FastDigest FspBI

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	Using the same primers and thermal cycling conditions described above, genotyping of g.52734272 in unrelated ABDs and other breeds was carried out through a restriction enzyme digest with 5 µL PCR product and either BfaI CutSmart (New England Biolabs, Ipswich, MA, USA) or FspBI FastDigest (Thermo Scientific) for total reaction volumes of 25 and 15 µL, respectively. BfaI (FspBI) recognizes and cleaves (^) the following sequence: 5′-C^TAG-3′. Digests were visualized on a 1.2 % agarose gel, where wild-type alleles are uncut (489 bp) and mutant alleles are cut once (248 and 241 bp).

Protocol tips

Using the same primers and thermal cycling conditions described above, genotyping of g.52734272 in unrelated ABDs and other breeds was carried out through a restriction enzyme digest with 5 µL PCR product and either BfaI CutSmart (New England Biolabs, Ipswich, MA, USA) or FspBI FastDigest (Thermo Scientific) for total reaction volumes of 25 and 15 µL, respectively. BfaI (FspBI) recognizes and cleaves (^) the following sequence: 5′-C^TAG-3′. Digests were visualized on a 1.2 % agarose gel, where wild-type alleles are uncut (489 bp) and mutant alleles are cut once (248 and 241 bp).

Publication protocol

"Primers for PCR were designed to capture a 489 bp region encompassing g.52734272 (Forward 5′-AAGTCCCAGCAGCAACATAA-3′, Reverse 5′-GTCCAAAGTGGTCGGTCCT-3′). Products were amplified using ReddyMix master mix (Thermo Scientific, Waltham, MA, USA) with 0.4 µM primers, 50 ng DNA, and water for a total volume of 25 µL. Thermal cycling conditions were as follows: 95 °C for 5 min; five touchdown cycles of 95 °C for 30 s, 58 °C for 30 s reducing 1 °C per cycle, and 72 °C for 1 min; 31 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min; and a 10-min final elongation at 72 °C. Direct sequencing was carried out with BigDye Terminator using an ABI 3730xl Genetic Analyzer to validate g.52734272.

Using the same primers and thermal cycling conditions described above, genotyping of g.52734272 in unrelated ABDs and other breeds was carried out through a restriction enzyme digest with 5 µL PCR product and either BfaI CutSmart (New England Biolabs, Ipswich, MA, USA) or FspBI FastDigest (Thermo Scientific) for total reaction volumes of 25 and 15 µL, respectively. BfaI (FspBI) recognizes and cleaves (^) the following sequence: 5′-C^TAG-3′. Digests were visualized on a 1.2 % agarose gel, where wild-type alleles are uncut (489 bp) and mutant alleles are cut once (248 and 241 bp)."

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