Primetime std qpcr assay

The PCR mixture for each primer/probe assay contained for each sample: 10 mL of 1X Supermix for Probes (Bio-Rad), 1 mL of primers/ probe mixture (PrimeTimeÒ Std qPCR Assay (500 rxn)) that yields a final concentration of 500 nM primers and 250 nM probe in the reaction, 7 mL of DNase free water and 2 mL of sample DNA. The PCR mixture for the duplex PCR reaction per sample contained: 10 mL of 1X Supermix for Probes (Bio-Rad), 1 mL of each primers/probe mixture (PrimeTimeÒ Std qPCR Assay (500 rxn)) that yields a final concentration of 500 nM primers and 250 nM probe, 6ul of DNase free water and 2 mL of sample DNA. To avoid sample losses, the MasterMix was directly pipetted into the tubes containing the total DNA contained in each spore. We used three positive controls per strain. Two positive controls were of homokaryotic strains (of each MAT-locus) and the last positive control was of the dikaryotic strain (whole culture extracted DNA).