Mitochondrial DNA amplification and sequencing
To amplify the 16S rRNA gene of samples from Osceola County, Florida, we
used the protocol described in Lv et al. 2014, using rDNA primers of
16S-F 5’-TTAAATTGCTGTRGTATT-3’ and 16S-R1 5’-CCGGTCTGAACTCASAWC-3’.
Amplification protocol was as followed: initial denaturation (94°C, 5
minutes); followed by five cycles of 94°C for 30 s, 49°C for 30 s, and
68°C for 30 s; five cycles of 94°C for 30 s, 47°C for 30 s, and 68°C for
30 s; five cycles of 94°C for 30 s, 45°C for 30 s, and 68°C for 30 s; 25
cycles of 94°C for 30 s, 43°C for 30 s, and 68°C for 30 s; followed by a
final extension step of 68°C for 5 minutes. All positive amplicons are
submitted for bi-directional sequencing at Genewiz Corporation (South
Plainfield, NJ).