Green gotaq 5 x flexi buffer

All PCRs were carried out using primers synthesized by Sigma and performed on a ³Prime Personal Thermal Cycler (Techne, UK) before being visualized on a 1.5% TAE agarose gel. To test for the presence of PaV1 in lobsters, a PCR was performed using known, specific primers for PaV1 (45aF: TTC CAG CCC AGG TAC GTA TC; and 543aR: AAC AGA TTT TCC AGC AGC GT) that amplify a region of 499 bp [47 (link)]. All PCR reactions were carried out in a total volume of 10 μl containing 1μl extracted DNA (50-200ng/ μl), 0.33 mM of each primer 45aF and 543aR, 2.5 mM MgCl2 (Promega), Green GoTaq® 5 x Flexi Buffer (Promega), 0.4 mM dNTP mixture (Promega), and 0.75 u GoTaq® Flexi DNA Polymerase (Promega) [16 ].

DNA was extracted from ~200 μl of hemolymph/ethanol mixture (~25 mg of hemolymph) with the Wizard genomic DNA purification kit (Promega) following a slightly modified manufacturer’s protocol [16 ]. DNA eluted was used as the template for PCR. Hemolymph DNA extraction was optimized to ensure detection of PaV1 by using known, positive controls initially derived from P. argus. DNA extractions were verified by running 1 μl DNA mixed with 4 μl Promega Green GoTaq® 5 x Flexi Buffer on a 1.5% TAE agarose gel.