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SfaNI > FAQ |  | SfaNI FAQ
See the Restriction Endonucleases FAQ also.
Q1: Is extended digestion using SfaNI recommended?
Q2: Why do the bands smear after SfaNI digestion when running an average agarose gel?
Q3: Does SfaNI cleave ssDNA?
Q1: Is extended digestion using SfaNI recommended?
A1: No, the DNA may get degraded. TypeIIs enzymes such as SfaNI will often yield worse results if more enzyme is added than is necessary or if digestion longer than one hour is attempted. This effect is not due to a contaminating nuclease, but instead to inherent additional activities of the SfaNI enzyme. Because this enzyme is composed of distinct recognition and cleavage moieties, random degradation of the protein during a long digest will occasionally release an active cleavage moiety which is no longer regulated by the recognition moiety. Thus, long digests will result in the creation of nuclease. This property is not the same as Star Activity.
Q2: Why do the bands smear after SfaNI digestion when running an average agarose gel?
A2: This is due to binding of the enzyme to the DNA, which will alter the migration rate (and/or cause smearing). This can be remedied by adding an ionic detergent (such as 0.1% SDS) to the sample before loading on the gel. Alternatively, extracting the sample with phenol, or using a `quick spin´ DNA purification column can help. This second cause relates to the tendency of some restriction enzymes to remain bound to their substrate after cleavage. All restriction enzymes have a very tight binding affinity for their recognition sites. And, they all have secondary, and relatively lower affinities for DNA in general, and for the half-site which results from cleavage. The strength of this secondary affinity varies greatly among restriction enzymes. For most restriction enzymes, this secondary affinity is sufficiently low enough to not be observed when digestion products are run on a gel. But, for several restriction enzymes, the secondary binding will cause an alteration of migration rate of the DNA fragments.
Q3: Does SfaNI cleave ssDNA?
A3: No.
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