Molecular Identification of Aquatic Organisms

A similarly-sized small piece of kidney and/or gill tissue was cut off and lysed overnight at 56 °C in 100 μl of Mole® lysis buffer and 10 μl of Proteinase K solution (Sigma-Aldrich, Hamburg, Germany). DNA from each piece was then extracted on a GenMole (Mole Genetics, Oslo, Norway) using the Mole Genetics DNA Tissue Kit and the DNA tissue protocol. Approximately 900 bp of rDNA was amplified using the primers and protocol of Freeman et al. [22 (link)] as described below. All PCR reactions were done using Illustra PuReTaq Ready-To-Go ™ PCR Beads (GE Healthcare, Oslo, Norway). Each reaction contained 1 μl of the forward primer, 1 μl of the reverse primer, 3 μl of the template DNA and 20 μl of sterile water. PCR products were sent to Macrogen for sequencing (Macrogen, Amsterdam, The Netherlands). All products were sequenced using the PCR primers. Sequences were proofread in VectorNTI ver. 11 (Invitrogen, Oslo, Norway) and subjected to a GenBank BLASTn search to search for identity with known sequences. The same samples were also subjected to RT-PCR analyses following the protocol of Nylund et al. [29 (link)], but targeting the rRNA gene and each analysis was repeated once. Average values for replicates were calculated.