Genotyping

Genotyping of both zebrafish mutant lines was completed using PCR and an enzyme digestion assay. DNA was extracted from a 3 mm section of tail fin clipping using 50 µl lysis buffer (1 ml of 1 M Tris-HCl at pH 8.4, 5 ml of 1 M KCl, 150 µl of 1 M MgCl₂, 1.5 ml 20% Tween-20, 3 ml 10% NP-40 and 89.35 ml deionized water) incubated at 95°C for 20 min. Then, 5 µl proteinase K (03115828001; Roche) was added to the lysis buffer and incubated at 55°C for 1 h; the reaction was inactivated by incubating at 95°C for 20 min.

The kcnh6a^s290 line is identified by a T>A point mutation. Primers s290 kcnh6 MUT T7 F1 (5′-TAATACGACTCACTATAGGGAGCCAGAACAGAACGAAACG-3′) and s290 kcnh6 MUT T7 R1 (5′-ACCCCAGTGTTTTGAATGGT-3′) were designed to flank the site of the point mutation for a total product size of 753 bp. To amplify this site, 5 µl GoTaq Green Master Mix (M7123; Promega Corporation) was mixed with 0.625 µl of 10 µM upstream primer, 0.625 µl of 10 µM downstream primer, 2 µl genomic DNA and 21.75 µl nuclease-free water, for a total reaction volume of 50 µl. The PCR conditions were set as follows: an initial denaturing step was run for 2 min at 95°C followed by 35 cycles of 30 s of denaturation at 95°C, 30 s of annealing at 60°C and 30 s of extension at 72°C. An additional 10 min of extension at 72°C was done after the final cycle was complete. Following PCR, 10 µl of the PCR product was combined with 1 µl restriction enzyme smlI (R0597L; New England Biolabs), 3 µl CutSmart 10× Buffer (B7204S; New England Biolabs) and 16 µl nuclease-free water for a 30 µl reaction. This reaction was incubated at 55°C for 1 h before being run on a 2% agarose gel. Results from this enzyme digestion will show cut sites at 405 bp and 348 bp only when the wild-type sequence is present.

A similar assay was used to genotype kcnh6a^tb218. Following the same PCR mix and cycling conditions as listed above, we used primers tb218 T7 geno F3 (5′-TAATACGACTCACTATAGGGTTGGTGGGTGAGGCTAAAGA-3′) and tb219 T7 geno R3 (5′-ATGCACTGGGTCTCTGCAA-3′) to amplify an 853 bp region around a T>G point mutation. After PCR, a 30 µl reaction was set up using the restriction enzyme sphI (R3182S; New England Biolabs). This reaction was incubated at 37°C for 1 h, followed by 20 min of heat inactivation at 65°C. Results from this enzyme reaction will show a 566 bp band when the wild-type sequence is present, whereas a 292 bp band appears if the mutant sequence is present.