problems with restriction digestion

Matthew Martino via methods%40net.bio.net (by martino from metabolix.com)
Mon May 11 14:50:15 EST 2009


I agree.  There is always the concern of Methylation, a cells natural defense of dna from restriction enzymes.  If the extra bands appear and they seem faint, for instance dimmer than the band above and the band below it, then you are having partials.  This can easily be explained by the fact that some  enzymes require exact salt conditions for activity and anything differing from the optimum will result in partial digests.  One way to check if this is the case is doing digests with the enzymes individually.  You should see linear DNA fragments in both cases (slight smiling of bands, but no streaking and definitely not multiple bands such as linear and supercoiled running together).  Whichever enzymes doesn't give you completely linear DNA is the problem.

Matt


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