Phosphatasing to prevent vector reclosures - (Nov/12/2008 )
I am subcloning an insert into a vector. I am digesting the insert and the vector with both BamHI and NotI. Since these are sticky and not compatible ends, do I need to add phosphatase after digestion?
Yes. Even with 2 different enzymes, I still had the self-ligated plasmid (I extracted only the linear vector), I really don't understand why, but the treatment with phosphatase improved this considerably.