Ceq 8800

PCR reactions were performed using a PTC-200 thermocycler (MJ Research, Watertown, Massachusetts, USA) in 20-μL solution containing 5 μL of genomic DNA, 10 mM Tris buffer (pH 8.0), 10 mM MgCl₂, 0.2 mM dNTPs, 0.4 μM primer of each primer, and 1 unit of Taq polymerase (Fermentas International Inc., Burlington, Ontario, Canada). The PCR program used included an initial step of 1 min of denaturation at 94°C; 35 cycles of 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C; and a final extension cycle of 5 min at 72°C. Genotyping was conducted using capillary electrophoresis on an automated genetic DNA analysis system (CEQ 8800; Beckman Coulter, Fullerton, California, USA). Four microliters of PCR product were mixed with 28 μL of formamide and 0.4 μL of 400-bp DNA size standard (GenomeLab, Beckman Coulter) for capillary electrophoresis. Fragments were identified on the basis of their size and according to their mobility in relation to the size standard using a cubic function.
Electrophoresis data were collected automatically using the GenomeLab GeXP Genetic Analysis System (Beckman Coulter). Once all of the data scoring was complete, random samples were reamplified and rerun to assess reproducibility and confirm the scoring and allele sizes.