AciI NEB#R0551

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	The PCR products were cut using the following restriction enzymes: fliC was cut with HhaI and Sau3AI; gnd with AciI and AluI; and mutS with AciI and HaeII. Restriction enzymes from New England Biolabs (Ipswich, MA) and Fermentas (Glen Burnie, MD) were used during the development of the molecular typing method. Single digestions were done by mixing 5 μl of the selected PCR product and 2.5U of NEB endonucleases or 1 Fast digest unit of the Fermentas endonucleases in final volume of 10 μl. NEB endonuclease mixtures were incubated for 1 h at 37°C. Fast digest mixtures were incubated for 10 min at 37°C. After incubation, DNA digestion was terminated by heat inactivation at 65°C for 20 or 10 min depending on the enzyme used or by the addition of 20 mM EDTA. In selected experiments, restriction digestions were cleaned using the MinElute Reaction Cleanup kit (Qiagen). Restriction digestions were repeated two to three times to test reproducibility of the restriction patterns. Restriction patterns were analyzed using the Agilent DNA 1000 kit and the 2100 Agilent Bioanalyzer (Agilent Technologies, Inc.).

Protocol tips

The PCR products were cut using the following restriction enzymes: fliC was cut with HhaI and Sau3AI; gnd with AciI and AluI; and mutS with AciI and HaeII. Restriction enzymes from New England Biolabs (Ipswich, MA) and Fermentas (Glen Burnie, MD) were used during the development of the molecular typing method. Single digestions were done by mixing 5 μl of the selected PCR product and 2.5U of NEB endonucleases or 1 Fast digest unit of the Fermentas endonucleases in final volume of 10 μl. NEB endonuclease mixtures were incubated for 1 h at 37°C. Fast digest mixtures were incubated for 10 min at 37°C. After incubation, DNA digestion was terminated by heat inactivation at 65°C for 20 or 10 min depending on the enzyme used or by the addition of 20 mM EDTA. In selected experiments, restriction digestions were cleaned using the MinElute Reaction Cleanup kit (Qiagen). Restriction digestions were repeated two to three times to test reproducibility of the restriction patterns. Restriction patterns were analyzed using the Agilent DNA 1000 kit and the 2100 Agilent Bioanalyzer (Agilent Technologies, Inc.).

Publication protocol

The PCR products were cut using the following restriction enzymes: fliC was cut with HhaI and Sau3AI; gnd with AciI and AluI; and mutS with AciI and HaeII. Restriction enzymes from New England Biolabs (Ipswich, MA) and Fermentas (Glen Burnie, MD) were used during the development of the molecular typing method. Single digestions were done by mixing 5 μl of the selected PCR product and 2.5U of NEB endonucleases or 1 Fast digest unit of the Fermentas endonucleases in final volume of 10 μl. NEB endonuclease mixtures were incubated for 1 h at 37°C. Fast digest mixtures were incubated for 10 min at 37°C. After incubation, DNA digestion was terminated by heat inactivation at 65°C for 20 or 10 min depending on the enzyme used or by the addition of 20 mM EDTA. In selected experiments, restriction digestions were cleaned using the MinElute Reaction Cleanup kit (Qiagen). Restriction digestions were repeated two to three times to test reproducibility of the restriction patterns. Restriction patterns were analyzed using the Agilent DNA 1000 kit and the 2100 Agilent Bioanalyzer (Agilent Technologies, Inc.).

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