Page 53 - Dedola2011

(COI and 16S) were used with the aim to compare results w ith those obtained by
Katsares (2008) and Ramboui (2011) for Aegean and Tunisian populations respectively.

Materials and Methods
Sampling

A total of 212 specimens belonging to 24 Mediterranean populations (Table l
and Fig. l) were collected. Where possible, at least ten individuals were analysed for
each sample. A non-lethal sampling method was applied by abscising a small portion of
mantle tissue in situ without sacrificing the individuai.

Analytical methods
DNA extraction: DNA was isolated using the Qiagen DNeasy tissue kit. The

DNA concentration was estimated by means of fluorimetric measurements (average
value per sample: l 00 ng/!J.l).

PCR amplification: COI and 16S rDNA mitochondrial regions were amplified
using specific primers designed by the Authors for the COI gene (L: 5'-
ggttgaactathtatccncc-3' and H: 5'-gaaatcatyccaaaagc-3 ') and pnmers provided by
Wilson and colleagues (200 l) for 16S gene. Each 25 !J.l PCR rnixture contained about
100 ng of total genomic DNA, 0.32 !J.M of each primer and 2.5 U of EuroTaq DNA
Polymerase (Euroclone) l x reaction buffer and 200 !J.M of each dNTP. The MgCh
concentration was set at 3 mM and 25 !J.g of BSA were added to the reaction mix.

PCR amplifications were performed according to the following steps: l cycle of
2 min at 94°C, 35 cycles of l min at 94°C, l min at 50°C and l min 30 s at 72°C each.
At the end, a post-treatment of 5 min at 72°C and a fina! cooling at 4°C were applied.
For all PCR reactions negative controls and replicates were included. Electrophoretic

53

Gìan Luca Dedola: ANALISI DELLA VARIABILITÀ GENETICA DI PATELLA FéRRUGINEA, PATEL.LA ULl'SSIPONENSIS (MOLLUSCA: GASTROPODA) E PINNA
NOBILIS (MOLLUSCA: BIVALVIA): lLCONTRJBUTO DEl DATI MOLECOLARl ALLA CONSERVAZIONE DI SPECIE MINACCIATE · Tesi di douorato in Scienze
della natura e delle sue risorse · indiri7.zo BK>logi.a Ambientale, Università degli Studi di Sassarl