Table 3

Primers used for specific PCR and direct sequencing, amplification conditions and temperature profiles.

Primer

Sequence

amplification conditions

temperature profile


COI universal [43]

5'-GGTCAACAATCATAAAGATATTGG-3' 5'-TAAACTTCAGGGTGACCAAAAAATCA-3'

total volume 25 μl with: 0.17 mM dNTPs 3 mM MgCl2 in 1 × PCR buffer 0.13 μM of each primer 1 unit Taq polymerase (Invitrogen)

1 cycle of 2.5 min at 94°C

40 cycle 30s at 90°C

1 min at 48°C

1 min at 72°C

1 cycle of 10 min at 72°C

16S universal [44]

5'-CGGCCGCCTGTTT ATCAAAAACAT-3' 5'-GGAGCTCCGGTTTGAACTCAGATC-3'

total volume 15 μl with: 0.1 mM dNTPs 2.5 mM MgCl2 in 1 × PCR buffer 0.2 μM of each primer 0.5 unit Taq polymerase (Invitrogen)

1 cycle of 2.5 min at 90°C

10 cycles of 50s at 92°C

30s at 44°C

40s at 72°C

36 cycles of 30s at 92°C

40s at 48°C

40s at 72°C

1 cycle of 3 min at 72°C

ITS-1 mollusc specific [45]

5'-TAACAAGGTTTCCGTAGGTGAA-3' 5'GCTGCGTTCTTCATCGATGC-3'

total volume 15 μl with: 0.3 mM dNTPs 2.5 mM MgCl2 in 1 × PCR buffer 0.18 μM of each primer 0.5 unit Taq polymerase (Invitrogen)

1 cycle of 3 min at 94°C

40 cycles of 30s at 92°C

30s at 52°C

1 min at 72°C

1 cycle of 5 min at 72°C


Pfenninger et al. BMC Evolutionary Biology 2005 5:59   doi:10.1186/1471-2148-5-59

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