Hi guys,

I am trying to insert a 20bp fragment into a vector regardless of the directionality of the fragment. The enzymatic sites I picked are XhoI and MluI. Therefore I added these two sites into my fragments and synthesized both strains, and annealed them together (top figure). The annealing starts from 95 degree to 25 degree, and decreases one degree for each cycle. After annealing, I phosphorylated the annealed products with T4 PNK from NEB.

I then cut the vector witch has a 1.5kb insert with XhoI and MluI and extracted the digested vectors (bottom figure, red box). Then I ligated the vector and fragment at room temp till the evening before I started the transformation. I only used 3ul of ligation products and I saw a decent amount of colonies the next day.

So far everything seems perfect, but then I extracted plasmids from those colonies and did the double digesting with XhoI and MluI, what's weird is I saw that 1.5kb band again, which should be cut out of the vector before the ligation (bottom figure, right). I then tested 7 extra colonies and they all have that 1.5kb insert...

There is definitely something I did wrong, so I would appreciate it if you can spot any mistakes.

Thanks in advance!