Page 106 - Dedola2011

2.4. RFLPs analysis
The effectiveness of the restriction enzyme Taqi in distinguishing P. ulissyponensis s.l.
was tested on COI gene amplification products obtained by the newly designed primers on
the 65 specimens of Patella sampled. To carry out the enzymatic digestions, an aliquot of
l O~-tl of each PCR product was mixed w ith a reaction solution containing l 111 of enzyme
(Taqi, FastDigest®by Fermentas~ and 0.67x Green Buffer®in a final volume of 30 111.
Samples were incubated at the temperature of 65° C for 5 min, and then separated on the
2% agarose gel for 90 min. In Figure 2, ali obtainable Taqi digestion fragments on a 437
bp long COI sequence of P. ulyssiponesis s.l., P. caerulea, P. vulgata and P. candei are
reported. Note that, for the analysis, we did not take into consideration fragments lower
than l 00 bp, since we chose to rely only on data unequivocally scorable. As well,
differences in gel migrations among fragments smaller than 50 bp were considered with
caution.

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