Colorless gotaq flexi reaction buffer

Genomic DNA was extracted from the fin clips with the DNeasy 96 Blood and Tissue Kit (Qiagen) according to the manufacturer’s protocol. PCR was carried out using Colorless GoTaq Flexi Reaction Buffer (Promega) with PCR dnd primers^{19 (link)}. The PCR product was purified with QIAquick PCR purification Kit (Qiagen) and measured via Qubit Fluorometric Quantitation (Invitrogen). Purified and quantified PCR products were cloned into PCR4-TOPO using the TOPO TA cloning kit for sequencing (Invitrogen). Cloned fragments were sequenced with M13 forward and reversed primers using BigDye 3.1.

DNA was amplified in the following PCR mixture: Colorless GoTaq Flexi Reaction Buffer (Promega, Madison, WI, USA), 2.5 mM MgCl₂, 0.2 mM dNTP, 10% of DMSO, 0.5 U/μL Taq DNA polymerase, 0.5 pmol/μL FAM-labeled Forward primer [(Fam)5′-TGGCGACCCTGGAAAAGCTGAT-3′] and 0.5 pmol/μL Reverse primer (5′-GGTGGCGGCTGTTGCTGCTGCTG-3′). The reactions were run on thermocycler C1000 (Bio-Rad) with the following thermal cycling program: 95 °C for 5 min; then 35 cycles of 95 °C for 15 s, 63 °C for 15 s, and 72 °C for 25 s; with final extension at 72 °C for 25 min. Capillary electrophoresis was performed using capillary genetic analyzer ABI Prism 3130 (Applied Biosystems, Waltham, MA, USA), with size standard Liz 600; the obtained data were analyzed in Data Collection software v3.0 and GeneMapper v.4.0 (Applied Biosystems, Waltham, MA, USA).