Restriction digestion by SpeI and SphI - (Aug/30/2006 )
Hi, I just joined the forum and have questions to ask again: if I wanna perform a double digestion with SpeI and SphI (Promega), how long shall I incubate the sample? Although I did not try it yet, I heard from my labmate that the digestion may take more than 20 hr. Is that true? anybody ever did this?
I follow the usage information that comes with the REs. Normally, 5U/ug DNA for 1 to 2 hours or more but less than 16 hrs. Have to make sure that DNA conc in the rxn mixture is not too high and viscous. If need be, increase final vol to 50ul or 100ul, esp. when you're digesting >10ug DNA. That'll increase digestion efficiency.
We usually follow the rule 1U/ug of DNA but we try to overdigest by 5-10x so we would add 5-10U/ug of DNA for 1-2 hrs.
Some enzymes even after overdigesting for 20hrs dont chew up the ends so its ok for them, but others u dont want to overdigest else u will have problems with ligation.