SacII digestion - (Aug/17/2007 )
I'm having some problems with a digestion using SacII. I searched the forum and the NEB site and it mentions that there are "slow sites" that can cause problems especially if you are trying to cleave a fragment with just one SacII recognition site (as I am trying to do). I was wondering if anyone has any experience with this and what they did to resolve the problem. So far I am able to "nick" some of the DNA but most remains supercoiled and I don't think any is fully cleaved. Any suggestions? I have tried digesting for 3 hours as suggested on NEB site but no luck.
I would try to digest over night at 4°C. I also find that SacII is not the best enzyme to use, but if you do not have a choice, then try to give it as much time as possible. Good luck!