digestion at 4 degrees - (Sep/27/2008 )

has anyone ever tried restriction digestion of PCR product overhang sites in the fridge overnight? i was thinking the cooler temperature would allow the enzymes to dock and cut better at the ends of these small PCR products. please let me know if this has worked or not worked for you, thanks!

I think this is unlikely to work. The length of the PCR product should have little effect on the activity of restriction enzymes. The length of the 5' primer overhang (if you are cutting added sites) will have a very major effect.

so the enzymes are basically not active at 4 degrees you're saying? ( know their optimal temperature is 37 degrees, but i've done cutting at room temperature too to reduce the kinetics for better docking and cutting at the overhang ends).
will restriction enzymes lose their activity if left in the fridge for ~ 20 hrs?

for cloning purposes I have digested my DNA overnight at 37 Celsius and have not experience any problem. i don't think you will need to trouble yourself with a 4 Celsius digestion. The length of bp around your restriction site is more important to the efficiency of digestions. The rule of the thumb say 6bp on both end of the restriction site. NEB technical guide (on NEB webpage) is useful.

I do not see much point in going for 4 degree digestion. I regularly clone PCR amplified fragments by using restriction enzymes. In general I leave 3-4 additional bp to the termini of the fragment. Leaving more does not make any sense as a number of experiments have proved.

Check out the following website for instance:

http://www.fermentas.com/techinfo/re/restrdigpcrii.htm

Below 10 degrees the enzyme activity would be so low that most of the DNA would remain uncut.

Each Restriction Enzyme have an optimum temp. and not always is 37C. Higher or lower temperatures will cause the enzyme to become inactive. I inactive mine by chilling the reaction. So check the literature of your enzyme and use it at that temp. For example I use a RE that the OT is 30C it could work at 28C or 33C but the eficiency will begin to lower.