BsgI requires 80 µM SAM in the reaction mixture. Incubation without SAM results in 25-50% activity. SAM should be fresh. We suggest making up the SAM just before usage and even aliquoting it out in the concentrated form so it does not have to undergo too many freeze-thaws. Even at -70°C it only lasts about six months. BsgI and SAM lose activity in overnight digests, so they are not recommended. It is better to add more units of BsgI and to spike once with fresh SAM than to do an overnight digest. BsgI is also inhibited by nucleotides and DNA contaminants. Drop dialysis of the DNA can increase cleavage.