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

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Problems with cell suspensions S S S S • Cell concentration is too high – DNA (dark bands) in wells incomplete cell lysis – thick “blurry” bands in lanes – more cell debris and more enzyme inhibitors requires more washing and more proteinase K – more DNA requires more units of enzyme and/or more time for complete digestion
Incomplete cell lysis Undigested, genomic DNA S S • Lyse in 5ml cell lysis buffer (50 mM Tris: 50 mM EDTA, pH 8.0; 1% sarcosyl; 100 µg/ml Proteinase K per sample) at 54ºC with constant and vigorous agitation • Some organisms lyse better than others – lysis times may vary, typically 1 – 4 hours – Gram+ organisms more difficult to lyse than Gram- organisms • Plugs typically clear as cells lyse • Incomplete lysis indicated by incomplete restriction, smearing, and significant fluorescence in the plug slice
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: Washing PFGE plugs • Washes - was
Page 15 and 16: High background • DNA degraded
Page 17 and 18: Enzyme digestion without BSA BSA BS
Page 19 and 20: Star activity looks like incomplete
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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