8. PFGE: Tips and Tricks to Success and Interpretation of Results for ...

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Poor / bad enzyme Vibrio cholerae digested with high fidelity NotI enzyme (NEB); not recommended for use with PFGE.
Star activity looks like incomplete • Star activity – a relaxation or alteration of the specificity of a restriction enzyme • Conditions that can lead to star activity – prolonged reaction time digestion – suboptimal buffer or buffer concentration – high (> 5% v/v) glycerol concentrations – high concentration of enzyme/µg of DNA ratio • Star activity is often misidentified as a lysis issue same plugs re-tested AscI AscI ApaI overnight incubation ApaI 2 hour incubation
Page 1 and 2: PFGE: Tips and Tricks to Success an
Page 3 and 4: Interpretation of PFGE Data • Tec
Page 5 and 6: Reproducibility 3 isolates of Salmo
Page 7 and 8: Artifacts • Salmonella Saintpaul
Page 9 and 10: “Ghost” or “Shadow” Bands
Page 11 and 12: Washing PFGE plugs • Washes - was
Page 13 and 14: Incomplete cell lysis Undigested, g
Page 15 and 16: High background • DNA degraded
Page 17: Enzyme digestion without BSA BSA BS
Page 21 and 22: Impact of run length on resolution
Page 23 and 24: Interpretation of PFGE Data • Tec
Page 25 and 26: Addition of a restriction site x x
Page 27 and 28: Mode of Transmission Contrasting Sh
Page 29: Acknowledgements All PulseNet parti

8. PFGE: Tips and Tricks to Success and Interpretation of Results for ...

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