Table 1. Primers and PCR profiles for amplification of enc1, glyt, cyt-b, and nd2.
| Gene | Source | Primer | Primer Sequencea | PCR Thermal Profileb |
|---|---|---|---|---|
| enc1 | Li et al. [53] | ENC1_F85 | 5'-GACATGCTGGAGTTTCAGGA-3' | (98 °C/20s, 57 °C/30s, 72 °C/45s) x 25 + (98 °C/20s, 55 °C/30s, 72 °C/45s) x 10 |
| ENC1_R982 | 5'-ACTTGTTRGCMACTGGGTCAAA-3' | |||
| ENC1_F88c | 5'-ATGCTGGAGTTTCAGGACAT-3' | (98 °C/20s, 57 °C/30s, 72 °C/45s) x 25 + (98 °C/20s, 55 °C/30s, 72 °C/45s) x 10 | ||
| ENC1_R975c | 5'-AGCMACTGGGTCAAACTGCTC-3' | |||
| glyt | Li et al. [53] | Glyt_F559 | 5'-GGACTGTCMAAGATGACCACMT-3' | (98 °C/20s, 57 °C/30s, 72 °C/45s) x 25 + (98 °C/20s, 55 °C/30s, 72 °C/45s) x 10 |
| Glyt_R1562 | 5'-CCCAAGAGGTTCTTGTTRAAGAT-3' | |||
| Glyt_F577c | 5'-ACATGGTACCAGTATGGCTTTGT-3' | (98 °C/20s, 57 °C/30s, 72 °C/45s) x 25 + (98 °C/20s, 55 °C/30s, 72 °C/45s) x 10 | ||
| Glyt_R1464c | 5'-GTAAGGCATATASGTGTTCTCTCC-3' | |||
| cyt-b | This study | cyb_Dist_f | ACAGGTCTTGGTTAGARTCCRGGYGGG | (95 °C/60s, 58 °C/60s, 72 °C/120s) x 35 |
| cyb_Dist_r | CCGGATTACAAGACCGGCGCT | |||
| nd2 | This study | nd2_Dist_f | AGCTTTTGGGCCCATACCCCA | (95 °C/60s, 58 °C/60s, 72 °C/120s) x 35 |
| nd2_Dist_r | AGGRACTAGGAGATTTTCACTCCTGCT |
a Listed from 5’ to 3’.
b Conditions for denaturation, annealing and extension steps for each cycle are listed in parenthesis, followed by the number of cycles. All reactions included a 5-minute initial denaturation at 95°C and a 7-minute final extension at 72°C.
c Primers used during a second (nested) PCR, required for successful amplification; 1:20 dilution between rounds.