FAQ: I am observing partial digestion when using MmeI. What could be the reason?
1. MmeI is sensitive to salt in the reaction and any salt carry-over from a DNA prep may inhibit the enzyme. Test the reaction on a commercial DNA to determine if this is the problem.
2. MmeI requires SAM for efficient cleavage, and SAM is extremely labile. Try to use a fresh tube of SAM as well as commercial DNA to see if this is the problem.
3. MmeI does not have much turn-over as an enzyme, unlike common Type IIP restriction enzymes. Thus it is important to have a 1:1 ratio of MmeI molecules to the number of sites to be cut in the reaction. This can be particularly important for short DNAs, since 1 microgram of a 100bp PCR product containing a single MmeI site represents approximately 10-fold more sites per microgram than the PhiX174 substrate used for MmeI unit definition.
4. MmeI requires two binding sites for efficient cleavage, and digestion of plasmids with a single site results in partial digestion of approximately 70% cutting. As MmeI requires 2 sites, addition of a double-strand oligo containing an MmeI site may help improve cleavage. The ds oligo should have 8 to 12 bp of flanking sequence on either side of the MmeI recognition sequence, but need not extend all the way to the point of cleavage.
