DdeI restriction enzyme

Protocol tips

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	A fragment that included the polymorphic nucleotide position was amplified and digested with endonuclease DdeI or Kpn2I (Takara Bio Inc., Shiga, Japan). Digested DNA fragments were separated on a 2.5% agarose gel and visualized after staining with ethidium bromide.

Protocol tips
A fragment that included the polymorphic nucleotide position was amplified and digested with endonuclease DdeI or Kpn2I (Takara Bio Inc., Shiga, Japan). Digested DNA fragments were separated on a 2.5% agarose gel and visualized after staining with ethidium bromide.

Publication protocol

Blood sample of each cattle, which were bled for medical check, were distributed from the Prefectural Livestock Experiment Stations and stored the samples at –20°C until we could extract DNA. We isolated genomic DNA from whole blood using a commercial DNA isolation kit (Takara Bio Inc., Shiga, Japan) and measured the purified DNA with a spectrophotometer (NanoDrop ND-2000c: Thermo Scientific, Inc) at the 260/280 nm absorbance ratio. Cattle were genotyped for the CRH and LEP genes with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), according to method by Buchanan et al. [7,8], using the following primers: CRH; forward (5’- GCG CCC GCT AAA ATG CGA CTG A -3’) and reverse (5’- CTG TGA TGC CTG CCG GGC AC -3’), LEP; forward (5’- GCG CCC GCT AAA ATG CGA CTG A -3’) and reverse (5’- CTG TGA TGC CTG CCG GGC AC -3’). A fragment that included the polymorphic nucleotide position was amplified and digested with endonuclease DdeI or Kpn2I (Takara Bio Inc., Shiga, Japan). Digested DNA fragments were separated on a 2.5% agarose gel and visualized after staining with ethidium bromide.

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Manufacturer protocol

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