ClaI restriction enzyme

Protocol tips

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	Two PCR products containing each mutation were digested by restriction endonucleases as follows. PCR products amplified by primer pair (iii) or (iv) were incubated with FokI (Takara‐Bio Co., Ltd, Kusatsu, Japan), and those amplified by primer pair (iv) were incubated with ClaI (Takara‐Bio Co.) for over 4 h, in order to identify the missense mutation in β3 exon 5 and exon 6. The digested PCR products were analyzed by electrophoresis using a 7% polyacrylamide gel. The PCR products after electrophoresis were visualized by staining with ethidium bromide solution (Wako Pure Chemical Industries).

Protocol tips

Two PCR products containing each mutation were digested by restriction endonucleases as follows. PCR products amplified by primer pair (iii) or (iv) were incubated with FokI (Takara‐Bio Co., Ltd, Kusatsu, Japan), and those amplified by primer pair (iv) were incubated with ClaI (Takara‐Bio Co.) for over 4 h, in order to identify the missense mutation in β3 exon 5 and exon 6. The digested PCR products were analyzed by electrophoresis using a 7% polyacrylamide gel. The PCR products after electrophoresis were visualized by staining with ethidium bromide solution (Wako Pure Chemical Industries).

Publication protocol

"In order to confirm two missense mutations in the b3 gene, PCR products for restriction fragment length polymorphism (RFLP) analysis were individually amplified from genomic DNA in the GeneAmp PCR System 9600 (Perkin-Elmer). Primer pairs (iii) H(+)/H(–) and (iv) I(+)/I(–) were used to detect a mutation in genomic exon 5 and exon 6, respectively. Thirty rounds of PCR were performed for genomic DNA under the following conditions: denaturation at 95 C for 30 s, annealing at 62 C (iii) or 60 C (iv) for 30 s, and extension at 72 C for 30 s. Two PCR products containing each mutation were digested by restriction endonucleases as follows. PCR products amplified by primer pair (iii) or (iv) were incubated with FokI (Takara-Bio Co., Ltd, Kusatsu, Japan), and those amplified by
primer pair (iv) were incubated with ClaI (Takara-Bio Co.) for
over 4 h, in order to identify the missense mutation in b3 exon 5 and exon 6. The digested PCR products were analyzed by electrophoresis using a 7% polyacrylamide gel. The PCR products after electrophoresis were visualized by staining with ethidium bromide solution (Wako Pure Chemical Industries)"

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