Candidate gene sequencing
PCR amplicons covering the coding sequence and partial 5' and 3' untranslated regions for each selected candidate gene were amplified, purified and sequenced from the CAST strain using protocols outlined in [57] with slight modifications. Total RNA from brain, liver, spleen, lung and testis was isolated from an adult CAST male mouse using Trizol (Ambion). cDNA was produced from total RNA using standard procedures. PCR primer sets for each gene were designed using Primer3 [58] (Additional File 5). The initial amplification was performed in 10 μl PCR reactions using the Invitrogen Platinum TaqPCRx amplification system (Invitrogen). The reactions contained 1X PCR buffer, 1X Enhancer solution, 1.5 mM MgSO4, 0.17 mM dNTPs, 1 μM each primer, 0.1 unit of Platinum Taq (Invitrogen) and approximately 25 ng of cDNA. Reactions were incubated for 5 min at 95°C, then cycled for 45 s at 95°C, 45 s at 55°C, 1 min at 72°C for 35 cycles with a final 72°C extension for 10 min on a MJ Research PTC-200. The products were visualized on 1.5% agarose gels containing 0.06 μg/ml EtBr. Bands of the correct size were excised and incubated in 100 μl of sterile H2O at 80°C for 10 min to elute DNA. Reamplification reactions consisted of 1X PCR buffer, 1.5 mM MgCl2, 0.17 mM dNTPs, 1 μM each primer, 0.1 unit of Taq (Promega) and 10 μl of eluate in a total volume of 50 μl. The same cycling parameters were used for reamplification reactions. Products were visualized on 1% 0.5X TBE agarose gels containing 0.06 μg/ml EtBr, excised and purfied using PCR purification columns (Promega). To quantity each fragment 1 μl of each purified product was run on a 1.0% agarose gel containing 0.06 μg/ml EtBr, along with a DNA mass ladder (Invitrogen). In a 96 well plate, 15 ng of each PCR product was added to 5 pM of primer for sequencing. The College of Agriculture and Environmental Sciences (CAES) Genomics Facility at the University of California, Davis, performed bidirectional sequencing of each amplicon.