmpSolution™ Reagent protects the amplification reaction against chemicals in the FTA® cards, SwabSolution™ and PunchSolution™ Reagents that would otherwise inhibit the PCR. The following direct amplification sample types were used from three donors each. 1. One 1.2 mm blood FTA® punch Unless specified otherwise, thermal cycling was performed on the GeneAmp® PCR System 9700 thermal cycler with a silver or gold-plated silver sample block (Life Technologies, Foster City, CA) using the cycling parameters described in the technical manuals [14], [15], [16] and [17]. These consisted

of an initial activation of the thermostable DNA polymerase at 96 °C for 1 minute, followed by 30 cycles (26 cycles for direct amplification) of dentauration at 96 °C for 5 s, annealing at 60 °C for 35 s and extension at 72 °C for 5 s. This was followed by a final extension at 60 °C for 2 min www.selleckchem.com/products/Staurosporine.html and a ramp down to a 4 °C soak. Max ramp mode was used on the GeneAmp® PCR System 9700 thermal cycler. The same cycling protocol was followed for experiments conducted

on the 96-well (0.2 mL) Veriti® thermal cycler (Life Technologies, Foster City, CA) and the GeneAmp® PCR System 2720 thermal cycler (Life Technologies, Foster City, CA). Ramp rate was left at “100%” on the 96-well (0.2 mL) Veriti® Thermal Cycler. Amplified samples and allelic ladder were processed according to the technical manuals [14], [15], [16] and [17]. One microliter of amplification product or allelic ladder was combined with 10 μL Hi-Di™ formamide and 2 μL of CC5-labelled Internal (-)-p-Bromotetramisole Oxalate Lane Standard 500 Pro (CC5 ILS 500 Pro). Samples were heated to 95 °C for 3 min prior to quick chilling in a crushed wet-ice bath for at least 3 min Samples were injected at 3 kV for 5 s on an Applied Biosystems 3130 or 3130xl Genetic Analyzer and at 1.2 kV for 24 seconds on the Applied Biosystems 3500xL Genetic Analyzer. Data generated on the Applied Biosystems 3130 or 3130xl Genetic Analyzer were analyzed using GeneMapper®ID 3.2.1 software (Life Technologies, Foster City, CA) and a 50 RFU detection threshold whereas data generated on the Applied Biosystems 3500xL Genetic Analyzer were analyzed using GeneMapper®ID-X software (Life Technologies, Foster City, CA) and a 175 RFU detection threshold.