Bacterial DNA for PFGE
rafael at howard.genetics.utah.edu
Wed Mar 26 14:45:25 EST 1997
On Mon, 24 Mar 1997, Yves Bertheau wrote:
> We started a comparison of the PFGE patterns of pectinolytic erwinias
> 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.
Rafael Maldonado | 'No te creas todo
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