SmaI restriction enzyme

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	After treatment with proteinase K (2 mg/ml) overnight at 52°C, the sample plugs were incubated three times (for 30 min each time) with 2 ml of washing buffer B (50 mM Tris-HCl, pH 7.5) containing 2 mM phenylmethylsulfonyl fluoride at 50°C and then rinsed three times (for 30 min each time) with 2 ml of washing buffer B. One-eighth of each plug was sliced off and digested with 10 U of SmaI (Takara Shuzo)

Protocol tips

After treatment with proteinase K (2 mg/ml) overnight at 52°C, the sample plugs were incubated three times (for 30 min each time) with 2 ml of washing buffer B (50 mM Tris-HCl, pH 7.5) containing 2 mM phenylmethylsulfonyl fluoride at 50°C and then rinsed three times (for 30 min each time) with 2 ml of washing buffer B. One-eighth of each plug was sliced off and digested with 10 U of SmaI (Takara Shuzo)

Publication protocol

Genomic DNA for the PFGE analysis was prepared by the procedure established by Hood (9) with some modifications. Disposable 100-μl scale plug molds (Bio-Rad) were used to prepare agarose plugs for each sample. Overnight cultures of the Hib isolates were resuspended at 5 × 108 cells/ml in washing buffer A (100 mM NaCl, 20 mM Tris-HCl, 20 mM EDTA, pH 7.6). Fifty microliters of each cell suspension was used to prepare each plug. After treatment with proteinase K (2 mg/ml) overnight at 52°C, the sample plugs were incubated three times (for 30 min each time) with 2 ml of washing buffer B (50 mM Tris-HCl, pH 7.5) containing 2 mM phenylmethylsulfonyl fluoride at 50°C and then rinsed three times (for 30 min each time) with 2 ml of washing buffer B. One-eighth of each plug was sliced off and digested with 10 U of SmaI (Takara Shuzo). The gels were processed with the contour-clamped homogeneous electric field DR II PFGE apparatus (Bio-Rad) under the following electrophoresis conditions: 7 h at 170 V with an initial time of 5 s and a final time of 20 s, followed by 14 h at 170 V with an initial time of 5 s and a final time of 80 s. Electrophoresis was performed in 0.5× TBE buffer at 11°C. A 48.5-kb lambda DNA ladder (FMC BioProducts) was used as a molecular size marker. Hib ATCC 10211 DNA was used for PFGE-RFLP as a universal standard marker. After electrophoresis, the gels were stained with ethidium bromide and photographed under UV light at 302 nm.

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