Blunt end is ligating to sticky end? - (Oct/27/2013 )
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?
When you generated your new insert, did you purify it or does it still contain the DNA polymerase. Could it be possible that your plasmid is recircularizing with the T4 ligase and then the polymerase is filling in the gap? The ligation would be energy favorable, but would your polymerase function?
Yes, everything (Vector and insert) were purified by gel purification.