Page 132 - Dedola2011

Materials and methods

Sampling and DNA extraction

A total of 478 specimens of Patella ulyssiponensis were collected from the intertidal
zones of40 localities over most ofthe species distribution range (see Table l , Fig. l for
details). Individuals were collected from each sampling site using the following a non
lethal protocol: the individuai was gently removed from the substrate by means of a
wood chisel, and a 30-60 mg sample of foot museie was excised using a sterilised
surgical forceps. The individuai was then repositioned in its so-called ' home scar', a
depression in the rock formed by abrasion by the shell, resulting in a tighter fit to the
rock and reduced risk of desiccation. Genomic DNA was extracted from the tissue using
the Macherey-Nagel NucleoSpin® Tissue.

ISSR amplifications

A set of22 primers was preliminarily assayed on a limited number ofindividuals
identify the primers that produced scorable and reproducible bands (Table l ). The PCR
reaction mixture (25 ).!l volume) contained 0.5 units ofTaq DNA Polymerase from
Thermus aquaticus with lOx reaction buffer without MgCh (Sigma-aldrich~, lx
reaction buffer (Sigma aldrich®), 2.5 mM MgCh, 0.2 ).!M primer, 200 ).!M of each dNTP
(Sigma-aldrich~, and up to 30 ng of genomic DNA. PCR amplification was performed
in a i-cycler Thermal Cycler (Biorad®) programmed for l cycle of3 min at 94° C, 35
cycles of 40 s at 94° C, 45 s at 50° C, and l min and 40 s at 72° C. At the end ofthese
cycles a post-treatment at 72° C for 5 min to complete partial amplification and a final

132

Gìan Luca Dedola: ANALISI DELLA VARIABILITÀ GENETICA DI PATELLA FéRRUGINEA, PATEL.LA ULl'SSIPONENSIS (MOLLUSCA: GASTROPODA) E PINNA
NOBILIS (MOLLUSCA: BIVALVIA): lLCONTRJBUTO DEl DATI MOLECOLARI 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