Bacterial DNA for PFGE

[Rafael Maldonado]

On Mon, 24 Mar 1997, Yves Bertheau wrote:
> We started a comparison of the PFGE patterns of pectinolytic erwinias
> (enterabacteria).
> We have troubles with some strains whose DNA get a smear in all
> tested  conditions (even with 1 min at 100  C before lysis).
> Does anybody know a protocol to avoid this DNA's degardation.

When do you get the degradation? During the digestion or before? If it is 
before, the only thing you can try is increasing the SDS and proteinase K 
concentrations (there is a limit in SDS concentration where proteinase K 
will work). Also, keep EDTA concentration high before digestion.

If it is after, you may have the restrictase contaminated. Sometimes, the 
batches are no good, so you may change the provider or try special 
enzymes certified for PFGE (even a certified enzyme did not work well for 
me). Try a control with the restrictase buffer without enzyme to test if 
the enzyme and its the contaminant are the culprits.

There is no much more you can do about it; maybe changing detergents 
(sarcosyl) or trying different proteases.

-Rafa

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