Mycycler gradient thermocycler

The E. garzetta mtDNA was obtained by polymerase chain reactions (PCR) using 28 primer sets reported by Sorenson et al. (1999) (link). The PCR products for each set of primers were < 1,500 bp in size and all fragment sequences overlapped each other by at least 200 bp. PCR amplifications were done with a Mycycler Gradient thermocycler (Bio-Rad) in a final volume of 50 μL, including 5 μL of 10x EXTaq buffer (Mg²⁺-free; Takara Biotech, Dalian, China), 2.5 mM of each dNTP, 75 mM MgCl₂, 10 μM of each primer, 1.5 U of ^EXTaq polymerase (Takara of Biotech, Dalian, China) and approximately 20–50 ng of total genomic DNA. The reaction included an initial denaturation at 94 °C for 3 min, followed by 35 cycles consisting of denaturation at 94 °C for 10 s, annealing at 50–56 °C for 30 s and extension at 72 °C for 2 min, with a final extension at 72 °C for 10 min. There was a negative control in each round of PCR to check for contamination. The products were electrophoresed on 1.5% agarose gels staining with ethidium bromide and visualized by ultraviolet transillumination. The PCR products were purified with a gel extraction kit (Sangon BioMedical, Shanghai, China) and directly sequenced (both directions) with an ABI 3730XL automatic sequencer (Perkin-Elmer) using an ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction kit (with AmpliTaq DNA polymerase FS, Applied Biosystems).