Problem in Vector-Insert digestion result - (Jun/07/2012 )
I cloned 1.4kb insert DNA into my 5.7kb vector. I got colonies and my colony PCR also showed positive results.
But when I digested the cloned plasmid, my result was strange. I got my insert (1.4kb) exactly but the vector was around 4kb.
I used pET15b vector and the restriction sites are overlapping one.
Please let me know what could be the problem?
Are you sure there is no other restriction site in the vector for the enzymes that you used?
If yes, it may undergone a non-specific digestion at some other place in the vector. You can try again by reducing the incubation time.