Sanger Sequencing of PCR Products

PCR reactions were prepared for Sanger sequencing by adding 10 units of exonuclease 1 and 1 unit of shrimp alkaline phosphatase to 10 μl of PCR reaction before incubation at 37°C for 30 min followed by 80°C for 10 min to inactivate the enzymes. One microliter of the cleaned product was used as sequencing template where the amplicon was 1 kb or less in size and 2 μl when over 1 kb. Sequencing reactions were in 10 μl volumes with 100 nM primer, 1.5 μl BigDye buffer and 1 μl BigDye 3.1, made up to 10 μl with water. After a denaturation step of 96°C for 2 min, 35 cycles of 96°C for 10 s/52°C for 15 s/60°C for 3 min were performed. Finally, reactions were held at 72°C for 1 min, sent for commercial data extraction and returned in the form of ABI files.