overnight restriction digestion- good or bad? - Should we modify amount of enzyme added for overnight digests? (Nov/25/2009 )

Thank you everyone!

I do a lot of overnight digestions especially if I am digesting 15 ug of plasmid SNa for subcloning so that I can ensure 100% digestion, but I always use a total volume of 50ul for overnight digestions. and I keep the enzyme volume under 10%. I never had any star activity problem and I did do a lot of subcloninig.

I also know some people who leave the digestions O/N at room temperature instead of 37 ºC, and they say it works fine.

The only time I left a digestion o/n, was with Eco91I, and I did it at 37 without having problems with star activity or anything.