I am cloning an 4kb mutation insert with a 5' blunt end and 3' sticky end into a 8kb vector (was 12kb, but had 4kb of wild-type removed) that was digested with the same enzymes (AleI and NdeI). This is essentially a "cut and paste" strategy. I am replacing 4kb of wild-type sequence with a 4kb insert that has some mutations. What is very interesting is that 100% of my ligation colonies are around 8Kb in length. This means I have successfully removed the wild-type 4kb and have NOT ligated the "new" 4kb into the vector.
The ONLY way to have a 8kb plasmid would be for the blunt end (AleI) to be ligated to the sticky end (NdeI). If one enzyme was not working I would still have a 12kb plasmid, but that is not the case and 100% of my ligation colonies yield 8kb plasmids.
I am planning on using alkaline phosphatase, but has anybody seen/heard of this? I was under the impression that blunt and sticky ends shouldnt be ligating. Could UV exposure cause this?